WNT3 promotes chemoresistance to oxaliplatin in oral squamous cell carcinoma via regulating ABCG2 expression

WNT3 promotes chemoresistance to oxaliplatin in oral squamous cell carcinoma via regulating ABCG2 expression | 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 WNT3 promotes chemoresistance to oxaliplatin in oral squamous cell carcinoma via regulating ABCG2 expression Youguang Lu, Kairui Sun, Xuyang Zhang, Ruihuan Gan, Shuoqi Lin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5267942/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jun, 2025 Read the published version in Cell & Bioscience → Version 1 posted 5 You are reading this latest preprint version Abstract Oxaliplatin (OXA) is a frontline therapeutic agent used in the treatment of oral squamous cell carcinoma (OSCC). However, the development of chemoresistance has emerged as a significant challenge, compromising the effectiveness of treatment strategies. Therefore, there is a critical need to unravel the mechanisms underpinning drug resistance and to identify potential therapeutic targets. In recent years, there has been a growing interest in understanding the role of drug efflux in cancer chemoresistance mechanisms. Despite this, the contribution of ABCG2, a member of the ATP-binding cassette (ABC) transporter family, to oxaliplatin resistance in OSCC remains unclear. In the current study, we aimed to investigate the involvement of ABCG2 in oxaliplatin resistance in OSCC and to elucidate the molecular mechanisms through which the Wingless and Int-1 (WNT) canonical signaling pathway upregulates ABCG2 to promote chemoresistance. To achieve this, we established oxaliplatin-resistant (OXA-R) OSCC cells as a model system. Our investigations revealed that the efflux ability of resistant cells was enhanced and the ABCG2 expression was up-regulated. Genetic silencing of ABCG2 significantly attenuated both efflux activity and chemoresistance in these resistant cells. Notably, we observed aberrant activation of the WNT canonical signaling pathway in resistant cells, accompanied by heightened expression of the WNT3 ligand. Additionally, overexpression of WNT3 in parental cells recapitulated the activation of the WNT canonical signaling cascade, resulting in increased chemoresistance, enhanced efflux function, and elevated ABCG2 expression levels. Conversely, inhibition of WNT3 in resistant cells resulted in reduced chemoresistance, suppression of efflux activity, and decreased ABCG2 expression. Finally, treatment with the WNT/β-catenin pathway inhibitor methyl 3-benzoate (MSAB) effectively reversed chemoresistance in resistant cells. Mechanistically, our studies revealed that the abnormal activation of the WNT canonical pathway promotes the recruitment of the transcription factor lymphoid enhancer-binding factor 1 (LEF1) to the ABCG2 promoter, thereby enhancing its transcriptional activity. In summary, our findings underscore the critical role of WNT3-mediated activation of the WNT canonical signaling pathway in upregulating ABCG2 expression, which enhances oxaliplatin efflux and contributes to the development of oxaliplatin resistance in OSCC. Oral squamous cell carcinoma Chemoresistance ABC transporters WNT canonical signaling pathway Oxaliplatin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 25 Figure 26 Background Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in oral and maxillofacial region[ 1 ]. Its treatment typically involves a multimodal approach including surgery, radiotherapy, and/or chemotherapy[ 2 , 3 ]. Oxaliplatin (OXA) is a frontline therapeutic agent in OSCC management, exerting its anticancer effects by inhibiting DNA synthesis[ 4 ]. Despite the initial efficacy of oxaliplatin, the emergence of chemoresistance poses a significant challenge, leading to therapeutic ineffectiveness. Current research on chemoresistance mechanisms focuses on several factors, such as drug inactivation[ 5 ], DNA damage repair[ 6 ], reduced drug accumulation[ 7 ], and enhanced drug efflux[ 8 ]. Among these, increased drug efflux has garnered considerable attention in recent research endeavors. ATP-binding cassette (ABC) transporters represent a class of ATP-dependent efflux pumps located in the plasma membrane of both prokaryotic and eukaryotic cells. Structurally, these transporters consist of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs)[ 9 ]. The TMDs and NBDs collectively through transmembrane channels facilitating the transport of diverse substrates, including anticancer drugs[ 10 ]. During the efflux process, substrates enter the channel from the membrane entrance site. Subsequently, ATP binding induces dimerization of the NBDs, resulting in a conformational change in the ABC transporters, thereby expelling the substrate from the TMDs to the extracellular environment. Among the recognized transporters implicated in cancer chemoresistance are ABCB1 (multidrug resistance protein 1, MDR1), ABCC1 (multidrug resistance-associated protein 1, MRP1), and ABCG2 (breast cancer resistance protein, BCRP)[ 9 , 11 , 12 ]. ABCG2, a member of the multidrug resistance proteins (MDRPs), has been implicated in drug resistance in head and neck squamous cell carcinoma (HNSCC) and OSCC. Lu X et al.[ 13 ] reported a 1.6-fold increase in ABCC1 and a 2.1-fold increase in ABCG2 expression in OSCC specimens treated with 5-fluorouracil (5-FU) and cisplatin compared to untreated specimens. Additionally, the Hedgehog signaling pathway has been implicated in the regulation of ABC transporters. Tonigold M et al.[ 14 ] demonstrated upregulation of ABCC2 and ABCG2 in cisplatin-resistant HNSCC cells, potentially mediated by P53 mutation. Furthermore, ABCG2 expression is heightened in multidrug-resistant HNSCC cells exhibiting cancer stem cell (CSC) properties. Knockdown of ABCG2 inhibits spheroid formation and drug efflux capacity, suggesting ABCG2 may serve as a potential prognostic marker for HNSCC[ 15 ]. These findings underscore the potential association between abnormal ABCG2 expression and OSCC chemoresistance, although the precise molecular mechanisms driving ABCG2 upregulation remain unclear. Through analysis and validation of RNA sequencing data from established oxaliplatin-resistant OSCC cells (OXA-R), we have identified the transporter ABCG2 as a potentially pivotal factor in OSCC chemoresistance. The upregulation of ABCG2 in resistant cells facilitates the efflux of oxaliplatin, consequently reducing intracellular drug concentrations. Subsequently, we elucidated the molecular mechanism underlying ABCG2 upregulation. Specifically, we found that WNT3 activates the WNT/β-catenin signaling pathway in OXA-R cells, thereby enhancing the binding of LEF1 to the ABCG2 promoter and ultimately elevating ABCG2 expression. Methods and Materials Cell culture The human OSCC cell lines (CAL27, HN30, and HN6) and the human embryonic kidney cell line (293T) were cultured in high-glucose DMEM (SH30243.01, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (10270-106, Gibco, USA) in a humidified incubator with 5% CO 2 at 37°C. Oxaliplatin-resistant (OXA-R) cell lines, including CAL27/OXA, HN30/OXA, and HN6/OXA, were established from the respective parental cells (CAL27, HN30, and HN6) through continuous exposure to incrementally increasing concentrations of oxaliplatin during cell culture. The final oxaliplatin concentration used for resistance induction was 10 µM. Construction of OSCC cells with stable Oxaliplatin resistance Parental CAL27 and HN30 cell lines were treated with increasing concentrations of oxaliplatin (MedChemExpress (MCE), China). Over an approximate 12-month period, the oxaliplatin -resistant cells (OXA-R) were successfully stabilized, resulting in the CAL27/OXA, HN30/OXA and HN6/OXA strains, which were then consistently nurtured in a medium supplemented with 10 µM oxaliplatin. CCK-8 assay The cells were seeded onto 96-well plates. After adhesion, drugs were added and incubated for 48 hours. Subsequently, Cell Counting Kit-8 (CCK-8) (CK04, Dojindo, Japan) was added to the cells and incubated for one hour. Absorbance values at 450 nm were measured using the SpectroMax iD3 microplate reader (Molecular Devices, USA). Half-maximal inhibitory concentration (IC 50 ) values were calculated using GraphPad Prism software (version 8.0). The combination index (CI) values were calculated using the Chou-Talalay method with CompuSyn software (version 1.0) and interpreted as follows: CI > 1 indicates antagonism, CI = 1represents an additive effect, and CI < 1 signifies synergism[ 16 , 17 ]. Colony formation assay Following inoculation, the cells were cultured in 6-well plates for a duration of 2 weeks. Subsequently, the colonies were fixed using 4% paraformaldehyde (PFA) and stained using crystal violet (C0121, Beyotime, China). The areas of the colonies were quantified using ImageJ software (version 1.8.0). Cell apoptosis assay Cells were seeded in 6-well plates and allowed to adhere overnight. After incubation, the cells were gently washed with phosphate-buffered saline (PBS) and resuspended in 1× binding buffer. Subsequently, the cells were stained with YF® 488 Annexin V and propidium iodide (PI) (Y6002L, UElandy, China) for 15 minutes at room temperature in the dark, according to the manufacturer’s instructions. After staining, the samples were immediately analyzed using an Accuri™ C6 Flow Cytometer (BD Biosciences, USA). Data analysis was performed using FlowJo software (version 10.8.1, Tree Star, USA) to quantify the percentage of apoptotic cells. Each experiment was conducted in triplicate to ensure statistical reliability. Wound-healing assay The cells were seeded in 12-well plates and allowed to grow until reaching more than 90% confluence. Subsequently, a wound was created using a sterile 200 µL pipette tip, and the cells were further cultured in serum-free medium. The progression of wound healing was monitored and recorded using a microscope. The healing percentage was quantified using ImageJ software. Transwell migration and invasion assays Migration and invasion assays were conducted using 24-well Transwell chambers equipped with 8-µm pore size polycarbonate membranes, either pre-coated with or without matrix (353097/354480, Corning Falcon, USA). The lower chamber was filled with medium containing 10% FBS, while serum-free cells were seeded into the upper chamber. Following incubation for 24 to 48 hours, cells were fixed and stained with crystal violet. Immunofluorescent staining and confocal laser scanning microscopy (CLSM) Cells were cultured on coverslips in 12-well plates until reaching 50% confluence. The cells were fixed with 4% paraformaldehyde solution (Biosharp, China) and permeabilized with 0.3% Triton X-100 (1139, Biofroxx, Germany). Subsequently, cells were blocked with 3% bovine serum albumin (BSA) (4240GR500, Biofroxx, Germany) at room temperature for 30 minutes and then incubated overnight at 4°C with the primary antibody targeting β-catenin (610154, BD Biosciences, USA). The following day, cells were incubated with a goat anti-mouse secondary antibody conjugated to Alexa Fluor® 488-conjugated goat anti-mouse secondary antibody (4408S, CST, USA). In a separate experiment, fixed and permeabilized cells were stained with YF®594-Phalloidin (YP0052S, UElandy, China).Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (C1002, Beyotime, China). Finally, cells were cleaned, mounted, and visualized using confocal laser scanning microscopy (CLSM) (Olympus, Japan). RNA sequencing (RNA-seq) analysis and bioinformatics analysis RNA sequencing (RNA-seq) analysis was conducted by Novogene Biotechnology (Beijing, China), with sequencing reads aligned to the GRCh38/hg38 reference genome. The dataset has been deposited in the Gene Expression Omnibus repository (GEO) under accession number GSE248792. Differential expression analysis of genes (DEGs) between parental and resistant strains was performed using the R software package (version 4.2.1), along with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The criteria for differential expression were set at adjusted P-value (P adj ) ≤ 0.05 and |log 2 (Fold Change)|>1. DEGs were sorted based on ascending P adj values and descending |log 2 (Fold Change)| values. Total RNA isolation and real-time quantitative PCR (RT-qPCR) Total RNA was extracted from cells using NucleoZol (740404.200, Macherey Nagel, Germany) and reverse-transcribed into cDNAs using the PrimeScript ™ RT Reagent Kit (RR047A, Takara Bio, Japan). Real-time PCR assays were conducted using the SYBR Green Kit (1725124, Bio-Rad, USA) and amplification was detected using a real-time PCR system (Applied Biosystems, USA). Relative mRNA expression levels were normalized using the 2 -ΔΔCt method with GAPDH serving as an internal reference. Primer sequences are provided in Table S1 . Protein extraction and western blotting (WB) Total protein was extracted using RIPA buffer (P0013B, Beyotime, China), and the protein concentration was determined using the BCA protein assay kit (P1102, Beyotime, China). Subsequently, the protein samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (ISEQ00010, Amersham, USA). The membranes were then blocked with 3% BSA and probed with primary antibodies. Following primary antibody incubation, the membranes were incubated with secondary antibodies. Protein bands were visualized using the ECL chemiluminescence kit (P0018M, Beyotime, China) and imaged with the ChemiDoc ™ XRS + Imaging System (Bio-Rad, USA). The antibodies used in the study are listed in Table S2 . Small interfering RNA (siRNA) transfection Resistant cells were seeded in 6-well plates and transiently transfected with siRNAs using the Lipofectamine RNAiMAX Transfection Reagent (13778150, Invitrogen, USA) following the manufacturer's instructions. The siRNA sequences targeting ABCG2 (siABCG2), WNT3 (siWNT3), and the negative control (siNC) are detailed in Table S3. In accordance with Dharmacon™ siRNA solutions ( https://horizondiscovery.com/en/gene-modulation/knockdown/sirna ), equal amounts of two specific siRNA sequences targeting each gene were combined to prepare the transfection mixture for the knockdown group. Transient cell transfection The WNT3 overexpression plasmid pcDNA3.1-WNT3-3×Flag-C (WNT3) and its vector plasmid pcDNA3.1 (Vector) were obtained from Dahong Biotechnology (Guangzhou, China). OSCC cells were seeded in 6-well plates and transiently transfected with plasmids using Lipofectamine 2000 reagent (11668019, Invitrogen, USA), followed by replacement with fresh culture medium. Overexpression efficiency was confirmed by post-transfection protein analysis, and the successfully transfected cells were subsequently utilized for further experiments. Luciferase assay To assess Wnt/β-catenin signaling activity, cells were co-transfected with either WNT3 or control Vector plasmids, along with the TopFlash/FOPFlash plasmid and the Renilla luciferase internal control plasmid (Addgene, USA). Following 48 hours of transfection, cells were lysed and collected. Luciferase activity was measured using the SpectraMax iD3 microplate reader according to the instructions provided with the Dual Luciferase Assay System (11402ES60, Yeason, China). CFDA-SE fluorescent staining Drug efflux capacity was assessed using carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) (C6034S, UElandy, China)[ 18 , 19 ]. For the fluorescence efflux experiment, cells were seeded in 6-well plates, and after adhesion, the medium was aspirated. Cells were then stained with CFDA-SE for 30 minutes and subsequently incubated with complete medium to terminate staining. The attenuation of fluorescence was observed using an inverted fluorescence microscope. In addition, the amount of fluorescence extrusion was quantified by flow cytometry. Prior to CFDA-SE staining, cells were harvested, and after staining termination, the medium was aspirated. Cells were resuspended in buffer solution and kept on ice. Fluorescence intensity was measured using a flow cytometer. High-performance liquid chromatography (HPLC) Sample processing involved collecting cells, counting, removing the medium, and subsequently lysing the cell precipitate. A standard solution of oxaliplatin was prepared in gradient concentrations. Chromatographic analysis was conducted using a TC-C18 column (4.6 mm × 250 mm, 5 µm) (Agilent, USA) with a mobile phase consisting of methanol and distilled water (methanol:water = 80:20) (vol/vol) at a flow rate of 1 mL/min. The column temperature was maintained at 25℃, and oxaliplatin was detected at a wavelength of 204 nm. Standard and sample solutions were filtered and injected into the Agilent 1260 Infinity II Prime LC system (Agilent, USA) at 10 µL each. Chromatin immunoprecipitation (ChIP) assay The DNA fragments bound to the transcription factor were captured using an anti-LEF1 antibody (76010S, CST, USA) through immunoprecipitation. Protein G Magnetic Beads (HY-K0204, MCE, China) were utilized for the immunoprecipitation process. Subsequently, proteases K and RNase A (RT405-02, TIANGEN, China) were employed to degrade proteins and RNA, facilitating the purification and precipitation of DNA fragments. The specificity of the DNA fragments bound to the target gene ABCG2 was assessed using quantitative PCR (qPCR) with specific primers. The immunoprecipitation efficiency of LEF1 was compared to that of the negative control IgG (A00002, Zen-Bio, China) to determine the fold enrichment. The primers for ChIP-qPCR were designed using the Cistrome Data Browser ( http://cistrome.org/db/#/ ) and the UCSC Genome Browser ( https://genome.ucsc.edu/cgi-bin/hgTracks ), as illustrated in Fig.S6. The ChIP primer sequence targeting ABCG2 is: forward: 5'-CACCCGGACCTTCCAAACAA-3', reverse: 5'-GAGATTGAGAGACGCGGCAA-3'. Statistical analysis All experiments were conducted in triplicate, and the results were presented as mean ± standard deviation (SD). Statistical analyses and graphical representations were performed using GraphPad Prism 8.0 (GraphPad Software, USA). Student’s t-tests were employed to determine statistical significance. A P-value less than 0.05 was considered statistically significant. The following significance marks were used: ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Results Establishment of oxaliplatin-resistant (OXA-R) cell lines in OSCC To investigate the mechanisms underlying oxaliplatin resistance in OSCC, we successfully established the oxaliplatin-resistant (OXA-R) cell lines through continuous induction with oxaliplatin. Compared to their parental cell lines, the OXA-R cells exhibited significantly higher IC 50 values ( P < 0.01), as shown in Fig. 1 A and Fig. S1 A. To investigate cellular morphological changes in greater detail, we performed phalloidin staining.We observed that CAL27/OXA cells displayed looser intercellular connections compared to their parental CAL27 cells, while HN30/OXA cells exhibited spindle-like alterations relative to HN30 cells (Fig. 1 B). Since changes in cellular morphology often indicate shifts in biological behaviors [ 20 ], the attenuated connections in cell-cell and spindle-shaped morphology alterations may enhance cellular motility. Consequently, we further investigated the phenotypic characteristics of resistant cells. Our findings revealed that upon oxaliplatin exposure, the colony-forming ability of resistant cells was augmented compared to that of parental cells (Fig. 1 C), while cellular apoptosis was diminished ( P < 0.01) (Fig. 1 D). In addition, the migratory and invasive potential of resistant cells was significantly enhanced ( P < 0.01) (Fig. 1 E-F). These observations suggest that the acquisition of oxaliplatin resistance in OSCC cells leads to significant morphological transformations and is associated with enhanced malignant phenotypes, suggesting a more aggressive cellular behavior. Enhanced drug efflux capacity mediated by ABCG2 upregulation in OSCC OXA-R cell lines Given that oxaliplatin induces DNA damage, we initially analyzed genes associated with DNA damage repair based on the sequencing results (Fig. 2 A-B). Interestingly, we observed no significant changes in genes related to DNA damage repair between parental and drug-resistant cells (Fig. S1 B). However, further analysis revealed upregulation of multiple ABC transporter family genes, which are associated with efflux function, in the resistant cells (Fig. 2 C). Pathway enrichment analysis also indicated activation of membrane transporter-related pathways in drug-resistant cells (Fig. 2 D). These results suggest that alterations in drug efflux mediated by ABC transporters may contribute to oxaliplatin resistance. To assess the efflux ability of cells, we utilized CFDA-SE, a fluorescent substrate of ABC transporters. CFDA-SE is converted into carboxyfluorescein succinimidyl ester (CFSE) inside living cells, emitting green fluorescence[ 21 , 22 ]. Therefore, changes in intracellular CFSE fluorescence intensity can serves as a reliable indicator of cellular efflux function. As depicted in Fig. 2 E-F and Fig. S1 C, the fluorescence efflux in OXA-R cells was accelerated compared to that in parental cells, leading to a more rapid attenuation of fluorescence intensity. Flow cytometry further confirmed that resistant cells expelled more fluorescence compared to parental cells (Fig. 2 G and Fig. S1 D). Additionally, we indirectly analyzed oxaliplatin efflux ability by measuring the relative intracellular oxaliplatin concentrations using HPLC. The peak location of oxaliplatin was determined based on the peak time and area of oxaliplatin standard with varying concentrations (Fig. 2 H and Fig. S1 E), and the relative intracellular oxaliplatin concentration was compared. As illustrated in Fig. 2 I and Fig. S1 F, the relative oxaliplatin concentrations in OXA-R cells was significantly lower than that in parental cells, providing further evidence of enhanced efflux function in OXA-R cells. To identify the key protein responsible for enhancing efflux function, we initially confirmed the upregulated ABC genes identified in the sequencing through RT-qPCR (Fig. S1 G). Subsequently, ABCG2, ABCB1, and ABCC9, which exhibited significant expression differences, were selected for protein-level verification by western blotting. Notably, we observed no discernible protein bands corresponding to ABCB1 and ABCC9 in either parental and resistant OSCC cells (Fig. S1 H). However, the expression of ABCG2 protein was notably elevated in the OXA-R cell lines (Fig. 2 J-K and Fig. S1 I). Furthermore, upon treatment with oxaliplatin at various concentrations for 48 hours, we observed an increase in ABCG2 expression in parental cells as well (Fig. 2 L). These findings suggest that the enhanced efflux capacity in OXA-R cells may be attributed to the upregulation of ABCG2. ABCG2 knockdown reduced oxaliplatin efflux and reversed chemoresistance in OSCC OXA-R cells To further substantiate that the pivotal role of ABCG2 in medidating enhanced drug efflux in OXA-R cells, we employed siRNA-mediated knockdown of ABCG2 and assessed the knockdown efficiency at both transcriptional and translational levels in OXA-R cells (Fig. 3 A-B and Fig. S2 A-B). We found that ABCG2 knockdown decelerated the CFSE efflux in OXA-R cells (Fig. 3 C and Fig. S2 C), which was confirmed by flow cytometry showing lower fluorescence output in the knockdown group compared to the control group (Fig. 3 D and Fig. S2 D). In addition, HPLC analysis demonstrated a substantial increase in intracellular oxaliplatin concentration following ABCG2 knockdown (Fig. 3 E and Fig. S2 E). These outcomes confirmed the crucial role of ABCG2 in modulating the efflux function of OXA-R cells. Subsequently, we delved into whether ABCG2 influenced chemoresistance phenotypes. It was observed that ABCG2 knockdown resulted in decreased IC 50 values of OXA-R cells (Fig. 3 F and Fig. S2 F), indicating restored drug sensitivity. Moreover, upon oxaliplatin stimulation, the knockdown group exhibited inhibited proliferation, colony formation, and anti-apoptotic abilities in comparison to the control group (Fig. 3 G-I and Fig. S2 G-H). We also explored the impact of ABCG2 on cell mobility and discovered that ABCG2 knockdown attenuated migration and invasion under oxaliplatin exposure (Fig. 3 J and Fig. S2 I). Collectively, these findings indicated that ABCG2 knockdown inhibited efflux, reduced intracellular oxaliplatin concentration, and increased sensitivity to oxaliplatin. Aberrant activation of the WNT canonical signaling pathway in OSCC OXA-R cells To elucidate the factors contributing to the up-regulation of ABCG2 expression, we conducted further analysis of the RNA sequencing data. Our investigation revealed the upregulation of key genes associated with the WNT canonical pathway in CAL27/OXA, including WNT ligands and downstream target genes such as Vimentin (VIM) and N-cadherin (CDH2), indicating the aberrant activation of the WNT canonical signaling pathway (Fig. 4 A). The WNT signaling pathways comprise both canonical and non-canonical pathways. Among these, the canonical pathway, also known as the β-catenin pathway, primarily regulates the transcription of target genes through the specific binding of the transcription factor LEF/TCF to the promoters of target genes[ 23 , 24 ]. To examine whether the up-regulation of ABCG2 expression is linked to the abnormal activation of the WNT canonical signaling pathway, we employed CiiiDER software[ 25 ] to predict the binding sites of transcription factors near the ABCG2 promoter. Notably, our analysis revealed there are binding sites near the promoter of ABCG2 to the transcription factor LEF1 (Fig. 4 B), suggesting potential transcriptional regulation of ABCG2 by the WNT/β-catenin pathway. Prior studies have established a close relationship between the WNT canonical pathway and chemoresistance, highlighting it as a promising therapeutic target. However, the specific regulatory mechanism underlying this association remains unclear[ 26 , 27 , 28 ]. Therefore, we needed to investigate the role of the WNT canonical signaling pathway in oxaliplatin resistance in OSCC. We first compared the basal β-catenin levels in several parental cell lines using western blotting and found that the basal β-catenin levels did not show significant differences among these three cell lines (Fig. S3A). Then we verified the activation of the WNT canonical signaling pathway in OXA-R cells. Immunofluorescence analysis demonstrated an increased nuclear translocation of β-catenin in resistant cells (Fig. 4 C), and WB analysis revealed elevated nuclear β-catenin accumulation in OXA-R cells compared to parental cells (Fig. 4 D). In the TOP/FOP flash luciferase reporter assay, the TOP/FOP activity was enhanced in the drug-resistant cells (Fig. 4 E). This suggests an increase in transcriptional activity in the drug-resistant cells. Furthermore, RT-qPCR analysis confirmed the upregulation of the target genes AXIN2 and CD44 in OXA-R cells (Fig. 4 F and Fig. S3B). WNT3 regulated oxaliplatin resistance in OSCC WNT ligands play the critical role in activating the WNT canonical pathway. To identify the key WNT ligand involved in the activation of the pathway, we screened for upregulated ligands in the sequencing results and those known to activate the canonical pathway in previous research[ 29 , 30 ]. Our RT-qPCR analysis revealed a significant difference in WNT3 expression between parental cells and OXA-R cells (Fig. S3C), with the protein level of WNT3 also found to be upregulated in the OXA-R cells (Fig. 4 G and Fig. S3D). We then evaluated whether WNT3 overexpression could activate the WNT canonical signaling pathway in OSCC. The effect of WNT3 overexpression was confirmed by WB analysis (Fig. 5 A). Following WNT3 overexpression in OSCC parental cells, immunofluorescence analysis demonstrated an increased translocation of β-catenin into the nucleus (Fig. 5 B), and WB experiment revealed upregulated levels of β-catenin in the nucleus (Fig. 5 C). Moreover, TOP/FOP flash luciferase reporter assays showed that overexpression of WNT3 significantly increased the TOP/FOP activity (Fig. 5 D and Fig. S3E), indicating enhanced transcriptional activity. Subsequently, we knocked down WNT3 in the drug-resistant cells and observed a reduction in TOP/FOP activity (Fig. S3F). These results collectively demonstrated the activating effect of WNT3 on the WNT canonical pathway. Having established the activation effect of WNT3 on the WNT canonical pathway, we then investigated its impact on OSCC oxaliplatin resistance by modulating WNT3 expression. We found that WNT3 overexpression in parental cells led to elevated IC 50 values (Fig. 5 E), indicating increased resistance to oxaliplatin. Additionally, under oxaliplatin treatment, the overexpression group exhibited enhanced proliferation, colony formation, and anti-apoptosis abilities compared to the control group (Fig. 5 F-H). Furthermore, WNT3 overexpression accelerated migration and invasion in cells exposed to oxaliplatin, highlighting its role in promoting cell motility under drug exposure (Fig. 5 I-J). Next, we employed siRNAs to knock down WNT3 expression to investigate its impact on OXA-R cells. RT-qPCR and western blot analyses confirmed a significant decrease in WNT3 expression following siRNA transfection (Fig. 6 A-B and Fig. S3G-H). Subsequently, we observed that WNT3 knockdown led to decreased IC 50 values in OXA-R cells (Fig. 6 C and Fig. S3I), suggesting restored oxaliplatin sensitivity. In addition, WNT3 knockdown inhibited the proliferation, colony formation, and anti-apoptotic capabilities of resistant cells upon exposure to oxaliplatin (Fig. 6 D-F and Fig. S3J-K). We also observed reduced migration and invasion of resistant cells under oxaliplatin exposure following WNT3 knockdown (Fig. 6 G-H and Fig. S3L). These results collectively indicated that WNT3 activated the WNT canonical signaling pathway and regulated oxaliplatin resistance in OSCC. WNT/β-catenin inhibitor reversed chemoresistance in OSCC OXA-R cells To further investigate the therapeutic potential of inhibiting WNT/β-catenin signaling in oxaliplatin-resistant OSCC, we used methyl 3-benzoate (MSAB), a small molecule inhibitor targeting β-catenin. We first measured the cell viability under different concentrations of MSAB, and selected 0.2 µg/mL MSAB (80% cell viability) as the concentration for subsequent work (Fig. 7 A and Fig. S4A). Then we verified that MSAB decreased the β-catenin expression in OXA-R cells (Fig. 7 B). In addition, under the stimulation of different concentrations of MSAB, the expression of apoptosis indicators increased in a concentration-dependent manner, suggesting that MSAB may exert inhibitory effects by activating the apoptotic pathway in OXA-R cells (Fig. 7 C). Subsequent studies on reversing chemotherapy resistance with MSAB showed that it could decrease the IC 50 values in OXA-R cells (Fig. 7 D). Furthermore, the combination of oxaliplatin and MSAB showed enhanced therapeutic effects compared to either treatment alone, with significant inhibition of cell proliferation and increased apoptosis (Fig. 7 E-F and Fig. S4B-C). To access whether MSAB and oxaliplatin had a synergistic effect, we calculated the combination index (CI) values. In OXA-R cells, the CI values of oxaliplatin combined with MSAB was below 1 (Fig. 7 G), indicating a synergistic effect. To sum up, MASB inhibited WNT canonical signaling pathway and reversed the chemoresistance in OSCC OXA-R cells. WNT3 upregulated ABCG2 expression by enhancing LEF1 binding to the ABCG2 promoter Based on the aforementioned results, the involvement of ABCG2 and the WNT canonical signaling pathway in oxaliplatin resistance prompted us to delve deeper into the relationship between ABCG2 and the WNT pathway. Initially, we analyzed the impact of WNT3 on the efflux function of OSCC. Overexpression of WNT3 in parental cells led to faster fluorescence efflux of CFSE, as visualized by fluorescence microscopy (Fig. 8 A) and quantitatively confirmed by flow cytometry showing increased fluorescence output (Fig. 8 B), and decreased