HIPK2 exhibits context dependent dualistic effects and combinational inhibition of HIPK2-NLK-MAPK11 axis manifests synergism against gastric cancer.

HIPK2 exhibits context dependent dualistic effects and combinational inhibition of HIPK2-NLK-MAPK11 axis manifests synergism against gastric cancer. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article HIPK2 exhibits context dependent dualistic effects and combinational inhibition of HIPK2-NLK-MAPK11 axis manifests synergism against gastric cancer. Gopal Gopisetty, Aathithya Rangarajan, Thirumoorthi Natarajan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7795753/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Homeodomain-interacting protein kinase 2 (HIPK2) can function either as a tumour suppressor or promoter in cancer. Understanding key features of HIPK2 context dependent effects in cancer could lead to novel therapeutic strategies. Our investigation of HIPK2 expression in Gastric Cancer (GC) showed it to be down regulated. HIPK2 expression manifested context dependent effects on cell proliferation, epithelial to mesenchymal transition and anti-apoptotic marker levels as observed in GC cell lines SNU638 and NUGC-3 (β-Catenin low , CD44 Hi ) and AGS (β-Catenin Hi , CD44 low ). The context dependent effects are plausible due to HIPK2's differential impacts on the Akt-mTOR pathway. The application of HIPK2 inhibitor (TBID) further supported context-dependent effect on mTOR signalling markers. To address the context dependent effects of HIPK2, gene expression combined with protein-protein interactions analysis was used to identify the HIPK2-NLK-MAPK11 axis linked by their interaction with transcription factor MYB in GC. Validation of the axis was shown by increased NLK and MAPK11 expression in GC cell lines concordant with lower HIPK2 expression. Similarly, HIPK2 ectopically expressing cells showed lower levels of NLK and MAPK11 expression. Combinational targeting of HIPK2-NLK/MAPK11 axis with (HIPK2i + NLKi) or (HIPK2i + MAPK11i) resulted in a synergistic effect suppressing GC cell proliferation and mTOR markers. Targeting HIPK2 in combination with NLK or MAPK11 can be a promising strategy to overcome context dependent effects of HIPK2. Gastric cancer HIPK2 NLK MAPK11 Tumor Suppressor Kinases Kinase Inhibitors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights of the study • HIPK2 is downregulated in gastric cancer. Its expression exhibits context dependent anti or pro tumorigenic effects in gastric cancer represented by AGS (β-Catenin, CD44) and SNU638 (β-Catenin, CD44) and NUGC3 (β-Catenin, CD44) cells. • HIPK2 expression showed dualistic effects on Akt-mTOR pathway highlighted by differential phosphorylation of Akt, inhibition of mTORC2 arm and activation of mTORC1.Inhibtion of mTOR signalling by HIPK2 inhibitor was context dependant. • A protein network involving HIPK2, NLK, and MAPK11 kinases was identified, linked by a common interaction with MYB in gastric cancer. • Combinational treatment with a HIPK2 inhibitor and either NLK or MAPK11 inhibitor manifests synergism in gastric cancer and inhibited mTOR signalling in both SNU638 and AGS cells. 1. Introduction Homeodomain-interacting protein kinase 2 (HIPK2) is a proline-directed serine/threonine kinase that belongs to the CMGC kinase family. Initially recognised as a homeoprotein transcriptional corepressor, HIPK2 has been found to play a role in various developmental and cellular processes, including neurogenesis, myogenesis, angiogenesis, DNA damage response, cell proliferation, differentiation and apoptosis [ 1 , 2 ]. The role of HIPK2 in cancer is complex, with studies showing it can act as both a tumour suppressor and a tumour promoter, depending on the context. For example, HIPK2 depletion can suppress the p53/E-cad axis, which inhibits metastasis in oral squamous cell carcinoma[ 3 ]. Additionally, its suppression can enhance angiogenesis and promote metastasis in hepatocellular carcinoma [ 4 ]. HIPK2 induction can downregulate vimentin, inhibiting breast cancer cell invasion[ 5 ]. Moreover, HIPK2 phosphorylates CtBP, a C-terminal binding protein that promotes tumorigenesis in osteosarcoma, facilitating its degradation [ 6 ]. HIPK2 also regulates aerobic glycolysis and inhibits cell proliferation in pancreatic cancer by destabilising cMYC [ 7 ]. In contrast, HIPK2 overexpression is associated with decreased overall survival as in HPV-positive tongue squamous cell carcinoma [ 8 ]. HIPK2 also phosphorylates FOXM1, a transcription factor that promotes cell proliferation in renal cell carcinoma [ 9 ]. Additionally, HIPK2 isoform 3 mediates the activation of the Hippo pathway, which promotes lung cancer progression [ 10 ]. HIPK2 expression has been found to be higher in ovarian and cervical cancers, suggesting its involvement in tumour progression [ 11 ]. One possible mechanism for HIPK2’s role in tumorigenesis involves the NRF2 pathway. NRF2 activates HIPK2 at the transcriptional level and activated HIPK2 facilitates the nuclear accumulation of NRF2 and activation of its targets. Under malignant conditions, this HIPK2-NRF2 axis can aberrantly activate cell survival pathways [ 12 ]. Gastric cancer is a significant global health concern, ranking fifth in frequently diagnosed cancers and fourth in cancer-related deaths [ 13 ]. Most patients present at an advanced stage, leading to poor survival rates [ 14 ]. Gastric cancer is categorised into intestinal and diffuse types, which differ in their aetiology, epidemiology, risk factors, clinical behaviour, and molecular alterations [ 15 ]. The PI3K/Akt signalling pathway and its downstream effector mTOR signalling are frequently altered in gastric cancer, contributing to disease progression, metastasis and poor survival [ 16 – 18 ]. This study investigated the expression of HIPK2 in gastric cancer, demonstrated context-dependent effects through full length HIPK2 expression and its inhibition with a HIPK2 kinase inhibitor. Mechanistically, context dependent activity of HIPK2 was found to be due to its differential effects on mTORC1 and mTORC2 arms of the mTOR pathway. The study identified a protein-protein interaction network involving HIPK2-NLK-MAPK11, linked by a common interaction with the transcription factor MYB in GC. The study also reports on the efficacy of targeting HIPK2, NLK and MAPK11, individually and in combinations. A combination of HIPK2 and NLK, and HIPK2 and MAPK11 inhibitors showed synergistic effects in inhibiting gastric cancer cell proliferation and inhibiting mTOR signalling. 2. Materials and Methods 2.1. Patient Samples: Gastric tumor (n = 23) paired normal (n = 17) and normal gastric mucosa (n = 8) were collected from the patients who underwent oesophagogastroduodenoscopy (OGD) at the Cancer Institute (WIA), Chennai for gene expression studies. Paired normal were obtained from adjacent normal tissues to the tumor cells, while normal gastric mucosa was collected from patients without stomach abnormalities. Patients were found to be treatment naive and without any co morbid illness. Archival FFPE Gastric tissue microarray (TMA) samples (Normal = 122, Tumor = 136) were procured from the department of Oncopathology for Immunohistochemical staining of HIPK2. Written informed consent has been obtained from the patients for their participation in the study, and they were found to be treatment naive. The study was conducted following approval from the Institute Ethics Committee (Reference no: IEC/2020/Dec 10). 2.2 Cell Lines: Human Gastric cancer cell lines AGS, SNU638 and NUGC-3 were cultured in RPMI-1640 medium (R-8758,Sigma Aldrich, USA) supplemented with 10% FBS (A5256701,Gibco,USA), 100 mg/L Streptomycin sulphate, and 100 mg/L penicillin G(Himedia, India) at 37°C with 5% CO 2 in a humidified incubator. Cell lines were found to be negative for mycoplasma contamination. 2.3. Immunohistochemical staining: For HIPK2 antibody staining, GC Tissur microarray (TMA) slides were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol (100, 90, 70, and 50% ethanol), followed by antigen retrieval with 10 mM citrate buffer (pH 6). The slides were incubated with 3% H 2 O 2 for 15 minutes, followed by blocking with 3% BSA for 1 hour, and overnight incubation with anti-HIPK2 primary antibody (1:200) at 4°C. Slides were washed with PBST (PBS + 0.1% Tween 20,pH 7.4),incubated with anti-rabbit HRP conjugate secondary antibody for 1 hour at RT, again washed with PBST and developed using the DAKO Envision detection system(K5007,US).The slides were counterstained with haematoxylin, mounted with DPX mountant and visualized under a Leica DMRXA fluorescence microscope. 2.4. RNA Isolation, cDNA Conversion and Quantitative Real Time PCR (qPCR): Total RNA from the cell lines and tissues were isolated using Tri Reagent (T9424,Sigma) following the manufacturer’s protocol. Briefly, GC tumors, paired normal, apparent normal, and GC cell lines were lysed with Tri reagent. For 1 ml of lysates, 100 ul of 1-Bromo-3-Chloropropane(BCP) was added and centrifuged at 10000 rpm for 20 min for phase separation. In the clear aqueous phase, isopropanol was added and centrifuged at 10000 rpm to precipitate the RNA, and the pellet was washed twice with 70% ethanol and resuspended in nuclease-free water. The quality and quantity of RNA were determined using a thermo scientific Nanodrop Lite spectrophotometer. For cDNA conversion, 1 ug of RNA was reverse transcribed into cDNA with random primers using cDNA synthesis kit with a genomic DNA (gDNA) eraser (RR047A,Takara). Briefly, to remove the contaminating DNA, 1 ug of RNAwastreated with a gDNA eraser at 42°C for 2 minutes, followed by reversetranscription. The conditions for reverse transcription were as follows: 37°C for 15 minutes and 85°C for 5 minutes. For gene expression studies, GoTaq SYBR green-based quantitative real-time PCR ( A6001,Promega) in triplicates were performed using the QuantStudio 6 Flex Real-Time PCR system. The PCR conditions were found to be initial heat activation at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 15 minutes, and combined annealing and extension at 60°C for 60 minutes. For the normalisation of genes, GAPDH was used as an internal control. Relative changes in the gene expression level were calculated using the 2–∆∆Ct method 2.5. Transfection: For ectopic expression ,50–70% confluent AGS, SNU638,NUGC-3 cells were transfected withpCMV3-N-FLAG-HIPK2(HG13216-NF) and pCMV3-N-FLAG-Negative control vector(CV016) (Sino biologicals, China) in complete RPMI 1640 medium using XFECT transfection Reagent (631317,Takara,USA) by following the manufacturer's protocol. For CRISPR RNP based genome editing of NLK, the RNP complex(20 pmol NLK gRNA exon 1 with 10 pmol cas9 nuclease )was delivered into AGS cells using lipofectamine RNAImax transfection reagent (13778100 ,Thermofisher scientific, USA) by following the manufacturer's protocol. 2.6.Western Blotting: Transiently transfected pCMV3-N-FLAG-Negative control / pCMV3-N-FLAG-HIPK2 or HIPK2/NLK/MAPK11 inhibitor-treated protein lysates were prepared in the RIPA lysis buffer. The total protein concentration in the lysates was estimated using a standard BCA assay(23227,Pierce BCA protein assay kit,Thermoscientific). Equal amounts of protein (30–50 ug) were resolved in 10% SDS PAGE, transferred into a PVDF membrane at 100 v for 60 mins, blocked with 5% BSA or skim milk for 1 hour, incubated overnight with the primary antibody at 4°C, followed by 1 hour of incubation with the secondary antibody, and developed with Clarity Western ECL substrate (1705060,Bio-Rad ,USA). The signal was then detected and documented using a BioRadChemiDoc MP Imaging System. The loading controls for the western blots which were consecutively probed with different antibodies have been displayed for the first instance for which the blot was probed. To avoid duplication of displaying the loading controls for subsequent antibodies reference to the respective loading control was mentioned in the figure legends. 2.7.Cell proliferation Assay: About 2x10^3 cells of transiently transfected pCMV3-N-FLAG-Negative control / pCMV3-N-FLAG-HIPK2 or HIPK2/ NLK/ MAPK11 inhibitor treated cells were seeded in 96 well plates and assessed for cell proliferation at definite time points in triplicates using AlamarBlue cell viability reagent (DAL1025, Invitrogen,US). Briefly,10 µl of alamarBlue was added into the 96 well plates and incubated at 37°C for 3–4 hours. After incubation, Fluorescence readings were taken at an excitation and emission wavelength of 530–560 and 590 nm. Percentage of cell viability was calculated as (Treatment-Blank)/(Control-Blank) *100.For ectopic HIPK2 expression studies, cell proliferation assay was performed after 48 hours of transient transfection. 2.8.Colony forming assay: The colony formation assay was performed as previously described by Franken et al.[ 19 ] with minor modifications. Briefly,2x10^3 cells of transiently transfected pCMV3-N-FLAG-Negative control / pCMV3-N-FLAG-HIPK2AGS ,SNU638,NUGC-3 cells were seeded in six well plates and incubated for a period of 2 weeks. After colony formation, cells were fixed in ice-cold 100% methanol and stained with 0.5% Crystal violet followed by counting the colonies using image J software. 2.9. Extraction and analysis of metabolites for targeted metabolomics: 1*10^6 cells of transiently transfected AGS and SNU638 cells with pCMV3-N-FLAG-HIPK2 and pCMV3-N-FLAG-Negative control vector (48 hrs) were homogenized in 500ul of methanol spiked with internal standards (10umol/L L-Methionine sulfone,10umol/ 2-(N-Morpholino) ethanesulfonicacid(MES) using MicroDistec TM homogenizer. To that 250ul of ultrapure water was added and thoroughly agitated. For metabolite extraction,400ul of chloroform was added into the 600ul of homogenized suspension solution and centrifuged at 15,000rpm for 15mins at 4°C. Upper clear layer was collected, dried with nitrogen evaporator and reconstituted in 50ul of ultra-pure water.5ul of extracted metabolites were analysed for 143 metabolomic components using Shim-pack (GIST)3uM PFPP (2.1X150mm (HSS)) column with a mobile phase A:0.1% formic acid in water and mobile phase B:0.1% formic acid in acetonitrile. A ten step gradient elution was employed.Separated metabolites were analysed using LCMS-8060 NX Liquid chromatogram and mass spectrometer (Kyoto, Shimadzu) with negative ion ESI. Data analysis was performed using labSolutions software and Multi-Omics data analysis package (Kyoto, Shimadzu). List of metabolites used for analysis were given in supplementary methods. 2.10. Gene expression analysis: Four datasets derived using different gene expression analysis platforms were analysed (E-MEXP-2694-Agilent array[ 20 ],E-GEOD-17154- Stanford array[ 21 ], E-MTAB-1338-Illumina HumanHT-12 V4.0 expression bead chip- Transcription profiling by array[ 22 ], E-GEOD-79973-Affymatrix array Expression data from gastric cancer and paired normal tissues[ 23 ]. Workflow used for analysis of data was that gene expression data sets were reprocessed using individual gene expression analysis algorithms specific for platforms in R .The expression sets were background adjusted, normalised, annotated. The differentially expressed genes were derived using the Linear Model of Microarray (LIMMA) algorithm following the construction of a contrast matrix. The differentially expressed gene set was extracted and searched for differential expression of Tumor suppressor kinase gene set. 2.11. Gene Co-expression analysis with Protein-Protein Interactions (PPI) using CEMi tool. The gene expression files along with phenotype data were assessed in CEMi tools ( https://cemitool.sysbio.tools/ ) to identify the gene expression modules differentiating cancer and normal gene expression profiles for the four gene expression datasets. The protein-protein interactions for the shortlisted expressed kinase genes and HIPK2 were extracted from Protein Interaction Network Online Tool (PINOT) a PPI database ( http://www.reading.ac.uk/bioinf/PINOT/PINOT_form.html ). The interactions were assessed individually for the kinase gene set and HIPK2 along with the gene expression and phenotype files. The analysis yielded a shortlist of interactions by mapping the PPI’s to gene expression. 2.12. Inhibitor treatment Studies: HIPK2 inhibitor TBID(HY-100464), NLK inhibitor Vx-702(S6005) and MAPK11 inhibitor SB202190(sc-222294) were purchased from MCE, USA; Selleck chemicals, USA and Santacruz, USA respectively. The stock solutions of the inhibitors were prepared according to the manufacturer’s instructions. For dose response studies, post 24 hours of seeding 2*10^3 cells of AGS,SNU638,NUGC-3 were treated with either individual or combinations of inhibitors: TBID, Vx-702 and SB202190. Cell viability was measured at definite time points by cell proliferation assay. To assess whether the combined effects of inhibitor treatment were synergistic (CI 1), the combination index (CI) was calculated using the Bliss independence model with Compusyn software. For protein and gene expression studies, post 24 hours of seeding, 3*10^5 cells of AGS and SNU638 were treated with HIPK2/NLK/MAPK11 inhibitor. To study the expression status of p-P70S6K, GC cells were grown in serum starved medium for 24 hours, treated with 100 ng/ml hIGF-1 for 10 mins, followed by combinational inhibitor treatment (24 hours). 2.13. Apoptosis detection by Flow cytometry: For apoptosis detection, 3x10 5 inhibitor treated cells were collected and washed with ice cold PBS followed by Annexin V FITC/PI staining for 15 mins in dark at room temperature(RT) by following the manufacture’s protocol (sc-4252 AK, Santacruz). About 20000 events per sample were acquired using Cytoflex Flow cytometer (Beckman Coulter). Gating was performed with forward scatter and side scatter of unstained cells. Data were processed using cytexpert 2.5 software to determine the percentage of early apoptotic (FITC + PI − ), late apoptotic/necrotic (FITC + PI + ) cells. 2.14. Statistical Analysis: Statistical analysis was conducted using GraphpadPrism(RRID:SCR_002798). Unpaired two tailed t-tests and one-way ANOVA followed by Bonferroni multiple comparison tests and chi-square test were employed to determine statistical significance (p < 0.05 *, p < 0.01 **, p < 0.001 *** ). Densitometric analysis of blot images was performed using ImageJ software. 2.15. Data Availability Statement. Data generated in this study are available within the article and its supplementary data files. 