FGFRL1 Modulates Notch Signaling and Glucose-Glycogen Homeostasis to Suppress Chemoresistance in Esophageal Carcinoma

FGFRL1 Modulates Notch Signaling and Glucose-Glycogen Homeostasis to Suppress Chemoresistance in Esophageal Carcinoma | 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 FGFRL1 Modulates Notch Signaling and Glucose-Glycogen Homeostasis to Suppress Chemoresistance in Esophageal Carcinoma Aprajita Aprajita, N.R Dash, Rinu Sharma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7525805/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Apr, 2026 Read the published version in Cell Biochemistry and Biophysics → Version 1 posted 10 You are reading this latest preprint version Abstract Chemoresistance remains a significant barrier to the treatment of esophageal cancer (EC), regulated by metabolic and signaling adaptations. Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) is a key regulator of cancer progression; however, its involvement in driving chemoresistance remains poorly understood. This study investigates the functional significance of FGFRL1 in chemo-resistant EC cells and its association with response to chemotherapy. FGFRL1 expression was analyzed in cisplatin-resistant EC cells using real-time PCR and Western blotting. FGFRL1 protein levels were examined in clinical specimens from EC patients post-neoadjuvant chemotherapy (NACT) to evaluate their correlation with treatment response using immunohistochemistry. Significantly decreased expression of FGFRL1 was observed in cisplatin-resistant EC cells (p < 0.05). Interestingly, overexpression of FGFRL1 suppressed proliferation, migration, and clonogenic potential (p < 0.05), while activating Notch signaling via JAG1, DLL1, DLL4, NOTCH1, NOTCH2, and HES1 (p < 0.05) in cisplatin-resistant EC cells. FGFRL1 overexpression also shifted glucose metabolism toward glycogen synthesis, involving regulators GFPT2, AQP3, and GALT5 (p < 0.05). In patient specimens, FGFRL1 upregulation was significantly associated with chemotherapy response, observed in 80% of complete responders versus 36.4% of non-responders (p = 0.000, OR = 8.61). We report for the first time that FGFRL1 regulates metabolic and signaling pathways in chemo-resistant EC, suggesting its potential as a drug target to counter resistance. FGFRL1 esophageal cancer chemoresistance Notch signaling glucose metabolism neoadjuvant chemotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Esophageal cancer (EC) cases are rising globally, with a large number of patients presenting at advanced stages. The standard treatment involves neoadjuvant chemotherapy followed by surgery. However, disease recurrence is frequent, and the 5-year survival rate (approximately 20%) has shown little improvement over the past several years (1). This is mainly attributed to the varied response to chemoradiotherapy (CRT), with many tumors being resistant and progressing during treatment (2). Understanding the underlying mechanism that leads to chemoresistance may be instrumental in designing strategies for combating it. Several mechanisms contribute to the development of chemoresistance, including enhanced activation of signaling pathways, drug inactivation, enhanced DNA repair mechanisms, triggering suppression of apoptosis, and epithelial-to-mesenchymal transition (EMT)(3,5). Moreover, altered metabolic reprogramming contributes significantly to drug resistance by elevating energy production, suppressing pharmacologically induced apoptosis, and promotion of proliferative signaling pathways. Enhanced aerobic glycolysis promotes lactic acid accumulation, resulting in a nutrient-starved tumor milieu, which in turn activates stress-response pathways that support cell survival and therapeutic resistance. Recently, FGFRs have garnered significant interest for their roles in driving cancer progression and resistance to therapy. Fibroblast growth factor receptors (FGFRs) are high-affinity tyrosine kinases that are critical for embryonic development, tissue homeostasis, and the progression of various cancers. FGFRs consist of four well-characterized members (FGFR1–4) and a newly identified member, FGFRL1, which lacks a kinase domain but regulates oncogenic pathways via PDZ and SH2 binding motifs in its short cytoplasmic domain (6-10). Several FGFs, including FGF1, FGF19, and FGF21, have been linked to metabolism. These factors are transcriptionally regulated by nuclear receptor superfamily members and mediate effects on glucose and lipid metabolism. FGF15/19 and FGF21 regulate metabolism by binding to the FGFR4/β-Klotho receptor complex, leading to metabolic effects such as gluconeogenesis repression and glycogen synthesis stimulation. FGFR1 signaling has also been implicated in glucose metabolism regulation, where its activation enhances glucose uptake, glycolysis, and lactate production through the MAPK and AKT/mTOR pathways. FGFR4 has been identified as a regulator of both glucose metabolism and chemoresistance in breast cancer. Increased glycolytic flux has been observed in Adriamycin-resistant breast cancer cells. Moreover, targeting the FGFR4-FRS2-ERK signaling pathway effectively blocks chemoresistance and the glycolytic phenotype (11). Selective FGFR inhibitors have demonstrated the ability to hinder glucose metabolism, cell proliferation, and viability, even under hypoxic conditions. Whereas dysregulation of FGFRL1 expression also has been associated with several cancers, including ovarian, lung, breast, and prostate cancer (12-13). Multiple reports have demonstrated the oncogenic role of FGFRL1-mediated signaling in cancer. (14-15). For example, FGFRL1 knockdown in ovarian cancer cells resulted in reduction in Gli1 and Gli2 levels, thereby modulating the Hedgehog (Hh) signaling pathway (12). Furthermore, decreased phosphorylation of MEK and ERK following FGFRL1 silencing suggests its involvement in the MAPK signaling pathway (13). However, few studies have explored the role of FGFRL1 in chemoresistance(16). These findings highlight the diverse roles of FGFRL1 in cancer progression, but its significance in metabolic regulation and chemoresistance is still not fully understood. Here, we investigate the role of FGFRL1 in chemoresistance and glucose-glycogen homeostasis in cisplatin-resistant EC cells. 2. Methodology Cell culture and Transfection: Cisplatin-resistant counterpart (CISR) was developed in our lab from KYSE 140(Pandey et.al 2023). KYSE140, a EC cell line, was kindly provided by Shimada.Y (Japan). Cells were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO₂. . For the overexpression studies, a human full-length FGFRL1 and C-domain deleted (FGFRL1ΔC) FGFRL1 sequence was amplified using PCR and inserted into the Sal I/BamHI site of the expression vector mEGFP-N1. The constructs were verified by restriction digestion and sequencing. CISR cells were transfected with pCMV-FGFRL1-EGFP/pCMV-FGFRL1ΔC-EGFP or empty vector via Lipofectamine 3000 (Invitrogen, CA, USA). Sample Collection: Sixteen surgically resected tissues were obtained from patients with esophageal cancer subjected to surgery post-neoadjuvant chemotherapy (NACT) at the Department of GI Surgery, AIIMS. One part of the tissue was fixed in 10% formalin, paraffin-embedded,, and prepared for hematoxylin and eosin staining as well as immunohistochemical evaluation. Clinicopathological information was documented using a predesigned proforma, which included details such as lesion site, histopathological differentiation (tumor grade), patient age, gender, and treatment response. The esophageal squamous cell carcinoma cases were categorized by location into upper, middle, and lower esophagus. Real-Time Quantitative Polymerase Chain Reaction (RTqPCR): RNA was isolated from KYSE140, CISR, and transfected cells (pCMV-FGFRL1-EGFP, pCMV-FGFRL1ΔC-EGFP, or empty vector) using the RNeasy Mini Kit (Qiagen, Copenhagen, Denmark). Complementary DNA was synthesized using 1 μg of total RNA . Gene expression was evaluated using two-step RTqPCR on a CFX96 Real-Time Thermal Cycler (Bio-Rad). The reaction mix consisted of SYBR® Premix Ex Taq (Takara, Japan) (12.5 µl), gene-specific primers (0.5 µM), and cDNA. The thermal cycling conditions included denaturation steps at 95 0 C and annealing at the gene-specific Tm, and extension at 72 °C, with fluorescence recorded at the end of each extension. Cycle threshold (Ct) values were determined for each sample. Comparative Ct (ΔΔCt) method was used for calcilation of relative expression leves using 5S RNA as the endogenous control for normalization. Protein isolation and Western blotting: CISR and KYSE140 cells were disrupted in RIPA buffer (Sigma) with 1× protease inhibitor cocktail added with 1× protease inhibitor cocktail and collected by scraping. Protein concentration was quantified using the Bradford assay (Sigma) using bovine serum albumin as a standard. Proteins of equal amounts were resolved by SDS–PAGE and transferred onto PVDF membranes. Skimmed milk (5%) was used for blocking the membranes before incubation with primary antibodies at 4 °C overnight followed by incubation with HRP-conjugated secondary antibodies: rabbit polyclonal anti-GAPDH (1:600, Novus Biologicals, USA; RRID: AB_1660142) and anti-FGFRL1 (1:200, Cloudclone). Enhanced chemiluminescence kit (Thermo