intracellular oxaliplatin concentration measured by HPLC (Fig. 8 C). Conversely, downregulation of WNT3 in OXA-R cells resulted in slower fluorescence efflux of CFSE, reduced fluorescence output, and increased intracellular oxaliplatin concentration (Fig. 8 D-F and Fig. S5A-C), indicating that the WNT pathway influenced the efflux ability of OSCC. Subsequently, we examined the effect of WNT3 on the expression of ABCG2. The results revealed that overexpression of WNT3 in parental cells upregulated ABCG2 expression (Fig. 8 G-H), while knockdown of WNT3 in resistant cells decreased ABCG2 expression (Fig. 8 I). Given that the WNT canonical pathway regulates target genes through β-catenin/TCF/LEF-mediated transcriptional activation[ 31 ], we conducted chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) to elucidate the mechanism of ABCG2 upregulation by WNT3. The findings indicated that overexpression of WNT3 enhanced the binding ability of the transcription factor LEF1 to the ABCG2 promoter (Fig. 8 J and Fig. S6A-B). Taken together, WNT3 upregulated ABCG2 expression by promoting the binding of LEF1 to the ABCG2 promoter, thereby enhancing oxaliplatin efflux. Discussion Oxaliplatin, a conventional chemotherapy agent utilized in the treatment of OSCC, has made some progress in retarding tumor progression and enhancing prognosis[ 32 ]. Nonetheless, the emergence of chemoresistance undermines its efficacy, necessitating a focused effort on addressing this issue. In this study, we explored mechanisms underlying oxaliplatin resistance in OSCC. Briefly, we established OXA-R OSCC cell lines in vitro, and observed an upregulation of ABCG2 transporter expression alongside aberrant activation of the WNT canonical signaling pathway in OXA-R cells. Mechanically, we discovered an augmented binding affinity of the WNT pathway transcription factor LEF1 to the ABCG2 promoter, resulting in the upregulation of ABCG2 expression and subsequent enhancement of drug efflux, thus facilitating chemoresistance. In this study, we have selected CAL27, HN30, and HN6, three widely used cell lines in OSCC research. CAL27, a highly aggressive cell line, commonly exhibits TP53 mutations[ 33 ]. HN30 exhibits typical OSCC features, derived from a primary tumor and is suitable for studying the impact of the tumor microenvironment[ 34 ]. HN6 cells are valuable for metastasis and drug resistance research, typically exhibiting TP53 and EGFR mutations[ 35 , 36 ]. These cell lines exhibit distinct genetic and epigenetic characteristics. Therefore, in our experimental design, we selected these cell lines to cover diverse biological behaviors and genetic information of OSCC, thereby comprehensively validating our research hypotheses. Although tumor treatment has entered a new era marked by targeted therapy and immunotherapy[ 37 ], platinum-based drugs remain the cornerstone of first-line adjuvant therapy in clinical practice, owing to considerations of efficacy, cost-effectiveness, and toxicity[ 32 ]. Platinum resistance, primarily attributed to DNA damage repair mechanisms, has garnered significant attention[ 6 , 38 ]. Interestingly, the sequencing results from this study revealed no significant alterations in DNA damage repair-related genes. Although DNA damage repair encompasses multiple pathways, and defects in one pathway can be compensated by others, this redundancy poses significant challenges for cancer treatment. However, certain tumors still exhibit inadequate DNA repair capacity[ 39 ]. The variability in DNA damage response (DDR) among different tumor types provides a plausible explanation for the initial drug sensitivity. This finding suggests that in our study, oxaliplatin resistance mechanisms may rely more on alternative molecular pathways rather than traditional changes in the expression of DNA damage repair-related genes. Cells may have developed other adaptive mechanisms to cope with DNA damage. The elucidation of various mechanisms contributing to platinum resistance includes abnormalities in drug transport, mutations in target genes[ 40 ], and effects arising from the tumor microenvironment[ 41 , 42 ]. Membrane transporters play a pivotal role in drug transport processes, with ATP-binding cassette (ABC) transporters, responsible for drug efflux, and solute carrier (SLC) transporters, facilitating drug uptake, being particularly noteworthy. Among these, ABC transporters have been extensively studied in the context of chemoresistance[ 43 ]. Notably, ABCG2, encoding the breast cancer resistance protein (BCRP), was initially identified in the human breast cancer MCF-7 cell line and has been implicated in multidrug resistance phenotypes in breast cancer[ 44 ]. ABCG2 functions by mediating substrate efflux in a manner akin to other ABC transporters. Upon ATP-driven conformational changes, the protein expels drugs from cells, thereby reducing intracellular drug concentrations and fostering drug resistance[ 45 ]. Increasing evidence suggests that elevated expression of ABCG2 is associated with chemoresistance in various cancers, including OSCC. Moreover, high ABCG2 expression has been observed in side population (SP) cells exhibiting stemness characteristics, alongside established stemness markers CD44 and CD133[ 46 , 47 ]. Sudo S et al.[ 48 ] demonstrated a dose-dependent upregulation of ABCG2 in OSCC HSC-2 and HSC-3 cells following direct stimulation with cisplatin for 24 hours, a process mediated by the activation of STAT1/3 expression. Similarly, Choi HS et al.[ 49 ] generated cisplatin-resistant OSCC cells and observed elevated expression of BCRP and MDR1. Functional assays utilizing Rhodamine 123 and BODIPY™ FL prazosin confirmed enhanced efflux activity in these resistant cells. Although the precise mechanism underlying BCRP and MDR1 upregulation was not fully elucidated, the observed efflux alterations align with our findings. In our investigation, we observed a significant enhancement in efflux capacity in oxaliplatin-resistant (OXA-R) cells, confirmed by CFDA-SE fluorescence efflux assay and HPLC. Additionally, RT-qPCR and Western blot analyses validated the upregulation of ABCG2 expression in OXA-R cells. Subsequent ABCG2 knockdown experiments resulted in weakened efflux capacity and chemoresistance, accompanied by inhibited proliferation and mobility, thus underscoring the pivotal role of ABCG2 in mediating oxaliplatin resistance in OSCC. Despite accumulating evidence implicating ABCG2 in tumor drug resistance, the precise mechanism underlying its upregulation remains elusive, particularly in the context of oxaliplatin resistance in OSCC. Zhang L et al.[ 50 ] utilized the PI3K inhibitor BAY-1082439 to target PI3K subunits P110α and P110β, resulting in reduced expression of ABCB1 and ABCG2 and reversal of chemoresistance in human epidermoid carcinoma and non-small cell lung cancer. However, inhibition of AKT failed to reverse ABCB1- or ABCG2-mediated multidrug resistance (MDR), suggesting an independent relationship between AKT and ABC transporters. In OXA-R colorectal cancer cells, upregulation of NF-κB phosphorylation was observed, leading to enhanced ABCG2 expression, attenuation of endoplasmic reticulum (ER) stress, and inhibition of apoptosis[ 51 ]. While these studies identified associations between ABCG2 upregulation and specific signaling pathways, they did not delve into the detailed regulatory mechanisms. In our investigation, we observed abnormal activation of the WNT canonical signaling pathway in OXA-R cells, suggesting a potential regulatory role of this pathway in ABCG2 expression. The WNT/β-catenin pathway is initiated when WNT ligands bind to co-receptors Frizzled (FZD) and low-density lipoprotein receptor-related proteins 5 (LRP5) or 6 (LRP6). This binding recruits effector proteins Dishevelled (DVL) and AXIN to the WNT-binding receptor, leading to the inhibition of glycogen synthase kinase 3β (GSK3β) activity. Consequently, GSK3β inhibition prevents the phosphorylation and subsequent degradation of β-catenin, resulting in its cytoplasmic accumulation and translocation into the nucleus[ 52 ]. Within the nucleus, β-catenin binds to the transcription factor T-cell factor/lymphoid enhancer factor (TCF/LEF), thereby activating downstream target genes. The pivotal role of WNT ligands in initiating the WNT canonical signaling pathway underscores their significance as potential therapeutic targets for interrupting signal transduction at its origin[ 53 , 54 ]. While our previous research has confirmed the role of WNT3 in 5-fluorouracil resistance in OSCC[ 55 ], research on WNT ligands' involvement in OSCC chemoresistance remains limited. In this study, we confirmed the promotion of oxaliplatin resistance in OSCC by WNT3. Initially, we validated the activation of the canonical signaling pathway by WNT3 in OSCC and assessed its impact on oxaliplatin resistance. Upregulation of WNT3 in parental cells enhanced resistance and increased proliferation and mobility under oxaliplatin treatment. Conversely, knockdown of WNT3 in oxaliplatin-resistant (OXA-R) cells reduced resistance and inhibited proliferation and mobility under oxaliplatin exposure. To facilitate the translation of laboratory findings into clinical practice, we employed MSAB, a small molecule inhibitor targeting β-catenin, to investigate its potential to reverse oxaliplatin resistance. MSAB selectively binds and degrades β-catenin, thereby inhibiting the transcription of downstream target genes. Our results demonstrated the inhibitory effect of MSAB in OXA-R cells, as evidenced by a decrease in total β-catenin levels with increasing MSAB concentration. Additionally, MSAB upregulated apoptosis indicators c-Caspase 3 and c-PARP, suggesting its potential to inhibit cell proliferation by activating the apoptotic pathway in OXA-R cells. Furthermore, MSAB decreased the IC 50 value of oxaliplatin in OXA-R cells, and when combined with oxaliplatin, it suppressed cell proliferation and anti-apoptotic ability. These findings indicated that MSAB could effectively reverse oxaliplatin resistance in OXA-R cells. calculation of the combined index (CI) value of MSAB and oxaliplatin revealed a synergistic effect, suggesting potential clinical utility. Previous studies have elucidated the role of the WNT pathway in promoting tumor drug resistance by upregulating the expression of ABC transporters. For instance, in multi-drug resistant hepatocellular carcinoma, scholars have observed upregulation of FZD7, ABCB1, ABCC1, and ABCC2 expression. Knockdown of the WNT pathway receptor FZD7 enhanced chemotherapy sensitivity and inhibited the expression of ABCB1, ABCC1, and ABCC2. Furthermore, treatment with the β-catenin inhibitor iCRT-3 downregulated the expression of ABCB1, ABCC1, and ABCC2, indicating an association between the WNT pathway and ABC transporters[ 56 ]. In chronic myeloid leukemia, the transcription factor TCF7 was recruited to ABCC2 promoter, leading to upregulation of ABCC2 expression and induction of imatinib resistance[ 57 ]. Similarly, in studies on the mechanism of nasopharyngeal carcinoma chemoresistance, TRIM11 was found to enhance WNT signal activity, increase β-catenin protein levels, and promote ABCC9 expression by binding to the ABCC9 promoter, thereby promoting drug resistance[ 58 ]. While these studies have demonstrated that the WNT pathway can upregulate the expression of ABC transporters, and some have provided initial insights into why the canonical WNT signaling pathway can upregulate ABCG2, there has been limited exploration into how key WNT molecules act on ABCG2. In this study, we conducted a comprehensive investigation into the mechanism by which WNT3 regulated ABCG2 expression and promoted drug resistance. We first confirmed the regulatory role of WNT pathway in efflux function and observed that WNT3 promoted efflux. Subsequently, we identified that WNT3 upregulated ABCG2 expression. Finally, we explored the mechanism by which WNT3 upregulated ABCG2 and found that WNT3 overexpression enhanced the binding ability of the transcription factor LEF1 to the ABCG2 promoter. In summary, we established OXA-R cell lines of OSCC through in vitro induction and identified ABCG2 as a key determinant of oxaliplatin resistance by augmenting efflux capacity and reducing intracellular drug concentrations. Mechanistically, upregulation of WNT3 ligand expression activated the WNT canonical signaling pathway, thereby enhancing the binding of LEF1 to the ABCG2 promoter and subsequently increasing ABCG2 expression (Fig. 9 ). This study contributes additional evidence towards addressing oxaliplatin resistance in OSCC and identifying potential therapeutic targets. Unfortunately, the key target of oxaliplatin chemoresistance identified in this study has not yet been validated in vivo or in clinical OSCC specimens. While our in vitro findings provide valuable insights, further validation in animal models and clinical samples is necessary to confirm the relevance and therapeutic potential of this target in a more physiologically relevant context. Future studies will focus on addressing this limitation to strengthen the translational impact of our findings. Conclusions The activation of the WNT canonical signaling pathway resulted in the upregulation of ABCG2 transporter expression, consequently leading to heightened efflux of oxaliplatin and promoted oxaliplatin resistance in OSCC. Declarations Acknowlegements We thank all the team members who participated in the study. Funding This study was supported by the National Natural Science Foundation of China (No.82272868 and No.82173180). Author information Authors and affiliations School and Hospital of Stomatology, Fujian Medical University, Fujian Key Laboratory of Oral Diseases, Biological Materials Engineering and Technology Center of Stomatology, 350004 Fuzhou, China. Department of Stomatology, The Affiliated Wuxi People's Hospital of Nanjing Medical University, 214023 Wuxi, China. Kairui Sun School and Hospital of Stomatology, Fujian Medical University, Fujian Key Laboratory of Oral Diseases, Fujian Provincial Biological Materials Engineering and Technology Center of Stomatology, 350004 Fuzhou, China. Xuyang Zhang, Shuoqi Lin & Dali Zheng Department of Preventive Dentistry, School and Hospital of Stomatology, Fujian Medical University, 350002 Fuzhou, China. Ruihuan Gan & Chen Yu School and Hospital of Stomatology, Fujian Medical University, Fujian Key Laboratory of Oral Diseases, Fujian Provincial Biological Materials Engineering and Technology Center of Stomatology, Department of Preventive Dentistry, Hospital of Stomatology, Fujian Medical University, 350002 Fuzhou, China. Youguang Lu Contributions The final manuscript was read and approved by all writers. The work has been sufficiently contributed to by each author. Kairui Sun: Conceptualization, Data curation, Investigation, Methodology, Data Analisis, Writing-original draft; Xuyang Zhang: Conceptualization, Data curation, Investigation, Methodology, Data Analisis, Writing-review & editing; Ruihuan Gan: Data curation; Investigation; Methodology; Writing-review & editing; Shuoqi Lin: Data curation; Investigation; Methodology; Writing-review & editing; Yu Chen: Data curation; Investigation; Methodology; Writing-review & editing; Dali Zheng: Conceptualization; Project administration; Resources; Supervision; Writing-review & editing; Youguang Lu: Conceptualization; Project administration; Funding acquisition; Resources; Supervision; Writing-review & editing. Corresponding authors Correspondence to Dali Zheng: [email protected] or Youguang Lu: [email protected] . Ethics declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. 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Supplementary Files Supplementaryfiguresandtables.docx SupplementaryfilesfororiginalimagesofWB.pdf Cite Share Download PDF Status: Published Journal Publication published 04 Jun, 2025 Read the published version in Cell & Bioscience → Version 1 posted Reviewers agreed at journal 03 Apr, 2025 Reviewers invited by journal 03 Apr, 2025 Editor assigned by journal 01 Apr, 2025 First submitted to journal 29 Mar, 2025 Editorial decision: Major revision 30 Dec, 2024 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. 