3. Results 3.1. HIPK2 is downregulated in gastric cancer. Putative tumor suppressor kinase gene expression (~ 40 genes) [ 24 ] was studied in gene expression datasets derived from gastric cancer. Analysis of transcription profiling in gastric cancer cell lines, normal gastric mucosa, malignant gastric tissues and adjacent non-cancerous mucosa, showed that HIPK2 gene expression was downregulated in both tumours and cell lines, when compared to normal gastric tissues (Supplementary Fig. 1A, Table.S1). To validate these findings, we examined a panel of 10 downregulated tumour suppressor (TS) kinases by Real-time PCR, of which 8 TS Kinases, including HIPK2, were confirmed to be downregulated in our sampleset (Supplementary Fig. 1B). For HIPK2, qPCR analysis was initially performed in gastric cancer cell lines (n = 3), treatment naive tumours (n = 23), paired normal tissues (n = 17), and apparent normal gastric mucosal biopsies (n = 8). Among these, only samples with high-quality RNA were included for analysis, resulting in a final cohort of GC cell lines (n = 3), tumour tissues (TR, n = 17), paired normal tissues (PN, n = 11), and apparent normal tissues (AN, n = 8).The Clinicopathologic characteristics of GC Patients used for gene expression analysis has been summarized in Table.1. Notably, it was observed that HIPK2 gene expression was found to be downregulated in both tumours and cell lines when compared to paired normal and apparent normal gastric tissues (Fig. 1 A). Additionally, HIPK2 protein expression was significantly lower in tumour tissue than in normal gastric epithelium (Fig. 1 B-C). Furthermore, HIPK2 was found to be located predominantly in the nucleus of gastric tumours, while it is located in the cytoplasm of normal gastric epithelium (Table.2). 3.2. HIPK2 expression elicits context dependent effects on GC cells. To investigate the role of HIPK2 in gastric cancer (GC), GC cell line models, AGS ,SNU638, NUGC-3 were studied. These cell lines differed in their expression of CD44 and β-catenin; AGS cells have low CD44 and high β-catenin levels, whereas SNU638 and NUGC-3 cells have high CD44 and low β-catenin levels (Fig. 1 D). The selection of the CD44 and β-catenin as markers for context was based on their mutually exclusive or inverse patterns of expression in GC marking context and tumor heterogenity. The effects of HIPK2 were examined through transient ectopic expression. Transient expression of FLAG-HIPK2 in GC cell lines were confirmed by western blotting. Ectopic expression of FLAG-HIPK2 induced p53 in both AGS and SNU638 and phospho-p53(ser46) in SNU638 cells indicating the functional status of FLAG/HIPK2 (Fig. 1 E-G). Transient HIPK2 expression decreased cell proliferation and significantly decreased colony forming ability in SNU638 and NUGC-3 cells but had no significant impact on AGS cells (Fig. 1 H-K). Next, the impact of HIPK2 on epithelial-mesenchymal transition (EMT) markers was studied. At the transcriptional level in SNU638 and NUGC-3 cells, compared to the pCMV3-N-FLAG control, ectopic expression of pCMV3-N-FLAG-HIPK2 led to the differential expression of EMT markers, characterized by a marked downregulation of mesenchymal marker-vimentin, while in AGS cells upon FLAG-HIPK2 ectopic expression, there was no differential expression between epithelial and mesenchymal markers suggesting that HIPK2 overexpression modulates EMT status prominently in SNU638, NUGC-3 cells than in AGS (Fig. 2 A). In SNU638 cells at protein level we observed increased expression of E-Cadherin, and decreased expression of mesenchymal markers (N-Cadherin, αSMA and Vimentin). Additionally, there was also a decreased expression of other markers such as β-catenin, α-catenin. Concomitant with decrease in β-catenin, HIPK2 expression decreased the level of cancer stem cell marker CD44 (Fig. 2 B-C), similarly in NUGC-3 cells at protein levels HIPK2 overexpression induces E-Cadherin expression, hence, based on our results it was determined that HIPK2 overexpression plausibly suppresses EMT in SNU638 and NUGC3 cells. AGS cells in contrast at protein expression level, there was a decrease in β-catenin levels however there were no significant change in the levels of α catenin or αSMA (Fig. 2 D-E) indicating that HIPK2 may not affect EMT status in AGS cells. Taken together all of this indicates that effects of HIPK2 on cell proliferation, colony formation, EMT status are context dependent in GC. Next, we selected AGS and SNU638 cells to assess for context based molecular effects of HIPK2 expression. 3.3 HIPK2 expression elicits dualistic effect on AKT/mTOR signalling in SNU638 cells. Since PI3K/Akt signalling pathway and its downstream effector mTOR signalling are frequently altered in gastric cancer, pCMV3-N-FLAG control or pCMV3-N-FLAG-HIPK2 expressing SNU638 lysates were assessed for markers of AKT/mTOR signalling. Increased HIPK2 expression lead to an increase in p-Akt (Thr308) and a decrease in p-Akt (Ser473). This was associated with a decrease in p-GSK-3β (Ser9), p-PTEN (Ser380), and p-PDK1 (Ser241), and an increased level of p-cRaf (Ser259) (Fig. 3 A-B). Additionally, HIPK2 expression resulted in reduction in anti-apoptotic proteins like Bcl-2, Bcl-xL, Mcl-1, and p-Bcl2 (Ser70), suggesting an increase in the apoptotic potential. This effect was further supported by an observed increase in PHLPP1 levels, a phosphatase that dephosphorylates p-Akt (Ser473) a marker for anti-apoptotic effects of Akt (Fig. 3 C). Next, we assessed how HIPK2 impacts the mTOR paṭhway. A decrease in RICTOR expression, relative to RAPTOR, indicated that the mTORC2 arm of the mTOR pathway is inhibited, consistent with decreased p-Akt (Ser473) levels and increased PHLPP1. Conversely, the mTORC1 arm appears active with increased pp70S6K(T389) level and a decrease in autophagy-related gene expression (Fig. 3 D-E). Thus, HIPK2 expression has a dualistic effect, increasing the apoptotic potential of cells while also promoting signals that support cell proliferation in SNU638 cells. 3.4. HIPK2 expression increases pro-tumorigenic AKT/mTOR signalling in AGS cells. HIPK2 expression in AGS led to an increase in the levels of p-Akt (Ser473), while p-Akt (Thr308) remained unchanged (Fig. 3 F). HIPK2 expression led to reduced PHLPP1 and increased RICTOR, indicating active mTORC2 (Fig. 3 G-H ). The activity of mTORC2 was further suggested by the induction of the anti-apoptotic marker Mcl-1 (Fig. 3 G ). There was also evidence that mTORC1 is active, with increased levels of pp70S6K(T389) and decreased expression of autophagy related genes (UVRAG,ATG9B,LAMP1,MCOLN1) (Fig. 3 G-H). In addition, increased levels of p-GSK-3β(Ser9) and p-c-Raf (Ser259) were observed, indicating an active Akt status. Conversely, decreased levels of p-PTEN(Ser380) suggested that HIPK2 suppressed upstream effectors of Akt signalling while maintaining active Akt (Fig. 3 I). These results suggested that HIPK2 expression in AGS cells may predominantly promote AKT/mTOR pathway. 3.5. Targeted metabolomics indicates activation of mTOR and HIPK2 inhibition exhibits context dependent suppression of mTOR signalling : Our data indicated that the expression of HIPK2 leads to the potential activation of mTORC1 in both AGS and SNU638 cells. Further in order to ascertain the potential activation status of mTOR we determined the levels of metabolite targets of mTOR such as in nucleotide metabolism and amino acid metabolism in AGS and SNU638 cells during transient ectopic expression of FLAG/HIPK2. In total 143 metabolites were analysed, under the conditions tested we identified differential response in the metabolite levels in both AGS and SNU638 cells ectopically expressing CMV-FLAG/HIPK2 relative CMV-FLAG alone expressing cells (Supplementary Fig. 2, Table.S2-3). In both AGS and SNU638 cells, we observed a significant increase in glutamine, leucine, guanosine, and guanosine monophosphate, known markers of mTORC1 [ 25 – 26 ], which indicates an active mTOR upon HIPK2 expression (Fig. 4 A). We further investigatedthe effects of HIPK2 kinase inhibition in GC cells using TBID, a HIPK2 inhibitor [ 27 ]. For this the levels of p53 and p-P53(Ser 46) were assessed following treatment with TBID and it was observed that increasing the concentration of TBID reduced HIPK2 kinase activity which was shown by downregulation of p-P53(Ser 46) though there was no significant changes in the levels of total p53 (Fig. 4 B) indicating that TBID treatment suppressed HIPK2 kinase under the conditions tested. In AGS cells, treatment with the HIPK2 inhibitor TBID increased the expression levels of HIPK2, RICTOR, RAPTOR, and phosphorylated p70S6K (T389), without affecting β-catenin levels. This suggests a possible activation of the mTOR pathway, potentially mediated by transcriptional upregulation of HIPK2 despite inhibition of its kinase activity. Whereas, in SNU638 cells, the TBID decreased the expression levels of HIPK2,RICTOR, RAPTOR, and p-p70S6k(T389) along with a concomitant reduction in CD44 levels which is known to vary with mTOR activity [ 28 ] this indicated mTOR inactivation upon HIPK2 inhibition (Fig. 4 C-E). Therefore, consistent with overexpression studies which demonstrated context-dependant effects, treatment with a HIPK2 inhibitor induced context-dependent activity by elicitng differential effects on mTOR signalling. 3.6. Identification of HIPK2, NLK and MAPK11 axis. Next, we wanted to assess kinases which are potentially linked to HIPK2 Kinase and expressed at higher levels that could possibly serve as targets in GC cells considering that HIPK2 was downregulated in GC. For which we implemented a bioinformatic analysis workflow which focuses on kinase interacting proteins and gene co-expression (Fig. 5 A). Bioinformatic analysis identified a protein-protein interaction network involving HIPK2, NLK, MAPK11, and TRIM28, all linked by a common interaction with MYB (Fig. 5 B,Supplementary Fig. 3-4A). Differential gene expression analysis revealed that HIPK2 expression was lower than MYB, NLK, MAPK11 in gastric cancer cell lines relative to normal gastric mucosa (Fig. 5 C). This suggested a potential negative regulatory role of HIPK2 on partner genes in the axis. Overexpression of HIPK2 in gastric cancer cells led to downregulation of NLK and MAPK11 at both protein and gene expression levels (Fig. 5 D, Supplementary Fig. 4B-C). Although cMYB expression was unaffected, two of its transcriptional targets, cMYC and STAT5A, were downregulated (Supplementary Fig. 4D). Knockdown of NLK using CRISPR CAS9 resulted in increased HIPK2 expression at both mRNA and protein expression levels, accompanied by elevated levels of p53 and pP53(Ser46) (Fig. 5 E-F). Additionally when treated with inhibitor of MAPK11(SB202190) or NLK(Vx-702) the treatment activated HIPK2 indicating that expression levels of HIPK2 on one hand and NLK and MAPK11 on the other are coordinately regulated (Fig. 5 G-H). 3.7. Effects of HIPK2 /MAPK11/ NLK Inhibition in Gastric Cancer. Treatment with HIPK2 inhibitor (TBID) induced a dose and time dependent inhibition of AGS and SNU638 cells which express HIPK2 protein albeit at lower levels, indicating that inhibition of HIPK2 kinase activity can suppress cell proliferation (Fig. 6 A). Further we determined the levels of anti-apoptotic markers upon inhibitor treatment, in both the cell lines we observed decreased Bcl-xL and increased Mcl-1 expression, suggesting that HIPK2 inhibition alone may not suppress anti-apoptotic signalling (Fig. 6 B). Since our observation of HIPK2, NLK and MAPK11 expression in gastric cancer celllines revealed that the expression levels of NLK and MAPK11 were higher than HIPK2 we wanted to assess the efficacy of targeting NLK and MAPK11 on GC cell proliferation. AGS and SNU638 cells were resistant to the NLK inhibitor(Vx-702) [ 29 ], but the MAPK11 inhibitor (SB202190) [ 30 ] significantly decreased cell proliferation in both cell lines (Fig. 6 C-F). Anti-apoptotic markers Bcl-xl and Mcl-1 were found to be decreased in both cell lines upon NLK inhibitor treatment (Fig. 6 G). The MAPK11 inhibitor decreased Mcl-1 levels and increased Bcl-xL in SNU638 cells, while in AGS cells, Mcl-1 levels decreased with no significant change in Bcl-xL (Fig. 6 H). 3.8. Combinational inhibitor treatment comprising of HIPK2-MAPK11/NLK Inhibitor manifests synergism in GC Our studies using individual inhibitors targeting NLK, MAPK11 and HIPK2 revealed that single agent treatments exhibit differential effects in GC cells. For instance, an NLK inhibitor decreased anti-apoptotic markers but did not affect cell proliferation, whereas a MAPK11 and HIPK2 inhibitor exhibited toxicity but also induced anti apoptotic marker levels. A previous study had shown that the sensitivity to the NLK inhibitor (Vx-702) increased upon mTOR inhibition [ 29 ]. Since TBID suppressed mTOR signaling in SNU638 cells, we explored whether TBID could enhance the sensitivity of Vx-702 in GC. Additionally, since induction of antiapoptotic markers were complementary between TBID and SB202190, a combination of TBID and SB202190 was tested. In both AGS and SNU638 cells combinational inhibitor testing showed that combining TBID with either Vx-702 or SB202190 inhibitors led to pronounced reductions in cell viability compared to individual inhibitor treatments. The combination index (CI) values, calculated using CompuSyn software [ 31 ], were consistently indicative of the synergistic effect for these combinations (Fig. 7 A-D,Table.3–6, Supplementary Fig. 5). This synergistic activity of the combinatorial inhibitor treatment comprising TBID with either Vx-702 or SB202190 was further demonstrated in NUGC-3 cells. (Supplementary Fig. 6,Table.S4-5). Furthermore, Annexin V/PI staining revealed that AGS cells treated with either individual or combinations of TBID (50uM) and Vx-702 (50uM) / SB202190(50uM) inhibitors for 24 hrs exhibited 2.62% (TBID), 0.23% (Vx-702 ), 1.44% (SB202190), 8.91% (TBID and Vx-702), 22.17% (TBID + SB202190) of early apoptotic cells (FITC + PI − ) compared to control, similarly SNU638 cells showed 4.53%(TBID), 5.09% (Vx-702 ),11.28% (TBID and Vx-702),17.89% (TBID + SB202190) of early apoptotic cells (FITC + PI − ) compared to control indicating effectiveness of these combinations over individual treatments (Fig. 7 E-G). In our analysis combinations of NLK and MAPK11 inhibitors did not show synergistic potential in cell proliferation assays (Supplementary Fig. 7A-B,Table.S6). In summary, our results suggest that targeting HIPK2 in combination with either NLK or MAPK11 offers a viable strategy to induce a synergistic effect in GC cells. 3.9. Combinational inhibitor treatment comprising of HIPK2 – NLK/MAPK11 Inhibitor mitigates mTOR signalling context independently. Next, we assessed the levels of molecular markers in response to combined treatment of HIPK2 with NLK or MAPK11 inhibitors in both AGS and SNU638 cells. Combining HIPK2 inhibitors with either NLK or MAPK11 inhibitors led to a reduction in the expression levels of genes such as cMYB, Myc, RICTOR, RAPTOR in both AGS and SNU638 cell lines(Fig. 8 A-B). Combined treatments of HIPK2 with NLK or MAPK11 inhibitors resulted in a reduction in total p70S6K levels in both AGS and SNU638 cells. Furthermore, in AGS cells treated with IGF1 known to activate mTOR to promote processes like protein synthesis and cell proliferation [ 32 ], the combination of HIPK2 and MAPK11 inhibitors decreased levels of p-p70S6K (Thr389), suggesting an impact on mTOR activity (Fig. 8 C-D). The study also demonstrated that in SNU638 cells, combined inhibitor treatment with HIPK2 and MAPK11 /NLK inhibitors resulted in a more pronounced reduction in CD44 levels when compared to individual inhibitor treatment.Together, this combined inhibitor treatment decreased β-Catenin levels in AGS cells compared to inifividual treatment (Fig. 8 E-F, Supplementary Fig. 7C).These markers β-Catenin [ 33 ] and CD44 are known to be associated with the status of mTOR signalling and their decrease could indicate that indeed the combination treatment suppresses mTOR under the two different contexts tested. Overall, the combinatorial approach possibly addressed the context-dependent nature of individual inhibitor effects representing a promising therapeutic approach for gastric cancer. 4. Discussion HIPK2 exhibits a complex, dual role in cancer, displaying both features of a tumour suppressor and a tumour promoter, and its effects being highly dependent on the context. Taking into consideration from the previous studies we assessed the context dependent role of HIPK2 based upon molecularly distinguishable expression levels of β-Catenin and CD44 in GC cell lines AGS, SNU638 and NUGC-3. CD44 a cell surface marker of Cancer Stem Cell (CSC) was found to promote tumorigenesis in several cancers such as gallbladder, breast and gastric cancer [ 34 – 36 ]. Similarly β-catenin promotes tumor progression in colon, glioma and gastric cancer[ 37 – 39 ]. In this study in both SNU638 and NUGC-3 cells, ectopic expression of HIPK2 lead to a decrease in cell proliferation, suppressed colony forming ability and epithelial to mesenchymal transition (EMT). These results were found to be consistent with previous studies which indicate that HIPK2 overexpression downregulates vimentin [ 5 ], and its knockdown increases N-cadherin, Fibronectin, decreases E-cadherin which potentiates EMT [ 40 ]. Similarly, Zhang, Na et al demonstrated the downregulation of HIPK2 in GC and potentially inhibited the EMT, migration, and invasive properties of GC[ 41 ]. Consistent with previous studies which showed that HIPK2 suppresses β-catenin [ 42 ] in our studies we found that the HIPK2 expression suppresses β-catenin and additionally α and γ catenin levels especially in SNU638 cells. Moreover, HIPK2 expression increased apoptotic potential by downregulating the mTORC2 while sustaining mTORC1 activity, indicating a probable dualistic effect. In AGS cells, the effects of HIPK2 are markedly different. The expression of HIPK2 did not alter the relative expression status of EMT markers and there was no change in the levels of mesenchymal marker α SMA, and activated Akt/mTOR pathway suggested pro-tumorigenic activity. Consistent with SNU638 cells HIPK2 expression decreased β-catenin levels which indicated HIPK2 activity in both type of cells since HIPK2 can reduce β-catenin levels by phosphorylation dependant degradation. Since stability of β-catenin affects CD44 levels [ 43 ] the loss of β-catenin possibly leads to a decrease in CD44 levels in SNU638 cells. A similar case of context dependent activity was previously observed with SP1 gene in gastric cancer. Knockdown of SP1 in intestinal type promoted migration and invasion whereas in diffuse type it was reduced [ 44 ]. Mechanistically, the context dependent effects could plausibly be explained by HIPK2's dualistic effects on the PI3K/AKT and mTOR pathways. In SNU638 cells, HIPK2 expression lead to differential phosphorylation of Akt whereas in AGS HIPK2 expression the Akt markers were indicative of an active Akt pathway. The mTOR pathway, which is critical for cell proliferation and survival [ 45 ], was also differentially affected. In SNU638 cells, HIPK2 expression downregulated the mTORC2 arm, increasing apoptotic potential, while sustaining mTORC1 activity conversely in AGS cells, HIPK2 expression activated both mTORC1 and mTORC2. The activity of mTORC1 was further confirmed by assessing for metabolite targets of mTOR in both SNU638 and AGS. In summary our findings demonstrated that HIPK2 overexpression activates mTORC1 signalling in AGS and SNU638 cells. These results led us to target mTOR by