Scientific, USA) was used to visualize the protein bands visualized. Protein levels were quantified as integrated density values (IDVs) using ImageJ and normalized to GAPDH. Analysis of Pathway and Functional Enrichment : We explored pathway-related functions of the overlapping genes between differentially expressed genes (DEGs) profile post-FGFRL1 knockdown expression and DEGs in cisplatin-resistant (CISR) vs parental cell lines of esophageal carcinoma (EC) (Prerna et.al, 2023). These were processed through Funrich and KEGG pathway analysis using Enricher. WST-1 assay: CISR cells (10,000 cells/well) were cultured in 96-well plates followed by transfection with pCMV-FGFRL1-EGFP, pCMV-FGFRL1ΔC-EGFP, or empty vector. Following 48 h of incubation, WST-1 reagent was added and incubated for 3 hours at 37 °C with 5% CO₂. Absorbance was then measured at 450 and 630 nm on a microplate reader. Percentage proliferation was calculated as a ratio of OD value of the experimental group to OD value of the control group multiplied by 100%. Colony formation assay: Following transfection with FGFRL1-EGFP or empty vector, CISR cells were trypsinized after 48 h to generate single-cell suspensions and seeded into 6-well plates at densities of 3 × 10³ or 6 × 10³ cells per well. Cells were incubated for 6 days at 37 °C, and media was changed at 2 days interval. After fixing colonies in pre-chilled methanol for 20 min, crystal violet staining and imaging was carried out. Colonies with over 50 cells were counted using ImageJ. Boyden Chamber Assay : CISR cells transfected with FGFRL1-EGFP or empty vector were harvested after 48 h. A total of 8 × 10⁴ cells in PBS were added into the insert upper chambers in presence or absence Matrigel (for migration and invasion, respectively). The lower chamber contained complete medium. 24 h post-incubation at 37°C, non-migrated cells were removed, while invaded cells on the membrane’s underside were methanol-fixed, stained, and quantified under an inverted fluorescence microscope. 3D droplet Invasion assay: CISR cells (50,000/drop), transfected with FGFRL1-EGFP, FGFRL1ΔC-EGFP, or empty vector, were resuspended in conditioned medium followed by centrifuged (1000 g, 5 min, 4°C). Cells were resuspended in cold Matrigel, pipetted into 24-well plates form drop-shaped constructs, and solidified in a 37 o C incubator with 5% CO 2 for 20 min. After the drops were solidified, 2 ml of 10% RPMI media was added to each well. Images were acquired daily, and invasion was quantified by measuring the area of migrating cells relative to the Matrigel drop using ImageJ. Glucose Assay : CISR Cell supernatant post-FGFRL1 overexpression was diluted with deionized water to achieve a concentration ranging from 0.05 to 5 mg of glucose per millilitre. Following this, a precise volume of solution containing 0.5-50 µg of glucose was carefully pipetted. The assay was then replicated, and the sample volume may have been adjusted if required to maintain a ΔA340 within the range of 0.03 to 1.6. To conduct the assay, the designated volumes of solutions were pipetted in labelled tubes, including the sample blank, reagent blank, and test tubes. After thorough mixing, the tubes were incubated at for 15 min at room temperature. Absorbance was then measured at 340 nm using deionized water as blank. Lactic Acid Assay : The CISR cell supernatant post-FGFRL1 overexpression was collected by centrifugation at 10,000 g for 10 minutes and preserved at -80°C for up to a month. For the assay, 20 μl of sample, blank, or standard (3 mmol/L lactic acid) was combined with 1000 μl of working solution of enzyme and 200 μl of reagent 3. After 10 min incubation at 37°C, 2000 μl reagent 4 was added. The spectrometer was zeroed with double-distilled water, and the optical density was measured at 530 nm within 30 minutes. Glycogen Assay : The extraction process for CISR cells post-FGFRL1 overexpression involved collecting 5 to 10 million cells into a centrifuge tube and resuspending them in 0.75 ml extraction solution ES30, followed by ultrasonication (30 cycles, 200 W, 3s pulses at 10s intervals,). After cooling, the tube was topped up to 5 ml with distilled water. Centrifugation was carried out at 8000 g, 10 min at 25 °C to obtain the supernatant for testing. For the measurement operation, the photometer was preheated for at least 30 minutes, with the wavelength adjusted to 620 nm and zeroed using distilled water The remaining reagent could be stored at 4 °C for a week. Standards were prepared from 10 mg/ml glucose solution diluted to 0.1 mg/ml. Reagents were prepared according to manufacturer's instructions. Immunohistochemistry: Paraffin-embedded EC tissues (5 µm), of surgically resected samples, were subjected to deparaffinization, rehydration, and antigen retrieval (Tris–EDTA buffer, pH 9.0). 0.3% H₂O₂ in methanol was used for quenching endogenous peroxidase, and blocking was carried out with 1% horse serum. Next, the sections were incubated with rabbit polyclonal anti-FGFRL1 antibody (1:50, Cloudclone) overnight, followed by treatment with HRP-conjugated secondary antibody (ImmPRESS, Vector Laboratories, USA). Staining was developed with DAB and counterstained with hematoxylin. FGFRL1 expression was scored semi-quantitatively by H-score (proportion: ≤10%=0; 11–20%=1; 21–40%=2; 41–60%=3; 61–80%=4; >81%=5; intensity: faint=1, moderate=2, strong=3). Final scores were the sum of both parameters, independently evaluated by three investigators. Statistical Data nalysis: Data analysis was carried out using IBM SPSS Statistics v26.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism . Data are presented as mean ± SD and all experiments were carried out in triplicate. For experimental comparisons, Student’s t-test was applied, while Mann–Whitney U test was used to evaluate FGFRL1 expression between responder and non-responder cohorts. p≤ 0.05 was the criteria used for assessing statistically significance. 3. Results 3.1 FGFRL1 Expression Is Reduced in Cisplatin-Resistant EC Cells and in Patients with No or Partial Response to NACT Cisplatin-resistant esophageal carcinoma cell line (CISR) developed in our laboratory showed a more than 96.8% reduction in FGFRL1 mRNA and a 95.6% reduction in protein expression (p < 0.05) (Figure 01 (a-c)) as compared to parental EC cells, KYSE140. As transcriptional regulation of Notch signalling markers is known to be regulated by FGFR signalling (17-18), we checked their levels and found decreased levels of Notch ligands such as JAG1, DLL1, NOTCH1, NOTCH2 and transcription factor (TF) HES1 in CISR relative to its parental esophageal cancer (EC) cell line, KYSE140. . Interestingly, after FGFRL1 overexpression, all Notch signalling ligands and TF HES1 were upregulated (Figure 01 (e-f)). Since FGFRL1 has a unique C-terminal domain instead of a tyrosine kinase domain, hence to delineate the cytoplasmic domain region of FGFRL1 which might be involved in downstream signalling, we constructed FGFRL1 C-domain deleted construct, FGFRL1ΔC (401-501aa). Next, we analyzed the effect of overexpressing the FGFRL1ΔC construct on the mRNA expression of Notch signalling markers. Herein, we noticed that in FGFRL1ΔC construct transfected cells, DLL1 and HES1 are affected post-FGFRL1 C-domain deletion (p < 0.05) (Figure 01-f). To further clarify whether the patients who do not respond to chemotherapy also have lower FGFRL1 levels, we checked FGFRL1 protein expression in available clinical specimens from patients who came for surgery post-NACT using immunohistochemistry. The patients were classified into responders, which includes complete responderz (n = 05), and non-responders (n = 11), i.e., partial or no response to NACT. A total of 16 specimens were analyzed and upregulation of FGFRL1 in 80% (4/5) complete response and non-responders 36.4% (4/11) EC tissues was found to be significant (p = 0.0012, OR = 6.08) (Figure 01 (h)). This emphasized the observation that FGFRL1 expression may be reduced after acquired resistance of platinum drugs e.g. cisplatin in cell lines as well as in patients post-chemotherapy. 3.2 FGFRL1 overexpression alone and in combination with Cisplatin decreases the clonogenic and invasive potential of cisplatin-resistant EC cells Next, the effect of FGFRL1 overexpression on cisplatin-resistant EC cell proliferation was examined via WST assay in the FGFRL1 overexpression group and the control group. After 48 h following transfection, data were measured. Rate of proliferation in the FGFRL1 overexpression group was found to be reduced by 49% in comparison to the control group (p < 0.001) (Figure02 (c)). We also overexpressed the FGFRL1ΔC (401-501aa) construct, wherein we noticed decrease in percentage proliferation post FGFRL1ΔCoverexpression (p<0.05) (Figure02 (b)). Moreover, a significantly reduced clonogenic potential of up to 66.3 % in CISR cells after FGFknoRL1 overexpression was observed (p <0.01) (Figure02 (a-b)). Also, if coupled with varying cisplatin doses (10μM-18μM) FGFRL1 overexpression results in reduced proliferation in CISR cell line(p=0.0244)(Figure02 (d)).For further validation of this effect on invasive potential, we performed a 3D droplet assay wherein after 24 hours of transfection, 50,000 cells /drop were plated with the Matrigel/ECM Gel. The results were captured from day 0 to day 6. At each time point the area covered by total invading drop and the area of non-invasive, propagating cells is calculated. We observed that the area of invasion by migrating cells is 62.5% more in Control as compared to post-FGFRL1 overexpression on day 6 th (Figure02 (e-f). All these observations confirm that FGFRL1 overexpression has a negative effect on cisplatin-resistant cells. 