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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-5267942","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":438115666,"identity":"89eae6e5-3bde-459c-9324-64d5c60cab79","order_by":0,"name":"Youguang Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie3QPQrCMBTA8Rci7RJwTakfV1C6CB28SrvYRZ07dHigZCp4FhHEzZRCF9tdb2EHoZOY2r3tKJj/kgTej5AA6HQ/mIkpAoTRxERZn2k3YTJRJM8cJvsTgkD21I+/ohchibBeaAQxua04hK6PZiHbCSTCHl9GmxiKM4c88JFtvVayrImVG5trTYhIfeRs1n2LJWjAvuTdk1iloF5DsB/ZueqT5+otp4WXBY5g6w7C0+RRhdGU8eJ4f0bu+GDm7QS4HNis2QB4ajXa51VDpGXVbDpndTqd7k/7AAgfS6/CzxgLAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1325-3790","institution":"Fujian Medical University Affiliated Hospital and School of Stomatology","correspondingAuthor":true,"prefix":"","firstName":"Youguang","middleName":"","lastName":"Lu","suffix":""},{"id":438115667,"identity":"ec4b1af0-b23c-4ed5-933f-a137e9135439","order_by":1,"name":"Kairui Sun","email":"","orcid":"","institution":"Fujian Medical University Affiliated Hospital and School of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Kairui","middleName":"","lastName":"Sun","suffix":""},{"id":438115668,"identity":"eaf07bb9-f432-44fa-a611-5249d6624069","order_by":2,"name":"Xuyang Zhang","email":"","orcid":"","institution":"Fujian Medical University Affiliated Hospital and School of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Xuyang","middleName":"","lastName":"Zhang","suffix":""},{"id":438115669,"identity":"5741ecfb-bd67-4c78-99e4-072fb1ee987c","order_by":3,"name":"Ruihuan Gan","email":"","orcid":"","institution":"Fujian Medical University Affiliated Hospital and School of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Ruihuan","middleName":"","lastName":"Gan","suffix":""},{"id":438115670,"identity":"6f335f7b-02d0-4fb6-99b2-74e8268638c2","order_by":4,"name":"Shuoqi Lin","email":"","orcid":"","institution":"Fujian Medical University Affiliated Hospital and School of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Shuoqi","middleName":"","lastName":"Lin","suffix":""},{"id":438115671,"identity":"3a4e4e76-f19f-4810-8b2c-0cffdf371e88","order_by":5,"name":"Yu Chen","email":"","orcid":"","institution":"Fujian Medical University Affiliated Hospital and School of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Chen","suffix":""},{"id":438115672,"identity":"e06c212b-f957-4b66-9f4f-d193bd62d355","order_by":6,"name":"Dali Zheng","email":"","orcid":"","institution":"Fujian Medical University Affiliated Hospital and School of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Dali","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2024-10-15 10:30:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5267942/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5267942/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13578-025-01414-w","type":"published","date":"2025-06-04T15:57:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80048497,"identity":"92369446-60b1-4caf-8ebf-c850e169c047","added_by":"auto","created_at":"2025-04-07 10:04:17","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1630651,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment and characterization of oxaliplatin-resistant (OXA-R) oral squamous cell carcinoma (OSCC) cells.\u003c/strong\u003e (A) The IC\u003csub\u003e50 \u003c/sub\u003evalues of parental and OXA-R cells from CAL27 and HN30 cell lines were determined using the CCK-8 assay over a 48-hour period. (B) Morphological differences between OSCC parental and OXA-R cells were visualized using confocal laser scanning microscopy (CLSM) following phalloidin staining (600×). Nuclei were counterstained with DAPI (blue), while phalloidin staining is shown in red. The white arrow highlights distinct morphological variances between the resistant and parental cells. (C) Colony formation experiments revealed an augmented colony formation ability in OXA-R cells under oxaliplatin treatment. (D) Flow cytometry was utilized to assess the apoptosis levels of OXA-R and parental cells under oxaliplatin treatment. (E) Wound healing assays demonstrated enhanced wound closure capacity in OXA-R cells (100×). (F) Transwell assays revealed an enhanced migration and invasion potential in OXA-R cells (100×). Data shown as mean ± SD (n = 3). Statistical significance was denoted as follows: **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, and ****, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/a9295dd75cf83474b64b6da7.jpeg"},{"id":80048501,"identity":"3f7053d7-7e6c-46c2-bbdd-caf12bfa540a","added_by":"auto","created_at":"2025-04-07 10:04:17","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1596630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced efflux capacity in OSCC OXA-R cells along with the up-regulation of the ABCG2 transporter.\u003c/strong\u003e (A) A volcano plot displayed the gene expression disparities between the parental CAL27 cell and the resistant CAL27/OXA cell of OSCC. (B) A heat map depicted the differential gene expression profile between the parental and resistant CAL27 cells. (C) Another heat map revealed no significant alterations in genes associated with DNA damage repair between the resistant and parental cells. (D) KEGG pathway enrichment analysis indicated the activation of membrane transporter pathways in OXA-R cells. (E-F) Fluorescence microscopy observation demonstrated that the fluorescence decay of CFSE in resistant cells occured more rapidly compared to parental cells over the same duration (100×). (G) Flow cytometry illustrated a significant increase in fluorescence output in OXA-R cells of OSCC. (H) High-performance liquid chromatography (HPLC) analysis of the peak area of oxaliplatin standard with gradient concentrations at approximately 3 minutes. (I) HPLC analysis indicated that the relative concentration of oxaliplatin in OXA-R cells is lower than that in parental cells. (J-K) Western blotting was utilized to assess the ABCG2 protein levels in OXA-R and parental cells. (L) Upregulation of ABCG2 mRNA expression was observed in parental cells treated with various concentrations of oxaliplatin (0 μM, 1 μM, 3 μM, 10 μM) for 48 hours by RT-qPCR. Data shown as mean ± SD (n = 3). Statistical significance was denoted as follows: *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001; and ****, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/61ac58612825ba1cef89acec.jpeg"},{"id":80048498,"identity":"81972409-dc0d-42d1-8191-8604bd3ac29f","added_by":"auto","created_at":"2025-04-07 10:04:17","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1872941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe impact of ABCG2 knockdown on reducing oxaliplatin efflux and inhibiting chemoresistance in OSCC OXA-R cells.\u003c/strong\u003e (A) RT-qPCR was employed to assess the mRNA level of ABCG2 knockdown in OXA-R cells. (B) Western blotting was utilized to detect the protein level of ABCG2 knockdown in OXA-R cells. (C) Fluorescence microscopy revealed a slower efflux of CFSE fluorescence following ABCG2 knockdown in resistant cells (100×). (D) Flow cytometry confirmed the decrease in CFSE efflux after ABCG2 knockdown in OXA-R cells. (E) After a 48-hour oxaliplatin treatment, the relative concentration of oxaliplatin in the ABCG2 knockdown group of resistant cells increased, as observed through HPLC analysis. (F) The IC\u003csub\u003e50\u003c/sub\u003e of oxaliplatin decreased in OXA-R cells after ABCG2 knockdown, as determined by the CCK-8 assay. (G) The CCK-8 assay further demonstrated that ABCG2 knockdown inhibited proliferation in OXA-R cells. (H) Colony formation assay results showed that ABCG2 knockdown reduced colony formation ability in OXA-R cells under oxaliplatin treatment. (I) Flow cytometry analysis indicated increased apoptosis of OXA-R cells under oxaliplatin treatment following ABCG2 knockdown. (J) Transwell chamber experiments revealed reduced migration and invasion of OXA-R cells under oxaliplatin treatment after ABCG2 knockdown (100×). Data shown as mean ± SD (n = 3). Statistical significance was denoted as follows: *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; and ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/961a2eb82f6d7955ccf1c4f2.jpeg"},{"id":80048509,"identity":"175b7a45-84be-4476-bf1e-bf5363964592","added_by":"auto","created_at":"2025-04-07 10:04:17","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7206618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAberrant activation of the WNT/β-catenin signaling pathway in OSCC OXA-R cells.\u003c/strong\u003e (A) Transcriptome sequencing of CAL27 and CAL27/OXA cells revealed the up-regulated expression of genes related to the WNT canonical pathway. (B) CiiiDER software was used to predict binding sites of transcription factors near the promoters of ABCG2. (C) Immunofluorescence staining was utilized to observe the nuclear translocation of β-catenin in parental and OXA-R cells (600×). DAPI staining is represented in blue, while β-catenin staining is shown in green. (D) The expression of β-catenin in the cytoplasm and nucleus of parental and OXA-R cells was analyzed via western blotting. (E) In the TOP/FOP flash luciferase reporter assay, the TOP/FOP activity was enhanced in the drug-resistant cells.\u003cu\u003e \u003c/u\u003e(F)RT-qPCR was employed to detect the expression of molecules related to the WNT canonical signaling pathway in OSCC parental and OXA-R cells. (G) The expression of WNT3 in parental and OXA-R cells was analyzed via western blotting. Data shown as mean ± SD (n = 3). Statistical significance was denoted as follows: *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/0d9ecdfc77be2a89d9b8044c.jpeg"},{"id":80049503,"identity":"f1726717-a37e-4eb2-84a2-ecb9b7280185","added_by":"auto","created_at":"2025-04-07 10:12:18","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6039049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of WNT3 overexpression on enhancing oxaliplatin resistance in OSCC parental cells.\u003c/strong\u003e (A) Western blotting confirmed the efficacy of WNT3 overexpression in parental cells. (B) Immunofluorescence staining illustrated that WNT3 overexpression increased the expression of β-catenin in the nucleus (600×). DAPI staining is represented in blue, while β-catenin staining is shown in green. (C) Western blotting revealed that WNT3 overexpression in parental cells up-regulates the expression of β-catenin in the nucleus. (D) Enhanced TOP/FOP activity was observed in the TOP/FOP luciferase reporter assay after WNT3 overexpression in HN6. (E) Overexpression of WNT3 increased the IC\u003csub\u003e50\u003c/sub\u003e values in parental cells. (F) The CCK-8 assay demonstrated that WNT3 overexpression promoted the proliferation of parental cells under oxaliplatin treatment. (G) Colony formation assay results indicated that WNT3 overexpression enhanced the colony formation ability of parental cells under oxaliplatin treatment. (H) Flow cytometry analysis detected that WNT3 overexpression inhibits apoptosis of parental cells under oxaliplatin treatment. (I) Wound healing experiments revealed that overexpression of WNT3 promoted the wound healing ability of parental cells under oxaliplatin treatment (100×). (J) Transwell chamber assays showed that overexpression of WNT3 promoted migration and invasion of parental cells under oxaliplatin treatment (100×). Data shown as mean ± SD (n = 3). Statistical significance was denoted as follows: *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001; and ****, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/ba5276c06c45ec9f64996a75.jpeg"},{"id":80050431,"identity":"f8f8d0ac-7a09-46d9-ae13-63c003d38ad9","added_by":"auto","created_at":"2025-04-07 10:20:17","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1611679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe impact of WNT3 knockdown on chemoresistance in OSCC OXA-R cells.\u003c/strong\u003e (A-B) The efficacy of WNT3 knockdown was confirmed through RT-qPCR (A) and western blotting analysis (B) following siRNA interference. (C) CCK-8 assay revealed a decreased IC\u003csub\u003e50\u003c/sub\u003e of oxaliplatin in OXA-R cells after WNT3 knockdown. (D-F) Transfection with siWNT3 resulted in inhibited proliferation (D), reduced colony formation (E), and decreased anti-apoptotic ability (F) upon exposure to oxaliplatin in OXA-R cells. (G-H) Diminished wound-healing (G) and reduced migration and invasion (H) were observed in OXA-R cells following WNT3 knockdown under oxaliplatin exposure. Data shown as mean ± SD (n = 3). Statistical significance was denoted as follows: *,\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ****, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/b7d5919b0a49ae696f45d0d2.jpeg"},{"id":80048504,"identity":"cdbedaa8-f43c-4075-937c-cbb464663838","added_by":"auto","created_at":"2025-04-07 10:04:17","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1271699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe reversal of chemoresistance in OSCC OXA-R cells by the WNT/β-catenin inhibitor MSAB.\u003c/strong\u003e (A) The activity of OXA-R cells under varying concentrations of MSAB was assessed using the CCK-8 assay. (B) Western blotting analysis demonstrated the expression levels of β-catenin in OXA-R cells following treatment with different concentrations of MSAB (0, 0.5, and 1 μg/mL). (C) The expression of apoptotic markers was evaluated via western blotting analysis under different concentrations of MSAB (0, 0.5, and 1 μg/mL for 48 hours). (D) A reduction in the IC\u003csub\u003e50\u003c/sub\u003e of oxaliplatin in OXA-R cells was observed upon combination with 0.2 μg/mL MSAB. (E-F) Proliferation curves (E) and apoptosis levels (F) of OXA-R cells were compared among the control group (Control), OXA (10 μM), MSAB (0.2 μg/mL), and the combination of OXA (10 μM) with MSAB (0.2 μg/mL). (G) The Combination Index (CI) of OXA-R cells treated with MSAB and OXA was found to be less than 1 in the Chou-Talalay assay, indicating a synergistic effect. Data shown as mean ± SD (n = 3). Statistical significance was denoted as follows: *, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ****, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/8eacee29f7fb9310d084253c.jpeg"},{"id":80049494,"identity":"2ce888d0-e35c-4be4-9212-2cfe2e135751","added_by":"auto","created_at":"2025-04-07 10:12:17","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1464904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe mechanism by which WNT3 upregulates ABCG2 expression through enhancing the binding of the transcription factor LEF1 to the ABCG2 promoter.\u003c/strong\u003e (A) Fluorescence microscopy revealed an acceleration in CFSE efflux following WNT3 overexpression within the same time frame (100×). (B) Flow cytometry data demonstrated that WNT3 overexpression increased CFSE efflux from parental cells. (C) HPLC analysis indicated a reduction in the relative concentration of oxaliplatin in the WNT3 overexpression group after 48 hours of oxaliplatin treatment. (D-E) Fluorescence microscopy (D) exhibited a deceleration in CFSE efflux after WNT3 knockdown in OXA-R cells (100×), which was further confirmed by flow cytometry (E) showing decreased CFSE efflux upon WNT3 knockdown. (F) HPLC analysis revealed an increase in the relative concentration of oxaliplatin in the WNT3 knockdown group after 48 hours of oxaliplatin treatment. (G-H) Overexpression of WNT3 in parental cells elevated the mRNA (G) and protein (H) expression of ABCG2. (I) Knockdown of WNT3 in OXA-R cells reduced the mRNA expression of ABCG2. (J) ChIP-qPCR experiments demonstrated that WNT3 overexpression in parental cells significantly enhanced the binding ability of the transcription factor LEF1 to the ABCG2 promoter. Data shown as mean ± SD (n = 3). Statistical significance was indicated as follows: *, \u003cem\u003eP\u003c/em\u003e \u0026lt;0.05; **, \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01; ***, \u003cem\u003eP \u003c/em\u003e\u0026lt;0.001; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/5fddf769148c1cb8a30e62f6.jpeg"},{"id":80048506,"identity":"f08476e4-0060-4a4e-bbef-22d20b710cf2","added_by":"auto","created_at":"2025-04-07 10:04:17","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":610843,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe mechanism of oxaliplatin resistance in OSCC.