The long isoform of PRLR promotes tumor progression by regulating CDK6 through MAPK signal pathway in SHH medulloblastoma

The long isoform of PRLR promotes tumor progression by regulating CDK6 through MAPK signal pathway in SHH medulloblastoma | 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 Article The long isoform of PRLR promotes tumor progression by regulating CDK6 through MAPK signal pathway in SHH medulloblastoma Ziwen Sun, Haishuang Li, Yu Peng, Yantao Liu, Yujia Wang, Jing Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5667015/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Medulloblastoma (MB) is the most common malignant brain tumors in children. Sonic Hedgehog (SHH) subgroup of MB accounts for about 25% of all MBs. SMO inhibitors are used for target therapy. However, drug resistance and toxicity occurred. New therapeutic targets are urgently needed to be developed. Here, through RNA-sequencing and Nanostring Assay analysis of primary MBs, we screened out prolactin receptor (PRLR) as a gene with higher expression level in SHH-MB compared with other subgroups of the tumor. Long isoform of PRLR (PRLR-LF) played a pivotal role in promoting SHH-MB tumor invasion, enhancing the proliferation and colony formation ability. KEGG analysis showed that PRLR-LF expression has close relationship with p53 signal pathway in SHH-MB cells. High expression of CDK6 downstream of the p53 pathway was observed to have a high correlation with PRLR expression, indicating a poor prognosis of the tumor. In addition, PRLR was demonstrated to promote cell proliferation by regulating CDK6 through Ras-MAPK signal pathway in vitro . Synthesized recombinant Δ1-11-G129R-PRL, a competitive inhibitor of PRLR, interfered PRL-PRLR binding, could inhibit the regulation to CDK6, and could and inhibit the proliferative ability of SHH-MB tumor cells. In conclusion, we unveiled PRLR promoted SHH-MB tumor progression through signaling pathway besides the canonical SHH pathway. PRLR inhibitor shed light on a potential therapeutic value for SHH-MB patients. Biological sciences/Cell biology/Mechanisms of disease Health sciences/Biomarkers/Prognostic markers Health sciences/Pathogenesis/Oncogenesis Medulloblastoma prolactin receptor CDK6 Sonic hedgehog Tumor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Medulloblastoma (MB) is the most common malignant brain tumors in childhood. It accounts for approximately 20% of all intracranial neoplasms in this age group. 1 , 2 As reported in 2021 World Health Organization classification of tumors, at least four consensus subgroups of MB are reported, including wingless (WNT), Sonic Hedgehog (SHH) with/without P53 mutation, and non-WNT/SHH (Groups 3 and 4). Different genetic signatures and DNA methylation status of each subgroup lead to different prognosis, histopathological features, and therapeutic strategy. 3 SHH-MB is characterized by the persistent activation of the SHH signaling pathway. 4 In the canonical SHH signaling pathway, the SHH ligand binds to the PTCH1 receptor on the cell membrane, inhibiting its blocking effect on the Smo. This ultimately leads to the transcription factor GLI entering the cell nucleus and initiating the transcription of SHH-related oncogenes. 5 , 6 SMO inhibitors, sonidegib and vismodegib, are used for targeted therapy of SHH-MB. 7 , 8 However, these drugs are challenging to use in clinical settings due to their high toxicity and drug resistance. 9 , 10 , 11 New adjunctive therapeutic target which is less toxic is needed to serve as clinical demands. SHH-MB differs from the other subgroups of MBs in that it originates from granule neuron precursor cells and has a greater number of gene characteristics associated with granule neurons. 12 , 13 Therefore, identifying the gene characteristics specific to SHH-MB, as compared to the other three subgroups, can help in discovering new drug treatment targets. Here, we analyzed the gene characteristics of various MB subtypes using RNA sequencing (n = 37) and Nanostring assay (n = 48), and identified PRLR as a potential biomarker for SHH-MB. As an oncogene, PRLR promotes the progression in many cancers, such as breast cancer, prostate cancer, etc. 14 , 15 PRL, as its natural ligand, binds to PRLR to activate various cancer-related signaling pathways, such as JAK/STAT1/STAT5, PI3K/AKT and Ras-MAPK. 16 , 17 , 18 The Ras-MAPK signaling pathway, as the main downstream signaling pathway of PRLR, regulates cell growth and proliferation. 19 Therefore, identifying key pathways and genes regulated by PRLR in SHH-MB can help to elucidate its cancer promoting function. In this study, we elucidated that the interaction between PRL and PRLR can promote tumor progression and enhance the proliferation ability of SHH-MB. The recombinant Δ1-11-G129R-PRL, an inhibitor of PRLR, served as a potential target inhibitor to suppress tumor progression. We also validated that high expression of PRLR or PRL-PRLR combination regulated CDK6 expression through the classic Ras-MAPK pathway, resulting in a stronger proliferative capacity of the tumor. Methods cell lines, antibodies, and reagents Human ONS-76 MB cell line was a generous gift from Prof. Xiu-Wu Bian (Southwest Hospital, Army Medical University). Human Daoy MB cell line were kindly provided by Prof. Ho Ho-Keung Ng (The Chinese University of Hong Kong). Human 293T cell were purchase from ATCC. RPMI 1640 (Gibico) with 10%FBS were used to culture ONS76 in a 37°C incubator with 5% (v/v) CO2. MEM (Gibico), DMEM(Gibico) with 10%FBS were used to culture Daoy and 293T in a 37°C incubator with 5% (v/v) CO2. Primary antibodies used for western blot, and immunohistochemistry stain are listed in Supplementary Table 1. plasmid construction The plasmids pLKO-puro, pLV-copGFP-puro, pGEX4T-1-puro were purchased from Addgene. PRLR-LF, PRLR-S1a, PRLR-S1b, Δ1-11-G129R-PRL,and short hairpin RNA sequences were inserted into those plasmid. All sequences constructed into those plasmids are listed in Supplementary Table 2. RNA extraction, RT-q-PCR analysis and RNA sequence analysis Cells RNA is extracted by TRIZOL reagent. RT-q-PCR were performed as done previously. RNA sequence analysis was performed by Majorbio (Shanghai). western blot analysis All experiments were performed on ice. Cells were washed 3 times by PBS at 4℃, then lysed in RIPA containing protease inhibitors and phosphatase inhibitors. Protein lysates were separated bysodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride membrane (ISEQ00010, Merck Millipore, 0.22 µm). After blocking with 5% skimmed milk, the membrane was incubated with antibodies exhibiting in Supplementary Table 1 at 4℃ overnight. Then incubated with secondary antibody (7074&7076, CST, 1:2000) at room temperature in 1h. Protein signals were visualized both with Super ECL Plus Substrate (Thermo Fisher) in Gel imaging system (Bio-Rad ChemiDoc MP) and in darkroom exposure. ImageJ (Ver.1.53e; NIH, Bethesda, MD) was used to quantify the protein bands, normalized to GAPDH, and determined as the percentage value. siRNA transfection and lentiviral infection si-RNA tRasfection were performed using jetPRIME transfection kit (polyplus) according to the manufacturer’s instructions. Lentiviral was packaged in 293T cell line by transfecting psPAX-2, pMD2.G and targeted plasmid at the same time. After 48h, lentiviral were purified by 100kd ultrafiltration tube (TIANGEN) and then add to infect target cell. cell counting kit-8(CCK8) assay Cells were plated into a 96-well plate (Thermo Fisher Scientific) at the density of 600 to 800 cells per well for determination of cell viability. CCK-8 reagent (Solarbo) was added into medium after the cells were cultured each 24h. After incubation for 2 h at 37°C, absorbance were detected by microplate reader (Thermo Fisher) at the light length of 450 nm. Each assay was repeated independently for 5 times. clone formation assay 500 cells were planted into a 6-well plate (Thermo Fisher Scientific). After 6–8 days culture until 50 cells per single cell colony, cells were fixed with methanol, stained with 0.1% crystal violet, and then quantified by ImageJ independently for 3 times. transwell assay Transwell migration assays were performed in a 6-well plate. In brief, cells in serum-free medium were seeded in the upper chamber, and the lower chamber was filled with complete media. After 24 h, cells in the upper chamber were gently removed. Migrated cells on the lower side of the membrane were fixed with methanol, stained with 0.1% crystal violet, and then counted at 10× magnification in three random fields per well. recombinant protein purification GST-tagged Δ1-11-G129R-PRL expressed in E. coli were purified using GST Fusion Protein Purification Kit (Beaver) according to the manufacturer’s instructions. Then purified product were digested by Thrombin (Solarbo) and then verified by Coomassie Brilliant Blue Staining. subcutaneous transplantation tumor in Balb/c nude mice Balb/c nude mice were bought from Dept. of Experimental Animal Science, Peking University Health Science Center, Beijing, 100191. Animal experiments have obtained ethical approval (LA2019121). All methods were performed in accordance with the relevant guidelines and regulations. Authors of this article have obtained a work permit for experimental animals. Under the premise of following the 3R principle of animal experimentation, 1×10 6 Daoy cells were injected subcutaneously into those nude mice 6 mice per group after anesthesia by pentobarbital sodium. After 24 days of measurement interval 3 days, the data showed statistical differences. To comply with the ethics of experimental animals, mice were executed. Tumor were