3.3 FGFRL1 play a major role in maintaining Glucose – Glycogen Homeostasis in EC Cell Lines and Cisplatin Resistant Cell Line We next compared the profile of differentially expressed genes (DEGs) post-FGFRL1 silencing (unpublished data) as well as the DEG profile of cisplatin-resistant EC cells (CISR) as compared to its parental counterpart (KYSE140) (17) In light of the aforementioned results, deregulated overlapping genes in both datasets were analysed. We found 5 common downregulated genes (ITGB6, COL15A1, CTSS, POF1B, GALNT5) and 2 common upregulated genes (AQP3, GFPT2) (Figure: 03(a)). Out of these, 2 common downregulated (CTSS, GALNT5) and 2 upregulated genes (AQP3, GFPT2) were selected for further validation, based on their known involvement in metabolic pathways like glycolysis and the Hexosamine Biosynthesis Pathway. Along with these, a major regulator of glycolysis, i.e., GLUT1 and HIF1α, was checked in both cell lines (Figure: 03(b), Figure 04). Upon validation, selected DEGs showed downregulation in cisplatin-resistant EC cells and FGFRL1 knockdown EC cell line KYSE410. Also, GLUT1 and HIF1αwas found to be upregulated specifically in the CISR cell line as compared to its parental. In FGFRL1-EGFP transfected CISR cells, significantly reduced levels of AQP3, GFPT2, GLUT1, and even CTSS were observed in comparison to the wild type (Figure: 03(c)(d)). Also, post-FGFRL1 C-domain deleted construct, FGFRL1ΔC transfection, the expression of AQP3, GFPT2, and GLUT1 increased as compared to wild type (p<0.05) (Figure: 03(e-g)). These findings suggest that altered expression of FGFRL1 leads to differential expression of AQP3, GFPT2, GALNT5, and GLUT1 specifically in cisplatin-resistant esophageal cancer cells (CISR). Since AQP3, GFPT2, GALNT5, and GLUT1 are all genes related to glycolysis, and also in our earlier studies we have observed reduced levels of phosphorylated GSK3β post-FGFRL1 silencing in esophageal cancer cell line KYSE410 (Unpublished) (Figure 04 (I)), this gave us a strong indication to analyse whether the alterations in FGFRL1 expression affect levels of glucose consumption, lactic acid, and glycogen production in cisplatin-resistant as well as esophageal cancer cells. Firstly, glucose consumption and production of lactic acid and glycogen in cisplatin-resistant (CISR) as well as esophageal cancer cells (KYSE140, KYSE410) were investigated. CISR cell line seems to consume more glucose, produce more lactic acid, and has less glycogen storage owing to its highly proliferative nature as compared to its parental cell line KYSE140 (Figure 04(II, a-c)). Whereas, esophageal cancer cell line KYSE410 shows consumption of glucose in similar amounts as CISR but produces less lactic acid and rather has high glycogen production. Furthermore, we also noted that FGFRL1 overexpression in both parental and cisplatin resistant esophageal cancer cells and significantly increased the glycogen production as compared to lactic acid (Figure 05(II, e-f)). Discussion FGFRL1 is a 504–amino-acid transmembrane protein with three Ig-like extracellular domains, a single transmembrane helix, and a cytoplasmic C-terminal tail (20,21). Recent studies indicate that specific motifs located within the C-terminal domain may be critical for mediating downstream signaling. Genomic analyses have revealed the potential presence of SH2- and PDZ-binding motifs, as well as immunoreceptor tyrosine-based activation (ITAM) and inhibitory motifs (ITIM) within the histidine-rich region of the C-terminal domain, suggesting a role in the regulation of signaling pathways(9). Furthermore, Silva et al. reported that an SH2 binding motif in the cytoplasmic domain of FGFRL1 interacts with SHP1, thereby promoting ERK1/2 signaling (10). In a 2006 study, Trueb and Taeschler examined FGFRL1 expression during embryonic development in mice and found that FGFRL1 mRNA is primarily expressed in cartilaginous structures, including the bone primordia and the permanent cartilage of the trachea, ribs, and nose, as well as in certain muscles, such as the tongue and diaphragm (14–15). Further research by Trueb and colleagues demonstrated that mice with targeted Fgfrl1 gene disruption exhibited neonatal lethality due to respiratory failure, attributed to a severely underdeveloped diaphragm, although the muscle fibers remained structurally differentiated, suggesting its role in diaphragm development(22). Beyond its role in development, FGFRL1 has gained attention in cancer biology due to its context-dependent dual roles. Several reports have shown the oncogenic potential of FGFRL1 in certain tumor types. For instance, FGFRL1 knockdown in ovarian cancer cells resulted in reduced Gli1 and Gli2 expression, implicating its role in regulating the Hedgehog (Hh) signaling pathway. Moreover, decreased phosphorylation of MEK and ERK following FGFRL1 silencing suggests that FGFRL1 may promote tumorigenesis through activation of the MAPK pathway (23). Conversely, other studies point to a tumor-suppressive role for FGFRL1. Trueb and colleagues have proposed FGFRL1 to function as a ligand trapping receptor that binds fibroblast growth factors (FGF2, FGF3, FGF4, FGF8, FGF10, FGF22) without initiating intracellular signaling, thereby inhibiting the FGF pathway by sequestering ligands away from functional FGFRs (20,21,25–26). This decoy activity was further supported by studies in Xenopus embryos, where injection of FGFRL1 mRNA led to developmental defects resembling those caused by a truncated, dominant-negative FGFR1, reinforcing the idea that FGFRL1 antagonizes FGF signaling. These findings illustrate the complex and context-specific nature of FGFRL1 in cancer, where its role in tumorigenesis can be either promoting or inhibitory, depending on the cellular and molecular environment (24). ​Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) has emerged as a significant player in esophageal cancer (EC), particularly esophageal squamous cell carcinoma (EC). Studies have reported that FGFRL1 expression is significantly upregulated in EC tissues (22). Additionally, tumor-suppressive miRNAs viz. miR-107 and miR-210 are reported to regulate FGFRL1 expression, indicating a complex regulatory mechanism that influences tumor progression.(13,23).​ Esophageal cancer (EC) remains an aggressive malignancy with poor survival rates, largely due to chemoresistance, which significantly limits the effectiveness of neoadjuvant chemotherapy (NACT)(24). Although NACT followed by surgery remains the established treatment approach for locally advanced EC, nearly half of the patients fail to respond, leading to disease progression, metastasis, and poor prognosis (24–25). Herein, we focused on FGFRL1 and its potential role in chemoresistance in EC, which has not been thoroughly explored. We observed reduced FGFRL1 mRNA and protein levels in cisplatin-resistant EC cells (CISR). Moreover, patients exhibiting partial or no response to chemotherapy showed significantly lower FGFRL1 protein expression compared to those with a complete response, indicating a potential correlation between FGFRL1 levels and chemotherapy response. Interestingly, further in-depth analysis demonstrates significantly reduced levels of JAG1/DLL ligands, Notch1-2 receptors, and the transcription factor HES1 in CISR cells compared to parental EC cells. Notch signaling has been extensively implicated in driving chemoresistance across multiple cancer types, such as head and neck squamous cell carcinoma, colorectal cancer, and ovarian cancer (27–30). Notch pathway activation has been shown in 5-FU-resistant colon cancer cells, with increased NICD protein and HES1 expression (31). High Notch2 and JAG1 expression is linked to gemcitabine resistance in pancreatic cancer cells (32–33). Although Notch signaling is often associated with chemoresistance, emerging evidence suggests that its downregulation, including decreased expression of Notch ligands and HES1, may contribute to adaptive survival mechanisms in certain cisplatin-resistant cancer models (34–35). Overexpressing FGFRL1 in CISR cells led to a significant reduction in cell proliferation, clonogenic potential, and migration. Additionally, FGFRL1 overexpression, combined with increasing cisplatin doses, reduced CISR cell proliferation, suggesting that FGFRL1 may regulate chemosensitivity in EC cells. To further explore the molecular mechanism, we identified and validated two commonly downregulated genes ( CTSS and GALNT5) and two upregulated genes (AQP3, GFPT2) in both CISR cells and FGFRL1-knockdown EC cells. Among CTSS and GALNT5 were downregulated, while AQP3 and GFPT2 were upregulated in metabolic pathways, including glycolysis and the hexosamine biosynthesis pathway (HBP). GLUT1, a key glycolysis regulator, was upregulated in CISR cells compared to parental cells. Notably, FGFRL1 overexpression in CISR cells affected AQP3 and GFPT2 mRNA levels