inhibiting HIPK2 with TBID. Intriguingly, treatment with TBID also activated HIPK2 expression and this was concomitant with the activation of markers for mTOR signalling in AGS cells. SNU638 cells in contrast upon TBID treatment showed a decrease in HIPK2 and markers for mTOR signalling. Intrestingly the treatment with TBID showed similar effects as HIPK2 overexpression in AGS cells. We hypothesise that the activation of mTOR marker expression in AGS cells despite HIPK2 kinase inhibition is due to the transcriptional upregulation of HIPK2 observed upon inhibitor treatment. This is plausible, since the domain structure of HIPK2 comprises a C-terminal Homeodomain-Interacting Domain (HID) for transcriptional factors which contributes to its transcriptional activity [ 46 ]. Additionally we observed that that β-catenin levels in AGS is unaltered which is due to inhibitor treatment indicating inhibtion of HIPK2 kinase activity. All of this indicates that targeting HIPK2 with TBID alone to suppress mTOR will be hindered by context dependent effects. In SNU638 cells, overexpression of HIPK2 suppresses cell proliferation, potentially through the degradation of CtBP1-a pro-tumorigenic transcriptional corepressor known to repress E-cadherin expression in gastric cancer [ 6 , 47 ]. Conversely, inhibition of HIPK2’s kinase activity also suppress cell proliferation, likely due to its role in facilitating KRAS signaling and tumor progression. Treatment with the HIPK2 inhibitor TBID attenuates KRAS–ERK pathway activation [ 48 ]. Thus, both HIPK2 over expression and kinase activity inhibition suppress cell proliferation, demonstrating its dual regulatory role in gastric cancer. A combined treatment approach was applied to overcome the context dependant effects of HIPK2 inhibition for this, we assessed kinases which are potentially linked to HIPK2 Kinase and serve as targets in GC cells. Using bioinformatic analysis we identified HIPK2 interaction with proteins involved in cancer-related processes, including NLK, MAPK11, MYB and TRIM28 in GC.The HIPK2-NLK-MAPK11 axis was marked by their common interaction with MYB. In a similar way Kanei-Ishii et al. identified a Wnt signalling interaction axis consisting of TAK1- HIPK2-NLK-MYB which facilitates cMYB proteosomal degradation[ 49 ]. All of this opened up the possibility that the HIPK2 /NLK/MAPK11 axis is a viable target in GC. Treatment with an inhibitor of HIPK2 or NLK or MAPK11 showed that the treatment elicited differential effects especially in the response of anti-apoptotic markers. The study further investigated the potential for combined treatment strategies. The case for a combination of HIPK2 and NLK inhibitors was based on the previous studies which showed that inhibition of mTOR can sensitise cells to NLK inhibitor. The case for HIPK2 and MAPK11 inhibitors was based on the complementary effects on the levels of anti-apoptotic markers Bcl-xl and Mcl-1. The results from our combination studies showed synergism for both the combinations of HIPK2/NLK and HIPK2/MAPK11 concomitant with reduction in the mTOR pathway markers in both cell lines tested. A combination approach is likely to be more effective considering that several studies have demonstrated the resistance to individual inhibitor treatment. Possibly due to the activation of an alternative signalling pathway or feedback loop which would possibly be counteracted with combinatorial treatment. For instance Thakuri, et al. demonstrated that the MAPK inhibitors (MAPKi) develops resistance in colorectal cancer due to the activation of PI3K/AKT/mTOR pathway, the combinatorial inhibitor treatment comprising of MAPKi with PI3K/mTOR inhibitor exhibits synergy in suppressing the growth of colorectal cancer cells [ 50 ], Dual inhibitor treatment with RAD001 and Volasertib, a PLK1 inhibitor, targeting mTOR and PLK demonstrated synergistic anti-tumor effects in non-small cell lung cancer (NSCLC) [ 51 ]. Zhu, Ruiqi et al. demonstrated that Targeting AML by FLT3 tyrosine kinase inhibitors (TKIs) together with Venetoclax, a Bcl-2 inhibitor exhibited synergy in inhibiting cell proliferation by counteracting the pro-survival pathways activated by FLT3 TKIs [ 52 ]. Consistent with previous studies in our study, unlike individual inhibitor treatments, the response to combinatorial treatment (HIPK2 inhibitor with either a NLK or MAPK11 inhibitor) was consistent across the cell lines tested, suggesting a potential to overcome the context-dependent activity of individual inhibitors. 5. Conclusion In summary, the dual role of HIPK2 in gastric cancer is influenced by the context and has impacts on cell proliferation, EMT, and key signalling pathways such as PI3K/AKT and mTOR. Significantly our study demonstrates the dualistic effects of HIPK2 on mTOR singalling. The understanding of context dependent activity of HIPK2 and the identification of HIPK2-NLK-MAPK11 axis led us to target mTOR context independently using a combinational approach. Our study has demonstrated that targeting HIPK2 in combination with NLK or MAPK11 can be a promising strategy to overcome context dependent effects of HIPK2 inhibition. Declarations 8. Conflict of Interest: There was no conflict of interest. 7. Authors contributions: Gopal Gopisetty - conception and design; analysis and interpretation of data, drafting /revising the article(lead), Aathithya Rangarajan-acquisition of data; analysis and interpretation of data, drafting /revising the article (equal), Jayavelu Subramani-acquisition of LC-MS data, Priya Ramanathan- acquisition of Flow cytometry data,RamakrishnanAyloor Seshadri, Thirumoorthi Natarajan, Sujatha Lakshminarayanan – Sample resource, Shirley Sunder Singh- analysis and interpretation of IHC data. 6. Acknowledgements: This study was financially supported by the Department of Science and Technology's Science and Engineering Research Board (DST-SERB) grant (CRG/2020/000877). The targeted metabolomics study was possible due to the kind donation of mass spectrometry equipment and related software by SPINCO technologies (Chennai, India) and Shimadzu (Kyoto, Japan) to the Cancer Institute (WIA). References Agnew C, Liu L, Liu S, Xu W, You L, Yeung W, Kannan N, Jablons D, Jura N. The crystal structure of the protein kinase HIPK2 reveals a unique architecture of its CMGC-insert region. J Biol Chem. 2019;294(37):13545–59. 10.1074/jbc.ra119.009725 . Zhou Q, Meng D, Li F, Zhang X, Liu L, Zhu Y, et al. Inhibition of HIPK2 protects stress-induced pathological cardiac remodeling. EBioMedicine. 2022;85:104274. 10.1016/j.ebiom.2022.104274 . Zheng X, Pan Y, Chen X, Xia S, Hu Y, Zhou Y, et al. Inactivation of homeodomain-interacting protein kinase 2 promotes oral squamous cell carcinoma metastasis through inhibition of P53-dependent E-cadherin expression. Cancer Sci. 2021;112(1):117–32. 10.1111/cas.14691 . Chen P, Duan X, Li X, Li J, Ba Q, Wang H. HIPK2 suppresses tumor growth and progression of hepatocellular carcinoma through promoting the degradation of HIF-1α. Oncogene. 2020;39(14):2863–76. 10.1038/s41388-020-1190-y . Nodale C, Sheffer M, Jacob-Hirsch J, Folgiero V, Falcioni R, Aiello A, et al. HIPK2 downregulates vimentin and inhibits breast cancer cell invasion. Cancer Biol Ther. 2012;13(4):198–205. 10.4161/cbt.13.4.18694 . Duan N, Zhang W, Li Z, Sun L, Song T, Yu Z, et al. Overexpression of HIPK2 removes the transrepression of proapoptotic genes mediated by the CtBP1-p300-FOXO3a complex and increases the chemosensitivity in osteosarcoma cells. J Cancer. 2021;12(6):1826–37. 10.7150/jca.52115 . Qin Y, Hu Q, Ji S, Xu J, Dai W, Liu W, et al. Homeodomain-interacting protein kinase 2 suppresses proliferation and aerobic glycolysis via ERK/cMyc axis in pancreatic cancer. Cell Prolif. 2019;52(3):e12603. 10.1111/cpr.12603 . Kwon MJ, Kang SY, Nam ES, Cho SJ, Rho YS. HIPK2 overexpression and its prognostic role in human papillomavirus-positive tonsillar squamous cell carcinoma. Biomed Res Int. 2017;1056427. 10.1155/2017/1056427 . Liu F, Li N, Liu Y, Zhang J, Zhang J, Wang Z. Homeodomain interacting protein kinase-2 phosphorylates FOXM1 and promotes FOXM1-mediated tumor growth in renal cell carcinoma. J Cell Biochem. 2019;120(6):10391–401. 10.1002/jcb28323 . Dai Y, Kyoyama H, Yang YL, Wang Y, Liu S, Wang Y, et al. A novel isoform of homeodomain-interacting protein kinase-2 promotes YAP/TEAD transcriptional activity in NSCLC cells. Oncotarget. 2021;12(3):173–84. 10.18632/oncotarget27871 . Conte A, Valente V, Paladino S, Pierantoni GM. HIPK2 in cancer biology and therapy: Recent findings and future perspectives. Cell Signal. 2023;101:110491. 10.1016/j.cellsig.2022.110491 . Torrente L, Sanchez C, Moreno R, Chowdhry S, Cabello P, Isono K, et al. Crosstalk between NRF2 and HIPK2 shapes cytoprotective responses. Oncogene. 2017;36(44):6204–12. 10.1038/onc.2017.221 . Ilic M, Ilic I. Epidemiology of stomach cancer. World J Gastroenterol. 2022;28(12):1187–203. 10.3748/wjg.v28.i12.1187 . Dikshit RP, Mathur G, Mhatre S, Yeole BB. Epidemiological review of gastric cancer in India. Indian J Med Paediatr Oncol. 2011;32(01):3–11. 10.4103/0971-5851.81883 . Petrelli F, Berenato R, Turati L, Mennitto A, Steccanella F, Caporale M, et al. Prognostic value of diffuse versus intestinal histotype in patients with gastric cancer: a systematic review and meta-analysis.JGastrointest. Oncol. 2017;8(1):148–63. 10.21037/jgo.2017 . .01. 10. Singh SS, Yap WN, Arfuso F, et al. Targeting the PI3K/Akt signaling pathway in gastric carcinoma: A reality for personalized medicine? World J Gastroenterol. 2015;21(43):12261–73. 10.3748/wjg.v21.i43.12261 . Marques-Ramos A, Cervantes R. Expression of mTOR in normal and pathological conditions. Mol Cancer. 2023;22(1):112. 10.1186/s12943-023-01820-z . Advani SH. Targeting mTOR pathway: A new concept in cancer therapy. Indian J Med Paediatr Oncol. 2010;31(04):132–6. 10.4103/0971-5851.76197 . Franken N, Rodermond H, Stap J, et al. Clonogenic assay of cells in vitro . Nat Protoc. 2006;1:2315–9. https://doi.org/10.1038/nprot.2006.339 . Tong J. Transcription profiling by array of human gastric cancer cell lines and normal gastricmucosa.BioStudies.2011.E-MEXP-2694.Retrieved fromhttps://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MEXP-2694 Rajkumar T, Rajkumar T. Gene expression studies in apparently normal paired normal and malignant gastric tissues. BioStudies .2010.E-GEOD-17154.Retrieved from https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-GEOD-17154 FlatbergA. Gene expression profiling of human gastric tumors and adjacent non-cancerous mucosa. BioStudies .2014.E-MTAB-1338.Retrieved from https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-1338 Shao Q, Yao H, He J. Expression data from gastric cancer and paired normal tissues. BioStudies .2016.E-GEOD-79973.Retrieved from https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-GEOD-79973 An E, Brognard J. Orange is the new black: Kinases are the new master regulators of tumor suppression. IUBMB Life. 2019;71(6):738–48. 10.1002/iub.1981 . Durán RV, Oppliger W, Robitaille AM, Heiserich L, Skendaj R, Gottlieb E, et al. Glutaminolysis activates RAG-MTORC1 signaling. Mol Cell. 2012;47(3):349–58. 10.1016/j.molcel.2012.05.043 . Emmanuel N, Ragunathan S, Shan Q, Wang F, Giannakou A, Huser N, et al. Purine nucleotide availability regulates mTORC1 activity through the Rheb GTPase. Cell Rep. 2017;19(13):2665–80. 10.1016/j.celrep.2017.05.043 . Cozza G, Zanin S, Determann R, Ruzzene M, Kunick C, Pinna LA. Synthesis and properties of a selective inhibitor of homeodomain-interacting protein kinase 2 (HIPK2). PLoS ONE. 2014;9(2):e89176. 10.1371/journal.pone.0089176 . Published 2014 Feb 24. Yang C, Zhang Y, Zhang Y, Zhang Z, Peng J, Li Z, et al. Downregulation of cancer stem cell properties via mTOR signaling pathway inhibition by rapamycin in nasopharyngeal carcinoma. Int J Oncol. 2015;47(3):909–17. 10.3892/ijo.2015.3100 . Wang X, Veeraraghavan J, Liu CC, Cao X, Qin L, Kim JA, et al. Therapeutic targeting of nemo-like kinase in primary and acquired endocrine-resistant breast cancer.Clin. Cancer Res. 2021;27(9):2648–62. 10.1158/1078-0432.CCR-20-2961 . Roche O, Fernández-Aroca DM, ,Arconada-Luque E, García-Flores N, Mellor LF, Ruiz-Hidalgo MJ, et al. p38β and Cancer: The Beginning of the Road. Int J Mol Sci. 2020;21(20):7524. 10.3390/ijms21207524 . Duarte D, Guerreiro I, Vale N. Novel strategies for cancer combat: Drug combination using repurposed drugs induces synergistic growth inhibition of MCF-7 breast and HT-29 colon cancer cells. Curr Issues Mol Biol. 2022;44(10):4930–49. 10.3390/cimb44100335 . Davaadelger B, Duan L, Perez RE, Gitelis S, Maki CG. Crosstalk between the IGF-1R/AKT/mTORC1 pathway and the tumor suppressors p53 and p27 determines cisplatin sensitivity and limits the effectiveness of an IGF-1R pathway inhibitor. Oncotarget. 2016; 7: 27511–27526. Retrieved from https://www.oncotarget.com/article/8484/text/ Semlali A, Papadakos S, Contant C, Zouaoui I, Rouabhia M. Rapamycin inhibits oral cancer cell growth by promoting oxidative stress and suppressing ERK1/2, NF-κB and beta-catenin pathways. Front Oncol. 2022;12:873447. https://doi.org/10.3389/fonc.2022.87344 . He Y, Xue C, Yu Y, Chen J, Chen X, Ren F, et al. CD44 is overexpressed and correlated with tumor progression in gallbladder cancer. Cancer Manag Res. 2018;10:3857–65. 10.2147/cmar.s175681 . McFarlane S, Coulter JA, Tibbits P, O’Grady A, McFarlane C, Montgomery N, et al. CD44 increases the efficiency of distant metastasis of breast cancer. Oncotarget. 2015;6(13):11465–76. 10.18632/oncotarget.3410 . Watanabe T, Okumura T, Hirano K, Yamaguchi T, Sekine S, Nagata T, et al. Circulating tumor cells expressing cancer stem cell marker CD44 as a diagnostic biomarker in patients with gastric cancer. Oncol Lett. 2017;13(1):281–8. 10.3892/ol.2016.5432 . Thaker AI, Rao MS, Bishnupuri KS, Kerr TA, Foster L, Marinshaw JM, et al. IDO1 metabolites activate β-catenin signaling to promote cancer cell proliferation and colon tumorigenesis in mice. Gastroenterology. 2013;145(2):416–25. 10.1053/j.gastro.2013 . e254.doi . Zhang N, Wei P, Gong A, Chiu WT, Lee HT, Colman H, et al. FoxM1 promotes β-Catenin nuclear localization and controls WNT target-gene expression and glioma tumorigenesis. Cancer Cell. 2011;20(4):427–42. 10.1016/j.ccr.2011 . Dong L, Deng J, Sun ZM, Pan AP, Xiang XJ, Zhang L, et al. Interference with the β-catenin gene in gastric cancer induces changes to the miRNA expression profile. Tumor Biol. 2015;36(9):6973–83. 10.1007/s13277-015-3415-1 . Tan M, Gong H, Zeng Y, Tao L, Wang J, Jiang J, et al. Downregulation of homeodomain-interacting protein kinase-2 contributes to bladder cancer metastasis by regulating WNT signaling. J Cell Biochem. 2014;115(10):1762–7. 10.1002/jcb.24842 . Zhang N, Meng H, Tian A, Chen Y. Expression of HIPK2 in gastric cancer and its effects on cell function in vitro. Minerva Gastroenterol. 2022;68(2). 10.23736/s2724-5985.21.02969-7 . Kim EA, Kim JE, Sung KS, Choi DW, Lee BJ, Choi CY. Homeodomain-interacting protein kinase 2 (HIPK2) targets β-catenin for phosphorylation and proteasomal degradation. BiochemBiophys Res Commun. 2010;394(4):966–71. 10.1016/j.bbrc.2010.03.099 . Li P, Zhang Z, Sun P. DOT1L promotes expression of CD44 through the Wnt/β-catenin signaling pathway in early gastric carcinoma. J Cancer. 2024;15(8):2276–91. https://doi.org/10.7150/jca.90170 . Lee HS, Park CK, Oh E, Erkin ÖC, Jung HS, Cho MH, et al. Low SP1 expression differentially affects intestinal-type compared with diffuse-type gastric adenocarcinoma. PLoS ONE. 2013;8(2):e55522. 10.1371/journal.pone.0055522 . Panwar V, Singh A, Bhatt M et al. Multifaceted role of mTOR (mammalian target of rapamycin) signaling pathway in human health and disease. Signal Transduct Target Ther . 2023;8(1):375. Published 2023 Oct 2. 10.1038/s41392-023-01608-z Kuwano Y, Nishida K, Akaike Y, Kurokawa K, Nishikawa T, Masuda K, Rokutan K. Homeodomain-Interacting Protein Kinase-2: A Critical Regulator of the DNA Damage Response and the Epigenome. Int J Mol Sci. 2016;17(10):1638. https://doi.org/10.3390/ijms17101638 . Li Y, Yan X, Ren L, Li Y. miR-644a Inhibits Cellular Proliferation and Invasion via Suppression of CtBP1 in Gastric Cancer Cells. Oncol Res. 2018;26(1):1–8. https://doi.org/10.3727/096504016X14772410356982 . Di Segni M, Virdia I, Verdina A, Amoreo CA, Baldari S, Toietta G, Diodoro MG, Mottolese M, Sperduti I, Moretti F, Buglioni S, Soddu S, Rocco D, G. HIPK2 Cooperates with KRAS Signaling and Associates with Colorectal Cancer Progression. Mol cancer research: MCR. 2022;20(5):686–98. https://doi.org/10.1158/1541-7786.MCR-21-0628 . Kanei-Ishii C, Ninomiya-Tsuji J, Tanikawa J, Nomura T, Ishitani T, Kishida S et al. Wnt-1 signal induces phosphorylation and degradation of c-Myb protein via TAK1, HIPK2 and NLK.Genes Dev. 2004;18(7):816–2910.1101/gad.1170604 Thakuri PS, Gupta M, Joshi R, Singh S, Tavana H. Synergistic inhibition of kinase pathways overcomes resistance of colorectal cancer spheroids to cyclic targeted therapies. ACSPharmacolTransl Sci. 2019;2(4):275–84. 10.1021/acsptsci.9b00042 . MontaudonE BRE, Vacher S. DéasO, NaguezA, Chateau-Joubert S, High in vitro and in vivo synergistic activity between mTORC1 and PLK1 inhibition in adenocarcinoma NSCLC.Oncotarget.2021;12(8):859–72. 10.18632/oncotarget.27930 Zhu R, Li L, Nguyen B, Seo J, Wu M, Seale T, et al. FLT3 tyrosine kinase inhibitors synergize with BCL-2 inhibition to eliminate FLT3/ITD acute leukemia cells through BIM activation.SignalTransduct. Target Ther. 2021;6(1):186. 10.1038/s41392-021-00578-4 . Tables Tables are available in the Supplementary Files section. Supplementary Files Tables.docx supplementarymethod.docx supplementaryresults1.