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/8878a101ec72e73b5e3d56f0.jpeg"},{"id":80048511,"identity":"0eb01d44-dcf8-4bf2-b620-b0d941d2b66a","added_by":"auto","created_at":"2025-04-07 10:04:17","extension":"jpg","order_by":25,"title":"Figure 25","display":"","copyAsset":false,"role":"figure","size":7206618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAberrant activation of the WNT/β-catenin signaling pathway in OSCC OXA-R cells.\u003c/strong\u003e (A) Transcriptome sequencing of CAL27 and CAL27/OXA cells revealed the up-regulated expression of genes related to the WNT canonical pathway. (B) CiiiDER software was used to predict binding sites of transcription factors near the promoters of ABCG2. (C) Immunofluorescence staining was utilized to observe the nuclear translocation of β-catenin in parental and OXA-R cells (600×). DAPI staining is represented in blue, while β-catenin staining is shown in green. (D) The expression of β-catenin in the cytoplasm and nucleus of parental and OXA-R cells was analyzed via western blotting. (E) In the TOP/FOP flash luciferase reporter assay, the TOP/FOP activity was enhanced in the drug-resistant cells.\u003cu\u003e \u003c/u\u003e(F)RT-qPCR was employed to detect the expression of molecules related to the WNT canonical signaling pathway in OSCC parental and OXA-R cells. (G) The expression of WNT3 in parental and OXA-R cells was analyzed via western blotting. Data shown as mean ± SD (n = 3). Statistical significance was denoted as follows: *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/4240582a7a7d033341b99925.jpg"},{"id":80048522,"identity":"96061a8c-aa43-47d6-9b1c-931ae22ba727","added_by":"auto","created_at":"2025-04-07 10:04:18","extension":"jpg","order_by":26,"title":"Figure 26","display":"","copyAsset":false,"role":"figure","size":6039049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of WNT3 overexpression on enhancing oxaliplatin resistance in OSCC parental cells.\u003c/strong\u003e (A) Western blotting confirmed the efficacy of WNT3 overexpression in parental cells. (B) Immunofluorescence staining illustrated that WNT3 overexpression increased the expression of β-catenin in the nucleus (600×). DAPI staining is represented in blue, while β-catenin staining is shown in green. (C) Western blotting revealed that WNT3 overexpression in parental cells up-regulates the expression of β-catenin in the nucleus. (D) Enhanced TOP/FOP activity was observed in the TOP/FOP luciferase reporter assay after WNT3 overexpression in HN6. (E) Overexpression of WNT3 increased the IC\u003csub\u003e50\u003c/sub\u003e values in parental cells. (F) The CCK-8 assay demonstrated that WNT3 overexpression promoted the proliferation of parental cells under oxaliplatin treatment. (G) Colony formation assay results indicated that WNT3 overexpression enhanced the colony formation ability of parental cells under oxaliplatin treatment. (H) Flow cytometry analysis detected that WNT3 overexpression inhibits apoptosis of parental cells under oxaliplatin treatment. (I) Wound healing experiments revealed that overexpression of WNT3 promoted the wound healing ability of parental cells under oxaliplatin treatment (100×). (J) Transwell chamber assays showed that overexpression of WNT3 promoted migration and invasion of parental cells under oxaliplatin treatment (100×). Data shown as mean ± SD (n = 3). Statistical significance was denoted as follows: *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001; and ****, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/963d8da9c015abba69641b8f.jpg"},{"id":84242534,"identity":"a135f0a5-fb35-4a13-8381-2619b2ac90e8","added_by":"auto","created_at":"2025-06-09 16:09:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":38097500,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/8b32bcf8-826d-43b9-91f1-1c9650bdd847.pdf"},{"id":80049496,"identity":"f1da636f-7a45-4dd0-9c70-5166ee4d7c18","added_by":"auto","created_at":"2025-04-07 10:12:17","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":16384414,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiguresandtables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/c59b3c0b618c12e5e6706023.docx"},{"id":80048529,"identity":"54e73b0b-3299-4af5-88a6-39992f5c9678","added_by":"auto","created_at":"2025-04-07 10:04:18","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":59505716,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfilesfororiginalimagesofWB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5267942/v1/baece6f5875fd4d98ad93666.pdf"}],"financialInterests":"","formattedTitle":"WNT3 promotes chemoresistance to oxaliplatin in oral squamous cell carcinoma via regulating ABCG2 expression","fulltext":[{"header":"Background","content":"\u003cp\u003eOral squamous cell carcinoma (OSCC) is the most common malignant tumor in oral and maxillofacial region[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Its treatment typically involves a multimodal approach including surgery, radiotherapy, and/or chemotherapy[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Oxaliplatin (OXA) is a frontline therapeutic agent in OSCC management, exerting its anticancer effects by inhibiting DNA synthesis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite the initial efficacy of oxaliplatin, the emergence of chemoresistance poses a significant challenge, leading to therapeutic ineffectiveness. Current research on chemoresistance mechanisms focuses on several factors, such as drug inactivation[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], DNA damage repair[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], reduced drug accumulation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and enhanced drug efflux[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among these, increased drug efflux has garnered considerable attention in recent research endeavors.\u003c/p\u003e \u003cp\u003eATP-binding cassette (ABC) transporters represent a class of ATP-dependent efflux pumps located in the plasma membrane of both prokaryotic and eukaryotic cells. Structurally, these transporters consist of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The TMDs and NBDs collectively through\u003c/p\u003e \u003cp\u003etransmembrane channels facilitating the transport of diverse substrates, including anticancer drugs[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. During the efflux process, substrates enter the channel from the membrane entrance site. Subsequently, ATP binding induces dimerization of the NBDs, resulting in a conformational change in the ABC transporters, thereby expelling the substrate from the TMDs to the extracellular environment. Among the recognized transporters implicated in cancer chemoresistance are ABCB1 (multidrug resistance protein 1, MDR1), ABCC1 (multidrug resistance-associated protein 1, MRP1), and ABCG2 (breast cancer resistance protein, BCRP)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eABCG2, a member of the multidrug resistance proteins (MDRPs), has been implicated in drug resistance in head and neck squamous cell carcinoma (HNSCC) and OSCC. Lu X et al.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] reported a 1.6-fold increase in ABCC1 and a 2.1-fold increase in ABCG2 expression in OSCC specimens treated with 5-fluorouracil (5-FU) and cisplatin compared to untreated specimens. Additionally, the Hedgehog signaling pathway has been implicated in the regulation of ABC transporters. Tonigold M et al.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] demonstrated upregulation of ABCC2 and ABCG2 in cisplatin-resistant HNSCC cells, potentially mediated by P53 mutation. Furthermore, ABCG2 expression is heightened in multidrug-resistant HNSCC cells exhibiting cancer stem cell (CSC) properties. Knockdown of ABCG2 inhibits spheroid formation and drug efflux capacity, suggesting ABCG2 may serve as a potential prognostic marker for HNSCC[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These findings underscore the potential association between abnormal ABCG2 expression and OSCC chemoresistance, although the precise molecular mechanisms driving ABCG2 upregulation remain unclear.\u003c/p\u003e \u003cp\u003eThrough analysis and validation of RNA sequencing data from established oxaliplatin-resistant OSCC cells (OXA-R), we have identified the transporter ABCG2 as a potentially pivotal factor in OSCC chemoresistance. The upregulation of ABCG2 in resistant cells facilitates the efflux of oxaliplatin, consequently reducing intracellular drug concentrations. Subsequently, we elucidated the molecular mechanism underlying ABCG2 upregulation. Specifically, we found that WNT3 activates the WNT/β-catenin signaling pathway in OXA-R cells, thereby enhancing the binding of LEF1 to the ABCG2 promoter and ultimately elevating ABCG2 expression.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe human OSCC cell lines (CAL27, HN30, and HN6) and the human embryonic kidney cell line (293T) were cultured in high-glucose DMEM\u003c/p\u003e \u003cp\u003e(SH30243.01, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (10270-106, Gibco, USA) in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Oxaliplatin-resistant (OXA-R) cell lines, including CAL27/OXA, HN30/OXA, and HN6/OXA, were established from the respective parental cells (CAL27, HN30, and HN6) through continuous exposure to incrementally increasing concentrations of oxaliplatin during cell culture. The final oxaliplatin concentration used for resistance induction was 10 \u0026micro;M.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eConstruction of OSCC cells with stable Oxaliplatin resistance\u003c/h3\u003e\n\u003cp\u003eParental CAL27 and HN30 cell lines were treated with increasing concentrations of oxaliplatin (MedChemExpress (MCE), China). Over an approximate 12-month period, the oxaliplatin -resistant cells (OXA-R) were successfully stabilized, resulting in the CAL27/OXA, HN30/OXA and HN6/OXA strains, which were then consistently nurtured in a medium supplemented with 10 \u0026micro;M oxaliplatin.\u003c/p\u003e\n\u003ch3\u003eCCK-8 assay\u003c/h3\u003e\n\u003cp\u003eThe cells were seeded onto 96-well plates. After adhesion, drugs were added and incubated for 48 hours. Subsequently, Cell Counting Kit-8 (CCK-8) (CK04, Dojindo, Japan) was added to the cells and incubated for one hour. Absorbance values at 450 nm were measured using the SpectroMax iD3 microplate reader (Molecular Devices, USA). Half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values were calculated using GraphPad Prism software (version 8.0). The combination index (CI) values were calculated using the Chou-Talalay method with CompuSyn software (version 1.0) and interpreted as follows: CI\u0026thinsp;\u0026gt;\u0026thinsp;1 indicates antagonism, CI\u0026thinsp;=\u0026thinsp;1represents an additive effect, and CI\u0026thinsp;\u0026lt;\u0026thinsp;1 signifies synergism[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eColony formation assay\u003c/h3\u003e\n\u003cp\u003eFollowing inoculation, the cells were cultured in 6-well plates for a duration of 2 weeks. Subsequently, the colonies were fixed using 4% paraformaldehyde (PFA) and stained using crystal violet (C0121, Beyotime, China). The areas of the colonies were quantified using ImageJ software (version 1.8.0).\u003c/p\u003e\n\u003ch3\u003eCell apoptosis assay\u003c/h3\u003e\n\u003cp\u003eCells were seeded in 6-well plates and allowed to adhere overnight. After incubation, the cells were gently washed with phosphate-buffered saline (PBS) and resuspended in 1\u0026times; binding buffer. Subsequently, the cells were stained with YF\u0026reg; 488 Annexin V and propidium iodide (PI) (Y6002L, UElandy, China) for 15 minutes at room temperature in the dark, according to the manufacturer\u0026rsquo;s instructions. After staining, the samples were immediately analyzed using an Accuri\u0026trade; C6 Flow Cytometer (BD Biosciences, USA). Data analysis was performed using FlowJo software (version 10.8.1, Tree Star, USA) to quantify the percentage of apoptotic cells. Each experiment was conducted in triplicate to ensure statistical reliability.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWound-healing assay\u003c/h2\u003e \u003cp\u003eThe cells were seeded in 12-well plates and allowed to grow until reaching more than 90% confluence. Subsequently, a wound was created using a sterile 200 \u0026micro;L pipette tip, and the cells were further cultured in serum-free medium. The progression of wound healing was monitored and recorded using a microscope. The healing percentage was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTranswell migration and invasion assays\u003c/h3\u003e\n\u003cp\u003eMigration and invasion assays were conducted using 24-well Transwell chambers equipped with 8-\u0026micro;m pore size polycarbonate membranes, either pre-coated with or without matrix (353097/354480, Corning Falcon, USA). The lower chamber was filled with medium containing 10% FBS, while serum-free cells were seeded into the upper chamber. Following incubation for 24 to 48 hours, cells were fixed and stained with crystal violet.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescent staining and confocal laser scanning microscopy (CLSM)\u003c/h3\u003e\n\u003cp\u003eCells were cultured on coverslips in 12-well plates until reaching 50% confluence. The cells were fixed with 4% paraformaldehyde solution (Biosharp, China) and permeabilized with 0.3% Triton X-100 (1139, Biofroxx, Germany). Subsequently, cells were blocked with 3% bovine serum albumin (BSA) (4240GR500, Biofroxx, Germany) at room temperature for 30 minutes and then incubated overnight at 4\u0026deg;C with the primary antibody targeting β-catenin (610154, BD Biosciences, USA). The following day, cells were incubated with a goat anti-mouse secondary antibody conjugated to Alexa Fluor\u0026reg; 488-conjugated goat anti-mouse secondary antibody (4408S, CST, USA). In a separate experiment, fixed and permeabilized cells were stained with YF\u0026reg;594-Phalloidin (YP0052S, UElandy, China).Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (C1002, Beyotime, China). Finally, cells were cleaned, mounted, and visualized using confocal laser scanning microscopy (CLSM) (Olympus, Japan).