but not in the KYSE410 EC cell line. GFPT2, a key enzyme in the hexosamine biosynthetic pathway (HBP), is upregulated in mesenchymal cells, particularly during partial epithelial–mesenchymal transition (EMT) (36). The HBP leads to O-GlcNAcylation of proteins, including Sp1, which promotes AQP3 expression. AQP3, responsible for glycerol influx, compensates for reduced glucose uptake(37). The increased AQP3 and GFPT2 levels in CISR cells suggest that glucose and lactic acid homeostasis are altered to support enhanced glycolysis and amino acid biosynthesis, which are critical for meeting cellular energy demands. Furthermore, the high glucose consumption, increased lactic acid production, and low glycogen levels in CISR cells support this hypothesis. Interestingly, AQP3 and GFPT2 levels were downregulated post FGFRL1 overexpression, further supporting this hypothesis. FGFRL1 overexpression in CISR cells altered this balance, increasing glycogen levels while reducing lactic acid production. In our previous studies, we observed that FGFRL1 knockdown in EC cells resulted in reduced Ser9 phosphorylation of glycogen synthase kinase 3 beta (GSK3β) which is a key regulator of glycogen synthesis (Unpublished). Active GSK3β maintains glycogen synthase (GS) in an inactive state by phosphorylation, and functionally inactive Ser9-phosphorylated GSK3β fails to phosphorylate GS, leading to increased glycogen production. In CISR cells, reduced FGFRL1 levels may enhance GS activity and glycogen synthesis. FGFRL1 overexpression in CISR cells disrupted the glucose–lactic acid balance by influencing glycogen synthesis, thereby restoring a more balanced metabolic state. In conclusion, our study provides evidence that FGFRL1 modulates key signaling pathways, including Notch, and metabolic pathways, which contribute to chemoresistance in EC. Conclusion Our study highlights FGFRL1 as a critical regulator of chemoresistance and metabolic reprogramming in cisplatin-resistant esophageal cancer cells. FGFRL1 overexpression suppressed cell proliferation, migration, and colony formation while concomitantly enhancing glycogen production, suggesting a metabolic shift in esophageal cancer cells. These results indicate that targeting FGFRL1 expression could represent a novel therapeutic approach to overcome chemoresistance and metabolic adaptations in esophageal cancer. Declarations CRediT authorship contribution statement The study was conceived and designed with contributions from all authors. Investigation and data curation were carried out by Aprajita. Conceptualization, investigation, and supervision were provided by Dr. Rinu Sharma. The initial manuscript draft was written by Aprajita, with all authors reviewing and commenting on previous versions. Dr. N.R. Dash served as the clinician, providing tissue samples and performing the surgical procedures. The final manuscript was read and approved by all authors. Data availability Data generated in the current study will be made available on request by contacting the corresponding author. Ethics approval This study ethicaly cleared from the Institutional Research Ethics Committee of the A.I.I.M.S, New Delhi (Ref. No.: IEC-545/06.08.2021), as well as from the Institutional Research Ethics Committee of GGSIP University, Dwarka, New Delhi (Ref. No.: GGSIPU/IEC/2021-A1). All procedures where human participants were involved were conducted in compliance with the ethical guidelines of both committees, 1964 Declaration of Helsinki principles and its subsequent amendments. Consent to participate All participants provided written informed consent prior to enrollment in the study. Declaration of Competing Interest The author(s) declare no conflicts of interest related to the content of this manuscript. They further confirm that no financial or personal relationships exist that influenced the work presented here. Consent for publication Not applicable Acknowledgement : Funding: Financial support for this study was provided by Guru Gobind Singh Indraprastha University (GGSIPU; Grant No. GGSIPU/DRC/FRGS/2021/594/17) and SERB, DST (Grant No. CRG/2022/003194). References Herskovic A, Russell W, et al. Esophageal carcinoma: advances in treatment results for locally advanced disease. Ann Oncol. 2012;23(5):1095-1103. doi:10.1093/annonc/mdr602 Ajani JA, D’Amico TA, et al. Esophageal and esophagogastric junction cancers: clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2011;9(8):830-887. doi:10.6004/jnccn.2011.0075 Housman G, Byler S, et al. 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Identification of a new fibroblast growth factor receptor, FGFR5. Gene. 2001;271(2):171-182. doi:10.1016/S0378-1119(01)00473-4 Silva EA, Yu C, et al. FGFRL1 promotes ERK1/2 signaling and regulates cell proliferation. J Biol Chem. 2012;287(30):25206-25215. doi:10.1074/jbc.M112.355016 Cui Y, Yao H, et al. Downregulation of FGFRL1 suppresses proliferation and migration of ovarian cancer cells via the Hedgehog signaling pathway. Biochem Biophys Res Commun. 2019;516(3):977-982. doi:10.1016/j.bbrc.2019.06.127 Wiedemann M, Trueb B, et al. The fibroblast growth factor receptor-like protein is a novel FGF binding protein that inhibits FGF signaling in Xenopus embryos. J Biol Chem. 2000;275(30):21970-21975. doi:10.1074/jbc.275.30.21970 Trueb B, et al. Fibroblast growth factor receptor-like 1 interacts with FGFs and modulates development. Int J Mol Med. 2006;17(4):617-620. Chen J, Yin J, et al. MiR-210 promotes esophageal squamous cell carcinoma progression by targeting FGFRL1. Oncol Rep. 2020;43(3):897-908. doi:10.3892/or.2020.7483 Shi Q, Xue C, et al. Notch signaling pathway in cancer: from mechanistic insights to targeted therapies. Signal Transduct Target Ther. 2024;9:128. doi:10.1038/s41392-024-01828-x Bazzoni R, Bentivegna A, et al. Context-dependent roles of Notch signaling in tumor progression and therapy resistance. Biomed Res Int. 2016;2016:4280904. doi:10.1155/2016/4280904 Meng RD, Shelton CC, et al. Gamma-secretase inhibitors abrogate oxaliplatin-induced activation of the Notch-1 signaling pathway in colon cancer cells resulting in enhanced chemosensitivity. Cancer Res. 2009;69(2):573-582. doi:10.1158/0008-5472.CAN-08-3010 Wang Z, Li Y, et al. Down-regulation of Notch-1 contributes to cell growth inhibition and apoptosis in pancreatic cancer cells. Mol Cancer Ther. 2006;5(3):483-493. doi:10.1158/1535-7163.MCT-05-0360 Sun L, Ke J, et al. HES1 promotes colorectal cancer cell resistance to 5-FU by inducing EMT and ABC transporter proteins. J Cancer. 2017;8(14):2802-2808. doi:10.7150/jca.19142 Lee J, Lee J, et al. Association of Jagged1 expression with malignancy and prognosis in human pancreatic cancer. Cell Oncol (Dordr). 2020;43(5):821-834. doi:10.1007/s13402-020-00527-3 Güngör C, Zander H, et al. Notch signaling activated by replication stress–induced expression of Midkine drives epithelial–mesenchymal transition and chemoresistance in pancreatic cancer. Cancer Res. 2011;71(14):5009-5019. doi:10.1158/0008-5472.CAN-11-0036 Hanlon L, Avila JL, et al. Notch1 functions as a tumor suppressor in a model of K-ras-induced pancreatic ductal adenocarcinoma. Cancer Res. 2010;70(11):4280-4286. doi:10.1158/0008-5472.CAN-09-4645 Han M, Wang S, et al. Esophageal cancer cells resist cisplatin via alteration of Notch signaling pathway and epithelial-to-mesenchymal transition. Cancer Cell Int. 2020;20:145. doi:10.1186/s12935-020-01274-w Briem E, Nielsen TO, et al. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) is upregulated in breast epithelial–mesenchymal transition and responds to oxidative stress. Mol Cell Proteomics. 2021;20:100157. doi:10.1016/j.mcpro.2021.100157 Zhang H, Qi J, et al. O-GlcNAc modification mediates aquaporin 3 to coordinate endometrial cell glycolysis and affects embryo implantation. J Adv Res. 2023;47:153-166. doi:10.1016/j.jare.2022.11.008 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 07 Apr, 2026 Read the published version in Cell Biochemistry and Biophysics → Version 1 posted Editorial decision: Revision requested 19 Sep, 2025 Reviews received at journal 13 Sep, 2025 Reviews received at journal 13 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers agreed at journal 05 Sep, 2025 Reviewers invited by journal 05 Sep, 2025 Editor assigned by journal 05 Sep, 2025 Submission checks completed at journal 05 Sep, 2025 First submitted to journal 03 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7525805","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513599354,"identity":"59623045-981c-4001-a78c-f5f724688089","order_by":0,"name":"Aprajita Aprajita","email":"","orcid":"","institution":"University School of Biotechnology, Guru Gobind Singh Indraprastha University","correspondingAuthor":false,"prefix":"","firstName":"Aprajita","middleName":"","lastName":"Aprajita","suffix":""},{"id":513599355,"identity":"bb70dbb5-1f6f-4025-be6d-f855a0b88c18","order_by":1,"name":"N.R Dash","email":"","orcid":"","institution":"All India Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"N.R","middleName":"","lastName":"Dash","suffix":""},{"id":513599356,"identity":"3e6d9d71-4daa-418d-b921-b298276314f1","order_by":2,"name":"Rinu