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 20 Oct, 2025 Reviewers invited by journal 13 Oct, 2025 Editor assigned by journal 07 Oct, 2025 First submitted to journal 06 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7795753","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":529128466,"identity":"72aebf35-f13f-4615-baaf-d56819c2eef5","order_by":0,"name":"Gopal Gopisetty","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-7869-4343","institution":"Cancer Institute (WIA)","correspondingAuthor":true,"prefix":"","firstName":"Gopal","middleName":"","lastName":"Gopisetty","suffix":""},{"id":529128467,"identity":"dcccce2d-46fa-421f-9d2d-0edf708361b9","order_by":1,"name":"Aathithya Rangarajan","email":"","orcid":"","institution":"Cancer Institute (WIA)","correspondingAuthor":false,"prefix":"","firstName":"Aathithya","middleName":"","lastName":"Rangarajan","suffix":""},{"id":529128468,"identity":"f4f35dc9-d1cf-4d97-851c-0b0fe8dd0547","order_by":2,"name":"Thirumoorthi Natarajan","email":"","orcid":"","institution":"Cancer Institute (WIA)","correspondingAuthor":false,"prefix":"","firstName":"Thirumoorthi","middleName":"","lastName":"Natarajan","suffix":""},{"id":529128469,"identity":"384aa61e-7675-4d88-9511-8bd7547a0b2d","order_by":3,"name":"Sujatha Lakshminarayanan","email":"","orcid":"","institution":"Cancer Institute (WIA)","correspondingAuthor":false,"prefix":"","firstName":"Sujatha","middleName":"","lastName":"Lakshminarayanan","suffix":""},{"id":529128470,"identity":"44e3a7a5-b916-4edf-ba65-f9b9216bd628","order_by":4,"name":"Shirley Sunder Singh","email":"","orcid":"","institution":"Cancer Institute (WIA)","correspondingAuthor":false,"prefix":"","firstName":"Shirley","middleName":"Sunder","lastName":"Singh","suffix":""},{"id":529128471,"identity":"c73e3e9c-faec-4f2d-bd2b-5a6037d7bb13","order_by":5,"name":"Jayavelu Subramani","email":"","orcid":"","institution":"Cancer Institute (WIA)","correspondingAuthor":false,"prefix":"","firstName":"Jayavelu","middleName":"","lastName":"Subramani","suffix":""},{"id":529128472,"identity":"7293ae50-c0f7-4a59-a7ca-9798c64626f0","order_by":6,"name":"Priya Ramanathan","email":"","orcid":"","institution":"Cancer Institute (WIA)","correspondingAuthor":false,"prefix":"","firstName":"Priya","middleName":"","lastName":"Ramanathan","suffix":""},{"id":529128473,"identity":"5e3e7a94-4156-4e34-b5a9-d5a64cf23f82","order_by":7,"name":"Ramakrishnan Ayloor Seshadri","email":"","orcid":"","institution":"Cancer Institute (WIA)","correspondingAuthor":false,"prefix":"","firstName":"Ramakrishnan","middleName":"Ayloor","lastName":"Seshadri","suffix":""}],"badges":[],"createdAt":"2025-10-07 04:50:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7795753/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7795753/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94598023,"identity":"39124db2-23dc-4b5e-8325-cb7138837ea3","added_by":"auto","created_at":"2025-10-28 18:50:58","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11558,"visible":true,"origin":"","legend":"","description":"","filename":"huceHUCED2500561.xml","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/0e1057662a6ca969b0115420.xml"},{"id":94597691,"identity":"eccd314b-1203-49f2-9c76-4ee891d6d494","added_by":"auto","created_at":"2025-10-28 18:48:30","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1364,"visible":true,"origin":"","legend":"","description":"","filename":"HUCED250056114719.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/a99b457f28de625ef03465da.xml"},{"id":94598149,"identity":"0d53147b-2921-4e26-bcb7-4b5029dbf460","added_by":"auto","created_at":"2025-10-28 18:51:30","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":831,"visible":true,"origin":"","legend":"","description":"","filename":"HUCED2500561Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/1b925a4861ae2f70a759e8e4.xml"},{"id":94598078,"identity":"ae10ae0e-b3d7-461d-8c22-1d2a05383eff","added_by":"auto","created_at":"2025-10-28 18:51:17","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172645,"visible":true,"origin":"","legend":"","description":"","filename":"HUCED25005610enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/c68e4f1b4d3a2032bda1cc0f.xml"},{"id":94597859,"identity":"ec558374-c7f9-4a2c-a5ba-e2856b19df4e","added_by":"auto","created_at":"2025-10-28 18:50:07","extension":"jpg","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":320027,"visible":true,"origin":"","legend":"","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/49879dd896207716427c6dc2.jpg"},{"id":94598279,"identity":"0b2b2320-719b-4f7a-9a62-e7717657ca22","added_by":"auto","created_at":"2025-10-28 18:52:17","extension":"jpg","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151175,"visible":true,"origin":"","legend":"","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/1c67d08b35c2523afb8a6cb3.jpg"},{"id":94597361,"identity":"4e0bb8e0-c1e5-473e-9bd6-31c9b93bc8bb","added_by":"auto","created_at":"2025-10-28 18:47:05","extension":"jpg","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":282234,"visible":true,"origin":"","legend":"","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/d715d2590bc6ccaedcdfc46b.jpg"},{"id":94598077,"identity":"32b26bd5-3c23-468d-bd2c-7446ca405734","added_by":"auto","created_at":"2025-10-28 18:51:15","extension":"jpg","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186564,"visible":true,"origin":"","legend":"","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/812169d01a2cd815bca877e9.jpg"},{"id":94597847,"identity":"fb8fc1ff-c4bf-4d44-a552-0713b4b501fe","added_by":"auto","created_at":"2025-10-28 18:49:55","extension":"jpg","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":267597,"visible":true,"origin":"","legend":"","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/9c2403d651388986a6b0beb4.jpg"},{"id":94597894,"identity":"cb24b7e8-0f13-4e32-b4e6-47d98a8ac337","added_by":"auto","created_at":"2025-10-28 18:50:13","extension":"jpg","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":318247,"visible":true,"origin":"","legend":"","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/dca15212b58109d0387c7155.jpg"},{"id":94598274,"identity":"bd8b66a1-1a57-4c12-8eb8-00a5cf5d8736","added_by":"auto","created_at":"2025-10-28 18:52:12","extension":"jpg","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":389821,"visible":true,"origin":"","legend":"","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/2ccbf42f7e2e2212d689fe9e.jpg"},{"id":94597818,"identity":"0c43caf6-bfdf-4b96-bf76-1885aaa035d0","added_by":"auto","created_at":"2025-10-28 18:49:38","extension":"jpg","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":289031,"visible":true,"origin":"","legend":"","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/b6996d3ac58d074607c6a79c.jpg"},{"id":94598273,"identity":"317bae1c-502f-4d3c-930d-fe72685be69f","added_by":"auto","created_at":"2025-10-28 18:52:12","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172580,"visible":true,"origin":"","legend":"","description":"","filename":"Online1.png","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/77c28f4a9862daefe0d8bb23.png"},{"id":94598148,"identity":"a788caa8-ed3a-48db-9305-34872d761746","added_by":"auto","created_at":"2025-10-28 18:51:29","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":54436,"visible":true,"origin":"","legend":"","description":"","filename":"Online2.png","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/864441dc58784d9d5fba8ab5.png"},{"id":94597955,"identity":"2ad21125-645c-40ab-af8c-e40b05ce3e33","added_by":"auto","created_at":"2025-10-28 18:50:29","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98809,"visible":true,"origin":"","legend":"","description":"","filename":"Online3.png","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/e990b7cdbfda076f28b380fe.png"},{"id":94598154,"identity":"db241a35-c6f9-40d5-9c3a-b1abe82d2f56","added_by":"auto","created_at":"2025-10-28 18:51:32","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":50879,"visible":true,"origin":"","legend":"","description":"","filename":"Online4.png","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/30a363956c39c18969ad092a.png"},{"id":94597790,"identity":"9b763f2f-a3d0-4113-a849-8587493f32a2","added_by":"auto","created_at":"2025-10-28 18:49:15","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67021,"visible":true,"origin":"","legend":"","description":"","filename":"Online5.png","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/d95e3102551f07f88aa3d62d.png"},{"id":94597956,"identity":"0f0c1cf7-9f43-49bf-9eb6-9448653a5044","added_by":"auto","created_at":"2025-10-28 18:50:30","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125249,"visible":true,"origin":"","legend":"","description":"","filename":"Online6.png","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/127d11fea5fb5b36e8935e6b.png"},{"id":94598283,"identity":"002a6ab2-21b4-4b8c-a4ad-cc4448c67be7","added_by":"auto","created_at":"2025-10-28 18:52:20","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173117,"visible":true,"origin":"","legend":"","description":"","filename":"Online7.png","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/16e4c2ab3a2d1465c152af25.png"},{"id":94598082,"identity":"c647c601-8915-404c-bbdd-0950a8f11f8a","added_by":"auto","created_at":"2025-10-28 18:51:20","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113932,"visible":true,"origin":"","legend":"","description":"","filename":"Online8.png","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/0e0e26d65695d0346ebe36e4.png"},{"id":94597701,"identity":"d0b538a7-d02e-4be7-ba76-5e1f54ec73d7","added_by":"auto","created_at":"2025-10-28 18:48:39","extension":"xml","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":170918,"visible":true,"origin":"","legend":"","description":"","filename":"HUCED25005610structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/dbd51173dceb85f2fdf4f126.xml"},{"id":94598079,"identity":"58dbb786-35eb-49f9-9459-bcf4c7f9cfd4","added_by":"auto","created_at":"2025-10-28 18:51:17","extension":"html","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":182063,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/ddb189f57f9c2883cb8ccde9.html"},{"id":94598281,"identity":"582e4968-b4df-49de-8467-b7a960ae3ca8","added_by":"auto","created_at":"2025-10-28 18:52:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":320027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHIPK2 a downregulated kinase, predominantly exhibits nuclear expression and displays context dependent activity in GC\u003c/strong\u003e :Representativewesternblot images shown were obtained from pooled protein lysates of two independent transient transfection experiments performed in gastric cancer (GC) cell lines:SNU638,AGS,NUGC-3. Normalised protein expression levels in the blots were calculated with ImageJ software. (A)Gene expression levels of HIPK2 in Gastric tumor(TR),PairedNormal(PN) and cell lines relative to apparent normal(AN) were determined by quantitative Real time PCR. Downregulation of HIPK2 in TR and cell lines with corresponds to PN were statistically significant(p\u0026lt;0.05).(B-C) At protein levels HIPK2 expression was determined by immunohistochemical staining of GC TMA comprises of 122 Normal and 136 TR tissues. (B) Representative 20X IHC image corresponds to downregulation of HIPK2 in TR ,(C) plot represents TMA scoring(+1,+2 and +3) calculated based on the intensity of HIPK2 staining in Gastric tumor and normal.(D) blot image corresponds to β catenin and CD44 expression in GC cell lines.(E-G)GC cell lines were transfected with pCMV3-N-FLAG-HIPK2(FLAG-HIPK2) and pCMV3-N-FLAG-Negative control vector(FLAG) after 48-72 hrs of transfection ,overexpression of HIPK2 and p53 levels were determined by by western blotting,(H-K) Cell proliferation and colony forming assay were performed after 48 hours of transient transfection of pCMV3-N-FLAG-Negative control (control) and pCMV3-N-FLAG-HIPK2(HIPK2) in GC cell lines.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/dc1da330f4509405009445db.jpg"},{"id":94598265,"identity":"96c07901-eba1-4e12-adcc-8db0f2090e34","added_by":"auto","created_at":"2025-10-28 18:52:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":151175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHIPK2 Expression induces differential effects on EMT markers in GC cells.\u003c/strong\u003eRepresentative westernblot images shown were obtained from pooled protein lysates of two independent transient transfection experiments performed in gastric cancer (GC) cell lines. Normalised protein expression levels in the blots were calculated with ImageJ software.(A) Quantitative real-time PCR analysis of E-cadherin and Vimentin expression in gastric cancer cell lines (SNU638, NUGC-3, and AGS) transiently transfected for 48 h with pCMV3-N-FLAG-HIPK2 or pCMV3-N-FLAG-Negative control (control). Relative expression levels were normalized to the housekeeping gene. Asterisks indicate statistically significant differences compared to Control (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).(B-E) EMT status upon HIPK2 overexpression in GC cell lines as determined by western blotanalysis.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/f3b3d7bd4c1bb98d9d5848cd.jpg"},{"id":94598280,"identity":"6094308e-a9e4-4f9f-9cc2-d380814c6d2c","added_by":"auto","created_at":"2025-10-28 18:52:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":282234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHIPK2 expression induces dualistic effect on AKT/mTOR signalling in SNU638 cells while it activates pro-tumorigenic AKT/mTOR signalling in AGS cells.\u003c/strong\u003eRepresentative blots and qPCR plots corresponds to protein and RNA lysates of two independent transient transfection experiment pooled together. Normalised protein expression levels in the blots were calculated with ImageJ software. Transiently transfected SNU638 and AGS pCMV3-N-FLAG-HIPK2 (FLAG-HIPK2) or pCMV3-N-FLAG Negative control vector (FLAG) protein and RNA lysates were analysed for PI3-Akt-mTOR signalling pathway.(A-C)Effect of HIPK2 overexpression on PI3-Akt signalling in SNU638 cells as determined by western blot analysis .(D) Quantitative real-time PCR analysis of mTOR and autophagy marker gene expression in SNU638 \u0026nbsp;cells transiently transfected for 48 h with either pCMV3-N-FLAG-HIPK2 (FLAG-HIPK2) or the pCMV3-N-FLAG negative control vector (Control). Relative expression levels were normalized to the housekeeping gene. Asterisks indicate statistically significant differences compared to Control(p\u0026lt;0.05-*,p\u0026lt;0.01-**,p\u0026lt;0.001-***),(E)Increased expression of pP-70S6k(T389) indicates an active mTORC1in SNU638 cells as determined by western blot analysis. (F-G) Effect of HIPK2 overexpression on PI3-Akt signalling in AGS cells as determined by western blot analysis(H)Quantitative real-time PCR analysis of mTOR and autophagy marker gene expression in AGS cells transiently transfected for 48 h with either pCMV3-N-FLAG-HIPK2 (FLAG-HIPK2) or the pCMV3-N-FLAG negative control vector (Control). Relative expression levels were normalized to the housekeeping gene. Asterisks indicate statistically significant differences compared to Control(p\u0026lt;0.05-*,p\u0026lt;0.01-**,p\u0026lt;0.001-***),(I) Effect of HIPK2 overexpression on Akt downstream and upstream signalling markers determined by western blotting.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/4c8a7693ea976f9c5ac86ab2.jpg"},{"id":94597555,"identity":"440dde9f-47c3-47d6-8eaa-08efe55b61da","added_by":"auto","created_at":"2025-10-28 18:47:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":186564,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHIPK2 expression activates mTORC1 signalling and its inhibition exhibits context dependent suppression of mTOR signalling.\u003c/strong\u003e(A)After 48 hrs of transient transfection , 1x10^6 cells of CMV-FLAG /CMV-FLAG HIPK2 transfected AGS and SNU638 cells were analysed for 143 metabolomic components by LC-MS based targeted metabolomics.HIPK2 expression significantly induces the levels of Glutamine,Leucine,Guanosine,Guanosine monophosphate in both AGS and SNU638 cells.(B)3*10^5 cells of SNU638 were treated with HIPK2 inhibitor(TBID 50uM) for 24 hours, inhibitor treatment decreases p-P53(ser 46) with no changes in the levels of total p53 indicates that inhibitor treatment inhibits the activity of HIPK2.(C-D)Gene expression levels of HIPK2, RICTOR and RAPTOR after TBID inhibitor treatment(24hrs) in AGS and SNU638 cells.(E) TBID inhibitor(24hrs) treated Protein lysates were analysed for the expression levels of p-P70S6K(T 389),CD44, β catenin by western blotting. Corresponding loading control for Fig.4E SNU638 TBID blot has been given in Fig.4B.Control represents vehicle control.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/049d3f9ac27601685956698e.jpg"},{"id":94597960,"identity":"3d1b86a6-3138-413c-83c8-f11b050230ff","added_by":"auto","created_at":"2025-10-28 18:50:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":267597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of HIPK2, NLK and MAPK11 signalling axis .\u003c/strong\u003eNormalised protein expression levels in the blots were calculated with ImageJ software (A)Workflow for the identification of HIPK2-associated signaling module comprising HIPK2, MYB, NLK, and MAPK11 through combined analysis of protein–protein interactions (PPIs) and gene expression data using the CEMitool.InEGEOD-79973 expression data set we assessed the gene expression changes ingastrictumor relative to normal gastric epithelium to arrive a set of significantly differentially expressed genes. We divided the differentially expressed set of kinases into over-expressed kinase genes and down regulated kinase genes. We next identified protein-protein interactions of all the overexpressed (positive Log 2 fold change in expression) kinase gene set and similarly for the down regulated kinase HIPK2. Next, we individually assessed the overexpressed kinase PPI’s or HIPK2 kinase PPI’s along with the gene expression using the CEMi tool to arrive at a subset of genes which are expressed and are also linked by protein-protein interactions. By combined analysis of the overexpressed kinase genes PPI’s and HIPK2 PPI’s we can derive a subset of PPI’s which link either directly the overexpressed kinases and HIPK2 kinase or through intermediary PPI between HIPK2 and the overexpressed kinase.(B)Cytoscape network reconstruction based on PPI’s of expressed kinase genes and HIPK2.(C)Gene expression analysis by quantitative Realtime PCR shows that HIPK2 expression was lower than c MYB, NLK, MAPK11in GCcell lines relative to normal gastric mucosa,(D)Ectopic expression of FLAG-HIPK2 decreases the levels of NLK and MAPK11 in AGS and SNU638 cells as determined by western blot analysis.(E)At gene expression levels NLK KD results in significantly increased expression of HIPK2(p\u0026lt;0.001).Control represents Wild type(WT) AGS cells (F)In AGS cells NLK knockdown (NLK KD) and the expression levels of HIPK2, total p53, and phosphorylated p53 (Ser46) in NLK KD cells were analysed by western blotting.(G-H) 3*10^5 cells of AGS were treated with MAPK11(SB202190) and NLK(Vx-702) inhibitors for 24 hours. corresponding blot image represents induction of HIPK2 upon MAPK11 inhibitor and NLK inhibitor treatment.Control represents vehicle control.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/ff5d779cd60606caccf7c298.jpg"},{"id":94598021,"identity":"c1d17c2b-62f8-4e29-8b5a-4ceb76c03a5d","added_by":"auto","created_at":"2025-10-28 18:50:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":318247,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of HIPK2,NLK,MAPK11 inhibition in GC.\u003c/strong\u003e (A)For cell proliferation assay 2*10^3 cells of AGS and SNU638 cells were treated with HIPK2 inhibitor(TBID) and proliferation rate was determined at different time points by Alamarblue assay together with protein expression levels of HIPK2 in GC cell lines : AGS,SNU 638.(B) 3*10^5 cells of AGS and SNU638 were treated with HIPK2 inhibitor(TBID) for 24 hours. In both AGS and SNU638 cells , TBID treatment decreases Bcl-xl but increases Mcl-1 in a dose dependent manner analyzed by western blotting. Corresponding loading control for (B) AGS TBID has been given in Fig.4E vinculin , for SNU638 TBID Corresponding loading control has been given in Fig.4B.