removed for quantification. Results High expression PRLR in SHH medulloblastoma To explore potential therapeutic target for SHH-MBs, RNA sequence analysis and Nanostring assay were performed covering four subtypes of MB, WNT activated (WNT), Sonic hedgehog pathway activated (SHH), Group3 (G3) and Group4 (G4)(Fig. 1 A). ‘Deseq2’ and ‘pheatmap’ package in R were used to screen out genes with differential expression. Totally 125 genes were detected with higher expression in SHH-MBs compared to other subtypes of MB by RNA-seq (Fig. 1 B). In our previous study, 20 , 21 nanostring assay of 48 MB patients were analyzed focusing on differential genes from SHH-MB to other 3 subtypes MB. Six genes that are significantly different from the other three subtypes were screened out. By intersecting the 125 genes enriched from RNA-seq sequencing with the differentially expressed genes obtained from the Nanostring assay, five candidates were identified, TGFB2, CXCL4, PRLR, SOX2, and SFRP1. (Fig. 1 A). Survival analysis by R2 dataset ( https://hgserver1.amc.nl/ ) revealed that PRLR expression level in SHH-MB may be correlated with female patients’ prognosis (Fig. 1 C, Supplementary Fig. 1A). GSE datasets analysis also showed PRLR expression level in SHH-MB is significantly higher than other 3 subtypes of MB (Fig. 1 D). Volcano plots revealed a significant difference of PRLR expression in SHH-MB compared to other three subtypes (Supplementary Fig. 1B). These data showed that PRLR may serve as a potential oncogene, which was correlated with poor prognosis of female patients of SHH-MB. Long isoform of PRLR (PRLR-LF) functions as the main component of PRLR in SHH-MB To validate whether PRLR is highly expressed in SHH-MB, RT-q-PCR and immunohistochemistry staining were performed in 4 subgroups of primary MBs. Higher expresson of PRLR on both RNA and protein level were confirmed in SHH-MBs compared to other 3 subtypes, respectively (Fig. 2 A,B). It has been reported that the PRLR gene has different splicing variants due to alternative splicing. 22 The full-length transcript PRLR-LF can generate transcripts such as PRLR-IF, PRLR-S1a, and PRLR-S1b through alternative splicing and deletions. 23 , 24 Among them, PRLR-LF and PRLR-SF(PRLR-S1a, PRLR-S1b) are currently well studied. To ivestigate the expression of the PRLR isoforms in SHH-MB patients and SHH-MB cell lines, the alternative splicing variants of PRLR in SHH-MB were tested to start with RT-q-PCR analysis in SHH-MB tumor tissue from patients. The result confirmed that long isoform of PRLR (PRLR-LF 80 kD) is the main component of PRLR spliceosome in the tumor (Fig. 2 C), not PRLR-S1a (40 kD), nor PRLR-S1b (32 kD). Similar result was obtained in the SHH-MB cell lines (Daoy and ONS-76) (Fig. 2 D). Furthermore, westernblot was performed to identify the gene’s expression on protein level. The data showed the same trend as the RNA level (Fig. 2 E). In order to further elucidate the possible function of PRLR-LF in SHH-MB, shRNA sequences were inserted into pCDH-copGFP-PURO and knockdown efficiency was tested on both RNA and protein level (Figure.2F,G). PRLR-LF CDS sequence cloning from 293T’s cDNA was inserted into pLVX-CMV-PURO to overexpress PRLR-LF in Daoy and ONS-76, respectively. Overexpression efficiency was tested by westernblot (Fig. 2 G) PRLR-LF overexpression regulated cell proliferation through p53 signal pathway In order to further elucidate the function of PRLR-LF in SHH-MB, differential expression was performed by RNA-seq in PRLR-LF overexpressed Daoy and wild type Daoy cells. About 3000 genes were screened out (Fig. 3 A) using R package ‘DEseq2’. KEGG analysis revealed high expression of PRLR-LF was significantly correlated with the p53 signaling pathway (Fig. 3 C). Further heatmap showed overexpression of PRLR-LF was associated with downstream genes of the p53 signaling pathway rather than p53 and p21 themselves (Fig. 3 B). The correlation between PRLR-LF and downstream genes of the P53 pathway was confirmed by RT-q-PCR assay (Fig. 3 D). Considering that many downstream genes of the p53 signaling pathway are involved in regulating cell proliferation, the phenotype of cell proliferation was verified by clone formation assay (Fig. 3 E), transwell assay (Fig. 3 F), and CCK-8 prolifiation assay (Figure.3G), respectively. The results indicated that overexpressed PRLR-LF in SHH-MB cell lines increased cell proliferation, migration, and cell colonization abilities, which may promote malignancy of SHH-MB tumor cells. Δ1-11-G129R-PRL, as a competitive inhibitor of PRL, suppressed SHH-MB’s proliferation by regulating CDK6 PRLR-LF as a cell membrane protein, participates in the transmission process of downstream signaling pathways. 25 PRL, as a natural ligand of PRLR, can promote tumor develpoment through PRLR-LF. 26 However, several modified ligand can serve as a competitive inhibitor to block its function. 27 Among them, PRL analogues reported to inhibit the function of PRLR have been used in the treatment of breast cancer and glioblastoma. 28 We hypothesized that Δ1-11-G129R-PRL as a inhibitor of PRL, may competitively bind to PRLR and inhibit tumor progression. To explore whether PRLR could directly regulate p53 and p21, then regulate the downstream signal pathway of p53 to promote tumor development, western blot were performed to detect the correlation between PRLR-LF and p53, p21 (Fig. 4 A). However, no direct correlation between them was observed by knockout of P53 using lenti-CRISPR-v2 in ONS-76 (p53 wild type) cell line (Fig. 4 B). It suggested that other genes involved in the p53 pathway may be regulated by PRLR-LF. R2 dataset screened out 7 genes positively correlated with PRLR-LF expression., among which CDK6 is located downstream of the p53 signaling pathway (Fig. 4 C). To exclude the possibility that PRLR-SF may be involved in regulating those genes’ expression, RT-q-PCR were performed to exclude the effect of PRLR short variant on CDK6 and other genes frequently involved in the p53 pathway (Fig. 4 D). Whereas, a significant positive correlation between CDK6 and PRLR in SHH-MB patients was identified by R2 correlation analysis (Fig. 4 E). Meanwhile, prognostic survival analysis showed high expression of CDK6 had positive relationship with SHH-MB patients’ prognosis (Fig. 4 F). To verify that PRLR can regulate CDK6 to promote tumor progression, recombinate Δ1-11-G129R-PRL was synthesized (Supplementary Fig. 2A) and was added as an inhibitor to cell culture medium. A siignificant inhibition effect on PRL was observed. Expression of CDK6 was rescued to normal level in both wild-type cells or PRLR-LF overexpressing cells (Fig. 4 G). Western blot also revealed significant inhibition of Δ1-11-G129R-PRLon PRL in Daoy and ONS-76 cells (Fig. 4 H). Furthermore, CCK-8 proliferation and clone formation assay showed that Δ1-11-G129R-PRL significantly inhibits the proliferation ability of SHH-MB cells (Fig. 4 I-J). CDK6 regulated by PRLR through Ras-MAPK pathway promotes SHH-MB tumor progression Due to the fact that PRLR can regulate the expression of cyclinD through Ras-MAPK signaling pathway, 19 CDK6 may be regulated similarly through this signal pathway. To valid this hypothesis, si-Ras and si-ERK1/2 were transfection into Daoy and ONS-76 cells. Westernblot revealed that CDK6 expression were upregulated by PRLR through Ras-Raf-MEK-ERK signal pathway activation in SHH-MB cells (Fig. 5 A-B). Whereas other 2 MAPK subfamily members JNK and p38 showed no difference after Ras-ERK signal pathway treatment (Fig. 5 A-B). To explore whether PRLR-LF overexpression may cause tumor progression, PRLR-LF overexpressed Daoy cells were injected to Balb/c nude mice after anesthesia by pentobarbital sodium (n = 5). After 24 days of measurement interval 3 days, PRLR-LF overexpressed cells showed a noticeable proliferation vitality compared to control cells after 21 days(about 60 mm 3 vs 30 mm 3 ) (Figure.5C). Under the premise of following the 3R principle, mice were executed and the tumor was extracted (Supplementary Fig. 3A). The tumor volume of PRLR-LF overexpressing cells significantly increased compared to control cells(about 60 mg vs 38mg) (Figure.5D). To further validate the expression correlation between PRLR and CDK6 in SHH-MB patients, immunohistochemistry staining was performed using FFPE SHH-MB samples (n = 40) (Figure.5F). CDK6 showed a significant correlation with PRLR in SHH-MB patients (Figure.5E). Meanwhile, we defined 2 groups by the expression of PRLR evaluated by two pathologists independently. We found that higher expression level of PRLR was accompanied with higher CDK6 expression level and higher Ki-67 proliferation index, which may cause a malignant progression in SHH-MB patients. (Figure.5G). Conclusions Given the persistent activation of the SHH signaling pathway in SHH-MB, most studies target the SHH signaling pathway itself. To identify transcriptional signatures specific to SHH-MB beyond the SHH signaling pathway, we explored the transcriptional profiles of SHH-MB compared to the other three subtypes at the whole transcriptome level through Nanostring and RNA sequencing analyses. Here, we screened out prolactin receptor (PRLR) as a gene with higher expression level in SHH-MB compared with other subgroups of the tumor. Long isoform of PRLR (PRLR-LF) played a pivotal role in promoting SHH-MB tumor invasion, enhancing the proliferation and colony formation ability. PRLR-LF expression has positive correlation with CDK6 through Ras-MAPK signal pathway, causing a poor prognosis in SHH-MB patients (Fig. 1 A). Mitogen-activated protein kinase (MAPK) signaling pathway is a classic cancer-related pathway that has been extensively studied. 