Sharma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDACZjB5AEQwPkiACh4gVguzAXFakNSwSRDlLvN23mMSDH/uyJtPO/ys4uGOwwz87QcYDxfg0SJzmC9NgrHtmeGc22lmNxLPHGaQOJPAcHgGHi0SzDxmEowNhxlnSCcAtbQdZmC4wcBwmIeQFoY/h+1nSKd/KwBpkSdOC9vhxBnSOWYMIC0GRGgxtgCqTAZqKZZIPJPOY3gmsQG/Fv4zhjc+/DlsC3TYxo8/d1jLyR0/fPgzPi1gkABjMDYw8IBJ4gFJikfBKBgFo2DEAAAatUlR9OtdiAAAAABJRU5ErkJggg==","orcid":"","institution":"University School of Biotechnology, Guru Gobind Singh Indraprastha University","correspondingAuthor":true,"prefix":"","firstName":"Rinu","middleName":"","lastName":"Sharma","suffix":""}],"badges":[],"createdAt":"2025-09-03 10:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7525805/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7525805/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12013-026-02065-5","type":"published","date":"2026-04-07T15:58:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91816590,"identity":"cddab7c2-1f6e-439e-966b-11fd6d2703df","added_by":"auto","created_at":"2025-09-22 06:52:03","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":393350,"visible":true,"origin":"","legend":"","description":"","filename":"Figure01.tif","url":"https://assets-eu.researchsquare.com/files/rs-7525805/v1/4b51e04d0e643f4bdd3348b5.tif"},{"id":91816889,"identity":"8c6146ed-d28d-460d-a180-68f1ca2663b2","added_by":"auto","created_at":"2025-09-22 06:52:53","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1074,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7525805/v1/1fb69c1119e70dfdb51340ef.jpeg"},{"id":91816660,"identity":"ab7added-e950-4305-a470-0c30ff94b9ae","added_by":"auto","created_at":"2025-09-22 06:52:27","extension":"xml","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":70267,"visible":true,"origin":"","legend":"","description":"","filename":"1276bee9f771407db788d6bdf36849a61structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7525805/v1/7567e813039c041d387160ea.xml"},{"id":91488939,"identity":"d024887e-a7df-47b6-9ab2-cec68f01f3ff","added_by":"auto","created_at":"2025-09-17 05:10:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":357333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) mRNA expression of FGFRL1 in the cisplatin-resistant EC cells(CisR). (b–c) Protein expression levels of FGFRL1 in the cisplatin-resistant EC cells(CisR).Lane 1, cisplatin resistant EC cells (CisR); Lane 2, parental esophageal cancer cell line (KYSE140).(d) Diagrammatic representation of pCMV-FGFRL1-EGFP and pCMV-FGFRL1ΔC-EGFP (e) Expression of total FGFRL1, Notch ligands; JAG1, DLL1, Notch1, Notch2, DLL4 and transcription factors HES1 was quantified by qRT-PCR in in CISR relative to its parental esophageal cancer (EC) cell line, KYSE140. (**P ≤ 0.0001) (f) Expression of total FGFRL1, Notch ligands and transcription factors HES1 was quantified by qRT-PCR FGFRL1/FGFRL1ΔC transfected cisplatin resistant esophageal cancer cells(CisR). (g)Representative section of esophageal tissue immunostained for FGFRL1in (I) responder tissue and (II) non-responder tissue (e) Boxplot showing differential expression of FGFRL1 in patients with response to therapy vs non-responder (p=0.000).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure01.png","url":"https://assets-eu.researchsquare.com/files/rs-7525805/v1/af8f7422c98c6073e683e3ea.png"},{"id":91485931,"identity":"e811e63d-d7cd-40b7-8f0a-4b77d5aa142a","added_by":"auto","created_at":"2025-09-17 04:54:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":449288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Effect of FGFRL1 overexpression on colony formation in CISR cells. Representative images of the colony formation assay following transfection with FGFRL1-EGFP or empty vector for 6 days. (b) Assessment of relative colony forming ability in FGFRL1-EGFP– and empty vector–transfected cells, normalized to untreated controls. (c) Histogram showing the percentage of cell proliferation in CISR cells 48 h after FGFRL1 overexpression. d) Effect of FGFRL1 overexpression combined with cisplatin on the proliferation of CISR cells. d) ) 3D drop Invasion assay: area invasion by migrating cells decreases post FGFRL1 overexpression as compared to control in CISR cells (*p \u0026lt; 0.05). ***indicates P \u0026lt; 0.001 and Values are presented as means ± standard error.)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure02.png","url":"https://assets-eu.researchsquare.com/files/rs-7525805/v1/b5d18dfab08683efa01068cf.png"},{"id":91487343,"identity":"209c3c20-29b4-40d2-aff8-b54ecc86c781","added_by":"auto","created_at":"2025-09-17 05:02:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":176271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea) Venn diagram illustrating overlapping differentially expressed genes between NGS profile post FGFRL1 silencing\u003c/strong\u003e \u003cstrong\u003ethe NGS profile of CISR (cisplatin-resistant EC cells) relative to the parental KYSE140 cell line. b) Pathway analysis of common dysregulated genes. c)\u0026amp;d)Validation of AQP3, GFPT2, CTSS, GALNT5, HIF1α and GLUT1 in\u003c/strong\u003e \u003cstrong\u003ecisplatin resistance esophageal cancer cells (*P ≤ 0.05), \u0026nbsp;e)-g) Expression levels of AQP3, GFPT2, CTSS, GALNT5, HIF1α and GLUT1 in\u003c/strong\u003e \u003cstrong\u003ecisplatin resistance EC cells was quantified by qRT-PCR 48hrs post of FGFRL1overexpression.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure03.png","url":"https://assets-eu.researchsquare.com/files/rs-7525805/v1/00adf1327dc3f2f6b0e009ff.png"},{"id":91487345,"identity":"da690998-4852-417e-8f5a-2c4dce7e8421","added_by":"auto","created_at":"2025-09-17 05:02:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":236654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eI) Diagrammatic representation of role of AQP3, GFPT2, and GLUT1 in Glycolysis and HBP pathway. II) (a-c)Lactic Acid production, glucose consumption (measured as glucose in supernatant) and glycogen production in cisplatin resistance esophageal cancer cells and parental cell line (KYSE410and KYSR140) (***P ≤ 0.001) (d-f) Lactic Acid production, glucose consumption (measured as glucose in supernatant) and glycogen production in post FGFRL1 overexpression in cisplatin resistance esophageal cancer cells(***P ≤ 0.001).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure04.png","url":"https://assets-eu.researchsquare.com/files/rs-7525805/v1/cbfe49f204fb00f237ba9234.png"},{"id":106809169,"identity":"654b8ce7-9c07-4d8d-bd94-7cb880155936","added_by":"auto","created_at":"2026-04-13 16:07:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2882432,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7525805/v1/375d3bc2-3232-41c1-891e-3ceb20cb38c9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"FGFRL1 Modulates Notch Signaling and Glucose-Glycogen Homeostasis to Suppress Chemoresistance in Esophageal Carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEsophageal cancer (EC) cases are rising globally,\u0026nbsp;with a large number of patients presenting at advanced stages. The standard treatment involves neoadjuvant chemotherapy followed by surgery. However, disease recurrence is frequent, and the 5-year survival rate (approximately 20%) has shown little improvement over the past several years (1). This is mainly attributed to the varied response to chemoradiotherapy (CRT), with many tumors being resistant and progressing during treatment (2). Understanding the underlying mechanism that leads to chemoresistance may be instrumental in designing strategies for combating it. Several mechanisms contribute to the development of chemoresistance, including enhanced activation of signaling pathways, drug inactivation, enhanced DNA repair mechanisms, triggering suppression of apoptosis, and epithelial-to-mesenchymal transition (EMT)(3,5). Moreover, altered metabolic reprogramming contributes significantly to drug resistance by elevating energy production, suppressing pharmacologically induced apoptosis, and promotion of proliferative signaling pathways. Enhanced aerobic glycolysis promotes lactic acid accumulation, resulting in a nutrient-starved tumor milieu, which in turn activates stress-response pathways that support cell survival and therapeutic resistance. Recently, FGFRs have garnered significant interest for their roles in driving cancer progression and resistance to therapy.