(C-F) For cell proliferation assay 2*10^3 cells of AGS and SNU638 cells were treated with NLK and MAPK11 inhibitor and proliferation rate was determined at different time points.(G-H) 3*10^5 cells of SNU638 and AGS were treated with NLK(Vx-702)and MAPK11(SB202190) inhibitor for 24 hours, the levels of anti-apoptotic markers Mcl-1 and Bcl-xL was determined by western blotting.Control represents Vehicle control.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/49f81f146c6a97660b981206.jpg"},{"id":94597559,"identity":"f43e1010-2c53-431b-8608-9f88e57ee247","added_by":"auto","created_at":"2025-10-28 18:47:56","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":389821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombinational inhibitor treatment comprising of HIPK2-NLK/MAPK11 Inhibitor shows potential synergism in GC.\u003c/strong\u003e For combinatorial inhibitor treatment cell proliferation assay, 2*10^3 cells ofAGS and SNU638 were treated with TBID,Vx-702 ,SB202190 and combinations of TBID and Vx-702/SB202190 inhibitor for 24 hrs and compusyn software was employed to determine whether the combinational treatment exhibits synergy(CI\u0026lt;1), additive(CI=1) and antagonistic(CI\u0026gt;1) effect. Fig.7A-D-* indicates Synergistic effect.Control represents vehicle control.(A-D)combinations of TBID and Vx-702/SB202190 inhibitor exhibits synergy in reducing cell viability inboth AGS and SNU638 cells.(E) % of early apoptotic cells (FITC+PI-) for inhibitor treated AGS and SNU 638 cells for 24 hours. % of early apoptotic cells calculated using the formula:% of early apoptotic cells in treatment-% of early apoptotic cells in control.(F-G) Representative flow cytometry analyses of Annexin V/PI-stained inhibitor (either individual or combination of TBID(50uM) ,Vx-702(50uM),SB202190(50uM)) treated AGS and SNU638 cells for 24 hours.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/8513f425cfe6319d78553a68.jpg"},{"id":94597700,"identity":"74f901e4-178e-4d51-8ba9-1caf0ae26836","added_by":"auto","created_at":"2025-10-28 18:48:38","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":289031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombinational inhibitor treatment comprising of HIPK2-MAPK11/NLK Inhibitor mitigates mTOR signallingcontext independently.\u003c/strong\u003e3\u003cstrong\u003e*\u003c/strong\u003e10^5 cells of AGS and SNU638 were treated with either individual or combinations of TBID and Vx-702/SB202190 for 24 hours. Control denotes vehicle control(A-B) Inhibitor treated AGS and SNU638 cell lysates were analysed for the gene expression levels of cMYB, Myc, mTOR markers(RICTOR,RAPTOR) by quantitative Real time PCR.To avoid redundancy, previously reported RICTOR and RAPTOR expression levels upon TBID (50 µM) treatment (Fig.4D) were used for comparative analysis with combination inhibitor treatments(C-F)Inhibitor treated AGS and SNU638 cell lysates were analysed for the protein expression levels of mTOR marker(Total P70S6k,p-P70S6k), pro-tumorigenic markers(CD44 and β-catenin) as determined by western blot analysis.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/ce827da9e21e8585df1cbdb5.jpg"},{"id":94599247,"identity":"c41d3d13-4e22-4dea-9437-b67fe93a1449","added_by":"auto","created_at":"2025-10-28 19:04:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3680850,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/d867fb2f-2dd3-4b4a-9a04-5c020aa88662.pdf"},{"id":94598269,"identity":"fc980ab2-ffd2-46d6-9c22-d0f4ea273f86","added_by":"auto","created_at":"2025-10-28 18:52:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19953,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/79f83477f0056b979dbd81e9.docx"},{"id":94597692,"identity":"2e2287c9-3c8f-4025-9aae-ff8b3ac73241","added_by":"auto","created_at":"2025-10-28 18:48:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":50797,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymethod.docx","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/48d176c0fa7009987cd686b8.docx"},{"id":94598270,"identity":"ef5331f7-3fbe-4cc6-b654-de300436ad2c","added_by":"auto","created_at":"2025-10-28 18:52:11","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8392344,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryresults1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7795753/v1/49d01e8fb894d83497ae0561.docx"}],"financialInterests":"","formattedTitle":"HIPK2 exhibits context dependent dualistic effects and combinational inhibition of HIPK2-NLK-MAPK11 axis manifests synergism against gastric cancer.","fulltext":[{"header":"Highlights of the study","content":"\u003cp\u003e\u0026bull; HIPK2 is downregulated in gastric cancer. Its expression exhibits context dependent anti or pro tumorigenic effects in gastric cancer represented by AGS (β-Catenin, CD44) and SNU638 (β-Catenin, CD44) and NUGC3 (β-Catenin, CD44) cells.\u003c/p\u003e\u003cp\u003e\u0026bull; HIPK2 expression showed dualistic effects on Akt-mTOR pathway highlighted by differential phosphorylation of Akt, inhibition of mTORC2 arm and activation of mTORC1.Inhibtion of mTOR signalling by HIPK2 inhibitor was context dependant.\u003c/p\u003e\u003cp\u003e\u0026bull; A protein network involving HIPK2, NLK, and MAPK11 kinases was identified, linked by a common interaction with MYB in gastric cancer.\u003c/p\u003e\u003cp\u003e\u0026bull; Combinational treatment with a HIPK2 inhibitor and either NLK or MAPK11 inhibitor manifests synergism in gastric cancer and inhibited mTOR signalling in both SNU638 and AGS cells.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eHomeodomain-interacting protein kinase 2 (HIPK2) is a proline-directed serine/threonine kinase that belongs to the CMGC kinase family. Initially recognised as a homeoprotein transcriptional corepressor, HIPK2 has been found to play a role in various developmental and cellular processes, including neurogenesis, myogenesis, angiogenesis, DNA damage response, cell proliferation, differentiation and apoptosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The role of HIPK2 in cancer is complex, with studies showing it can act as both a tumour suppressor and a tumour promoter, depending on the context. For example, HIPK2 depletion can suppress the p53/E-cad axis, which inhibits metastasis in oral squamous cell carcinoma[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, its suppression can enhance angiogenesis and promote metastasis in hepatocellular carcinoma [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. HIPK2 induction can downregulate vimentin, inhibiting breast cancer cell invasion[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, HIPK2 phosphorylates CtBP, a C-terminal binding protein that promotes tumorigenesis in osteosarcoma, facilitating its degradation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. HIPK2 also regulates aerobic glycolysis and inhibits cell proliferation in pancreatic cancer by destabilising cMYC [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In contrast, HIPK2 overexpression is associated with decreased overall survival as in HPV-positive tongue squamous cell carcinoma [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. HIPK2 also phosphorylates FOXM1, a transcription factor that promotes cell proliferation in renal cell carcinoma [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, HIPK2 isoform 3 mediates the activation of the Hippo pathway, which promotes lung cancer progression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. HIPK2 expression has been found to be higher in ovarian and cervical cancers, suggesting its involvement in tumour progression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. One possible mechanism for HIPK2\u0026rsquo;s role in tumorigenesis involves the NRF2 pathway. NRF2 activates HIPK2 at the transcriptional level and activated HIPK2 facilitates the nuclear accumulation of NRF2 and activation of its targets. Under malignant conditions, this HIPK2-NRF2 axis can aberrantly activate cell survival pathways [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGastric cancer is a significant global health concern, ranking fifth in frequently diagnosed cancers and fourth in cancer-related deaths [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Most patients present at an advanced stage, leading to poor survival rates [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Gastric cancer is categorised into intestinal and diffuse types, which differ in their aetiology, epidemiology, risk factors, clinical behaviour, and molecular alterations [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The PI3K/Akt signalling pathway and its downstream effector mTOR signalling are frequently altered in gastric cancer, contributing to disease progression, metastasis and poor survival [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study investigated the expression of HIPK2 in gastric cancer, demonstrated context-dependent effects through full length HIPK2 expression and its inhibition with a HIPK2 kinase inhibitor. Mechanistically, context dependent activity of HIPK2 was found to be due to its differential effects on mTORC1 and mTORC2 arms of the mTOR pathway. The study identified a protein-protein interaction network involving HIPK2-NLK-MAPK11, linked by a common interaction with the transcription factor MYB in GC. The study also reports on the efficacy of targeting HIPK2, NLK and MAPK11, individually and in combinations. A combination of HIPK2 and NLK, and HIPK2 and MAPK11 inhibitors showed synergistic effects in inhibiting gastric cancer cell proliferation and inhibiting mTOR signalling.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Patient Samples:\u003c/h2\u003e\u003cp\u003eGastric tumor (n\u0026thinsp;=\u0026thinsp;23) paired normal (n\u0026thinsp;=\u0026thinsp;17) and normal gastric mucosa (n\u0026thinsp;=\u0026thinsp;8) were collected from the patients who underwent oesophagogastroduodenoscopy (OGD) at the Cancer Institute (WIA), Chennai for gene expression studies. Paired normal were obtained from adjacent normal tissues to the tumor cells, while normal gastric mucosa was collected from patients without stomach abnormalities. Patients were found to be treatment naive and without any co morbid illness. Archival FFPE Gastric tissue microarray (TMA) samples (Normal\u0026thinsp;=\u0026thinsp;122, Tumor\u0026thinsp;=\u0026thinsp;136) were procured from the department of Oncopathology for Immunohistochemical staining of HIPK2. Written informed consent has been obtained from the patients for their participation in the study, and they were found to be treatment naive. The study was conducted following approval from the Institute Ethics Committee (Reference no: IEC/2020/Dec 10).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell Lines:\u003c/h2\u003e\u003cp\u003eHuman Gastric cancer cell lines AGS, SNU638 and NUGC-3 were cultured in RPMI-1640 medium (R-8758,Sigma Aldrich, USA) supplemented with 10% FBS (A5256701,Gibco,USA), 100 mg/L Streptomycin sulphate, and 100 mg/L penicillin G(Himedia, India) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator. Cell lines were found to be negative for mycoplasma contamination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Immunohistochemical staining:\u003c/h2\u003e\u003cp\u003eFor HIPK2 antibody staining, GC Tissur microarray (TMA) slides were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol (100, 90, 70, and 50% ethanol), followed by antigen retrieval with 10 mM citrate buffer (pH 6). The slides were incubated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 15 minutes, followed by blocking with 3% BSA for 1 hour, and overnight incubation with anti-HIPK2 primary antibody (1:200) at 4\u0026deg;C. Slides were washed with PBST (PBS\u0026thinsp;+\u0026thinsp;0.1% Tween 20,pH 7.4),incubated with anti-rabbit HRP conjugate secondary antibody for 1 hour at RT, again washed with PBST and developed using the DAKO Envision detection system(K5007,US).The slides were counterstained with haematoxylin, mounted with DPX mountant and visualized under a Leica DMRXA fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. RNA Isolation, cDNA Conversion and Quantitative Real Time PCR (qPCR):\u003c/h2\u003e\u003cp\u003eTotal RNA from the cell lines and tissues were isolated using Tri Reagent (T9424,Sigma) following the manufacturer\u0026rsquo;s protocol. Briefly, GC tumors, paired normal, apparent normal, and GC cell lines were lysed with Tri reagent. For 1 ml of lysates, 100 ul of 1-Bromo-3-Chloropropane(BCP) was added and centrifuged at 10000 rpm for 20 min for phase separation. In the clear aqueous phase, isopropanol was added and centrifuged at 10000 rpm to precipitate the RNA, and the pellet was washed twice with 70% ethanol and resuspended in nuclease-free water. The quality and quantity of RNA were determined using a thermo scientific Nanodrop Lite spectrophotometer. For cDNA conversion, 1 ug of RNA was reverse transcribed into cDNA with random primers using cDNA synthesis kit with a genomic DNA (gDNA) eraser (RR047A,Takara). Briefly, to remove the contaminating DNA, 1 ug of RNAwastreated with a gDNA eraser at 42\u0026deg;C for 2 minutes, followed by reversetranscription. The conditions for reverse transcription were as follows: 37\u0026deg;C for 15 minutes and 85\u0026deg;C for 5 minutes. For gene expression studies, GoTaq SYBR green-based quantitative real-time PCR ( A6001,Promega) in triplicates were performed using the QuantStudio 6 Flex Real-Time PCR system. The PCR conditions were found to be initial heat activation at 95\u0026deg;C for 2 minutes, followed by 40 cycles of denaturation at 95\u0026deg;C for 15 minutes, and combined annealing and extension at 60\u0026deg;C for 60 minutes. For the normalisation of genes, GAPDH was used as an internal control. Relative changes in the gene expression level were calculated using the 2\u0026ndash;∆∆Ct method\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Transfection:\u003c/h2\u003e\u003cp\u003eFor ectopic expression ,50\u0026ndash;70% confluent AGS, SNU638,NUGC-3 cells were transfected withpCMV3-N-FLAG-HIPK2(HG13216-NF) and pCMV3-N-FLAG-Negative control vector(CV016) (Sino biologicals, China) in complete RPMI 1640 medium using XFECT transfection Reagent (631317,Takara,USA) by following the manufacturer's protocol. For CRISPR RNP based genome editing of NLK, the RNP complex(20 pmol NLK gRNA exon 1 with 10 pmol cas9 nuclease )was delivered into AGS cells using lipofectamine RNAImax transfection reagent (13778100 ,Thermofisher scientific, USA) by following the manufacturer's protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6.Western Blotting:\u003c/h2\u003e\u003cp\u003eTransiently transfected pCMV3-N-FLAG-Negative control / pCMV3-N-FLAG-HIPK2 or HIPK2/NLK/MAPK11 inhibitor-treated protein lysates were prepared in the RIPA lysis buffer. The total protein concentration in the lysates was estimated using a standard BCA assay(23227,Pierce BCA protein assay kit,Thermoscientific). Equal amounts of protein (30\u0026ndash;50 ug) were resolved in 10% SDS PAGE, transferred into a PVDF membrane at 100 v for 60 mins, blocked with 5% BSA or skim milk for 1 hour, incubated overnight with the primary antibody at 4\u0026deg;C, followed by 1 hour of incubation with the secondary antibody, and developed with Clarity Western ECL substrate (1705060,Bio-Rad ,USA). The signal was then detected and documented using a BioRadChemiDoc MP Imaging System. The loading controls for the western blots which were consecutively probed with different antibodies have been displayed for the first instance for which the blot was probed. To avoid duplication of displaying the loading controls for subsequent antibodies reference to the respective loading control was mentioned in the figure legends.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7.Cell proliferation Assay:\u003c/h2\u003e\u003cp\u003eAbout 2x10^3 cells of transiently transfected pCMV3-N-FLAG-Negative control / pCMV3-N-FLAG-HIPK2 or HIPK2/ NLK/ MAPK11 inhibitor treated cells were seeded in 96 well plates and assessed for cell proliferation at definite time points in triplicates using AlamarBlue cell viability reagent (DAL1025, Invitrogen,US). Briefly,10 \u0026micro;l of alamarBlue was added into the 96 well plates and incubated at 37\u0026deg;C for 3\u0026ndash;4 hours. After incubation, Fluorescence readings were taken at an excitation and emission wavelength of 530\u0026ndash;560 and 590 nm. Percentage of cell viability was calculated as (Treatment-Blank)/(Control-Blank) *100.For ectopic HIPK2 expression studies, cell proliferation assay was performed after 48 hours of transient transfection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8.Colony forming assay:\u003c/h2\u003e\u003cp\u003eThe colony formation assay was performed as previously described by Franken et al.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] with minor modifications. Briefly,2x10^3 cells of transiently transfected pCMV3-N-FLAG-Negative control / pCMV3-N-FLAG-HIPK2AGS ,SNU638,NUGC-3 cells were seeded in six well plates and incubated for a period of 2 weeks. After colony formation, cells were fixed in ice-cold 100% methanol and stained with 0.5% Crystal violet followed by counting the colonies using image J software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Extraction and analysis of metabolites for targeted metabolomics:\u003c/h2\u003e\u003cp\u003e1*10^6 cells of transiently transfected AGS and SNU638 cells with pCMV3-N-FLAG-HIPK2 and pCMV3-N-FLAG-Negative control vector (48 hrs) were homogenized in 500ul of methanol spiked with internal standards (10umol/L L-Methionine sulfone,10umol/ 2-(N-Morpholino) ethanesulfonicacid(MES) using MicroDistec\u003csup\u003eTM\u003c/sup\u003ehomogenizer. To that 250ul of ultrapure water was added and thoroughly agitated. For metabolite extraction,400ul of chloroform was added into the 600ul of homogenized suspension solution and centrifuged at 15,000rpm for 15mins at 4\u0026deg;C. Upper clear layer was collected, dried with nitrogen evaporator and reconstituted in 50ul of ultra-pure water.5ul of extracted metabolites were analysed for 143 metabolomic components using Shim-pack (GIST)3uM PFPP (2.1X150mm (HSS)) column with a mobile phase A:0.1% formic acid in water and mobile phase B:0.1% formic acid in acetonitrile. A ten step gradient elution was employed.Separated metabolites were analysed using LCMS-8060 NX Liquid chromatogram and mass spectrometer (Kyoto, Shimadzu) with negative ion ESI. Data analysis was performed using labSolutions software and Multi-Omics data analysis package (Kyoto, Shimadzu). List of metabolites used for analysis were given in supplementary methods.