29 Activation of the Ras-Raf-MEK-ERK signaling pathway leads to the activation of cell cycle-related genes associated with proliferation, promoting the progression of the tumor cell cycle and enabling tumor growth. 30 PRLR, as an upstream molecule in the classic MAPK pathway, mediates the activation of various cancer-related signaling pathways. 25 Previous reports indicated that activation of PRLR can activate the Ras-Raf-MEK-ERK pathway, regulating cyclin D1 expression to advance the cell cycle. 19 Recent studies also suggested that PRLR-SF, a variant of PRLR, has the ability to inhibit tumor cell proliferation through the NEK9-Hippo signaling pathway. 31 Due to the presence of different transcriptional variants of PRLR with distinct signal functions, 25 , 31 we conducted experiments and found that the PRLR mainly act as the long isoform (PRLR-LF) in SHH-MB. We confirmed that PRLR-LF is highly expressed in SHH-MB compared to other 3 subtypes MB patients and cell lines. It promotes cell viability, proliferation, and colony formation ability of the tumor cells, which is contrary to the function of PRLR-SF in pancreatic cancer. 31 We validated in vitro that a positive relationship between CDK6, an oncogene associated with tumor growth, and PRLR in SHH-MB was mediated by the PRL-PRLR binding followed by activation of the RAS-MAPK signaling pathway. Immunohistochemistry (IHC) results from primary SHH-MBs also demonstrated a positive correlation among PRLR, CDK6 and the proliferation index of the tumor (Fig. 5 G). It has been shown that hG129R-PRL, a PRL analog, can occupy the active site of PRLR, blocking the binding between PRLR and PRL and thereby inhibiting the activation of downstream signaling pathways in breast cancer cell lines and glioblastoma cell lines. 27 , 28 Our data showed the first time that Δ1-11-G129R-PRL inhibited the function of PRLR through reducing CDK6 expression in SHH-MB cells (Fig. 4 G-H). PRLR and CDK6 could serve as potential therapeutic targets for SHH-MBs, especially for female SHH-MB patients with PRL high expression. However, urgent research is needed on the potential application of combining Δ1-11-G129R-PRL and Smo inhibitors in the treatment of SHH-MB mouse model. In summary, we selected PRLR as a highly expressed gene specifically in SHH-MBs. We elucidated that the interaction between PRL and PRLR can promote tumor progression and enhance the proliferation ability of SHH-MB. We validated the high expression of PRLR regulates CDK6 expression through the classic MAPK signal pathway, promoting proliferative capacity of the tumor. Δ1-11-G129R-PRL may serve as a potential target inhibitor to suppress tumor progression. Declarations Supplemental information Supplementary Figure 1-3, Supplementary Table 1-2 Data availability statement All data generated or analyzed during this study are included in this published article and its supplementary information files. Further information and requests for reagents should be directed to and will be fulfilled by the Lead Contact Qing Chang ( [email protected] ). Funding statement This study was supported by the Beijing Natural Science Foundation (No.7232098, No.7192095), Beijing Health Foundation Commission (11000023T000002044300-4) and the National Natural Science Foundation of China (No.81972353, No.81101900, No.30540008) to Qing Chang. Conflict of interest disclosure The authors declare no conflict of interest. Ethics approval statement All animal experiments were reported in accordance with ARRIVE guidelines . Experiments referred to Ethics were permitted by Animal Experimentation Ethics Committee No. LA2019121. Patient consent statement Investigators have to obtain informed consent before using participants’ FFPE for IHC staining Permission to reproduce material from other sources No reproduce figure or material from other sources was quoted in this study. Clinical trial registration No clinical trial was carried out in this study. Acknowledgments We appreciate XiuWu Bian's lab for providing the ONS-76 cell line. Author contributions Conceptualization, Z.S. and H.L.; methodology, Z.S., Y.L. and H.L.; software, Z.S.; validation, Y.W., H.L. and Q.C.; formal analysis, Z.S., D.Z.; investigation, Z.S., J.W. and Y.P.; resources, H.L. and Q.C.; data curation, Y.W., Y.P. and Z.S..; writing—original draft preparation, Y.W. and Z.S.; writing—review and editing, Z.S., H.L., X.L. and Q.C.; visualization, Z.S., D.F. and X.L..; supervision, H.L. and Q.C.; project administration, Q.C.; funding acquisition, Q.C.. All authors have read and agreed to the published version of the manuscript. References Ostrom QT, Cioffi G, Gittleman H, Patil N, Waite K, Kruchko C, Barnholtz-Sloan JS. CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2012-2016. Neuro Oncol. 2019 , 21, 1-100. Khanna V, Achey RL, Ostrom QT, Block-Beach H, Kruchko C, Barnholtz-Sloan JS, de Blank PM. Incidence and survival trends for medulloblastomas in the United States from 2001 to 2013. J Neurooncol. 2017 , 135, 433-441. Perreault S, Ramaswamy V, Achrol AS, Chao K, Liu TT, Shih D, et al. MRI surrogates for molecular subgroups of medulloblastoma. Am J Neuroradiol. 2014 , 35, 1263–1269. Lospinoso Severini L, Ghirga F, Bufalieri F, Quaglio D, Infante P, Di Marcotullio L. The SHH/GLI signaling pathway: a therapeutic target for medulloblastoma. Expert Opin Ther Targets 2020 , 24, 1159-1181. Carballo GB, Honorato JR, de Lopes GPF, Spohr TCLSE. A highlight on Sonic hedgehog pathway. Cell Commun Signal 2018 ,16,11. Waszak SM, Robinson GW, Gudenas BL, Smith KS, Forget A, Kojic M, et al. Germline elongator mutations in Sonic Hedgehog medulloblastoma. Nature 2020 , 580, 396–401. Sekulic A, Von Hoff D. Hedgehog Pathway Inhibition. Cell 2016 , 164, 831. Espinosa-Bustos C, Mella J, Soto-Delgado J, Salas CO. State of the art of Smo antagonists for cancer therapy: advances in the target receptor and new ligand structures. Future Med Chem. 2019 , 11, 617-638. Rudin CM, Hann CL, Laterra J, et al. Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N Engl J Med 2009 , 361, 1173-1178 Yauch RL, Dijkgraaf GJ, Alicke B, et al. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science 2009 , 326, 572-4 Metcalfe C, de Sauvage FJ. Hedgehog fights back: mechanisms of acquired resistance against smoothened antagonists. Cancer Res. 2011 , 71, 5057-5061 Wechsler-Reya RJ, Scott MP. Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 1999 , 22, 103-114. Oliver TG, Read TA, Kessler JD, Mehmeti A, Wells JF, Huynh TT, Lin SM, Wechsler-Reya RJ. Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma. Development 2005 , 132, 2425-2439. Kavarthapu R, Anbazhagan R, Dufau ML. Crosstalk between PRLR and EGFR/HER2 Signaling Pathways in Breast Cancer. Cancers (Basel). 2021 , 13, 4685. Damiano JS, Wasserman E. Molecular pathways: blockade of the PRLR signaling pathway as a novel antihormonal approach for the treatment of breast and prostate cancer. Clin Cancer Res. 2013 , 19, 1644-1650. Huang Y., Li X., Jiang J., Frank S.J. Prolactin modulates phosphorylation, signaling and trafficking of epidermal growth factor receptor in human T47D breast cancer cells. Oncogene 2006 , 25, 7565–7576. Frank S.J. Mechanistic aspects of crosstalk between GH and PRL and ErbB receptor family signaling. J. Mammary Gland Biol. Neoplasia. 2008 , 13, 119–129. Kavarthapu R., Dufau M.L. Role of EGF/ERBB1 in the tr anscriptional regulation of the prolactin receptor independent of estrogen and prolactin in breast cancer cells. Oncotarget 2016 , 7, 65602–65613. Harari D., Yarden Y. Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. Oncogene 2000 , 19, 6102–6114. Zhu L, Yang Y, Li H, Xu L, You H, Liu Y, Liu Z, Liu X, Zheng D, Bie J, Li J, Song C, Yang B, Luo J, Chang Q. Exosomal microRNAs induce tumor-associated macrophages via PPARγ during tumor progression in SHH medulloblastoma. Cancer Lett. 2022 , 535, 215630. Li H, Liu Y, Liu Y, Xu L, Sun Z, Zheng D, Liu X, Song C, Zhang Y, Liang H, Yang B, Tian X, Luo J, Chang Q. Tumor-associated astrocytes promote tumor progression of Sonic Hedgehog medulloblastoma by secreting lipocalin-2. Brain Pathol. 2024 , 34, e13212. Hu ZZ, Meng J, Dufau ML. Isolation and characterization of two novel forms of the human prolactin receptor generated by alternative splicing of a newly identified exon 11. J Biol Chem. 2001 , 276, 41086-41094. Clevenger CV, Furth PA, Hankinson SE, Schuler LA. The role of prolactin in mammary carcinoma. Endocr Rev. 2003 , 24, 1-27. Kline JB, Clevenger CV. Identification and characterization of the prolactin-binding protein in human serum and milk. J Biol Chem. 2001 , 276, 24760-24766. Ben-Jonathan N, LaPensee CR, LaPensee EW. What can we learn from rodents about prolactin in humans? Endocr Rev. 2008 , 29, 1-41. Yang M, Yang M, Lin Y, Meng F, Du X, Zeng X, Bu G. Characterization of prolactin (PRL) and PRL receptor (PRLR) in Chinese soft-shelled turtle: Molecular identification, ligand-receptor interaction and tissue distribution. Gen Comp Endocrinol. 