\u003c/p\u003e\n\u003cp\u003eFibroblast growth factor receptors (FGFRs) are high-affinity tyrosine kinases that are critical for embryonic development, tissue homeostasis, and the progression of various cancers. FGFRs consist of four well-characterized members (FGFR1\u0026ndash;4) and a newly identified member, FGFRL1, which lacks a kinase domain but regulates oncogenic pathways via PDZ and SH2 binding motifs in its short cytoplasmic domain (6-10). Several FGFs, including FGF1, FGF19, and FGF21, have been linked to metabolism. These factors are transcriptionally regulated by nuclear receptor superfamily members and mediate effects on glucose and lipid metabolism. FGF15/19 and FGF21 regulate metabolism by binding to the FGFR4/\u0026beta;-Klotho receptor complex, leading to metabolic effects such as gluconeogenesis repression and glycogen synthesis stimulation. FGFR1 signaling has also been implicated in glucose metabolism regulation, where its activation enhances glucose uptake, glycolysis, and lactate production through the MAPK and AKT/mTOR pathways. FGFR4 has been identified as a regulator of both glucose metabolism and chemoresistance in breast cancer. Increased glycolytic flux has been observed \u0026nbsp;in Adriamycin-resistant breast cancer cells. Moreover, targeting the FGFR4-FRS2-ERK signaling pathway effectively blocks chemoresistance and the glycolytic phenotype (11). Selective FGFR inhibitors have demonstrated the ability to hinder glucose metabolism, cell proliferation, and viability, even under hypoxic conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhereas dysregulation of FGFRL1 expression also has been associated with several cancers, including ovarian, lung, breast, and prostate cancer (12-13). Multiple reports have demonstrated the oncogenic role of FGFRL1-mediated signaling in cancer. (14-15). For example, FGFRL1 knockdown in ovarian cancer cells resulted in reduction in Gli1 and Gli2 levels, thereby modulating the Hedgehog (Hh) signaling pathway (12). Furthermore, decreased phosphorylation of MEK and ERK following FGFRL1 silencing suggests its involvement in the MAPK signaling pathway (13). However, few studies have explored the role of FGFRL1 in chemoresistance(16). These findings highlight the diverse roles of FGFRL1 in cancer progression, but its significance in metabolic regulation and chemoresistance is still not fully understood. Here, we investigate the role of FGFRL1 in chemoresistance and glucose-glycogen homeostasis in cisplatin-resistant EC cells.\u003c/p\u003e"},{"header":"2. Methodology ","content":"\u003cp\u003e\u003cstrong\u003eCell culture and Transfection: \u003c/strong\u003eCisplatin-resistant counterpart (CISR) was developed in our lab from KYSE 140(Pandey et.al 2023). KYSE140, a EC cell line, was kindly provided by Shimada.Y (Japan). Cells were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37 \u0026deg;C with 5% CO₂. . For the overexpression studies, a human full-length FGFRL1 and C-domain deleted (FGFRL1\u0026Delta;C) FGFRL1 sequence was amplified using PCR and inserted into the Sal I/BamHI site of the expression vector mEGFP-N1. The constructs were verified by restriction digestion and sequencing. CISR cells were transfected with pCMV-FGFRL1-EGFP/pCMV-FGFRL1\u0026Delta;C-EGFP or empty vector via Lipofectamine 3000 (Invitrogen, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Collection: \u003c/strong\u003eSixteen surgically resected tissues were obtained from patients with esophageal cancer subjected to surgery post-neoadjuvant chemotherapy (NACT) at the Department of GI Surgery, AIIMS. One part of the tissue was fixed in 10% formalin, paraffin-embedded,, and prepared for hematoxylin and eosin staining as well as immunohistochemical evaluation. Clinicopathological information was documented using a predesigned proforma, which included details such as lesion site, histopathological differentiation (tumor grade), patient age, gender, and treatment response. The esophageal squamous cell carcinoma cases were categorized by location into upper, middle, and lower esophagus.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-Time Quantitative Polymerase Chain Reaction (RTqPCR): \u003c/strong\u003eRNA was isolated from KYSE140, CISR, and transfected cells (pCMV-FGFRL1-EGFP, pCMV-FGFRL1\u0026Delta;C-EGFP, or empty vector) using the RNeasy Mini Kit (Qiagen, Copenhagen, Denmark). Complementary DNA was synthesized using 1 \u0026mu;g of total RNA . Gene expression was evaluated using two-step RTqPCR on a CFX96 Real-Time Thermal Cycler (Bio-Rad). The reaction mix consisted of SYBR\u0026reg; Premix Ex Taq (Takara, Japan) (12.5 \u0026micro;l), gene-specific primers (0.5 \u0026micro;M), and cDNA. The thermal cycling conditions included denaturation steps at 95\u003csup\u003e0\u003c/sup\u003eC and annealing at the gene-specific Tm, and extension at 72 \u0026deg;C, with fluorescence recorded at the end of each extension. Cycle threshold (Ct) values were determined for each sample. Comparative Ct (\u0026Delta;\u0026Delta;Ct) method was used for calcilation of relative expression leves using 5S RNA as the endogenous control for normalization. \u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein isolation and Western blotting:\u003c/strong\u003e CISR and KYSE140 cells were disrupted in RIPA buffer (Sigma) with 1\u0026times; protease inhibitor cocktail added with 1\u0026times; protease inhibitor cocktail and collected by scraping. Protein concentration was quantified using the Bradford assay (Sigma) using bovine serum albumin as a standard. Proteins of equal amounts were resolved by SDS\u0026ndash;PAGE and transferred onto PVDF membranes. Skimmed milk (5%) was used for blocking the membranes before incubation with primary antibodies at 4 \u0026deg;C overnight followed by incubation with HRP-conjugated secondary antibodies: rabbit polyclonal anti-GAPDH (1:600, Novus Biologicals, USA; RRID: AB_1660142) and anti-FGFRL1 (1:200, Cloudclone). Enhanced chemiluminescence kit (Thermo Scientific, USA) was used to visualize the protein bands visualized. Protein levels were quantified as integrated density values (IDVs) using ImageJ and normalized to GAPDH.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAnalysis of Pathway and Functional Enrichment\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eWe explored pathway-related functions of the overlapping genes between differentially expressed genes (DEGs) profile post-FGFRL1 knockdown expression and DEGs in cisplatin-resistant (CISR) vs parental cell lines of esophageal carcinoma (EC) (Prerna et.al, 2023). These were processed through Funrich and KEGG pathway analysis using Enricher.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWST-1 assay: \u003c/strong\u003eCISR cells (10,000 cells/well) were cultured in 96-well plates followed by transfection with pCMV-FGFRL1-EGFP, pCMV-FGFRL1\u0026Delta;C-EGFP, or empty vector. Following 48 h of incubation, WST-1 reagent was added and incubated for 3 hours at 37 \u0026deg;C with 5% CO₂. Absorbance was then measured at 450 and 630 nm on a microplate reader. Percentage proliferation was calculated as a ratio of OD value of the experimental group to OD value of the control group multiplied by 100%.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony formation assay:\u003c/strong\u003e Following transfection with FGFRL1-EGFP or empty vector, CISR cells were trypsinized after 48 h to generate single-cell suspensions and seeded into 6-well plates at densities of 3 \u0026times; 10\u0026sup3; or 6 \u0026times; 10\u0026sup3; cells per well. Cells were incubated for 6 days at 37 \u0026deg;C, and media was changed at 2 days interval. After fixing colonies in pre-chilled methanol for 20 min, crystal violet staining and imaging was carried out. Colonies with over 50 cells were counted using ImageJ. \u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBoyden Chamber Assay\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eCISR cells transfected with FGFRL1-EGFP or empty vector were harvested after 48 h. A total of 8 \u0026times; 10⁴ cells in PBS were added into the insert upper chambers in presence or absence Matrigel (for migration and invasion, respectively). The lower chamber contained complete medium. 24 h post-incubation at 37\u0026deg;C, non-migrated cells were removed, while invaded cells on the membrane\u0026rsquo;s underside were methanol-fixed, stained, and quantified under an inverted fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3D droplet Invasion assay:\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eCISR cells (50,000/drop), transfected with FGFRL1-EGFP, FGFRL1\u0026Delta;C-EGFP, or empty vector, were resuspended in conditioned medium followed by centrifuged (1000 g, 5 min, 4\u0026deg;C). Cells were resuspended in cold Matrigel, pipetted into 24-well plates form drop-shaped constructs, and solidified in a 37\u003csup\u003eo\u003c/sup\u003eC incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e for 20 min. After the drops were solidified, 2 ml of 10% RPMI media was added to each well. Images were acquired daily, and invasion was quantified by measuring the area of migrating cells relative to the Matrigel drop using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlucose Assay\u003c/strong\u003e: CISR Cell supernatant post-FGFRL1 overexpression was diluted with deionized water to achieve a concentration ranging from 0.05 to 5 mg of glucose per millilitre. Following this, a precise volume of solution containing 0.5-50 \u0026micro;g of glucose was carefully pipetted. The assay was then replicated, and the sample volume may have been adjusted if required to maintain a \u0026Delta;A340 within the range of 0.03 to 1.6. To conduct the assay, the designated volumes of solutions were pipetted in labelled tubes, including the sample blank, reagent blank, and test tubes. After thorough mixing, the tubes were incubated at for 15 min at room temperature. Absorbance was then measured at 340 nm using deionized water as blank.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLactic Acid Assay\u003c/strong\u003e: The CISR cell supernatant post-FGFRL1 overexpression was collected by centrifugation at 10,000 g for 10 minutes and preserved at -80\u0026deg;C for up to a month. \u003c/p\u003e\n\u003cp\u003eFor the assay, 20 \u0026mu;l of sample, blank, or standard (3 mmol/L lactic acid) was combined with 1000 \u0026mu;l of working solution of enzyme and 200 \u0026mu;l of reagent 3. After 10 min incubation at 37\u0026deg;C, 2000 \u0026mu;l reagent 4 was added. The spectrometer was zeroed with double-distilled water, and the optical density was measured at 530 nm within 30 minutes.