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Gene expression analysis:\u003c/h2\u003e\u003cp\u003eFour datasets derived using different gene expression analysis platforms were analysed (E-MEXP-2694-Agilent array[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e],E-GEOD-17154- Stanford array[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], E-MTAB-1338-Illumina HumanHT-12 V4.0 expression bead chip- Transcription profiling by array[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], E-GEOD-79973-Affymatrix array Expression data from gastric cancer and paired normal tissues[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Workflow used for analysis of data was that gene expression data sets were reprocessed using individual gene expression analysis algorithms specific for platforms in R .The expression sets were background adjusted, normalised, annotated. The differentially expressed genes were derived using the Linear Model of Microarray (LIMMA) algorithm following the construction of a contrast matrix. The differentially expressed gene set was extracted and searched for differential expression of Tumor suppressor kinase gene set.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Gene Co-expression analysis with Protein-Protein Interactions (PPI) using CEMi tool.\u003c/h2\u003e\u003cp\u003eThe gene expression files along with phenotype data were assessed in CEMi tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cemitool.sysbio.tools/\u003c/span\u003e\u003cspan address=\"https://cemitool.sysbio.tools/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e to identify the gene expression modules differentiating cancer and normal gene expression profiles for the four gene expression datasets. The protein-protein interactions for the shortlisted expressed kinase genes and HIPK2 were extracted from Protein Interaction Network Online Tool (PINOT) a PPI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.reading.ac.uk/bioinf/PINOT/PINOT_form.html\u003c/span\u003e\u003cspan address=\"http://www.reading.ac.uk/bioinf/PINOT/PINOT_form.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The interactions were assessed individually for the kinase gene set and HIPK2 along with the gene expression and phenotype files. The analysis yielded a shortlist of interactions by mapping the PPI\u0026rsquo;s to gene expression.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Inhibitor treatment Studies:\u003c/h2\u003e\u003cp\u003eHIPK2 inhibitor TBID(HY-100464), NLK inhibitor Vx-702(S6005) and MAPK11 inhibitor SB202190(sc-222294) were purchased from MCE, USA; Selleck chemicals, USA and Santacruz, USA respectively. The stock solutions of the inhibitors were prepared according to the manufacturer\u0026rsquo;s instructions. For dose response studies, post 24 hours of seeding 2*10^3 cells of AGS,SNU638,NUGC-3 were treated with either individual or combinations of inhibitors: TBID, Vx-702 and SB202190. Cell viability was measured at definite time points by cell proliferation assay. To assess whether the combined effects of inhibitor treatment were synergistic (CI\u0026thinsp;\u0026lt;\u0026thinsp;1), additive (CI\u0026thinsp;=\u0026thinsp;1), or antagonistic(CI\u0026thinsp;\u0026gt;\u0026thinsp;1), the combination index (CI) was calculated using the Bliss independence model with Compusyn software. For protein and gene expression studies, post 24 hours of seeding, 3*10^5 cells of AGS and SNU638 were treated with HIPK2/NLK/MAPK11 inhibitor. To study the expression status of p-P70S6K, GC cells were grown in serum starved medium for 24 hours, treated with 100 ng/ml hIGF-1 for 10 mins, followed by combinational inhibitor treatment (24 hours).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Apoptosis detection by Flow cytometry:\u003c/h2\u003e\u003cp\u003eFor apoptosis detection, 3x10\u003csup\u003e5\u003c/sup\u003e inhibitor treated cells were collected and washed with ice cold PBS followed by Annexin V FITC/PI staining for 15 mins in dark at room temperature(RT) by following the manufacture\u0026rsquo;s protocol (sc-4252 AK, Santacruz). About 20000 events per sample were acquired using Cytoflex Flow cytometer (Beckman Coulter). Gating was performed with forward scatter and side scatter of unstained cells. Data were processed using cytexpert 2.5 software to determine the percentage of early apoptotic (FITC\u003csup\u003e+\u003c/sup\u003ePI\u003csup\u003e\u0026minus;\u003c/sup\u003e), late apoptotic/necrotic (FITC\u003csup\u003e+\u003c/sup\u003ePI\u003csup\u003e+\u003c/sup\u003e) cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14. Statistical Analysis:\u003c/h2\u003e\u003cp\u003eStatistical analysis was conducted using GraphpadPrism(RRID:SCR_002798). Unpaired two tailed t-tests and one-way ANOVA followed by Bonferroni multiple comparison tests and chi-square test were employed to determine statistical significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 *, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 **, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 ***\u003cb\u003e).\u003c/b\u003e Densitometric analysis of blot images was performed using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15. Data Availability Statement.\u003c/h2\u003e\u003cp\u003eData generated in this study are available within the article and its supplementary data files.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.1. HIPK2 is downregulated in gastric cancer.\u003c/h2\u003e\u003cp\u003ePutative tumor suppressor kinase gene expression (~\u0026thinsp;40 genes) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] was studied in gene expression datasets derived from gastric cancer. Analysis of transcription profiling in gastric cancer cell lines, normal gastric mucosa, malignant gastric tissues and adjacent non-cancerous mucosa, showed that HIPK2 gene expression was downregulated in both tumours and cell lines, when compared to normal gastric tissues (Supplementary Fig.\u0026nbsp;1A, Table.S1). To validate these findings, we examined a panel of 10 downregulated tumour suppressor (TS) kinases by Real-time PCR, of which 8 TS Kinases, including HIPK2, were confirmed to be downregulated in our sampleset (Supplementary Fig.\u0026nbsp;1B). For HIPK2, qPCR analysis was initially performed in gastric cancer cell lines (n\u0026thinsp;=\u0026thinsp;3), treatment naive tumours (n\u0026thinsp;=\u0026thinsp;23), paired normal tissues (n\u0026thinsp;=\u0026thinsp;17), and apparent normal gastric mucosal biopsies (n\u0026thinsp;=\u0026thinsp;8). Among these, only samples with high-quality RNA were included for analysis, resulting in a final cohort of GC cell lines (n\u0026thinsp;=\u0026thinsp;3), tumour tissues (TR, n\u0026thinsp;=\u0026thinsp;17), paired normal tissues (PN, n\u0026thinsp;=\u0026thinsp;11), and apparent normal tissues (AN, n\u0026thinsp;=\u0026thinsp;8).The Clinicopathologic characteristics of GC Patients used for gene expression analysis has been summarized in Table.1. Notably, it was observed that HIPK2 gene expression was found to be downregulated in both tumours and cell lines when compared to paired normal and apparent normal gastric tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, HIPK2 protein expression was significantly lower in tumour tissue than in normal gastric epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). Furthermore, HIPK2 was found to be located predominantly in the nucleus of gastric tumours, while it is located in the cytoplasm of normal gastric epithelium (Table.2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.2. HIPK2 expression elicits context dependent effects on GC cells.\u003c/h2\u003e\u003cp\u003eTo investigate the role of HIPK2 in gastric cancer (GC), GC cell line models, AGS ,SNU638, NUGC-3 were studied. These cell lines differed in their expression of CD44 and β-catenin; AGS cells have low CD44 and high β-catenin levels, whereas SNU638 and NUGC-3 cells have high CD44 and low β-catenin levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The selection of the CD44 and β-catenin as markers for context was based on their mutually exclusive or inverse patterns of expression in GC marking context and tumor heterogenity. The effects of HIPK2 were examined through transient ectopic expression. Transient expression of FLAG-HIPK2 in GC cell lines were confirmed by western blotting. Ectopic expression of FLAG-HIPK2 induced p53 in both AGS and SNU638 and phospho-p53(ser46) in SNU638 cells indicating the functional status of FLAG/HIPK2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G). Transient HIPK2 expression decreased cell proliferation and significantly decreased colony forming ability in SNU638 and NUGC-3 cells but had no significant impact on AGS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-K). Next, the impact of HIPK2 on epithelial-mesenchymal transition (EMT) markers was studied. At the transcriptional level in SNU638 and NUGC-3 cells, compared to the pCMV3-N-FLAG control, ectopic expression of pCMV3-N-FLAG-HIPK2 led to the differential expression of EMT markers, characterized by a marked downregulation of mesenchymal marker-vimentin, while in AGS cells upon FLAG-HIPK2 ectopic expression, there was no differential expression between epithelial and mesenchymal markers suggesting that HIPK2 overexpression modulates EMT status prominently in SNU638, NUGC-3 cells than in AGS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In SNU638 cells at protein level we observed increased expression of E-Cadherin, and decreased expression of mesenchymal markers (N-Cadherin, αSMA and Vimentin). Additionally, there was also a decreased expression of other markers such as β-catenin, α-catenin. Concomitant with decrease in β-catenin, HIPK2 expression decreased the level of cancer stem cell marker CD44 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C), similarly in NUGC-3 cells at protein levels HIPK2 overexpression induces E-Cadherin expression, hence, based on our results it was determined that HIPK2 overexpression plausibly suppresses EMT in SNU638 and NUGC3 cells. AGS cells in contrast at protein expression level, there was a decrease in β-catenin levels however there were no significant change in the levels of α catenin or αSMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E) indicating that HIPK2 may not affect EMT status in AGS cells. Taken together all of this indicates that effects of HIPK2 on cell proliferation, colony formation, EMT status are context dependent in GC. Next, we selected AGS and SNU638 cells to assess for context based molecular effects of HIPK2 expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.3 HIPK2 expression elicits dualistic effect on AKT/mTOR signalling in SNU638 cells.\u003c/h2\u003e\u003cp\u003eSince PI3K/Akt signalling pathway and its downstream effector mTOR signalling are frequently altered in gastric cancer, pCMV3-N-FLAG control or pCMV3-N-FLAG-HIPK2 expressing SNU638 lysates were assessed for markers of AKT/mTOR signalling. Increased HIPK2 expression lead to an increase in p-Akt (Thr308) and a decrease in p-Akt (Ser473). This was associated with a decrease in p-GSK-3β (Ser9), p-PTEN (Ser380), and p-PDK1 (Ser241), and an increased level of p-cRaf (Ser259) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Additionally, HIPK2 expression resulted in reduction in anti-apoptotic proteins like Bcl-2, Bcl-xL, Mcl-1, and p-Bcl2 (Ser70), suggesting an increase in the apoptotic potential. This effect was further supported by an observed increase in PHLPP1 levels, a phosphatase that dephosphorylates p-Akt (Ser473) a marker for anti-apoptotic effects of Akt (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Next, we assessed how HIPK2 impacts the mTOR paṭhway. A decrease in RICTOR expression, relative to RAPTOR, indicated that the mTORC2 arm of the mTOR pathway is inhibited, consistent with decreased p-Akt (Ser473) levels and increased PHLPP1. Conversely, the mTORC1 arm appears active with increased pp70S6K(T389) level and a decrease in autophagy-related gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). Thus, HIPK2 expression has a dualistic effect, increasing the apoptotic potential of cells while also promoting signals that support cell proliferation in SNU638 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.4. HIPK2 expression increases pro-tumorigenic AKT/mTOR signalling in AGS cells.\u003c/h2\u003e\u003cp\u003eHIPK2 expression in AGS led to an increase in the levels of p-Akt (Ser473), while p-Akt (Thr308) remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). HIPK2 expression led to reduced PHLPP1 and increased RICTOR, indicating active mTORC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H ). The activity of mTORC2 was further suggested by the induction of the anti-apoptotic marker Mcl-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG ). There was also evidence that mTORC1 is active, with increased levels of pp70S6K(T389) and decreased expression of autophagy related genes (UVRAG,ATG9B,LAMP1,MCOLN1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). In addition, increased levels of p-GSK-3β(Ser9) and p-c-Raf (Ser259) were observed, indicating an active Akt status. Conversely, decreased levels of p-PTEN(Ser380) suggested that HIPK2 suppressed upstream effectors of Akt signalling while maintaining active Akt (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). These results suggested that HIPK2 expression in AGS cells may predominantly promote AKT/mTOR pathway.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5. Targeted metabolomics indicates activation of mTOR and HIPK2 inhibition exhibits context dependent suppression of mTOR signalling\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eOur data indicated that the expression of HIPK2 leads to the potential activation of mTORC1 in both AGS and SNU638 cells. Further in order to ascertain the potential activation status of mTOR we determined the levels of metabolite targets of mTOR such as in nucleotide metabolism and amino acid metabolism in AGS and SNU638 cells during transient ectopic expression of FLAG/HIPK2. In total 143 metabolites were analysed, under the conditions tested we identified differential response in the metabolite levels in both AGS and SNU638 cells ectopically expressing CMV-FLAG/HIPK2 relative CMV-FLAG alone expressing cells (Supplementary Fig.\u0026nbsp;2, Table.S2-3). In both AGS and SNU638 cells, we observed a significant increase in glutamine, leucine, guanosine, and guanosine monophosphate, known markers of mTORC1 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], which indicates an active mTOR upon HIPK2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We further investigatedthe effects of HIPK2 kinase inhibition in GC cells using TBID, a HIPK2 inhibitor [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For this the levels of p53 and p-P53(Ser 46) were assessed following treatment with TBID and it was observed that increasing the concentration of TBID reduced HIPK2 kinase activity which was shown by downregulation of p-P53(Ser 46) though there was no significant changes in the levels of total p53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) indicating that TBID treatment suppressed HIPK2 kinase under the conditions tested. In AGS cells, treatment with the HIPK2 inhibitor TBID increased the expression levels of HIPK2, RICTOR, RAPTOR, and phosphorylated p70S6K (T389), without affecting β-catenin levels. This suggests a possible activation of the mTOR pathway, potentially mediated by transcriptional upregulation of HIPK2 despite inhibition of its kinase activity. Whereas, in SNU638 cells, the TBID decreased the expression levels of HIPK2,RICTOR, RAPTOR, and p-p70S6k(T389) along with a concomitant reduction in CD44 levels which is known to vary with mTOR activity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] this indicated mTOR inactivation upon HIPK2 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E). Therefore, consistent with overexpression studies which demonstrated context-dependant effects, treatment with a HIPK2 inhibitor induced context-dependent activity by elicitng differential effects on mTOR signalling.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Identification of HIPK2, NLK and MAPK11 axis.\u003c/h2\u003e\u003cp\u003eNext, we wanted to assess kinases which are potentially linked to HIPK2 Kinase and expressed at higher levels that could possibly serve as targets in GC cells considering that HIPK2 was downregulated in GC. For which we implemented a bioinformatic analysis workflow which focuses on kinase interacting proteins and gene co-expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Bioinformatic analysis identified a protein-protein interaction network involving HIPK2, NLK, MAPK11, and TRIM28, all linked by a common interaction with MYB (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,Supplementary Fig.\u0026nbsp;3-4A). Differential gene expression analysis revealed that HIPK2 expression was lower than MYB, NLK, MAPK11 in gastric cancer cell lines relative to normal gastric mucosa (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This suggested a potential negative regulatory role of HIPK2 on partner genes in the axis. Overexpression of HIPK2 in gastric cancer cells led to downregulation of NLK and MAPK11 at both protein and gene expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, Supplementary Fig.\u0026nbsp;4B-C). Although cMYB expression was unaffected, two of its transcriptional targets, cMYC and STAT5A, were downregulated (Supplementary Fig.\u0026nbsp;4D). Knockdown of NLK using CRISPR CAS9 resulted in increased HIPK2 expression at both mRNA and protein expression levels, accompanied by elevated levels of p53 and pP53(Ser46) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F). Additionally when treated with inhibitor of MAPK11(SB202190) or NLK(Vx-702) the treatment activated HIPK2 indicating that expression levels of HIPK2 on one hand and NLK and MAPK11 on the other are coordinately regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-H).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Effects of HIPK2 /MAPK11/ NLK Inhibition in Gastric Cancer.