2022 , 316, 113941. Standing D, Dandawate P, Anant S. Prolactin receptor signaling: A novel target for cancer treatment - Exploring anti-PRLR signaling strategies. Front Endocrinol (Lausanne) 2023 , 13, 1112987. Asad AS, Nicola Candia AJ, Gonzalez N, Zuccato CF, Abt A, Orrillo SJ, Lastra Y, De Simone E, Boutillon F, Goffin V, Seilicovich A, Pisera DA, Ferraris MJ, Candolfi M. Prolactin and its receptor as therapeutic targets in glioblastoma multiforme. Sci Rep. 2019 , 9, 19578 Degirmenci U, Wang M, Hu J. Targeting Aberrant RAS/RAF/MEK/ERK Signaling for Cancer Therapy. Cells 2020 , 9, 198. Song Y, Bi Z, Liu Y, Qin F, Wei Y, Wei X. Targeting RAS-RAF-MEK-ERK signaling pathway in human cancer: Current status in clinical trials. Genes Dis. 2022 , f10, 76-88. Nie H, Huang PQ, Jiang SH, Yang Q, Hu LP, Yang XM, Li J, Wang YH, Li Q, Zhang YF, Zhu L, Zhang YL, Yu Y, Xiao GG, Sun YW, Ji J, Zhang ZG. The short isoform of PRLR suppresses the pentose phosphate pathway and nucleotide synthesis through the NEK9-Hippo axis in pancreatic cancer. Theranostics 2021 , 11, 3898-3915. Additional Declarations No competing interests reported. Supplementary Files supplfig.zip originaldata.zip supplementaryfile.rar Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-5667015","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":396431749,"identity":"9514bf3f-d77a-4d56-8d03-57601f14f8ab","order_by":0,"name":"Ziwen Sun","email":"","orcid":"","institution":"Peking University Third Hospital, Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Ziwen","middleName":"","lastName":"Sun","suffix":""},{"id":396431750,"identity":"5499d206-cbce-4372-9fed-7d4045c0743d","order_by":1,"name":"Haishuang Li","email":"","orcid":"","institution":"Peking University Third Hospital, Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Haishuang","middleName":"","lastName":"Li","suffix":""},{"id":396431751,"identity":"7c68ad90-4be4-487f-bb59-cc1d68cc1f82","order_by":2,"name":"Yu Peng","email":"","orcid":"","institution":"Peking University Third Hospital, Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Peng","suffix":""},{"id":396431752,"identity":"5201677b-583f-42ba-a0dd-5f2608ea7ae4","order_by":3,"name":"Yantao Liu","email":"","orcid":"","institution":"Peking University Third Hospital, Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Yantao","middleName":"","lastName":"Liu","suffix":""},{"id":396431753,"identity":"20782dfa-4cd2-4034-8266-9acf5c6eea80","order_by":4,"name":"Yujia Wang","email":"","orcid":"","institution":"Peking University Third Hospital, Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Yujia","middleName":"","lastName":"Wang","suffix":""},{"id":396431754,"identity":"8ea9fc98-d3d3-4f22-9082-9011f4aa4609","order_by":5,"name":"Jing Wang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wang","suffix":""},{"id":396431755,"identity":"1c2b31ea-5473-4e76-8791-5862795e380d","order_by":6,"name":"Xiaodan Liu","email":"","orcid":"","institution":"Peking University Third Hospital, Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Xiaodan","middleName":"","lastName":"Liu","suffix":""},{"id":396431756,"identity":"200da048-35ba-49ee-94df-8a562c1ba9d1","order_by":7,"name":"Danfeng Zheng","email":"","orcid":"","institution":"Peking University Third Hospital, Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Danfeng","middleName":"","lastName":"Zheng","suffix":""},{"id":396431757,"identity":"714b90e7-f2d7-4fde-8616-a9570bfdcae8","order_by":8,"name":"Hui Liang","email":"","orcid":"","institution":"Peking University Third Hospital, Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Liang","suffix":""},{"id":396431758,"identity":"dfad5311-e753-47b5-91e4-b4c490d736e6","order_by":9,"name":"Qing Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYDACCRjJzP/8w8cGNgY24rWw97AxziRBCxDwnGFj5m0gwl38s5ufPfxSZpEnH5F77LHtDr48PgbmYx+/MNjl4bTkzjFzY5lzEsWGN/LSjXPPsBUDnZY8W4YhuRiXFgOJBDNpyTaJxI0zEgykc9vYEtsYeIyZJRgOJOJypIFE+jeEFkvitOSYSX4EapnPc8ZMmhGqhfEDHi0SN3LKpBnOSSRuYG9LNuw9A9TCzJbMzGCQjFML/4z0bZI/yuoS5zczH3zwc8exxPntzYcZf1TY4dQCAsw8wOgzOABmHwNyQSIGeNQDAeMPoBZ5iKE1UBH8OkbBKBgFo2BkAQBFcFMzAn1ASgAAAABJRU5ErkJggg==","orcid":"","institution":"Capital Medical University","correspondingAuthor":true,"prefix":"","firstName":"Qing","middleName":"","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2024-12-18 07:24:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5667015/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5667015/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72800811,"identity":"8bdd8f9f-7c7f-44b4-8089-2973e5799761","added_by":"auto","created_at":"2025-01-02 09:26:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7190685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRLR is high expression in SHH medulloblastoma.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) RNA-seq result of 37 clinical patients derived tumor tissues reveals 125 differential genes between SHH MB and 3 other subtypes. (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap of those 125 differential genes edited by R package ‘pheatmap’ showed PRLR as a higher expression gene in SHH MB. (\u003cstrong\u003eC\u003c/strong\u003e) Survival analysis displayed higher expression level of PRLR was correlated with poorer prognosis of female SHH MB patients. (\u003cstrong\u003eD\u003c/strong\u003e) PRLR gene expression analysis in GSE dataset. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, NS, no significant, by Wilcox test.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5667015/v1/5908e662d27d572ac00b5c14.jpg"},{"id":72800810,"identity":"47e1756c-31c4-4beb-a12b-a1d69ab6d1a2","added_by":"auto","created_at":"2025-01-02 09:26:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7242786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRLR long isoform(PRLR-LF) function as the main component of PRLR in SHH MB.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) PRLR expression of MB patients in 4 subgroups. Data shows as mean fold change compared to WNT subgroup, with n = 3 replicates. (B) immunohistochemical analysis of FFPE samples derived from each subgroup MB patients shows higher expression level of PRLR in SHH subgroup. Positive control, breast ductal adenocarcinoma incubated with PRLR antibody. Negative control, breast ductal adenocarcinoma incubated with 4% goat serum.(C) Realtime qPCR(RT-qPCR) analyses for expression level of PRLR long isoform(LF), short isoform(S1a and S1b) in SHH patients derived tumor tissues. (D) RT-qPCR analyses for expression level of each PRLR isoforms in SHH MB cell lines Daoy and ONS-76. (E) immunoblotting(western blot) results to verify the expression level of each PRLR isoforms in SHH MB cell lines Daoy and ONS-76. (F) RT-qPCR to verify the efficiency of RNAi and the infection efficiency of sh-PRLR lentivirus. (G) western blot to verify the efficiency of sh-PRLR vector and OE-PRLR-LF vector. Data are presented as means ±SD from three independent experiments. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, NS, no significant.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5667015/v1/376ce2144b7b3a87d4837ab0.jpg"},{"id":72800819,"identity":"8ea12854-b277-43e9-bf95-e1012f797b2e","added_by":"auto","created_at":"2025-01-02 09:26:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7920968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRLR-LF overexpression regulated cell proliferation through p53 signal pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) RNA-seq result of OE-PRLR-LF verses OE-control in Daoy cell line showed about 3000 genes with differential expression. (\u003cstrong\u003eB\u003c/strong\u003e) KEGG analysis of whole-genome expression profiles demonstrated differential expressed genes enriched in p53 pathway. (\u003cstrong\u003eC\u003c/strong\u003e) Genes cluster analysis revealed that OE-PRLR-LF regulated cell proliferation progress. (\u003cstrong\u003eD\u003c/strong\u003e) RT-q-PCR assay verified OE-PRLR-LF may regulate cell proliferation through G1/S and G2/S checkpoints in Daoy and ONS-76. (\u003cstrong\u003eE-G\u003c/strong\u003e) Clone formation assay, trans-well migration assay and CCK8 proliferation assay with overexpressed PRLR-LF in Daoy and ONS-76. ±SD from three independent experiments. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, NS, no significant.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5667015/v1/c4e199a4bb11e62116749a6c.jpg"},{"id":72800820,"identity":"cfc3d481-2b34-42fc-9429-e1a5c6b25cb9","added_by":"auto","created_at":"2025-01-02 09:26:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7573396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eΔ1-11-G129R-PRL as a competitive inhibitor of PRL suppressed SHH-MB’s proliferation by regulating CDK6.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot indicated lack of direct regulation between PRLR and p53 pathway. (B) Knockout of p53 in ONS-76 cell (p53 wild type SHH-MB) showed little correlation between PRLR and p53 pathway. (D) RT-q-PCR assay revealed correlation between PRLR-LF and downstream genes of p53 pathway. (C) Venn diagram displayed 7 significant genes correlated with PRLR in SHH-MB. (E-F) gene correlation analysis and survival analysis (Kaplan-Meier) showed CDK6 was an important oncogene positively correlated with PRLR in SHH-MB. (G-H) RT-q-PCR and western blot revealed significant inhibitory effect of Δ1-11-G129R-PRL on PRLR activation in Daoy and ONS76. (I-J) CCK8 proliferation assay and clone formation experiment showed remarkable inhibitory effect on proliferation ability.\u003cem\u003e \u003c/em\u003e±SD from three independent experiments. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, NS, no significant.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5667015/v1/14880fd88913fd0e238aee24.jpg"},{"id":72800838,"identity":"e8176d29-558e-45be-8671-680135e50277","added_by":"auto","created_at":"2025-01-02 09:26:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10325445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRLR regulating CDK6 by Ras-MAPK pathway promotes SHH-MB tumor progression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) western blot shows PRLR regulates CDK6 by Ras-Raf-MEK-ERK signal pathway, which can be blocked by Ras or ERK RNA silence. (C) tumor proliferation curve in vivo exhibits a stronger proliferation ability on Daoy OE-PRLR-LF cell. (E) tumor volume statistical chart, p-value was calculated by t-test. (F-G) immunohistochemical analysis of FFPE samples derived from SHH-MB patients shows relavence between PRLR, CDK6, and Ki-67. IHC score and Ki-67 proliferation index were evaluated by two pathologists independently. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, NS, no significant, by t-test.\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5667015/v1/3014dcf8d6e322dc99bc7de9.jpg"},{"id":83425131,"identity":"1770af07-c719-4acf-80fe-81c1865fede4","added_by":"auto","created_at":"2025-05-26 04:21:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":41332603,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5667015/v1/611555d1-e9ad-4317-ba8e-135fa2adea00.pdf"},{"id":72800825,"identity":"49f0b1b3-0c90-4132-a033-03542154c584","added_by":"auto","created_at":"2025-01-02 09:26:16","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":27049036,"visible":true,"origin":"","legend":"","description":"","filename":"supplfig.zip","url":"https://assets-eu.researchsquare.com/files/rs-5667015/v1/7fea292bdd7738020b798986.zip"},{"id":72800814,"identity":"6c84659d-fc2e-48a9-bfc3-b95341fcd79a","added_by":"auto","created_at":"2025-01-02 09:26:15","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3460070,"visible":true,"origin":"","legend":"","description":"","filename":"originaldata.zip","url":"https://assets-eu.researchsquare.com/files/rs-5667015/v1/750c41c3a0083a2981b1893d.zip"},{"id":72800812,"identity":"442b4fa9-b3b7-4fc1-9236-0290330d7ed2","added_by":"auto","created_at":"2025-01-02 09:26:15","extension":"rar","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5094636,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.rar","url":"https://assets-eu.researchsquare.com/files/rs-5667015/v1/6219f83d2c42e1ba5ae2cd51.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"The long isoform of PRLR promotes tumor progression by regulating CDK6 through MAPK signal pathway in SHH medulloblastoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMedulloblastoma (MB) is the most common malignant brain tumors in childhood. It accounts for approximately 20% of all intracranial neoplasms in this age group.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e As reported in 2021 World Health Organization classification of tumors, at least four consensus subgroups of MB are reported, including wingless (WNT), Sonic Hedgehog (SHH) with/without P53 mutation, and non-WNT/SHH (Groups 3 and 4). Different genetic signatures and DNA methylation status of each subgroup lead to different prognosis, histopathological features, and therapeutic strategy.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e SHH-MB is characterized by the persistent activation of the SHH signaling pathway.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e In the canonical SHH signaling pathway, the SHH ligand binds to the PTCH1 receptor on the cell membrane, inhibiting its blocking effect on the Smo. This ultimately leads to the transcription factor GLI entering the cell nucleus and initiating the transcription of SHH-related oncogenes.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e SMO inhibitors, sonidegib and vismodegib, are used for targeted therapy of SHH-MB.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e However, these drugs are challenging to use in clinical settings due to their high toxicity and drug resistance.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e New adjunctive therapeutic target which is less toxic is needed to serve as clinical demands.\u003c/p\u003e \u003cp\u003eSHH-MB differs from the other subgroups of MBs in that it originates from granule neuron precursor cells and has a greater number of gene characteristics associated with granule neurons.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Therefore, identifying the gene characteristics specific to SHH-MB, as compared to the other three subgroups, can help in discovering new drug treatment targets. Here, we analyzed the gene characteristics of various MB subtypes using RNA sequencing (n\u0026thinsp;=\u0026thinsp;37) and Nanostring assay (n\u0026thinsp;=\u0026thinsp;48), and identified PRLR as a potential biomarker for SHH-MB. As an oncogene, PRLR promotes the progression in many cancers, such as breast cancer, prostate cancer, etc.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e PRL, as its natural ligand, binds to PRLR to activate various cancer-related signaling pathways, such as JAK/STAT1/STAT5, PI3K/AKT and Ras-MAPK.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e The Ras-MAPK signaling pathway, as the main downstream signaling pathway of PRLR, regulates cell growth and proliferation.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Therefore, identifying key pathways and genes regulated by PRLR in SHH-MB can help to elucidate its cancer promoting function.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn this study, we elucidated that the interaction between PRL and PRLR can promote tumor progression and enhance the proliferation ability of SHH-MB. The recombinant Δ1-11-G129R-PRL, an inhibitor of PRLR, served as a potential target inhibitor to suppress tumor progression. We also validated that high expression of PRLR or PRL-PRLR combination regulated CDK6 expression through the classic Ras-MAPK pathway, resulting in a stronger proliferative capacity of the tumor.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ecell lines, antibodies, and reagents\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHuman ONS-76 MB cell line was a generous gift from Prof. Xiu-Wu Bian (Southwest Hospital, Army Medical University). Human Daoy MB cell line were kindly provided by Prof. Ho Ho-Keung Ng (The Chinese University of Hong Kong). Human 293T cell were purchase from ATCC. RPMI 1640 (Gibico) with 10%FBS were used to culture ONS76 in a 37\u0026deg;C incubator with 5% (v/v) CO2. MEM (Gibico), DMEM(Gibico) with 10%FBS were used to culture Daoy and 293T in a 37\u0026deg;C incubator with 5% (v/v) CO2. Primary antibodies used for western blot, and immunohistochemistry stain are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eplasmid construction\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe plasmids pLKO-puro, pLV-copGFP-puro, pGEX4T-1-puro were purchased from Addgene. PRLR-LF, PRLR-S1a, PRLR-S1b, Δ1-11-G129R-PRL,and short hairpin RNA sequences were inserted into those plasmid. All sequences constructed into those plasmids are listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eRNA extraction, RT-q-PCR analysis and RNA sequence analysis\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCells RNA is extracted by TRIZOL reagent. RT-q-PCR were performed as done previously. RNA sequence analysis was performed by Majorbio (Shanghai).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ewestern blot analysis\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll experiments were performed on ice. Cells were washed 3 times by PBS at 4℃, then lysed in RIPA containing protease inhibitors and phosphatase inhibitors. Protein lysates were separated bysodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride membrane (ISEQ00010, Merck Millipore, 0.22 \u0026micro;m). After blocking with 5% skimmed milk, the membrane was incubated with antibodies exhibiting in Supplementary Table\u0026nbsp;1 at 4℃ overnight. Then incubated with secondary antibody (7074\u0026amp;7076, CST, 1:2000) at room temperature in 1h. Protein signals were visualized both with Super ECL Plus Substrate (Thermo Fisher) in Gel imaging system (Bio-Rad ChemiDoc MP) and in darkroom exposure. ImageJ (Ver.1.53e; NIH, Bethesda, MD) was used to quantify the protein bands, normalized to GAPDH, and determined as the percentage value.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003esiRNA transfection and lentiviral infection\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e si-RNA tRasfection were performed using jetPRIME transfection kit (polyplus) according to the manufacturer\u0026rsquo;s instructions. Lentiviral was packaged in 293T cell line by transfecting psPAX-2, pMD2.G and targeted plasmid at the same time. After 48h, lentiviral were purified by 100kd ultrafiltration tube (TIANGEN) and then add to infect target cell.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ecell counting kit-8(CCK8) assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCells were plated into a 96-well plate (Thermo Fisher Scientific) at the density of 600 to 800 cells per well for determination of cell viability. CCK-8 reagent (Solarbo) was added into medium after the cells were cultured each 24h. After incubation for 2 h at 37\u0026deg;C, absorbance were detected by microplate reader (Thermo Fisher) at the light length of 450 nm. Each assay was repeated independently for 5 times.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eclone formation assay\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e500 cells were planted into a 6-well plate (Thermo Fisher Scientific). After 6\u0026ndash;8 days culture until 50 cells per single cell colony, cells were fixed with methanol, stained with 0.1% crystal violet, and then quantified by ImageJ independently for 3 times.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003etranswell assay\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTranswell migration assays were performed in a 6-well plate. In brief, cells in serum-free medium were seeded in the upper chamber, and the lower chamber was filled with complete media. After 24 h, cells in the upper chamber were gently removed. Migrated cells on the lower side of the membrane were fixed with methanol, stained with 0.1% crystal violet, and then counted at 10\u0026times; magnification in three random fields per well.