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eGlycogen Assay\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eThe extraction process for CISR cells post-FGFRL1 overexpression involved collecting 5 to 10 million cells into a centrifuge tube and resuspending them in 0.75 ml extraction solution ES30, followed by ultrasonication (30 cycles, 200 W, 3s pulses at 10s intervals,). After cooling, the tube was topped up to 5 ml with distilled water. Centrifugation was carried out at 8000 g, 10 min at 25 \u0026deg;C to obtain the supernatant for testing. For the measurement operation, the photometer was preheated for at least 30 minutes, with the wavelength adjusted to 620 nm and zeroed using distilled water The remaining reagent could be stored at 4 \u0026deg;C for a week. Standards were prepared from 10 mg/ml glucose solution diluted to 0.1 mg/ml. Reagents were prepared according to manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry: \u003c/strong\u003eParaffin-embedded EC tissues (5 \u0026micro;m), of surgically resected samples, were subjected to deparaffinization, rehydration, and antigen retrieval (Tris\u0026ndash;EDTA buffer, pH 9.0). 0.3% H₂O₂ in methanol was used for quenching endogenous peroxidase, and blocking was carried out with 1% horse serum. Next, the sections were incubated with rabbit polyclonal anti-FGFRL1 antibody (1:50, Cloudclone) overnight, followed by treatment with HRP-conjugated secondary antibody (ImmPRESS, Vector Laboratories, USA). Staining was developed with DAB and counterstained with hematoxylin. FGFRL1 expression was scored semi-quantitatively by H-score (proportion: \u0026le;10%=0; 11\u0026ndash;20%=1; 21\u0026ndash;40%=2; 41\u0026ndash;60%=3; 61\u0026ndash;80%=4; \u0026gt;81%=5; intensity: faint=1, moderate=2, strong=3). Final scores were the sum of both parameters, independently evaluated by three investigators.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Data nalysis:\u003c/strong\u003eData analysis was carried out using IBM SPSS Statistics v26.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism . Data are presented as mean \u0026plusmn; SD and all experiments were carried out in triplicate. For experimental comparisons, Student\u0026rsquo;s t-test was applied, while Mann\u0026ndash;Whitney U test was used to evaluate FGFRL1 expression between responder and non-responder cohorts. p\u0026le; 0.05 was the criteria used for assessing statistically significance.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 FGFRL1 Expression Is Reduced in Cisplatin-Resistant EC Cells and in Patients with No or Partial Response to NACT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCisplatin-resistant esophageal carcinoma cell line (CISR) developed in our laboratory showed a more than 96.8% reduction in FGFRL1 mRNA and a 95.6% reduction in protein expression (p \u0026lt; 0.05) (Figure 01 (a-c)) as compared to parental EC cells, KYSE140. As transcriptional regulation of Notch signalling markers is known to be regulated by FGFR signalling (17-18), we checked their levels and found decreased levels of Notch ligands such as JAG1, DLL1, NOTCH1, NOTCH2 and transcription factor (TF) HES1 in CISR relative to its parental esophageal cancer (EC) cell line, KYSE140. . Interestingly, after FGFRL1 overexpression, all Notch signalling ligands and TF HES1 were upregulated (Figure 01 (e-f)). Since FGFRL1 has a unique C-terminal domain instead of a tyrosine kinase domain, hence to delineate the cytoplasmic domain region of FGFRL1 which might be involved in downstream signalling, we constructed FGFRL1 C-domain deleted construct, FGFRL1\u0026Delta;C (401-501aa). Next, we analyzed the effect of overexpressing the FGFRL1\u0026Delta;C construct on the mRNA expression of Notch signalling markers. Herein, we noticed that in FGFRL1\u0026Delta;C construct transfected cells, DLL1 and HES1 are affected post-FGFRL1 C-domain deletion (p \u0026lt; 0.05) (Figure 01-f). To further clarify whether the patients who do not respond to chemotherapy also have lower FGFRL1 levels, we checked FGFRL1 protein expression in available clinical specimens from patients who came for surgery post-NACT using immunohistochemistry. The patients were classified into responders, which includes complete responderz (n = 05), and non-responders (n = 11), i.e., partial or no response to NACT. A total of 16 specimens were analyzed and upregulation of FGFRL1 in 80% (4/5) complete response and non-responders 36.4% (4/11) EC tissues was found to be significant (p = 0.0012, OR = 6.08) (Figure 01 (h)). This emphasized the observation that FGFRL1 expression may be reduced after acquired resistance of platinum drugs e.g. cisplatin in cell lines as well as in patients post-chemotherapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 FGFRL1 overexpression alone and in combination with Cisplatin decreases the clonogenic and invasive potential of cisplatin-resistant EC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, the effect of FGFRL1 overexpression on cisplatin-resistant EC cell proliferation was examined \u0026nbsp;via WST assay in the FGFRL1 overexpression group and the control group. After 48 h following transfection, \u0026nbsp;data were measured. Rate of proliferation in the FGFRL1 overexpression group was found to be reduced by 49% in comparison \u0026nbsp;to the control group (p \u0026lt; 0.001) (Figure02 (c)). We also overexpressed the FGFRL1\u0026Delta;C (401-501aa) construct, wherein we noticed decrease in percentage proliferation post FGFRL1\u0026Delta;Coverexpression (p\u0026lt;0.05) (Figure02 (b)). Moreover, a significantly reduced clonogenic potential of up to 66.3 % in CISR cells after FGFknoRL1 overexpression was observed (p \u0026lt;0.01) (Figure02 (a-b)). Also, if coupled with varying cisplatin doses (10\u0026mu;M-18\u0026mu;M) FGFRL1 overexpression results in reduced proliferation in CISR cell line(p=0.0244)(Figure02 (d)).For further validation of this effect on invasive potential, we performed a 3D droplet assay wherein after 24 hours of transfection, 50,000 cells /drop were plated with the Matrigel/ECM Gel. The results were captured from day 0 to day 6. At each time point the area covered by total invading drop and the area of non-invasive, propagating cells is calculated. We observed that the area of invasion by migrating cells is 62.5% more in Control as compared to post-FGFRL1 overexpression on day 6\u003csup\u003eth\u003c/sup\u003e (Figure02 (e-f). All these observations confirm that FGFRL1 overexpression has a negative effect on cisplatin-resistant cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 FGFRL1 play a major role in maintaining Glucose \u0026ndash; Glycogen Homeostasis in EC Cell Lines and Cisplatin Resistant Cell Line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next compared the profile of differentially expressed genes (DEGs) post-FGFRL1 silencing (unpublished data) as well as the DEG profile of cisplatin-resistant EC cells (CISR) as compared to its parental counterpart (KYSE140) (17) In light of the aforementioned results, deregulated overlapping genes in both datasets were analysed. We found 5 common downregulated genes (ITGB6, COL15A1, CTSS, POF1B, GALNT5) and 2 common upregulated genes (AQP3, GFPT2) (Figure: 03(a)). Out of these, 2 common downregulated (CTSS, GALNT5) and 2 upregulated genes (AQP3, GFPT2) were selected for further validation, based on their known involvement in metabolic pathways like glycolysis and the Hexosamine Biosynthesis Pathway. Along with these, a major regulator of glycolysis, i.e., GLUT1 and HIF1\u0026alpha;, was checked in both cell lines (Figure: 03(b), Figure 04).\u003c/p\u003e\n\u003cp\u003eUpon validation, selected DEGs showed downregulation in cisplatin-resistant EC cells and FGFRL1 knockdown EC cell line KYSE410. Also, GLUT1 and HIF1\u0026alpha;was found to be upregulated specifically in the CISR cell line as compared to its parental. In FGFRL1-EGFP transfected CISR cells, significantly reduced levels of AQP3, GFPT2, GLUT1, and even CTSS were observed in comparison to the wild type (Figure: 03(c)(d)). Also, post-FGFRL1 C-domain deleted construct, FGFRL1\u0026Delta;C transfection, the expression of AQP3, GFPT2, and GLUT1 increased as compared to wild type (p\u0026lt;0.05) (Figure: 03(e-g)). These findings suggest that altered expression of FGFRL1 leads to differential expression of AQP3, GFPT2, GALNT5, and GLUT1 specifically in cisplatin-resistant esophageal cancer cells (CISR). Since AQP3, GFPT2, GALNT5, and GLUT1 are all genes related to glycolysis, and also in our earlier studies we have observed reduced levels of phosphorylated GSK3\u0026beta; post-FGFRL1 silencing in esophageal cancer cell line KYSE410 (Unpublished) (Figure 04 (I)), this gave us a strong indication to analyse whether the alterations in FGFRL1 expression \u0026nbsp;affect levels of glucose consumption, lactic acid, and glycogen production in cisplatin-resistant as well as esophageal cancer cells. Firstly, glucose consumption and production of lactic acid and glycogen in cisplatin-resistant (CISR) as well as esophageal cancer cells (KYSE140, KYSE410) were investigated. CISR cell line seems to consume more glucose, produce more lactic acid, and has less glycogen storage owing to its highly proliferative nature as compared to its parental cell line KYSE140 (Figure 04(II, a-c)). Whereas, esophageal cancer cell line KYSE410 shows consumption of glucose in similar amounts as CISR but produces less lactic acid and rather has high glycogen production. Furthermore, we also noted that FGFRL1 overexpression in both parental and cisplatin resistant esophageal cancer cells \u0026nbsp;and significantly increased the glycogen production as compared to lactic acid (Figure 05(II, e-f)).