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing (RNA-seq) analysis and bioinformatics analysis\u003c/h2\u003e \u003cp\u003eRNA sequencing (RNA-seq) analysis was conducted by Novogene Biotechnology (Beijing, China), with sequencing reads aligned to the GRCh38/hg38 reference genome. The dataset has been deposited in the Gene Expression Omnibus repository (GEO) under accession number GSE248792. Differential expression analysis of genes (DEGs) between parental and resistant strains was performed using the R software package (version 4.2.1), along with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The criteria for differential expression were set at adjusted P-value (P\u003csub\u003eadj\u003c/sub\u003e)\u0026thinsp;\u0026le;\u0026thinsp;0.05 and |log\u003csub\u003e2\u003c/sub\u003e(Fold Change)|\u0026gt;1. DEGs were sorted based on ascending P\u003csub\u003eadj\u003c/sub\u003e values and descending |log\u003csub\u003e2\u003c/sub\u003e(Fold Change)| values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA isolation and real-time quantitative PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells using NucleoZol (740404.200, Macherey Nagel, Germany) and reverse-transcribed into cDNAs using the PrimeScript\u003csup\u003e\u0026trade;\u003c/sup\u003e RT Reagent Kit (RR047A, Takara Bio, Japan). Real-time PCR assays were conducted using the SYBR Green Kit (1725124, Bio-Rad, USA) and amplification was detected using a real-time PCR system (Applied Biosystems, USA). Relative mRNA expression levels were normalized using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method with GAPDH serving as an internal reference. Primer sequences are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and western blotting (WB)\u003c/h2\u003e \u003cp\u003eTotal protein was extracted using RIPA buffer (P0013B, Beyotime, China), and the protein concentration was determined using the BCA protein assay kit (P1102, Beyotime, China). Subsequently, the protein samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (ISEQ00010, Amersham, USA). The membranes were then blocked with 3% BSA and probed with primary antibodies. Following primary antibody incubation, the membranes were incubated with secondary antibodies. Protein bands were visualized using the ECL chemiluminescence kit (P0018M, Beyotime, China) and imaged with the ChemiDoc\u003csup\u003e\u0026trade;\u003c/sup\u003e XRS\u0026thinsp;+\u0026thinsp;Imaging System (Bio-Rad, USA). The antibodies used in the study are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSmall interfering RNA (siRNA) transfection\u003c/h2\u003e \u003cp\u003eResistant cells were seeded in 6-well plates and transiently transfected with siRNAs using the Lipofectamine RNAiMAX Transfection Reagent (13778150, Invitrogen, USA) following the manufacturer's instructions. The siRNA sequences targeting ABCG2 (siABCG2), WNT3 (siWNT3), and the negative control (siNC) are detailed\u003c/p\u003e \u003cp\u003ein Table S3. In accordance with Dharmacon\u0026trade; siRNA solutions (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://horizondiscovery.com/en/gene-modulation/knockdown/sirna\u003c/span\u003e\u003cspan address=\"https://horizondiscovery.com/en/gene-modulation/knockdown/sirna\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), equal amounts of two specific siRNA sequences targeting each gene were combined to prepare the transfection mixture for the knockdown group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTransient cell transfection\u003c/h2\u003e \u003cp\u003eThe WNT3 overexpression plasmid pcDNA3.1-WNT3-3\u0026times;Flag-C (WNT3) and its vector plasmid pcDNA3.1 (Vector) were obtained from Dahong Biotechnology (Guangzhou, China). OSCC cells were seeded in 6-well plates and transiently transfected with plasmids using Lipofectamine 2000 reagent (11668019, Invitrogen, USA), followed by replacement with fresh culture medium. Overexpression efficiency was confirmed by post-transfection protein analysis, and the successfully transfected cells were subsequently utilized for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase assay\u003c/h2\u003e \u003cp\u003eTo assess Wnt/β-catenin signaling activity, cells were co-transfected with either WNT3 or control Vector plasmids, along with the TopFlash/FOPFlash plasmid and the Renilla luciferase internal control plasmid (Addgene, USA). Following 48 hours of transfection, cells were lysed and collected. Luciferase activity was measured using the SpectraMax iD3 microplate reader according to the instructions provided with the Dual Luciferase Assay System (11402ES60, Yeason, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCFDA-SE fluorescent staining\u003c/h2\u003e \u003cp\u003eDrug efflux capacity was assessed using carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) (C6034S, UElandy, China)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For the fluorescence efflux experiment, cells were seeded in 6-well plates, and after adhesion, the medium was aspirated. Cells were then stained with CFDA-SE for 30 minutes and subsequently incubated with complete medium to terminate staining. The attenuation of fluorescence was observed using an inverted fluorescence microscope.\u003c/p\u003e \u003cp\u003eIn addition, the amount of fluorescence extrusion was quantified by flow cytometry. Prior to CFDA-SE staining, cells were harvested, and after staining termination, the medium was aspirated. Cells were resuspended in buffer solution and kept on ice. Fluorescence intensity was measured using a flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHigh-performance liquid chromatography (HPLC)\u003c/h2\u003e \u003cp\u003eSample processing involved collecting cells, counting, removing the medium, and subsequently lysing the cell precipitate. A standard solution of oxaliplatin was prepared in gradient concentrations. Chromatographic analysis was conducted using a TC-C18 column (4.6 mm \u0026times; 250 mm, 5 \u0026micro;m) (Agilent, USA) with a mobile phase consisting of methanol and distilled water (methanol:water\u0026thinsp;=\u0026thinsp;80:20) (vol/vol) at a flow rate of 1 mL/min. The column temperature was maintained at 25℃, and oxaliplatin was detected at a wavelength of 204 nm. Standard and sample solutions were filtered and injected into the Agilent 1260 Infinity II Prime LC system (Agilent, USA) at 10 \u0026micro;L each.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP) assay\u003c/h2\u003e \u003cp\u003eThe DNA fragments bound to the transcription factor were captured using an anti-LEF1 antibody (76010S, CST, USA) through immunoprecipitation. Protein G Magnetic Beads (HY-K0204, MCE, China) were utilized for the immunoprecipitation process. Subsequently, proteases K and RNase A (RT405-02, TIANGEN, China) were employed to degrade proteins and RNA, facilitating the purification and precipitation of DNA fragments. The specificity of the DNA fragments bound to the target gene ABCG2 was assessed using quantitative PCR (qPCR) with specific primers. The immunoprecipitation efficiency of LEF1 was compared to that of the negative control IgG (A00002, Zen-Bio, China) to determine the fold enrichment. The primers for ChIP-qPCR were designed using the Cistrome Data Browser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cistrome.org/db/#/\u003c/span\u003e\u003cspan address=\"http://cistrome.org/db/#/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the UCSC Genome Browser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ucsc.edu/cgi-bin/hgTracks\u003c/span\u003e\u003cspan address=\"https://genome.ucsc.edu/cgi-bin/hgTracks\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), as illustrated in Fig.S6. The ChIP primer sequence targeting ABCG2 is: forward: 5'-CACCCGGACCTTCCAAACAA-3', reverse: 5'-GAGATTGAGAGACGCGGCAA-3'.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted in triplicate, and the results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses and graphical representations were performed using GraphPad Prism 8.0 (GraphPad Software, USA). Student\u0026rsquo;s t-tests were employed to determine statistical significance. A P-value less than 0.05 was considered statistically significant. The following significance marks were used: ns, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.05; *, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eEstablishment of oxaliplatin-resistant (OXA-R) cell lines in OSCC\u003c/h2\u003e\n \u003cp\u003eTo investigate the mechanisms underlying oxaliplatin resistance in OSCC, we successfully established the oxaliplatin-resistant (OXA-R) cell lines through continuous induction with oxaliplatin. Compared to their parental cell lines, the OXA-R cells exhibited significantly higher IC\u003csub\u003e50\u003c/sub\u003e values (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA. To investigate cellular morphological changes in greater detail, we performed phalloidin staining.We observed that CAL27/OXA cells displayed looser intercellular connections compared to their parental CAL27 cells, while HN30/OXA cells exhibited spindle-like alterations relative to HN30 cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Since changes in cellular morphology often indicate shifts in biological behaviors [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], the attenuated connections in cell-cell and spindle-shaped morphology alterations may enhance cellular motility. Consequently, we further investigated the phenotypic characteristics of resistant cells. Our findings revealed that upon oxaliplatin exposure, the colony-forming ability of resistant cells was augmented compared to that of parental cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC), while cellular apoptosis was diminished (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, the migratory and invasive potential of resistant cells was significantly enhanced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). These observations suggest that the acquisition of oxaliplatin resistance in OSCC cells leads to significant morphological transformations and is associated with enhanced malignant phenotypes, suggesting a more aggressive cellular behavior.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eEnhanced drug efflux capacity mediated by ABCG2 upregulation in OSCC OXA-R cell lines\u003c/h2\u003e\n \u003cp\u003eGiven that oxaliplatin induces DNA damage, we initially analyzed genes associated with DNA damage repair based on the sequencing results (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Interestingly, we observed no significant changes in genes related to DNA damage repair between parental and drug-resistant cells (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eB). However, further analysis revealed upregulation of multiple ABC transporter family genes, which are associated with efflux function, in the resistant cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Pathway enrichment analysis also indicated activation of membrane transporter-related pathways in drug-resistant cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). These results suggest that alterations in drug efflux mediated by ABC transporters may contribute to oxaliplatin resistance.\u003c/p\u003e\n \u003cp\u003eTo assess the efflux ability of cells, we utilized CFDA-SE, a fluorescent substrate of ABC transporters. CFDA-SE is converted into carboxyfluorescein succinimidyl ester (CFSE) inside living cells, emitting green fluorescence[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, changes in intracellular CFSE fluorescence intensity can serves as a reliable indicator of cellular efflux function. As depicted in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE-F and Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eC, the fluorescence efflux in OXA-R cells was accelerated compared to that in parental cells, leading to a more rapid attenuation of fluorescence intensity. Flow cytometry further confirmed that resistant cells expelled more fluorescence compared to parental cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG and Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eD). Additionally, we indirectly analyzed oxaliplatin efflux ability by measuring the relative intracellular oxaliplatin concentrations using HPLC. The peak location of oxaliplatin was determined based on the peak time and area of oxaliplatin standard with varying concentrations (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH and Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eE), and the relative intracellular oxaliplatin concentration was compared. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI and Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eF, the relative oxaliplatin concentrations in OXA-R cells was significantly lower than that in parental cells, providing further evidence of enhanced efflux function in OXA-R cells.\u003c/p\u003e\n \u003cp\u003eTo identify the key protein responsible for enhancing efflux function, we initially confirmed the upregulated ABC genes identified in the sequencing through RT-qPCR (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eG). Subsequently, ABCG2, ABCB1, and ABCC9, which exhibited significant expression differences, were selected for protein-level verification by western blotting. Notably, we observed no discernible protein bands corresponding to ABCB1 and ABCC9 in either parental and resistant OSCC cells (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eH). However, the expression of ABCG2 protein was notably elevated in the OXA-R cell lines (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eJ-K and Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eI). Furthermore, upon treatment with oxaliplatin at various concentrations for 48 hours, we observed an increase in ABCG2 expression in parental cells as well (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eL). These findings suggest that the enhanced efflux capacity in OXA-R cells may be attributed to the upregulation of ABCG2.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eABCG2 knockdown reduced oxaliplatin efflux and reversed chemoresistance in OSCC OXA-R cells\u003c/h2\u003e\n \u003cp\u003eTo further substantiate that the pivotal role of ABCG2 in medidating enhanced drug efflux in OXA-R cells, we employed siRNA-mediated knockdown of ABCG2 and assessed the knockdown efficiency at both transcriptional and translational levels in OXA-R cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-B and Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eA-B). We found that ABCG2 knockdown decelerated the CFSE efflux in OXA-R cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC and Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eC), which was confirmed by flow cytometry showing lower fluorescence output in the knockdown group compared to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD and Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eD). In addition, HPLC analysis demonstrated a substantial increase in intracellular oxaliplatin concentration following ABCG2 knockdown (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE and Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eE). These outcomes confirmed the crucial role of ABCG2 in modulating the efflux function of OXA-R cells.\u003c/p\u003e\n \u003cp\u003eSubsequently, we delved into whether ABCG2 influenced chemoresistance phenotypes. It was observed that ABCG2 knockdown resulted in decreased IC\u003csub\u003e50\u003c/sub\u003e values of OXA-R cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF and Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eF), indicating restored drug sensitivity. Moreover, upon oxaliplatin stimulation, the knockdown group exhibited inhibited proliferation, colony formation, and anti-apoptotic abilities in comparison to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG-I and Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eG-H). We also explored the impact of ABCG2 on cell mobility and discovered that ABCG2 knockdown attenuated migration and invasion under oxaliplatin exposure (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eJ and Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eI). Collectively, these findings indicated that ABCG2 knockdown inhibited efflux, reduced intracellular oxaliplatin concentration, and increased sensitivity to oxaliplatin.