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003erecombinant protein purification\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGST-tagged Δ1-11-G129R-PRL expressed in \u003cem\u003eE. coli\u003c/em\u003e were purified using GST Fusion Protein Purification Kit (Beaver) according to the manufacturer\u0026rsquo;s instructions. Then purified product were digested by Thrombin (Solarbo) and then verified by Coomassie Brilliant Blue Staining.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003esubcutaneous transplantation tumor in Balb/c nude mice\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBalb/c nude mice were bought from Dept. of Experimental Animal Science, Peking University Health Science Center, Beijing, 100191. Animal experiments have obtained ethical approval (LA2019121). All methods were performed in accordance with the relevant guidelines and regulations. Authors of this article have obtained a work permit for experimental animals. Under the premise of following the 3R principle of animal experimentation, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e Daoy cells were injected subcutaneously into those nude mice 6 mice per group after anesthesia by pentobarbital sodium. After 24 days of measurement interval 3 days, the data showed statistical differences. To comply with the ethics of experimental animals, mice were executed. Tumor were removed for quantification.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHigh expression PRLR in SHH medulloblastoma\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo explore potential therapeutic target for SHH-MBs, RNA sequence analysis and Nanostring assay were performed covering four subtypes of MB, WNT activated (WNT), Sonic hedgehog pathway activated (SHH), Group3 (G3) and Group4 (G4)(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). \u0026lsquo;Deseq2\u0026rsquo; and \u0026lsquo;pheatmap\u0026rsquo; package in R were used to screen out genes with differential expression. Totally 125 genes were detected with higher expression in SHH-MBs compared to other subtypes of MB by RNA-seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In our previous study,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e nanostring assay of 48 MB patients were analyzed focusing on differential genes from SHH-MB to other 3 subtypes MB. Six genes that are significantly different from the other three subtypes were screened out. By intersecting the 125 genes enriched from RNA-seq sequencing with the differentially expressed genes obtained from the Nanostring assay, five candidates were identified, TGFB2, CXCL4, PRLR, SOX2, and SFRP1. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Survival analysis by R2 dataset (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hgserver1.amc.nl/\u003c/span\u003e\u003cspan address=\"https://hgserver1.amc.nl/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) revealed that PRLR expression level in SHH-MB may be correlated with female patients\u0026rsquo; prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;1A). GSE datasets analysis also showed PRLR expression level in SHH-MB is significantly higher than other 3 subtypes of MB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Volcano plots revealed a significant difference of PRLR expression in SHH-MB compared to other three subtypes (Supplementary Fig.\u0026nbsp;1B). These data showed that PRLR may serve as a potential oncogene, which was correlated with poor prognosis of female patients of SHH-MB.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLong isoform of PRLR (PRLR-LF) functions as the main component of PRLR in SHH-MB\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo validate whether PRLR is highly expressed in SHH-MB, RT-q-PCR and immunohistochemistry staining were performed in 4 subgroups of primary MBs. Higher expresson of PRLR on both RNA and protein level were confirmed in SHH-MBs compared to other 3 subtypes, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). It has been reported that the PRLR gene has different splicing variants due to alternative splicing.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e The full-length transcript PRLR-LF can generate transcripts such as PRLR-IF, PRLR-S1a, and PRLR-S1b through alternative splicing and deletions.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Among them, PRLR-LF and PRLR-SF(PRLR-S1a, PRLR-S1b) are currently well studied. To ivestigate the expression of the PRLR isoforms in SHH-MB patients and SHH-MB cell lines, the alternative splicing variants of PRLR in SHH-MB were tested to start with RT-q-PCR analysis in SHH-MB tumor tissue from patients. The result confirmed that long isoform of PRLR (PRLR-LF 80 kD) is the main component of PRLR spliceosome in the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), not PRLR-S1a (40 kD), nor PRLR-S1b (32 kD). Similar result was obtained in the SHH-MB cell lines (Daoy and ONS-76) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Furthermore, westernblot was performed to identify the gene\u0026rsquo;s expression on protein level. The data showed the same trend as the RNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In order to further elucidate the possible function of PRLR-LF in SHH-MB, shRNA sequences were inserted into pCDH-copGFP-PURO and knockdown efficiency was tested on both RNA and protein level (Figure.2F,G). PRLR-LF CDS sequence cloning from 293T\u0026rsquo;s cDNA was inserted into pLVX-CMV-PURO to overexpress PRLR-LF in Daoy and ONS-76, respectively. Overexpression efficiency was tested by westernblot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG)\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePRLR-LF overexpression regulated cell proliferation through p53 signal pathway\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn order to further elucidate the function of PRLR-LF in SHH-MB, differential expression was performed by RNA-seq in PRLR-LF overexpressed Daoy and wild type Daoy cells. About 3000 genes were screened out (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) using R package \u0026lsquo;DEseq2\u0026rsquo;. KEGG analysis revealed high expression of PRLR-LF was significantly correlated with the p53 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Further heatmap showed overexpression of PRLR-LF was associated with downstream genes of the p53 signaling pathway rather than p53 and p21 themselves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The correlation between PRLR-LF and downstream genes of the P53 pathway was confirmed by RT-q-PCR assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Considering that many downstream genes of the p53 signaling pathway are involved in regulating cell proliferation, the phenotype of cell proliferation was verified by clone formation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), transwell assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), and CCK-8 prolifiation assay (Figure.3G), respectively. The results indicated that overexpressed PRLR-LF in SHH-MB cell lines increased cell proliferation, migration, and cell colonization abilities, which may promote malignancy of SHH-MB tumor cells.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eΔ1-11-G129R-PRL, as a competitive inhibitor of PRL, suppressed SHH-MB\u0026rsquo;s proliferation by regulating CDK6\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePRLR-LF as a cell membrane protein, participates in the transmission process of downstream signaling pathways.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e PRL, as a natural ligand of PRLR, can promote tumor develpoment through PRLR-LF.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e However, several modified ligand can serve as a competitive inhibitor to block its function.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Among them, PRL analogues reported to inhibit the function of PRLR have been used in the treatment of breast cancer and glioblastoma.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e We hypothesized that Δ1-11-G129R-PRL as a inhibitor of PRL, may competitively bind to PRLR and inhibit tumor progression.\u003c/p\u003e \u003cp\u003eTo explore whether PRLR could directly regulate p53 and p21, then regulate the downstream signal pathway of p53 to promote tumor development, western blot were performed to detect the correlation between PRLR-LF and p53, p21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, no direct correlation between them was observed by knockout of P53 using lenti-CRISPR-v2 in ONS-76 (p53 wild type) cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). It suggested that other genes involved in the p53 pathway may be regulated by PRLR-LF. R2 dataset screened out 7 genes positively correlated with PRLR-LF expression., among which CDK6 is located downstream of the p53 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). To exclude the possibility that PRLR-SF may be involved in regulating those genes\u0026rsquo; expression, RT-q-PCR were performed to exclude the effect of PRLR short variant on CDK6 and other genes frequently involved in the p53 pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Whereas, a significant positive correlation between CDK6 and PRLR in SHH-MB patients was identified by R2 correlation analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Meanwhile, prognostic survival analysis showed high expression of CDK6 had positive relationship with SHH-MB patients\u0026rsquo; prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). To verify that PRLR can regulate CDK6 to promote tumor progression, recombinate Δ1-11-G129R-PRL was synthesized (Supplementary Fig.