\u003c/h2\u003e\u003cp\u003eTreatment with HIPK2 inhibitor (TBID) induced a dose and time dependent inhibition of AGS and SNU638 cells which express HIPK2 protein albeit at lower levels, indicating that inhibition of HIPK2 kinase activity can suppress cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Further we determined the levels of anti-apoptotic markers upon inhibitor treatment, in both the cell lines we observed decreased Bcl-xL and increased Mcl-1 expression, suggesting that HIPK2 inhibition alone may not suppress anti-apoptotic signalling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Since our observation of HIPK2, NLK and MAPK11 expression in gastric cancer celllines revealed that the expression levels of NLK and MAPK11 were higher than HIPK2 we wanted to assess the efficacy of targeting NLK and MAPK11 on GC cell proliferation. AGS and SNU638 cells were resistant to the NLK inhibitor(Vx-702) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], but the MAPK11 inhibitor (SB202190) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] significantly decreased cell proliferation in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-F). Anti-apoptotic markers Bcl-xl and Mcl-1 were found to be decreased in both cell lines upon NLK inhibitor treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). The MAPK11 inhibitor decreased Mcl-1 levels and increased Bcl-xL in SNU638 cells, while in AGS cells, Mcl-1 levels decreased with no significant change in Bcl-xL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.8. Combinational inhibitor treatment comprising of HIPK2-MAPK11/NLK Inhibitor manifests synergism in GC\u003c/h2\u003e\u003cp\u003eOur studies using individual inhibitors targeting NLK, MAPK11 and HIPK2 revealed that single agent treatments exhibit differential effects in GC cells. For instance, an NLK inhibitor decreased anti-apoptotic markers but did not affect cell proliferation, whereas a MAPK11 and HIPK2 inhibitor exhibited toxicity but also induced anti apoptotic marker levels. A previous study had shown that the sensitivity to the NLK inhibitor (Vx-702) increased upon mTOR inhibition [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Since TBID suppressed mTOR signaling in SNU638 cells, we explored whether TBID could enhance the sensitivity of Vx-702 in GC. Additionally, since induction of antiapoptotic markers were complementary between TBID and SB202190, a combination of TBID and SB202190 was tested. In both AGS and SNU638 cells combinational inhibitor testing showed that combining TBID with either Vx-702 or SB202190 inhibitors led to pronounced reductions in cell viability compared to individual inhibitor treatments. The combination index (CI) values, calculated using CompuSyn software [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], were consistently indicative of the synergistic effect for these combinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D,Table.3\u0026ndash;6, Supplementary Fig.\u0026nbsp;5). This synergistic activity of the combinatorial inhibitor treatment comprising TBID with either Vx-702 or SB202190 was further demonstrated in NUGC-3 cells. (Supplementary Fig.\u0026nbsp;6,Table.S4-5). Furthermore, Annexin V/PI staining revealed that AGS cells treated with either individual or combinations of TBID (50uM) and Vx-702 (50uM) / SB202190(50uM) inhibitors for 24 hrs exhibited 2.62% (TBID), 0.23% (Vx-702 ), 1.44% (SB202190), 8.91% (TBID and Vx-702), 22.17% (TBID\u0026thinsp;+\u0026thinsp;SB202190) of early apoptotic cells (FITC\u003csup\u003e+\u003c/sup\u003ePI\u003csup\u003e\u0026minus;\u003c/sup\u003e) compared to control, similarly SNU638 cells showed 4.53%(TBID), 5.09% (Vx-702 ),11.28% (TBID and Vx-702),17.89% (TBID\u0026thinsp;+\u0026thinsp;SB202190) of early apoptotic cells (FITC\u003csup\u003e+\u003c/sup\u003ePI\u003csup\u003e\u0026minus;\u003c/sup\u003e) compared to control indicating effectiveness of these combinations over individual treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-G). In our analysis combinations of NLK and MAPK11 inhibitors did not show synergistic potential in cell proliferation assays (Supplementary Fig.\u0026nbsp;7A-B,Table.S6). In summary, our results suggest that targeting HIPK2 in combination with either NLK or MAPK11 offers a viable strategy to induce a synergistic effect in GC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.9. Combinational inhibitor treatment comprising of HIPK2 \u0026ndash; NLK/MAPK11 Inhibitor mitigates mTOR signalling context independently.\u003c/h2\u003e\u003cp\u003eNext, we assessed the levels of molecular markers in response to combined treatment of HIPK2 with NLK or MAPK11 inhibitors in both AGS and SNU638 cells. Combining HIPK2 inhibitors with either NLK or MAPK11 inhibitors led to a reduction in the expression levels of genes such as cMYB, Myc, RICTOR, RAPTOR in both AGS and SNU638 cell lines(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). Combined treatments of HIPK2 with NLK or MAPK11 inhibitors resulted in a reduction in total p70S6K levels in both AGS and SNU638 cells. Furthermore, in AGS cells treated with IGF1 known to activate mTOR to promote processes like protein synthesis and cell proliferation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], the combination of HIPK2 and MAPK11 inhibitors decreased levels of p-p70S6K (Thr389), suggesting an impact on mTOR activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-D). The study also demonstrated that in SNU638 cells, combined inhibitor treatment with HIPK2 and MAPK11 /NLK inhibitors resulted in a more pronounced reduction in CD44 levels when compared to individual inhibitor treatment.Together, this combined inhibitor treatment decreased β-Catenin levels in AGS cells compared to inifividual treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE-F, Supplementary Fig.\u0026nbsp;7C).These markers β-Catenin [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and CD44 are known to be associated with the status of mTOR signalling and their decrease could indicate that indeed the combination treatment suppresses mTOR under the two different contexts tested. Overall, the combinatorial approach possibly addressed the context-dependent nature of individual inhibitor effects representing a promising therapeutic approach for gastric cancer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHIPK2 exhibits a complex, dual role in cancer, displaying both features of a tumour suppressor and a tumour promoter, and its effects being highly dependent on the context. Taking into consideration from the previous studies we assessed the context dependent role of HIPK2 based upon molecularly distinguishable expression levels of β-Catenin and CD44 in GC cell lines AGS, SNU638 and NUGC-3. CD44 a cell surface marker of Cancer Stem Cell (CSC) was found to promote tumorigenesis in several cancers such as gallbladder, breast and gastric cancer [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Similarly β-catenin promotes tumor progression in colon, glioma and gastric cancer[\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study in both SNU638 and NUGC-3 cells, ectopic expression of HIPK2 lead to a decrease in cell proliferation, suppressed colony forming ability and epithelial to mesenchymal transition (EMT). These results were found to be consistent with previous studies which indicate that HIPK2 overexpression downregulates vimentin [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and its knockdown increases N-cadherin, Fibronectin, decreases E-cadherin which potentiates EMT [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Similarly, Zhang, Na et al demonstrated the downregulation of HIPK2 in GC and potentially inhibited the EMT, migration, and invasive properties of GC[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Consistent with previous studies which showed that HIPK2 suppresses β-catenin [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] in our studies we found that the HIPK2 expression suppresses β-catenin and additionally α and γ catenin levels especially in SNU638 cells. Moreover, HIPK2 expression increased apoptotic potential by downregulating the mTORC2 while sustaining mTORC1 activity, indicating a probable dualistic effect. In AGS cells, the effects of HIPK2 are markedly different. The expression of HIPK2 did not alter the relative expression status of EMT markers and there was no change in the levels of mesenchymal marker α SMA, and activated Akt/mTOR pathway suggested pro-tumorigenic activity. Consistent with SNU638 cells HIPK2 expression decreased β-catenin levels which indicated HIPK2 activity in both type of cells since HIPK2 can reduce β-catenin levels by phosphorylation dependant degradation. Since stability of β-catenin affects CD44 levels [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] the loss of β-catenin possibly leads to a decrease in CD44 levels in SNU638 cells. A similar case of context dependent activity was previously observed with SP1 gene in gastric cancer. Knockdown of SP1 in intestinal type promoted migration and invasion whereas in diffuse type it was reduced [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMechanistically, the context dependent effects could plausibly be explained by HIPK2's dualistic effects on the PI3K/AKT and mTOR pathways. In SNU638 cells, HIPK2 expression lead to differential phosphorylation of Akt whereas in AGS HIPK2 expression the Akt markers were indicative of an active Akt pathway. The mTOR pathway, which is critical for cell proliferation and survival [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], was also differentially affected. In SNU638 cells, HIPK2 expression downregulated the mTORC2 arm, increasing apoptotic potential, while sustaining mTORC1 activity conversely in AGS cells, HIPK2 expression activated both mTORC1 and mTORC2. The activity of mTORC1 was further confirmed by assessing for metabolite targets of mTOR in both SNU638 and AGS. In summary our findings demonstrated that HIPK2 overexpression activates mTORC1 signalling in AGS and SNU638 cells. These results led us to target mTOR by inhibiting HIPK2 with TBID.\u003c/p\u003e\u003cp\u003eIntriguingly, treatment with TBID also activated HIPK2 expression and this was concomitant with the activation of markers for mTOR signalling in AGS cells. SNU638 cells in contrast upon TBID treatment showed a decrease in HIPK2 and markers for mTOR signalling. Intrestingly the treatment with TBID showed similar effects as HIPK2 overexpression in AGS cells. We hypothesise that the activation of mTOR marker expression in AGS cells despite HIPK2 kinase inhibition is due to the transcriptional upregulation of HIPK2 observed upon inhibitor treatment. This is plausible, since the domain structure of HIPK2 comprises a C-terminal Homeodomain-Interacting Domain (HID) for transcriptional factors which contributes to its transcriptional activity [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Additionally we observed that that β-catenin levels in AGS is unaltered which is due to inhibitor treatment indicating inhibtion of HIPK2 kinase activity. All of this indicates that targeting HIPK2 with TBID alone to suppress mTOR will be hindered by context dependent effects.\u003c/p\u003e\u003cp\u003eIn SNU638 cells, overexpression of HIPK2 suppresses cell proliferation, potentially through the degradation of CtBP1-a pro-tumorigenic transcriptional corepressor known to repress E-cadherin expression in gastric cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Conversely, inhibition of HIPK2\u0026rsquo;s kinase activity also suppress cell proliferation, likely due to its role in facilitating KRAS signaling and tumor progression. Treatment with the HIPK2 inhibitor TBID attenuates KRAS\u0026ndash;ERK pathway activation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Thus, both HIPK2 over expression and kinase activity inhibition suppress cell proliferation, demonstrating its dual regulatory role in gastric cancer.\u003c/p\u003e\u003cp\u003eA combined treatment approach was applied to overcome the context dependant effects of HIPK2 inhibition for this, we assessed kinases which are potentially linked to HIPK2 Kinase and serve as targets in GC cells. Using bioinformatic analysis we identified HIPK2 interaction with proteins involved in cancer-related processes, including NLK, MAPK11, MYB and TRIM28 in GC.The HIPK2-NLK-MAPK11 axis was marked by their common interaction with MYB. In a similar way Kanei-Ishii et al. identified a Wnt signalling interaction axis consisting of TAK1- HIPK2-NLK-MYB which facilitates cMYB proteosomal degradation[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. All of this opened up the possibility that the HIPK2 /NLK/MAPK11 axis is a viable target in GC. Treatment with an inhibitor of HIPK2 or NLK or MAPK11 showed that the treatment elicited differential effects especially in the response of anti-apoptotic markers.\u003c/p\u003e\u003cp\u003eThe study further investigated the potential for combined treatment strategies. The case for a combination of HIPK2 and NLK inhibitors was based on the previous studies which showed that inhibition of mTOR can sensitise cells to NLK inhibitor. The case for HIPK2 and MAPK11 inhibitors was based on the complementary effects on the levels of anti-apoptotic markers Bcl-xl and Mcl-1. The results from our combination studies showed synergism for both the combinations of HIPK2/NLK and HIPK2/MAPK11 concomitant with reduction in the mTOR pathway markers in both cell lines tested. A combination approach is likely to be more effective considering that several studies have demonstrated the resistance to individual inhibitor treatment. Possibly due to the activation of an alternative signalling pathway or feedback loop which would possibly be counteracted with combinatorial treatment. For instance Thakuri, et al. demonstrated that the MAPK inhibitors (MAPKi) develops resistance in colorectal cancer due to the activation of PI3K/AKT/mTOR pathway, the combinatorial inhibitor treatment comprising of MAPKi with PI3K/mTOR inhibitor exhibits synergy in suppressing the growth of colorectal cancer cells [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], Dual inhibitor treatment with RAD001 and Volasertib, a PLK1 inhibitor, targeting mTOR and PLK demonstrated synergistic anti-tumor effects in non-small cell lung cancer (NSCLC) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Zhu, Ruiqi et al. demonstrated that Targeting AML by FLT3 tyrosine kinase inhibitors (TKIs) together with Venetoclax, a Bcl-2 inhibitor exhibited synergy in inhibiting cell proliferation by counteracting the pro-survival pathways activated by FLT3 TKIs [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Consistent with previous studies in our study, unlike individual inhibitor treatments, the response to combinatorial treatment (HIPK2 inhibitor with either a NLK or MAPK11 inhibitor) was consistent across the cell lines tested, suggesting a potential to overcome the context-dependent activity of individual inhibitors.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, the dual role of HIPK2 in gastric cancer is influenced by the context and has impacts on cell proliferation, EMT, and key signalling pathways such as PI3K/AKT and mTOR. Significantly our study demonstrates the dualistic effects of HIPK2 on mTOR singalling. The understanding of context dependent activity of HIPK2 and the identification of HIPK2-NLK-MAPK11 axis led us to target mTOR context independently using a combinational approach. Our study has demonstrated that targeting HIPK2 in combination with NLK or MAPK11 can be a promising strategy to overcome context dependent effects of HIPK2 inhibition.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003e8. Conflict of Interest:\u003c/h2\u003e\u003cp\u003eThere was no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003e7. Authors contributions:\u003c/h2\u003e\u003cp\u003eGopal Gopisetty - conception and design; analysis and interpretation of data, drafting /revising the article(lead), Aathithya Rangarajan-acquisition of data; analysis and interpretation of data, drafting /revising the article (equal), Jayavelu Subramani-acquisition of LC-MS data, Priya Ramanathan- acquisition of Flow cytometry data,RamakrishnanAyloor Seshadri, Thirumoorthi Natarajan, Sujatha Lakshminarayanan \u0026ndash; Sample resource, Shirley Sunder Singh- analysis and interpretation of IHC data.\u003c/p\u003e\u003ch2\u003e6. Acknowledgements:\u003c/h2\u003e\u003cp\u003eThis study was financially supported by the Department of Science and Technology's Science and Engineering Research Board (DST-SERB) grant (CRG/2020/000877). The targeted metabolomics study was possible due to the kind donation of mass spectrometry equipment and related software by SPINCO technologies (Chennai, India) and Shimadzu (Kyoto, Japan) to the Cancer Institute (WIA).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgnew C, Liu L, Liu S, Xu W, You L, Yeung W, Kannan N, Jablons D, Jura N. The crystal structure of the protein kinase HIPK2 reveals a unique architecture of its CMGC-insert region. J Biol Chem. 2019;294(37):13545\u0026ndash;59. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.ra119.009725\u003c/span\u003e\u003cspan address=\"10.1074/jbc.ra119.009725\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou Q, Meng D, Li F, Zhang X, Liu L, Zhu Y, et al. Inhibition of HIPK2 protects stress-induced pathological cardiac remodeling. EBioMedicine. 2022;85:104274. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ebiom.2022.104274\u003c/span\u003e\u003cspan address=\"10.1016/j.ebiom.2022.104274\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng X, Pan Y, Chen X, Xia S, Hu Y, Zhou Y, et al. Inactivation of homeodomain-interacting protein kinase 2 promotes oral squamous cell carcinoma metastasis through inhibition of P53-dependent E-cadherin expression. Cancer Sci. 2021;112(1):117\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/cas.14691\u003c/span\u003e\u003cspan address=\"10.1111/cas.14691\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen P, Duan X, Li X, Li J, Ba Q, Wang H. HIPK2 suppresses tumor growth and progression of hepatocellular carcinoma through promoting the degradation of HIF-1α. Oncogene. 2020;39(14):2863\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41388-020-1190-y\u003c/span\u003e\u003cspan address=\"10.1038/s41388-020-1190-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNodale C, Sheffer M, Jacob-Hirsch J, Folgiero V, Falcioni R, Aiello A, et al. HIPK2 downregulates vimentin and inhibits breast cancer cell invasion. Cancer Biol Ther. 2012;13(4):198\u0026ndash;205. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4161/cbt.13.4.18694\u003c/span\u003e\u003cspan address=\"10.4161/cbt.13.4.18694\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuan N, Zhang W, Li Z, Sun L, Song T, Yu Z, et al. Overexpression of HIPK2 removes the transrepression of proapoptotic genes mediated by the CtBP1-p300-FOXO3a complex and increases the chemosensitivity in osteosarcoma cells. J Cancer. 2021;12(6):1826\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7150/jca.52115\u003c/span\u003e\u003cspan address=\"10.7150/jca.52115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQin Y, Hu Q, Ji S, Xu J, Dai W, Liu W, et al. Homeodomain-interacting protein kinase 2 suppresses proliferation and aerobic glycolysis via ERK/cMyc axis in pancreatic cancer. Cell Prolif. 2019;52(3):e12603. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/cpr.12603\u003c/span\u003e\u003cspan address=\"10.1111/cpr.12603\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKwon MJ, Kang SY, Nam ES, Cho SJ, Rho YS. HIPK2 overexpression and its prognostic role in human papillomavirus-positive tonsillar squamous cell carcinoma. Biomed Res Int. 2017;1056427. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2017/1056427\u003c/span\u003e\u003cspan address=\"10.1155/2017/1056427\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu F, Li N, Liu Y, Zhang J, Zhang J, Wang Z. Homeodomain interacting protein kinase-2 phosphorylates FOXM1 and promotes FOXM1-mediated tumor growth in renal cell carcinoma. J Cell Biochem. 2019;120(6):10391\u0026ndash;401. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jcb28323\u003c/span\u003e\u003cspan address=\"10.1002/jcb28323\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDai Y, Kyoyama H, Yang YL, Wang Y, Liu S, Wang Y, et al. A novel isoform of homeodomain-interacting protein kinase-2 promotes YAP/TEAD transcriptional activity in NSCLC cells. Oncotarget. 