\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eAberrant activation of the WNT canonical signaling pathway in OSCC OXA-R cells\u003c/h2\u003e\n \u003cp\u003eTo elucidate the factors contributing to the up-regulation of ABCG2 expression, we conducted further analysis of the RNA sequencing data. Our investigation revealed the upregulation of key genes associated with the WNT canonical pathway in CAL27/OXA, including WNT ligands and downstream target genes such as Vimentin (VIM) and N-cadherin (CDH2), indicating the aberrant activation of the WNT canonical signaling pathway (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eThe WNT signaling pathways comprise both canonical and non-canonical pathways. Among these, the canonical pathway, also known as the \u0026beta;-catenin pathway, primarily regulates the transcription of target genes through the specific binding of the transcription factor LEF/TCF to the promoters of target genes[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. To examine whether the up-regulation of ABCG2 expression is linked to the abnormal activation of the WNT canonical signaling pathway, we employed CiiiDER software[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] to predict the binding sites of transcription factors near the ABCG2 promoter. Notably, our analysis revealed there are binding sites near the promoter of ABCG2 to the transcription factor LEF1 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting potential transcriptional regulation of ABCG2 by the WNT/\u0026beta;-catenin pathway. Prior studies have established a close relationship between the WNT canonical pathway and chemoresistance, highlighting it as a promising therapeutic target. However, the specific regulatory mechanism underlying this association remains unclear[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, we needed to investigate the role of the WNT canonical signaling pathway in oxaliplatin resistance in OSCC. We first compared the basal \u0026beta;-catenin levels in several parental cell lines using western blotting and found that the basal \u0026beta;-catenin levels did not show significant differences among these three cell lines (Fig. S3A). Then we verified the activation of the WNT canonical signaling pathway in OXA-R cells. Immunofluorescence analysis demonstrated an increased nuclear translocation of \u0026beta;-catenin in resistant cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC), and WB analysis revealed elevated nuclear \u0026beta;-catenin accumulation in OXA-R cells compared to parental cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). In the TOP/FOP flash luciferase reporter assay, the TOP/FOP activity was enhanced in the drug-resistant cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). This suggests an increase in transcriptional activity in the drug-resistant cells. Furthermore, RT-qPCR analysis confirmed the upregulation of the target genes AXIN2 and CD44 in OXA-R cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF and Fig. S3B).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003eWNT3 regulated oxaliplatin resistance in OSCC\u003c/h2\u003e\n \u003cp\u003eWNT ligands play the critical role in activating the WNT canonical pathway. To identify the key WNT ligand involved in the activation of the pathway, we screened for upregulated ligands in the sequencing results and those known to activate the canonical pathway in previous research[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our RT-qPCR analysis revealed a significant difference in WNT3 expression between parental cells and OXA-R cells (Fig. S3C), with the protein level of WNT3 also found to be upregulated in the OXA-R cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG and Fig. S3D).\u003c/p\u003e\n \u003cp\u003eWe then evaluated whether WNT3 overexpression could activate the WNT canonical signaling pathway in OSCC. The effect of WNT3 overexpression was confirmed by WB analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Following WNT3 overexpression in OSCC parental cells, immunofluorescence analysis demonstrated an increased translocation of \u0026beta;-catenin into the nucleus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), and WB experiment revealed upregulated levels of \u0026beta;-catenin in the nucleus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Moreover, TOP/FOP flash luciferase reporter assays showed that overexpression of WNT3 significantly increased the TOP/FOP activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD and Fig. S3E), indicating enhanced transcriptional activity. Subsequently, we knocked down WNT3 in the drug-resistant cells and observed a reduction in TOP/FOP activity (Fig. S3F). These results collectively demonstrated the activating effect of WNT3 on the WNT canonical pathway.\u003c/p\u003e\n \u003cp\u003eHaving established the activation effect of WNT3 on the WNT canonical pathway, we then investigated its impact on OSCC oxaliplatin resistance by modulating WNT3 expression. We found that WNT3 overexpression in parental cells led to elevated IC\u003csub\u003e50\u003c/sub\u003e values (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE), indicating increased resistance to oxaliplatin. Additionally, under oxaliplatin treatment, the overexpression group exhibited enhanced proliferation, colony formation, and anti-apoptosis abilities compared to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF-H). Furthermore, WNT3 overexpression accelerated migration and invasion in cells exposed to oxaliplatin, highlighting its role in promoting cell motility under drug exposure (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI-J).\u003c/p\u003e\n \u003cp\u003eNext, we employed siRNAs to knock down WNT3 expression to investigate its impact on OXA-R cells. RT-qPCR and western blot analyses confirmed a significant decrease in WNT3 expression following siRNA transfection (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA-B and Fig. S3G-H). Subsequently, we observed that WNT3 knockdown led to decreased IC\u003csub\u003e50\u003c/sub\u003e values in OXA-R cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC and Fig. S3I), suggesting restored oxaliplatin sensitivity. In addition, WNT3 knockdown inhibited the proliferation, colony formation, and anti-apoptotic capabilities of resistant cells upon exposure to oxaliplatin (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD-F and Fig. S3J-K). We also observed reduced migration and invasion of resistant cells under oxaliplatin exposure following WNT3 knockdown (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG-H and Fig. S3L). These results collectively indicated that WNT3 activated the WNT canonical signaling pathway and regulated oxaliplatin resistance in OSCC.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003eWNT/\u0026beta;-catenin inhibitor reversed chemoresistance in OSCC OXA-R cells\u003c/h2\u003e\n \u003cp\u003eTo further investigate the therapeutic potential of inhibiting WNT/\u0026beta;-catenin signaling in oxaliplatin-resistant OSCC, we used methyl 3-benzoate (MSAB), a small molecule inhibitor targeting \u0026beta;-catenin. We first measured the cell viability under different concentrations of MSAB, and selected 0.2 \u0026micro;g/mL MSAB (80% cell viability) as the concentration for subsequent work (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA and Fig. S4A). Then we verified that MSAB decreased the \u0026beta;-catenin expression in OXA-R cells (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). In addition, under the stimulation of different concentrations of MSAB, the expression of apoptosis indicators increased in a concentration-dependent manner, suggesting that MSAB may exert inhibitory effects by activating the apoptotic pathway in OXA-R cells (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). Subsequent studies on reversing chemotherapy resistance with MSAB showed that it could decrease the IC\u003csub\u003e50\u003c/sub\u003e values in OXA-R cells (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). Furthermore, the combination of oxaliplatin and MSAB showed enhanced therapeutic effects compared to either treatment alone, with significant inhibition of cell proliferation and increased apoptosis (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE-F and Fig. S4B-C). To access whether MSAB and oxaliplatin had a synergistic effect, we calculated the combination index (CI) values. In OXA-R cells, the CI values of oxaliplatin combined with MSAB was below 1 (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eG), indicating a synergistic effect. To sum up, MASB inhibited WNT canonical signaling pathway and reversed the chemoresistance in OSCC OXA-R cells.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003eWNT3 upregulated ABCG2 expression by enhancing LEF1 binding to the ABCG2 promoter\u003c/h2\u003e\n \u003cp\u003eBased on the aforementioned results, the involvement of ABCG2 and the WNT canonical signaling pathway in oxaliplatin resistance prompted us to delve deeper into the relationship between ABCG2 and the WNT pathway. Initially, we analyzed the impact of WNT3 on the efflux function of OSCC. Overexpression of WNT3 in parental cells led to faster fluorescence efflux of CFSE, as visualized by fluorescence microscopy (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA) and quantitatively confirmed by flow cytometry showing increased fluorescence output (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB), and decreased intracellular oxaliplatin concentration measured by HPLC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC). Conversely, downregulation of WNT3 in OXA-R cells resulted in slower fluorescence efflux of CFSE, reduced fluorescence output, and increased intracellular oxaliplatin concentration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD-F and Fig. S5A-C), indicating that the WNT pathway influenced the efflux ability of OSCC.\u003c/p\u003e\n \u003cp\u003eSubsequently, we examined the effect of WNT3 on the expression of ABCG2. The results revealed that overexpression of WNT3 in parental cells upregulated ABCG2 expression (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eG-H), while knockdown of WNT3 in resistant cells decreased ABCG2 expression (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eI). Given that the WNT canonical pathway regulates target genes through \u0026beta;-catenin/TCF/LEF-mediated transcriptional activation[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e], we conducted chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) to elucidate the mechanism of ABCG2 upregulation by WNT3. The findings indicated that overexpression of WNT3 enhanced the binding ability of the transcription factor LEF1 to the ABCG2 promoter (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eJ and Fig. S6A-B).\u003c/p\u003e\n \u003cp\u003eTaken together, WNT3 upregulated ABCG2 expression by promoting the binding of LEF1 to the ABCG2 promoter, thereby enhancing oxaliplatin efflux.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOxaliplatin, a conventional chemotherapy agent utilized in the treatment of OSCC, has made some progress in retarding tumor progression and enhancing prognosis[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Nonetheless, the emergence of chemoresistance undermines its efficacy, necessitating a focused effort on addressing this issue. In this study, we explored mechanisms underlying oxaliplatin resistance in OSCC. Briefly, we established OXA-R OSCC cell lines in vitro, and observed an upregulation of ABCG2 transporter expression alongside aberrant activation of the WNT canonical signaling pathway in OXA-R cells. Mechanically, we discovered an augmented binding affinity of the WNT pathway transcription factor LEF1 to the ABCG2 promoter, resulting in the upregulation of ABCG2 expression and subsequent enhancement of drug efflux, thus facilitating chemoresistance. In this study, we have selected CAL27, HN30, and HN6, three widely used cell lines in OSCC research. CAL27, a highly aggressive cell line, commonly exhibits TP53 mutations[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. HN30 exhibits typical OSCC features, derived from a primary tumor and is suitable for studying the impact of the tumor microenvironment[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. HN6 cells are valuable for metastasis and drug resistance research, typically exhibiting TP53 and EGFR mutations[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These cell lines exhibit distinct genetic and epigenetic characteristics. Therefore, in our experimental design, we selected these cell lines to cover diverse biological behaviors and genetic information of OSCC, thereby comprehensively validating our research hypotheses.\u003c/p\u003e \u003cp\u003eAlthough tumor treatment has entered a new era marked by targeted therapy and immunotherapy[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], platinum-based drugs remain the cornerstone of first-line adjuvant therapy in clinical practice, owing to considerations of efficacy, cost-effectiveness, and toxicity[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Platinum resistance, primarily attributed to DNA damage repair mechanisms, has garnered significant attention[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Interestingly, the sequencing results from this study revealed no significant alterations in DNA damage repair-related genes. Although DNA damage repair encompasses multiple pathways, and defects in one pathway can be compensated by others, this redundancy poses significant challenges for cancer treatment. However, certain tumors still exhibit inadequate DNA repair capacity[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The variability in DNA damage response (DDR) among different tumor types provides a plausible explanation for the initial drug sensitivity. This finding suggests that in our study, oxaliplatin resistance mechanisms may rely more on alternative molecular pathways rather than traditional changes in the expression of DNA damage repair-related genes. Cells may have developed other adaptive mechanisms to cope with DNA damage.\u003c/p\u003e \u003cp\u003eThe elucidation of various mechanisms contributing to platinum resistance includes abnormalities in drug transport, mutations in target genes[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and effects arising from the tumor microenvironment[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Membrane transporters play a pivotal role in drug transport processes, with ATP-binding cassette (ABC) transporters, responsible for drug efflux, and solute carrier (SLC) transporters, facilitating drug uptake, being particularly noteworthy. Among these, ABC transporters have been extensively studied in the context of chemoresistance[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Notably, ABCG2, encoding the breast cancer resistance protein (BCRP), was initially identified in the human breast cancer MCF-7 cell line and has been implicated in multidrug resistance phenotypes in breast cancer[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. ABCG2 functions by mediating substrate efflux in a manner akin to other ABC transporters. Upon ATP-driven conformational changes, the protein expels drugs from cells, thereby reducing intracellular drug concentrations and fostering drug resistance[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIncreasing evidence suggests that elevated expression of ABCG2 is associated with chemoresistance in various cancers, including OSCC. Moreover, high ABCG2 expression has been observed in side population (SP) cells exhibiting stemness characteristics, alongside established stemness markers CD44 and CD133[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Sudo S et al.[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] demonstrated a dose-dependent upregulation of ABCG2 in OSCC HSC-2 and HSC-3 cells following direct stimulation with cisplatin for 24 hours, a process mediated by the activation of STAT1/3 expression. Similarly, Choi HS et al.[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] generated cisplatin-resistant OSCC cells and observed elevated expression of BCRP and MDR1. Functional assays utilizing Rhodamine 123 and BODIPY\u0026trade; FL prazosin confirmed enhanced efflux activity in these resistant cells. Although the precise mechanism underlying BCRP and MDR1 upregulation was not fully elucidated, the observed efflux alterations align with our findings. In our investigation, we observed a significant enhancement in efflux capacity in oxaliplatin-resistant (OXA-R) cells, confirmed by CFDA-SE fluorescence efflux assay and HPLC. Additionally, RT-qPCR and Western blot analyses validated the upregulation of ABCG2 expression in OXA-R cells. Subsequent ABCG2 knockdown experiments resulted in weakened efflux capacity and chemoresistance, accompanied by inhibited proliferation and mobility, thus underscoring the pivotal role of ABCG2 in mediating oxaliplatin resistance in OSCC.