\u0026nbsp;2A) and was added as an inhibitor to cell culture medium. A siignificant inhibition effect on PRL was observed. Expression of CDK6 was rescued to normal level in both wild-type cells or PRLR-LF overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Western blot also revealed significant inhibition of Δ1-11-G129R-PRLon PRL in Daoy and ONS-76 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Furthermore, CCK-8 proliferation and clone formation assay showed that Δ1-11-G129R-PRL significantly inhibits the proliferation ability of SHH-MB cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-J).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCDK6 regulated by PRLR through Ras-MAPK pathway promotes SHH-MB tumor progression\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDue to the fact that PRLR can regulate the expression of cyclinD through Ras-MAPK signaling pathway,\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e CDK6 may be regulated similarly through this signal pathway. To valid this hypothesis, si-Ras and si-ERK1/2 were transfection into Daoy and ONS-76 cells. Westernblot revealed that CDK6 expression were upregulated by PRLR through Ras-Raf-MEK-ERK signal pathway activation in SHH-MB cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Whereas other 2 MAPK subfamily members JNK and p38 showed no difference after Ras-ERK signal pathway treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). To explore whether PRLR-LF overexpression may cause tumor progression, PRLR-LF overexpressed Daoy cells were injected to Balb/c nude mice after anesthesia by pentobarbital sodium (n\u0026thinsp;=\u0026thinsp;5). After 24 days of measurement interval 3 days, PRLR-LF overexpressed cells showed a noticeable proliferation vitality compared to control cells after 21 days(about 60 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e vs 30 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) (Figure.5C). Under the premise of following the 3R principle, mice were executed and the tumor was extracted (Supplementary Fig.\u0026nbsp;3A). The tumor volume of PRLR-LF overexpressing cells significantly increased compared to control cells(about 60 mg vs 38mg) (Figure.5D). To further validate the expression correlation between PRLR and CDK6 in SHH-MB patients, immunohistochemistry staining was performed using FFPE SHH-MB samples (n\u0026thinsp;=\u0026thinsp;40) (Figure.5F). CDK6 showed a significant correlation with PRLR in SHH-MB patients (Figure.5E). Meanwhile, we defined 2 groups by the expression of PRLR evaluated by two pathologists independently. We found that higher expression level of PRLR was accompanied with higher CDK6 expression level and higher Ki-67 proliferation index, which may cause a malignant progression in SHH-MB patients. (Figure.5G).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGiven the persistent activation of the SHH signaling pathway in SHH-MB, most studies target the SHH signaling pathway itself. To identify transcriptional signatures specific to SHH-MB beyond the SHH signaling pathway, we explored the transcriptional profiles of SHH-MB compared to the other three subtypes at the whole transcriptome level through Nanostring and RNA sequencing analyses. Here, we screened out prolactin receptor (PRLR) as a gene with higher expression level in SHH-MB compared with other subgroups of the tumor. Long isoform of PRLR (PRLR-LF) played a pivotal role in promoting SHH-MB tumor invasion, enhancing the proliferation and colony formation ability. PRLR-LF expression has positive correlation with CDK6 through Ras-MAPK signal pathway, causing a poor prognosis in SHH-MB patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eMitogen-activated protein kinase (MAPK) signaling pathway is a classic cancer-related pathway that has been extensively studied.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Activation of the Ras-Raf-MEK-ERK signaling pathway leads to the activation of cell cycle-related genes associated with proliferation, promoting the progression of the tumor cell cycle and enabling tumor growth.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e PRLR, as an upstream molecule in the classic MAPK pathway, mediates the activation of various cancer-related signaling pathways.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Previous reports indicated that activation of PRLR can activate the Ras-Raf-MEK-ERK pathway, regulating cyclin D1 expression to advance the cell cycle.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Recent studies also suggested that PRLR-SF, a variant of PRLR, has the ability to inhibit tumor cell proliferation through the NEK9-Hippo signaling pathway.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Due to the presence of different transcriptional variants of PRLR with distinct signal functions,\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e we conducted experiments and found that the PRLR mainly act as the long isoform (PRLR-LF) in SHH-MB. We confirmed that PRLR-LF is highly expressed in SHH-MB compared to other 3 subtypes MB patients and cell lines. It promotes cell viability, proliferation, and colony formation ability of the tumor cells, which is contrary to the function of PRLR-SF in pancreatic cancer.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e We validated \u003cem\u003ein vitro\u003c/em\u003e that a positive relationship between CDK6, an oncogene associated with tumor growth, and PRLR in SHH-MB was mediated by the PRL-PRLR binding followed by activation of the RAS-MAPK signaling pathway. Immunohistochemistry (IHC) results from primary SHH-MBs also demonstrated a positive correlation among PRLR, CDK6 and the proliferation index of the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eIt has been shown that hG129R-PRL, a PRL analog, can occupy the active site of PRLR, blocking the binding between PRLR and PRL and thereby inhibiting the activation of downstream signaling pathways in breast cancer cell lines and glioblastoma cell lines.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Our data showed the first time that Δ1-11-G129R-PRL inhibited the function of PRLR through reducing CDK6 expression in SHH-MB cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H). PRLR and CDK6 could serve as potential therapeutic targets for SHH-MBs, especially for female SHH-MB patients with PRL high expression. However, urgent research is needed on the potential application of combining Δ1-11-G129R-PRL and Smo inhibitors in the treatment of SHH-MB mouse model.\u003c/p\u003e \u003cp\u003eIn summary, we selected PRLR as a highly expressed gene specifically in SHH-MBs. We elucidated that the interaction between PRL and PRLR can promote tumor progression and enhance the proliferation ability of SHH-MB. We validated the high expression of PRLR regulates CDK6 expression through the classic MAPK signal pathway, promoting proliferative capacity of the tumor. Δ1-11-G129R-PRL may serve as a potential target inhibitor to suppress tumor progression.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplemental information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 1-3, Supplementary Table 1-2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. Further information and requests for reagents should be directed to and will be fulfilled by the Lead Contact Qing Chang ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Beijing Natural Science Foundation (No.7232098, No.7192095), Beijing Health Foundation Commission (11000023T000002044300-4) and the National Natural Science Foundation of China (No.81972353, No.81101900, No.30540008) to Qing Chang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were reported in accordance with\u0026nbsp;ARRIVE guidelines\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eExperiments referred to Ethics were permitted by\u0026nbsp;Animal Experimentation Ethics Committee No. LA2019121.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvestigators have to obtain informed consent before using participants’ FFPE for IHC staining\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePermission to reproduce material from other sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo reproduce figure or material from other sources was quoted in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo clinical trial was carried out in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate XiuWu Bian's lab for providing the ONS-76 cell line.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Z.S. and H.L.; methodology, Z.S., Y.L. and H.L.; software, Z.S.; validation, Y.W., H.L. and Q.C.; formal analysis, Z.S., D.Z.; investigation, Z.S., J.W. and Y.P.; resources, H.L. and Q.C.; data curation, Y.W., Y.P. and Z.S..; writing—original draft preparation, Y.W. and Z.S.; writing—review and editing, Z.S., H.L., X.L. and Q.C.; visualization, Z.S., D.F. and X.L..; supervision, H.L. and Q.C.; project administration, Q.C.; funding acquisition, Q.C.. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOstrom QT, Cioffi G, Gittleman H, Patil N, Waite K, Kruchko C, Barnholtz-Sloan JS. CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2012-2016. Neuro Oncol. \u003cstrong\u003e2019\u003c/strong\u003e, 21, 1-100. \u003c/li\u003e\n\u003cli\u003eKhanna V, Achey RL, Ostrom QT, Block-Beach H, Kruchko C, Barnholtz-Sloan JS, de Blank PM. Incidence and survival trends for medulloblastomas in the United States from 2001 to 2013. J Neurooncol. \u003cstrong\u003e2017\u003c/strong\u003e, 135, 433-441. \u003c/li\u003e\n\u003cli\u003ePerreault S, Ramaswamy V, Achrol AS, Chao K, Liu TT, Shih D, et al. MRI surrogates for molecular subgroups of medulloblastoma. Am J Neuroradiol. \u003cstrong\u003e2014\u003c/strong\u003e, 35, 1263\u0026ndash;1269.