2021;12(3):173\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget27871\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget27871\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eConte A, Valente V, Paladino S, Pierantoni GM. HIPK2 in cancer biology and therapy: Recent findings and future perspectives. Cell Signal. 2023;101:110491. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cellsig.2022.110491\u003c/span\u003e\u003cspan address=\"10.1016/j.cellsig.2022.110491\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTorrente L, Sanchez C, Moreno R, Chowdhry S, Cabello P, Isono K, et al. Crosstalk between NRF2 and HIPK2 shapes cytoprotective responses. Oncogene. 2017;36(44):6204\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/onc.2017.221\u003c/span\u003e\u003cspan address=\"10.1038/onc.2017.221\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIlic M, Ilic I. Epidemiology of stomach cancer. World J Gastroenterol. 2022;28(12):1187\u0026ndash;203. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3748/wjg.v28.i12.1187\u003c/span\u003e\u003cspan address=\"10.3748/wjg.v28.i12.1187\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDikshit RP, Mathur G, Mhatre S, Yeole BB. Epidemiological review of gastric cancer in India. Indian J Med Paediatr Oncol. 2011;32(01):3\u0026ndash;11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/0971-5851.81883\u003c/span\u003e\u003cspan address=\"10.4103/0971-5851.81883\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePetrelli F, Berenato R, Turati L, Mennitto A, Steccanella F, Caporale M, et al. Prognostic value of diffuse versus intestinal histotype in patients with gastric cancer: a systematic review and meta-analysis.JGastrointest. Oncol. 2017;8(1):148\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21037/jgo.2017\u003c/span\u003e\u003cspan address=\"10.21037/jgo.2017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. .01. 10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh SS, Yap WN, Arfuso F, et al. Targeting the PI3K/Akt signaling pathway in gastric carcinoma: A reality for personalized medicine? World J Gastroenterol. 2015;21(43):12261\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3748/wjg.v21.i43.12261\u003c/span\u003e\u003cspan address=\"10.3748/wjg.v21.i43.12261\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarques-Ramos A, Cervantes R. Expression of mTOR in normal and pathological conditions. Mol Cancer. 2023;22(1):112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12943-023-01820-z\u003c/span\u003e\u003cspan address=\"10.1186/s12943-023-01820-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdvani SH. Targeting mTOR pathway: A new concept in cancer therapy. Indian J Med Paediatr Oncol. 2010;31(04):132\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/0971-5851.76197\u003c/span\u003e\u003cspan address=\"10.4103/0971-5851.76197\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFranken N, Rodermond H, Stap J, et al. Clonogenic assay of cells \u003cem\u003ein vitro\u003c/em\u003e. Nat Protoc. 2006;1:2315\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nprot.2006.339\u003c/span\u003e\u003cspan address=\"10.1038/nprot.2006.339\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTong J. Transcription profiling by array of human gastric cancer cell lines and normal gastricmucosa.BioStudies.2011.E-MEXP-2694.Retrieved \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003efromhttps://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MEXP-2694\u003c/span\u003e\u003cspan address=\"http://fromhttps://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MEXP-2694\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRajkumar T, Rajkumar T. Gene expression studies in apparently normal paired normal and malignant gastric tissues.\u003cem\u003eBioStudies\u003c/em\u003e.2010.E-GEOD-17154.Retrieved from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-GEOD-17154\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-GEOD-17154\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFlatbergA. Gene expression profiling of human gastric tumors and adjacent non-cancerous mucosa.\u003cem\u003eBioStudies\u003c/em\u003e.2014.E-MTAB-1338.Retrieved from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-1338\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-1338\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShao Q, Yao H, He J. Expression data from gastric cancer and paired normal tissues.\u003cem\u003eBioStudies\u003c/em\u003e.2016.E-GEOD-79973.Retrieved from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-GEOD-79973\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-GEOD-79973\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAn E, Brognard J. Orange is the new black: Kinases are the new master regulators of tumor suppression. IUBMB Life. 2019;71(6):738\u0026ndash;48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/iub.1981\u003c/span\u003e\u003cspan address=\"10.1002/iub.1981\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDur\u0026aacute;n RV, Oppliger W, Robitaille AM, Heiserich L, Skendaj R, Gottlieb E, et al. Glutaminolysis activates RAG-MTORC1 signaling. Mol Cell. 2012;47(3):349\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2012.05.043\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2012.05.043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEmmanuel N, Ragunathan S, Shan Q, Wang F, Giannakou A, Huser N, et al. Purine nucleotide availability regulates mTORC1 activity through the Rheb GTPase. Cell Rep. 2017;19(13):2665\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.celrep.2017.05.043\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2017.05.043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCozza G, Zanin S, Determann R, Ruzzene M, Kunick C, Pinna LA. Synthesis and properties of a selective inhibitor of homeodomain-interacting protein kinase 2 (HIPK2). PLoS ONE. 2014;9(2):e89176. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0089176\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0089176\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Published 2014 Feb 24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang C, Zhang Y, Zhang Y, Zhang Z, Peng J, Li Z, et al. Downregulation of cancer stem cell properties via mTOR signaling pathway inhibition by rapamycin in nasopharyngeal carcinoma. Int J Oncol. 2015;47(3):909\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/ijo.2015.3100\u003c/span\u003e\u003cspan address=\"10.3892/ijo.2015.3100\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang X, Veeraraghavan J, Liu CC, Cao X, Qin L, Kim JA, et al. Therapeutic targeting of nemo-like kinase in primary and acquired endocrine-resistant breast cancer.Clin. Cancer Res. 2021;27(9):2648\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1078-0432.CCR-20-2961\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.CCR-20-2961\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoche O, Fern\u0026aacute;ndez-Aroca DM, ,Arconada-Luque E, Garc\u0026iacute;a-Flores N, Mellor LF, Ruiz-Hidalgo MJ, et al. p38β and Cancer: The Beginning of the Road. Int J Mol Sci. 2020;21(20):7524. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms21207524\u003c/span\u003e\u003cspan address=\"10.3390/ijms21207524\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuarte D, Guerreiro I, Vale N. Novel strategies for cancer combat: Drug combination using repurposed drugs induces synergistic growth inhibition of MCF-7 breast and HT-29 colon cancer cells. Curr Issues Mol Biol. 2022;44(10):4930\u0026ndash;49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cimb44100335\u003c/span\u003e\u003cspan address=\"10.3390/cimb44100335\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDavaadelger B, Duan L, Perez RE, Gitelis S, Maki CG. Crosstalk between the IGF-1R/AKT/mTORC1 pathway and the tumor suppressors p53 and p27 determines cisplatin sensitivity and limits the effectiveness of an IGF-1R pathway inhibitor. Oncotarget. 2016; 7: 27511\u0026ndash;27526. Retrieved from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.oncotarget.com/article/8484/text/\u003c/span\u003e\u003cspan address=\"https://www.oncotarget.com/article/8484/text/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSemlali A, Papadakos S, Contant C, Zouaoui I, Rouabhia M. Rapamycin inhibits oral cancer cell growth by promoting oxidative stress and suppressing ERK1/2, NF-κB and beta-catenin pathways. Front Oncol. 2022;12:873447. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fonc.2022.87344\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2022.87344\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe Y, Xue C, Yu Y, Chen J, Chen X, Ren F, et al. CD44 is overexpressed and correlated with tumor progression in gallbladder cancer. Cancer Manag Res. 2018;10:3857\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/cmar.s175681\u003c/span\u003e\u003cspan address=\"10.2147/cmar.s175681\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcFarlane S, Coulter JA, Tibbits P, O\u0026rsquo;Grady A, McFarlane C, Montgomery N, et al. CD44 increases the efficiency of distant metastasis of breast cancer. Oncotarget. 2015;6(13):11465\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.3410\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.3410\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatanabe T, Okumura T, Hirano K, Yamaguchi T, Sekine S, Nagata T, et al. Circulating tumor cells expressing cancer stem cell marker CD44 as a diagnostic biomarker in patients with gastric cancer. Oncol Lett. 2017;13(1):281\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/ol.2016.5432\u003c/span\u003e\u003cspan address=\"10.3892/ol.2016.5432\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThaker AI, Rao MS, Bishnupuri KS, Kerr TA, Foster L, Marinshaw JM, et al. IDO1 metabolites activate β-catenin signaling to promote cancer cell proliferation and colon tumorigenesis in mice. Gastroenterology. 2013;145(2):416\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1053/j.gastro.2013\u003c/span\u003e\u003cspan address=\"10.1053/j.gastro.2013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ee254.doi\u003c/span\u003e\u003cspan address=\"http://e254.doi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang N, Wei P, Gong A, Chiu WT, Lee HT, Colman H, et al. FoxM1 promotes β-Catenin nuclear localization and controls WNT target-gene expression and glioma tumorigenesis. Cancer Cell. 2011;20(4):427\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ccr.2011\u003c/span\u003e\u003cspan address=\"10.1016/j.ccr.2011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDong L, Deng J, Sun ZM, Pan AP, Xiang XJ, Zhang L, et al. Interference with the β-catenin gene in gastric cancer induces changes to the miRNA expression profile. Tumor Biol. 2015;36(9):6973\u0026ndash;83. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s13277-015-3415-1\u003c/span\u003e\u003cspan address=\"10.1007/s13277-015-3415-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTan M, Gong H, Zeng Y, Tao L, Wang J, Jiang J, et al. Downregulation of homeodomain-interacting protein kinase-2 contributes to bladder cancer metastasis by regulating WNT signaling. J Cell Biochem. 2014;115(10):1762\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jcb.24842\u003c/span\u003e\u003cspan address=\"10.1002/jcb.24842\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang N, Meng H, Tian A, Chen Y. Expression of HIPK2 in gastric cancer and its effects on cell function in vitro. Minerva Gastroenterol. 2022;68(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.23736/s2724-5985.21.02969-7\u003c/span\u003e\u003cspan address=\"10.23736/s2724-5985.21.02969-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim EA, Kim JE, Sung KS, Choi DW, Lee BJ, Choi CY. Homeodomain-interacting protein kinase 2 (HIPK2) targets β-catenin for phosphorylation and proteasomal degradation. BiochemBiophys Res Commun. 2010;394(4):966\u0026ndash;71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbrc.2010.03.099\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2010.03.099\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi P, Zhang Z, Sun P. DOT1L promotes expression of CD44 through the Wnt/β-catenin signaling pathway in early gastric carcinoma. J Cancer. 2024;15(8):2276\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7150/jca.90170\u003c/span\u003e\u003cspan address=\"10.7150/jca.90170\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee HS, Park CK, Oh E, Erkin \u0026Ouml;C, Jung HS, Cho MH, et al. Low SP1 expression differentially affects intestinal-type compared with diffuse-type gastric adenocarcinoma. PLoS ONE. 2013;8(2):e55522. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0055522\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0055522\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePanwar V, Singh A, Bhatt M et al. Multifaceted role of mTOR (mammalian target of rapamycin) signaling pathway in human health and disease. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e. 2023;8(1):375. Published 2023 Oct 2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41392-023-01608-z\u003c/span\u003e\u003cspan address=\"10.1038/s41392-023-01608-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKuwano Y, Nishida K, Akaike Y, Kurokawa K, Nishikawa T, Masuda K, Rokutan K. Homeodomain-Interacting Protein Kinase-2: A Critical Regulator of the DNA Damage Response and the Epigenome. Int J Mol Sci. 2016;17(10):1638. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms17101638\u003c/span\u003e\u003cspan address=\"10.3390/ijms17101638\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Y, Yan X, Ren L, Li Y. miR-644a Inhibits Cellular Proliferation and Invasion via Suppression of CtBP1 in Gastric Cancer Cells. Oncol Res. 2018;26(1):1\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3727/096504016X14772410356982\u003c/span\u003e\u003cspan address=\"10.3727/096504016X14772410356982\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDi Segni M, Virdia I, Verdina A, Amoreo CA, Baldari S, Toietta G, Diodoro MG, Mottolese M, Sperduti I, Moretti F, Buglioni S, Soddu S, Rocco D, G. HIPK2 Cooperates with KRAS Signaling and Associates with Colorectal Cancer Progression. Mol cancer research: MCR. 2022;20(5):686\u0026ndash;98. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/1541-7786.MCR-21-0628\u003c/span\u003e\u003cspan address=\"10.1158/1541-7786.MCR-21-0628\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKanei-Ishii C, Ninomiya-Tsuji J, Tanikawa J, Nomura T, Ishitani T, Kishida S et al. Wnt-1 signal induces phosphorylation and degradation of c-Myb protein via TAK1, HIPK2 and NLK.Genes Dev. 2004;18(7):816\u0026ndash;2910.1101/gad.1170604\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThakuri PS, Gupta M, Joshi R, Singh S, Tavana H. Synergistic inhibition of kinase pathways overcomes resistance of colorectal cancer spheroids to cyclic targeted therapies. ACSPharmacolTransl Sci. 2019;2(4):275\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acsptsci.9b00042\u003c/span\u003e\u003cspan address=\"10.1021/acsptsci.9b00042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMontaudonE BRE, Vacher S. D\u0026eacute;asO, NaguezA, Chateau-Joubert S, High in vitro and in vivo synergistic activity between mTORC1 and PLK1 inhibition in adenocarcinoma NSCLC.Oncotarget.2021;12(8):859\u0026ndash;72.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.27930\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.27930\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu R, Li L, Nguyen B, Seo J, Wu M, Seale T, et al. FLT3 tyrosine kinase inhibitors synergize with BCL-2 inhibition to eliminate FLT3/ITD acute leukemia cells through BIM activation.SignalTransduct. Target Ther. 2021;6(1):186. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41392-021-00578-4\u003c/span\u003e\u003cspan address=\"10.1038/s41392-021-00578-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gastric cancer, HIPK2, NLK, MAPK11, Tumor Suppressor Kinases, Kinase Inhibitors","lastPublishedDoi":"10.21203/rs.3.rs-7795753/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7795753/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHomeodomain-interacting protein kinase 2 (HIPK2) can function either as a tumour suppressor or promoter in cancer. Understanding key features of HIPK2 context dependent effects in cancer could lead to novel therapeutic strategies. Our investigation of HIPK2 expression in Gastric Cancer (GC) showed it to be down regulated. HIPK2 expression manifested context dependent effects on cell proliferation, epithelial to mesenchymal transition and anti-apoptotic marker levels as observed in GC cell lines SNU638 and NUGC-3 (β-Catenin\u003csup\u003elow\u003c/sup\u003e, CD44\u003csup\u003eHi\u003c/sup\u003e) and AGS (β-Catenin\u003csup\u003eHi\u003c/sup\u003e, CD44\u003csup\u003elow\u003c/sup\u003e). The context dependent effects are plausible due to HIPK2's differential impacts on the Akt-mTOR pathway. The application of HIPK2 inhibitor (TBID) further supported context-dependent effect on mTOR signalling markers. To address the context dependent effects of HIPK2, gene expression combined with protein-protein interactions analysis was used to identify the HIPK2-NLK-MAPK11 axis linked by their interaction with transcription factor MYB in GC. Validation of the axis was shown by increased NLK and MAPK11 expression in GC cell lines concordant with lower HIPK2 expression. Similarly, HIPK2 ectopically expressing cells showed lower levels of NLK and MAPK11 expression. Combinational targeting of HIPK2-NLK/MAPK11 axis with (HIPK2i\u0026thinsp;+\u0026thinsp;NLKi) or (HIPK2i\u0026thinsp;+\u0026thinsp;MAPK11i) resulted in a synergistic effect suppressing GC cell proliferation and mTOR markers. Targeting HIPK2 in combination with NLK or MAPK11 can be a promising strategy to overcome context dependent effects of HIPK2.\u003c/p\u003e","manuscriptTitle":"HIPK2 exhibits context dependent dualistic effects and combinational inhibition of HIPK2-NLK-MAPK11 axis manifests synergism against gastric cancer.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-28 18:08:14","doi":"10.21203/rs.3.rs-7795753/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-10-20T06:46:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-14T01:50:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-07T13:47:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2025-10-07T00:49:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"137966c3-df06-4534-9819-d5531d0eaa31","owner":[],"postedDate":"October 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-28T18:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-28 18:08:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7795753","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7795753","identity":"rs-7795753","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}