\u003c/p\u003e \u003cp\u003eDespite accumulating evidence implicating ABCG2 in tumor drug resistance, the precise mechanism underlying its upregulation remains elusive, particularly in the context of oxaliplatin resistance in OSCC. Zhang L et al.[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] utilized the PI3K inhibitor BAY-1082439 to target PI3K subunits P110α and P110β, resulting in reduced expression of ABCB1 and ABCG2 and reversal of chemoresistance in human epidermoid carcinoma and non-small cell lung cancer. However, inhibition of AKT failed to reverse ABCB1- or ABCG2-mediated multidrug resistance (MDR), suggesting an independent relationship between AKT and ABC transporters. In OXA-R colorectal cancer cells, upregulation of NF-κB phosphorylation was observed, leading to enhanced ABCG2 expression, attenuation of endoplasmic reticulum (ER) stress, and inhibition of apoptosis[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. While these studies identified associations between ABCG2 upregulation and specific signaling pathways, they did not delve into the detailed regulatory mechanisms. In our investigation, we observed abnormal activation of the WNT canonical signaling pathway in OXA-R cells, suggesting a potential regulatory role of this pathway in ABCG2 expression.\u003c/p\u003e \u003cp\u003eThe WNT/β-catenin pathway is initiated when WNT ligands bind to co-receptors Frizzled (FZD) and low-density lipoprotein receptor-related proteins 5 (LRP5) or 6 (LRP6). This binding recruits effector proteins Dishevelled (DVL) and AXIN to the WNT-binding receptor, leading to the inhibition of glycogen synthase kinase 3β (GSK3β) activity. Consequently, GSK3β inhibition prevents the phosphorylation and subsequent degradation of β-catenin, resulting in its cytoplasmic accumulation and translocation into the nucleus[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Within the nucleus, β-catenin binds to the transcription factor T-cell factor/lymphoid enhancer factor (TCF/LEF), thereby activating downstream target genes. The pivotal role of WNT ligands in initiating the WNT canonical signaling pathway underscores their significance as potential therapeutic targets for interrupting signal transduction at its origin[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. While our previous research has confirmed the role of WNT3 in 5-fluorouracil resistance in OSCC[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], research on WNT ligands' involvement in OSCC chemoresistance remains limited. In this study, we confirmed the promotion of oxaliplatin resistance in OSCC by WNT3. Initially, we validated the activation of the canonical signaling pathway by WNT3 in OSCC and assessed its impact on oxaliplatin resistance. Upregulation of WNT3 in parental cells enhanced resistance and increased proliferation and mobility under oxaliplatin treatment. Conversely, knockdown of WNT3 in oxaliplatin-resistant (OXA-R) cells reduced resistance and inhibited proliferation and mobility under oxaliplatin exposure.\u003c/p\u003e \u003cp\u003eTo facilitate the translation of laboratory findings into clinical practice, we employed MSAB, a small molecule inhibitor targeting β-catenin, to investigate its potential to reverse oxaliplatin resistance. MSAB selectively binds and degrades β-catenin, thereby inhibiting the transcription of downstream target genes. Our results demonstrated the inhibitory effect of MSAB in OXA-R cells, as evidenced by a decrease in total β-catenin levels with increasing MSAB concentration. Additionally, MSAB upregulated apoptosis indicators c-Caspase 3 and c-PARP, suggesting its potential to inhibit cell proliferation by activating the apoptotic pathway in OXA-R cells. Furthermore, MSAB decreased the IC\u003csub\u003e50\u003c/sub\u003e value of oxaliplatin in OXA-R cells, and when combined with oxaliplatin, it suppressed cell proliferation and anti-apoptotic ability. These findings indicated that MSAB could effectively reverse oxaliplatin resistance in OXA-R cells. calculation of the combined index (CI) value of MSAB and oxaliplatin revealed a synergistic effect, suggesting potential clinical utility.\u003c/p\u003e \u003cp\u003ePrevious studies have elucidated the role of the WNT pathway in promoting tumor drug resistance by upregulating the expression of ABC transporters. For instance, in multi-drug resistant hepatocellular carcinoma, scholars have observed upregulation of FZD7, ABCB1, ABCC1, and ABCC2 expression. Knockdown of the WNT pathway receptor FZD7 enhanced chemotherapy sensitivity and inhibited the expression of ABCB1, ABCC1, and ABCC2. Furthermore, treatment with the β-catenin inhibitor iCRT-3 downregulated the expression of ABCB1, ABCC1, and ABCC2, indicating an association between the WNT pathway and ABC transporters[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In chronic myeloid leukemia, the transcription factor TCF7 was recruited to ABCC2 promoter, leading to upregulation of ABCC2 expression and induction of imatinib resistance[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Similarly, in studies on the mechanism of nasopharyngeal carcinoma chemoresistance, TRIM11 was found to enhance WNT signal activity, increase β-catenin protein levels, and promote ABCC9 expression by binding to the ABCC9 promoter, thereby promoting drug resistance[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. While these studies have demonstrated that the WNT pathway can upregulate the expression of ABC transporters, and some have provided initial insights into why the canonical WNT signaling pathway can upregulate ABCG2, there has been limited exploration into how key WNT molecules act on ABCG2. In this study, we conducted a comprehensive investigation into the mechanism by which WNT3 regulated ABCG2 expression and promoted drug resistance. We first confirmed the regulatory role of WNT pathway in efflux function and observed that WNT3 promoted efflux. Subsequently, we identified that WNT3 upregulated ABCG2 expression. Finally, we explored the mechanism by which WNT3 upregulated ABCG2 and found that WNT3 overexpression enhanced the binding ability of the transcription factor LEF1 to the ABCG2 promoter.\u003c/p\u003e \u003cp\u003eIn summary, we established OXA-R cell lines of OSCC through in vitro induction and identified ABCG2 as a key determinant of oxaliplatin resistance by augmenting efflux capacity and reducing intracellular drug concentrations. Mechanistically, upregulation of WNT3 ligand expression activated the WNT canonical signaling pathway, thereby enhancing the binding of LEF1 to the ABCG2 promoter and subsequently increasing ABCG2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). This study contributes additional evidence towards addressing oxaliplatin resistance in OSCC and identifying potential therapeutic targets. Unfortunately, the key target of oxaliplatin chemoresistance identified in this study has not yet been validated in vivo or in clinical OSCC specimens. While our in vitro findings provide valuable insights, further validation in animal models and clinical samples is necessary to confirm the relevance and therapeutic potential of this target in a more physiologically relevant context. Future studies will focus on addressing this limitation to strengthen the translational impact of our findings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe activation of the WNT canonical signaling pathway resulted in the upregulation of ABCG2 transporter expression, consequently leading to heightened efflux of oxaliplatin and promoted oxaliplatin resistance in OSCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eAcknowlegements\u003c/h3\u003e\n\u003cp\u003eWe thank all the team members who participated in the study.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (No.82272868 and No.82173180).\u003c/p\u003e\n\u003ch3\u003eAuthor information\u003c/h3\u003e\n\u003cp\u003eAuthors and affiliations\u003c/p\u003e\n\u003cp\u003eSchool and Hospital of Stomatology, Fujian Medical University, Fujian Key Laboratory of Oral Diseases,\u0026nbsp;Biological Materials Engineering and Technology Center of Stomatology, 350004 Fuzhou, China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDepartment of Stomatology,\u0026nbsp;The Affiliated Wuxi People\u0026apos;s Hospital of Nanjing Medical University, 214023 Wuxi, China.\u003c/p\u003e\n\u003cp\u003eKairui Sun\u003c/p\u003e\n\u003cp\u003eSchool and Hospital of Stomatology, Fujian Medical University, Fujian Key Laboratory of Oral Diseases,\u0026nbsp;Fujian Provincial Biological Materials Engineering and Technology Center of Stomatology, 350004 Fuzhou, China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXuyang Zhang, Shuoqi Lin \u0026amp; Dali Zheng\u003c/p\u003e\n\u003cp\u003eDepartment of Preventive Dentistry, School and Hospital of Stomatology, Fujian Medical University, 350002 Fuzhou, China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRuihuan Gan \u0026amp; Chen Yu\u003c/p\u003e\n\u003cp\u003eSchool and Hospital of Stomatology, Fujian Medical University, Fujian Key Laboratory of Oral Diseases,\u0026nbsp;Fujian Provincial Biological Materials Engineering and Technology Center of Stomatology, Department of Preventive Dentistry, Hospital of Stomatology, Fujian Medical University, 350002 Fuzhou, China.\u003c/p\u003e\n\u003cp\u003eYouguang Lu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe final manuscript was read and approved by all writers. The work has been sufficiently contributed to by each author. Kairui Sun: Conceptualization, Data curation, Investigation, Methodology, Data Analisis, Writing-original draft; Xuyang Zhang: Conceptualization, Data curation, Investigation, Methodology, Data Analisis, Writing-review \u0026amp; editing; Ruihuan Gan: Data curation; Investigation; Methodology; Writing-review \u0026amp; editing; Shuoqi Lin: Data curation; Investigation; Methodology; Writing-review \u0026amp; editing; Yu Chen: Data curation; Investigation; Methodology; Writing-review \u0026amp; editing; Dali Zheng: Conceptualization; Project administration; Resources; Supervision; Writing-review \u0026amp; editing; Youguang Lu: Conceptualization; Project administration; Funding acquisition; Resources; Supervision; Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Dali Zheng: [email protected] or Youguang Lu: [email protected].\u003c/p\u003e\n\u003ch2\u003eEthics declarations\u003c/h2\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eSupplementary Information\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eSupplementary figures.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe RNA-seq dataset was uploaded to the GEO online repository with the number GSE248792.\u003c/p\u003e\n\u003cp\u003eAll the data presented in this study and information supporting the results can be found in the article or in the supplementary information files and are available upon reasonable request from the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJohnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR. 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Oncogenesis. 2020;9:45.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-bioscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbio","sideBox":"Learn more about [Cell \u0026 Bioscience](http://cellandbioscience.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cbio/default.aspx","title":"Cell \u0026 Bioscience","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Oral squamous cell carcinoma, Chemoresistance, ABC transporters, WNT canonical signaling pathway, Oxaliplatin","lastPublishedDoi":"10.21203/rs.3.rs-5267942/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5267942/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxaliplatin (OXA) is a frontline therapeutic agent used in the treatment of oral squamous cell carcinoma (OSCC). However, the development of chemoresistance has emerged as a significant challenge, compromising the effectiveness of treatment strategies. Therefore, there is a critical need to unravel the mechanisms underpinning drug resistance and to identify potential therapeutic targets. In recent years, there has been a growing interest in understanding the role of drug efflux in cancer chemoresistance mechanisms. Despite this, the contribution of ABCG2, a member of the ATP-binding cassette (ABC) transporter family, to oxaliplatin resistance in OSCC remains unclear. In the current study, we aimed to investigate the involvement of ABCG2 in oxaliplatin resistance in OSCC and to elucidate the molecular mechanisms through which the Wingless and Int-1 (WNT) canonical signaling pathway upregulates ABCG2 to promote chemoresistance. To achieve this, we established oxaliplatin-resistant (OXA-R) OSCC cells as a model system. Our investigations revealed that the efflux ability of resistant cells was enhanced and the ABCG2 expression was up-regulated. Genetic silencing of ABCG2 significantly attenuated both efflux activity and chemoresistance in these resistant cells. Notably, we observed aberrant activation of the WNT canonical signaling pathway in resistant cells, accompanied by heightened expression of the WNT3 ligand. Additionally, overexpression of WNT3 in parental cells recapitulated the activation of the WNT canonical signaling cascade, resulting in increased chemoresistance, enhanced efflux function, and elevated ABCG2 expression levels. Conversely, inhibition of WNT3 in resistant cells resulted in reduced chemoresistance, suppression of efflux activity, and decreased ABCG2 expression. Finally, treatment with the WNT/β-catenin pathway inhibitor methyl 3-benzoate (MSAB) effectively reversed chemoresistance in resistant cells. Mechanistically, our studies revealed that the abnormal activation of the WNT canonical pathway promotes the recruitment of the transcription factor lymphoid enhancer-binding factor 1 (LEF1) to the ABCG2 promoter, thereby enhancing its transcriptional activity. In summary, our findings underscore the critical role of WNT3-mediated activation of the WNT canonical signaling pathway in upregulating ABCG2 expression, which enhances oxaliplatin efflux and contributes to the development of oxaliplatin resistance in OSCC.\u003c/p\u003e","manuscriptTitle":"WNT3 promotes chemoresistance to oxaliplatin in oral squamous cell carcinoma via regulating ABCG2 expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 10:04:12","doi":"10.21203/rs.3.rs-5267942/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-03T19:07:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-03T19:06:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-01T05:31:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell \u0026 Bioscience","date":"2025-03-29T08:25:26+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revision","date":"2024-12-30T23:41:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-bioscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbio","sideBox":"Learn more about [Cell \u0026 Bioscience](http://cellandbioscience.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cbio/default.aspx","title":"Cell \u0026 Bioscience","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5ff69e99-3a57-41d8-9431-b4ffa0d573b2","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:00:32+00:00","versionOfRecord":{"articleIdentity":"rs-5267942","link":"https://doi.org/10.1186/s13578-025-01414-w","journal":{"identity":"cell-and-bioscience","isVorOnly":false,"title":"Cell \u0026 Bioscience"},"publishedOn":"2025-06-04 15:57:13","publishedOnDateReadable":"June 4th, 2025"},"versionCreatedAt":"2025-04-07 10:04:12","video":"","vorDoi":"10.1186/s13578-025-01414-w","vorDoiUrl":"https://doi.org/10.1186/s13578-025-01414-w","workflowStages":[]},"version":"v1","identity":"rs-5267942","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5267942","identity":"rs-5267942","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}