\u003c/li\u003e\n\u003cli\u003eLospinoso Severini L, Ghirga F, Bufalieri F, Quaglio D, Infante P, Di Marcotullio L. The SHH/GLI signaling pathway: a therapeutic target for medulloblastoma. Expert Opin Ther Targets \u003cstrong\u003e2020\u003c/strong\u003e, 24, 1159-1181.\u003c/li\u003e\n\u003cli\u003eCarballo GB, Honorato JR, de Lopes GPF, Spohr TCLSE. A highlight on Sonic hedgehog pathway. Cell Commun Signal \u003cstrong\u003e2018\u003c/strong\u003e,16,11. \u003c/li\u003e\n\u003cli\u003eWaszak SM, Robinson GW, Gudenas BL, Smith KS, Forget A, Kojic M, et al. Germline elongator mutations in Sonic Hedgehog medulloblastoma. Nature \u003cstrong\u003e2020\u003c/strong\u003e, 580, 396\u0026ndash;401.\u003c/li\u003e\n\u003cli\u003eSekulic A, Von Hoff D. Hedgehog Pathway Inhibition. Cell \u003cstrong\u003e2016\u003c/strong\u003e, 164, 831.\u003c/li\u003e\n\u003cli\u003eEspinosa-Bustos C, Mella J, Soto-Delgado J, Salas CO. State of the art of Smo antagonists for cancer therapy: advances in the target receptor and new ligand structures. Future Med Chem. \u003cstrong\u003e2019\u003c/strong\u003e, 11, 617-638.\u003c/li\u003e\n\u003cli\u003eRudin CM, Hann CL, Laterra J, et al. Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N Engl J Med \u003cstrong\u003e2009\u003c/strong\u003e, 361, 1173-1178\u003c/li\u003e\n\u003cli\u003eYauch RL, Dijkgraaf GJ, Alicke B, et al. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science \u003cstrong\u003e2009\u003c/strong\u003e, 326, 572-4\u003c/li\u003e\n\u003cli\u003eMetcalfe C, de Sauvage FJ. Hedgehog fights back: mechanisms of acquired resistance against smoothened antagonists. Cancer Res. \u003cstrong\u003e2011\u003c/strong\u003e, 71, 5057-5061\u003c/li\u003e\n\u003cli\u003eWechsler-Reya RJ, Scott MP. Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron \u003cstrong\u003e1999\u003c/strong\u003e, 22, 103-114.\u003c/li\u003e\n\u003cli\u003eOliver TG, Read TA, Kessler JD, Mehmeti A, Wells JF, Huynh TT, Lin SM, Wechsler-Reya RJ. Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma. Development \u003cstrong\u003e2005\u003c/strong\u003e, 132, 2425-2439. \u003c/li\u003e\n\u003cli\u003eKavarthapu R, Anbazhagan R, Dufau ML. Crosstalk between PRLR and EGFR/HER2 Signaling Pathways in Breast Cancer. Cancers (Basel). \u003cstrong\u003e2021\u003c/strong\u003e, 13, 4685. \u003c/li\u003e\n\u003cli\u003eDamiano JS, Wasserman E. Molecular pathways: blockade of the PRLR signaling pathway as a novel antihormonal approach for the treatment of breast and prostate cancer. Clin Cancer Res. \u003cstrong\u003e2013\u003c/strong\u003e, 19, 1644-1650. \u003c/li\u003e\n\u003cli\u003eHuang Y., Li X., Jiang J., Frank S.J. Prolactin modulates phosphorylation, signaling and trafficking of epidermal growth factor receptor in human T47D breast cancer cells. Oncogene \u003cstrong\u003e2006\u003c/strong\u003e, 25, 7565\u0026ndash;7576.\u003c/li\u003e\n\u003cli\u003eFrank S.J. Mechanistic aspects of crosstalk between GH and PRL and ErbB receptor family signaling. J. Mammary Gland Biol. Neoplasia. \u003cstrong\u003e2008\u003c/strong\u003e, 13, 119\u0026ndash;129.\u003c/li\u003e\n\u003cli\u003eKavarthapu R., Dufau M.L. Role of EGF/ERBB1 in the tr anscriptional regulation of the prolactin receptor independent of estrogen and prolactin in breast cancer cells. Oncotarget \u003cstrong\u003e2016\u003c/strong\u003e, 7, 65602\u0026ndash;65613.\u003c/li\u003e\n\u003cli\u003eHarari D., Yarden Y. Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. Oncogene \u003cstrong\u003e2000\u003c/strong\u003e, 19, 6102\u0026ndash;6114. \u003c/li\u003e\n\u003cli\u003eZhu L, Yang Y, Li H, Xu L, You H, Liu Y, Liu Z, Liu X, Zheng D, Bie J, Li J, Song C, Yang B, Luo J, Chang Q. Exosomal microRNAs induce tumor-associated macrophages via PPAR\u0026gamma; during tumor progression in SHH medulloblastoma. Cancer Lett. \u003cstrong\u003e2022\u003c/strong\u003e, 535, 215630.\u003c/li\u003e\n\u003cli\u003eLi H, Liu Y, Liu Y, Xu L, Sun Z, Zheng D, Liu X, Song C, Zhang Y, Liang H, Yang B, Tian X, Luo J, Chang Q. Tumor-associated astrocytes promote tumor progression of Sonic Hedgehog medulloblastoma by secreting lipocalin-2. Brain Pathol. \u003cstrong\u003e2024\u003c/strong\u003e, 34, e13212. \u003c/li\u003e\n\u003cli\u003eHu ZZ, Meng J, Dufau ML. Isolation and characterization of two novel forms of the human prolactin receptor generated by alternative splicing of a newly identified exon 11. J Biol Chem. \u003cstrong\u003e2001\u003c/strong\u003e, 276, 41086-41094.\u003c/li\u003e\n\u003cli\u003eClevenger CV, Furth PA, Hankinson SE, Schuler LA. The role of prolactin in mammary carcinoma. Endocr Rev. \u003cstrong\u003e2003\u003c/strong\u003e, 24, 1-27.\u003c/li\u003e\n\u003cli\u003eKline JB, Clevenger CV. Identification and characterization of the prolactin-binding protein in human serum and milk. J Biol Chem. \u003cstrong\u003e2001\u003c/strong\u003e, 276, 24760-24766.\u003c/li\u003e\n\u003cli\u003eBen-Jonathan N, LaPensee CR, LaPensee EW. What can we learn from rodents about prolactin in humans? Endocr Rev.\u003cstrong\u003e 2008\u003c/strong\u003e , 29, 1-41.\u003c/li\u003e\n\u003cli\u003eYang M, Yang M, Lin Y, Meng F, Du X, Zeng X, Bu G. Characterization of prolactin (PRL) and PRL receptor (PRLR) in Chinese soft-shelled turtle: Molecular identification, ligand-receptor interaction and tissue distribution. Gen Comp Endocrinol. \u003cstrong\u003e2022\u003c/strong\u003e, 316, 113941.\u003c/li\u003e\n\u003cli\u003eStanding D, Dandawate P, Anant S. Prolactin receptor signaling: A novel target for cancer treatment - Exploring anti-PRLR signaling strategies. Front Endocrinol (Lausanne) \u003cstrong\u003e2023\u003c/strong\u003e, 13, 1112987.\u003c/li\u003e\n\u003cli\u003eAsad AS, Nicola Candia AJ, Gonzalez N, Zuccato CF, Abt A, Orrillo SJ, Lastra Y, De Simone E, Boutillon F, Goffin V, Seilicovich A, Pisera DA, Ferraris MJ, Candolfi M. Prolactin and its receptor as therapeutic targets in glioblastoma multiforme. Sci Rep. \u003cstrong\u003e2019\u003c/strong\u003e, 9, 19578\u003c/li\u003e\n\u003cli\u003eDegirmenci U, Wang M, Hu J. Targeting Aberrant RAS/RAF/MEK/ERK Signaling for Cancer Therapy. Cells \u003cstrong\u003e2020\u003c/strong\u003e, 9, 198.\u003c/li\u003e\n\u003cli\u003eSong Y, Bi Z, Liu Y, Qin F, Wei Y, Wei X. Targeting RAS-RAF-MEK-ERK signaling pathway in human cancer: Current status in clinical trials. Genes Dis. \u003cstrong\u003e2022\u003c/strong\u003e, f10, 76-88. \u003c/li\u003e\n\u003cli\u003eNie H, Huang PQ, Jiang SH, Yang Q, Hu LP, Yang XM, Li J, Wang YH, Li Q, Zhang YF, Zhu L, Zhang YL, Yu Y, Xiao GG, Sun YW, Ji J, Zhang ZG. The short isoform of PRLR suppresses the pentose phosphate pathway and nucleotide synthesis through the NEK9-Hippo axis in pancreatic cancer. Theranostics\u003cstrong\u003e 2021\u003c/strong\u003e, 11, 3898-3915.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Medulloblastoma, prolactin receptor, CDK6, Sonic hedgehog, Tumor","lastPublishedDoi":"10.21203/rs.3.rs-5667015/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5667015/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMedulloblastoma (MB) is the most common malignant brain tumors in children. Sonic Hedgehog (SHH) subgroup of MB accounts for about 25% of all MBs. SMO inhibitors are used for target therapy. However, drug resistance and toxicity occurred. New therapeutic targets are urgently needed to be developed. Here, through RNA-sequencing and Nanostring Assay analysis of primary MBs, we screened out prolactin receptor (PRLR) as a gene with higher expression level in SHH-MB compared with other subgroups of the tumor. Long isoform of PRLR (PRLR-LF) played a pivotal role in promoting SHH-MB tumor invasion, enhancing the proliferation and colony formation ability. KEGG analysis showed that PRLR-LF expression has close relationship with p53 signal pathway in SHH-MB cells. High expression of CDK6 downstream of the p53 pathway was observed to have a high correlation with PRLR expression, indicating a poor prognosis of the tumor. In addition, PRLR was demonstrated to promote cell proliferation by regulating CDK6 through Ras-MAPK signal pathway \u003cem\u003ein vitro\u003c/em\u003e. Synthesized recombinant Δ1-11-G129R-PRL, a competitive inhibitor of PRLR, interfered PRL-PRLR binding, could inhibit the regulation to CDK6, and could and inhibit the proliferative ability of SHH-MB tumor cells. In conclusion, we unveiled PRLR promoted SHH-MB tumor progression through signaling pathway besides the canonical SHH pathway. PRLR inhibitor shed light on a potential therapeutic value for SHH-MB patients.\u003c/p\u003e","manuscriptTitle":"The long isoform of PRLR promotes tumor progression by regulating CDK6 through MAPK signal pathway in SHH medulloblastoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-02 09:26:10","doi":"10.21203/rs.3.rs-5667015/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"54079beb-5465-4b1d-94c6-1fb281b38770","owner":[],"postedDate":"January 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42237452,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":42237453,"name":"Health sciences/Biomarkers/Prognostic markers"},{"id":42237454,"name":"Health sciences/Pathogenesis/Oncogenesis"}],"tags":[],"updatedAt":"2025-05-26T04:13:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-02 09:26:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5667015","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5667015","identity":"rs-5667015","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}