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFGFRL1 is a 504\u0026ndash;amino-acid transmembrane protein with three Ig-like extracellular domains, a single transmembrane helix, and a cytoplasmic C-terminal tail (20,21). Recent studies indicate that specific motifs located within the C-terminal domain may be critical for mediating downstream signaling. Genomic analyses have revealed the potential presence of SH2- and PDZ-binding motifs, as well as immunoreceptor tyrosine-based activation (ITAM) and inhibitory motifs (ITIM) within the histidine-rich region of the C-terminal domain, suggesting a role in the regulation of signaling pathways(9). Furthermore, Silva et al. reported that an SH2 binding motif in the cytoplasmic domain of FGFRL1 interacts with SHP1, thereby promoting ERK1/2 signaling (10).\u003c/p\u003e\u003cp\u003eIn a 2006 study, Trueb and Taeschler examined FGFRL1 expression during embryonic development in mice and found that FGFRL1 mRNA is primarily expressed in cartilaginous structures, including the bone primordia and the permanent cartilage of the trachea, ribs, and nose, as well as in certain muscles, such as the tongue and diaphragm (14\u0026ndash;15). Further research by Trueb and colleagues demonstrated that mice with targeted Fgfrl1 gene disruption exhibited neonatal lethality due to respiratory failure, attributed to a severely underdeveloped diaphragm, although the muscle fibers remained structurally differentiated, suggesting its role in diaphragm development(22).\u003c/p\u003e\u003cp\u003eBeyond its role in development, FGFRL1 has gained attention in cancer biology due to its context-dependent dual roles. Several reports have shown the oncogenic potential of FGFRL1 in certain tumor types. For instance, FGFRL1 knockdown in ovarian cancer cells resulted in reduced Gli1 and Gli2 expression, implicating its role in regulating the Hedgehog (Hh) signaling pathway. Moreover, decreased phosphorylation of MEK and ERK following FGFRL1 silencing suggests that FGFRL1 may promote tumorigenesis through activation of the MAPK pathway (23). Conversely, other studies point to a tumor-suppressive role for FGFRL1. Trueb and colleagues have proposed FGFRL1 to function as a ligand trapping receptor that binds fibroblast growth factors (FGF2, FGF3, FGF4, FGF8, FGF10, FGF22) without initiating intracellular signaling, thereby inhibiting the FGF pathway by sequestering ligands away from functional FGFRs (20,21,25\u0026ndash;26). This decoy activity was further supported by studies in \u003cem\u003eXenopus\u003c/em\u003e embryos, where injection of FGFRL1 mRNA led to developmental defects resembling those caused by a truncated, dominant-negative FGFR1, reinforcing the idea that FGFRL1 antagonizes FGF signaling. These findings illustrate the complex and context-specific nature of FGFRL1 in cancer, where its role in tumorigenesis can be either promoting or inhibitory, depending on the cellular and molecular environment (24).\u003c/p\u003e\u003cp\u003e​Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) has emerged as a significant player in esophageal cancer (EC), particularly esophageal squamous cell carcinoma (EC). Studies have reported that FGFRL1 expression is significantly upregulated in EC tissues (22). Additionally, tumor-suppressive miRNAs \u003cem\u003eviz.\u003c/em\u003e miR-107 and miR-210 are reported to regulate FGFRL1 expression, indicating a complex regulatory mechanism that influences tumor progression.(13,23).​ Esophageal cancer (EC) remains an aggressive malignancy with poor survival rates, largely due to chemoresistance, which significantly limits the effectiveness of neoadjuvant chemotherapy (NACT)(24). Although NACT followed by surgery remains the established treatment approach for locally advanced EC, nearly half of the patients fail to respond, leading to disease progression, metastasis, and poor prognosis (24\u0026ndash;25). Herein, we focused on FGFRL1 and its potential role in chemoresistance in EC, which has not been thoroughly explored.\u003c/p\u003e\u003cp\u003eWe observed reduced FGFRL1 mRNA and protein levels in cisplatin-resistant EC cells (CISR). Moreover, patients exhibiting partial or no response to chemotherapy showed significantly lower FGFRL1 protein expression compared to those with a complete response, indicating a potential correlation between FGFRL1 levels and chemotherapy response.\u003c/p\u003e\u003cp\u003eInterestingly, further in-depth analysis demonstrates significantly reduced levels of JAG1/DLL ligands, Notch1-2 receptors, and the transcription factor HES1 in CISR cells compared to parental EC cells. Notch signaling has been extensively implicated in driving chemoresistance across multiple cancer types, such as head and neck squamous cell carcinoma, colorectal cancer, and ovarian cancer (27\u0026ndash;30). Notch pathway activation has been shown in 5-FU-resistant colon cancer cells, with increased NICD protein and HES1 expression (31). High Notch2 and JAG1 expression is linked to gemcitabine resistance in pancreatic cancer cells (32\u0026ndash;33). Although Notch signaling is often associated with chemoresistance, emerging evidence suggests that its downregulation, including decreased expression of Notch ligands and HES1, may contribute to adaptive survival mechanisms in certain cisplatin-resistant cancer models (34\u0026ndash;35). Overexpressing FGFRL1 in CISR cells led to a significant reduction in cell proliferation, clonogenic potential, and migration. Additionally, FGFRL1 overexpression, combined with increasing cisplatin doses, reduced CISR cell proliferation, suggesting that FGFRL1 may regulate chemosensitivity in EC cells.\u003c/p\u003e\u003cp\u003eTo further explore the molecular mechanism, we identified and validated two commonly downregulated genes ( CTSS and GALNT5) and two upregulated genes (AQP3, GFPT2) in both CISR cells and FGFRL1-knockdown EC cells. Among CTSS and GALNT5 were downregulated, while AQP3 and GFPT2 were upregulated in metabolic pathways, including glycolysis and the hexosamine biosynthesis pathway (HBP). GLUT1, a key glycolysis regulator, was upregulated in CISR cells compared to parental cells. Notably, FGFRL1 overexpression in CISR cells affected AQP3 and GFPT2 mRNA levels but not in the KYSE410 EC cell line.\u003c/p\u003e\u003cp\u003eGFPT2, a key enzyme in the hexosamine biosynthetic pathway (HBP), is upregulated in mesenchymal cells, particularly during partial epithelial\u0026ndash;mesenchymal transition (EMT) (36). The HBP leads to O-GlcNAcylation of proteins, including Sp1, which promotes AQP3 expression. AQP3, responsible for glycerol influx, compensates for reduced glucose uptake(37). The increased AQP3 and GFPT2 levels in CISR cells suggest that glucose and lactic acid homeostasis are altered to support enhanced glycolysis and amino acid biosynthesis, which are critical for meeting cellular energy demands. Furthermore, the high glucose consumption, increased lactic acid production, and low glycogen levels in CISR cells support this hypothesis. Interestingly, AQP3 and GFPT2 levels were downregulated post FGFRL1 overexpression, further supporting this hypothesis. FGFRL1 overexpression in CISR cells altered this balance, increasing glycogen levels while reducing lactic acid production. In our previous studies, we observed that FGFRL1 knockdown in EC cells resulted in reduced Ser9 phosphorylation of glycogen synthase kinase 3 beta (GSK3β) which is a key regulator of glycogen synthesis (Unpublished). Active GSK3β maintains glycogen synthase (GS) in an inactive state by phosphorylation, and functionally inactive Ser9-phosphorylated GSK3β fails to phosphorylate GS, leading to increased glycogen production. In CISR cells, reduced FGFRL1 levels may enhance GS activity and glycogen synthesis. FGFRL1 overexpression in CISR cells disrupted the glucose\u0026ndash;lactic acid balance by influencing glycogen synthesis, thereby restoring a more balanced metabolic state. In conclusion, our study provides evidence that FGFRL1 modulates key signaling pathways, including Notch, and metabolic pathways, which contribute to chemoresistance in EC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study highlights FGFRL1 as a critical regulator of chemoresistance and metabolic reprogramming in cisplatin-resistant esophageal cancer cells. FGFRL1 overexpression suppressed cell proliferation, migration, and colony formation while concomitantly enhancing glycogen production, suggesting a metabolic shift in esophageal cancer cells. These results indicate that targeting FGFRL1 expression could represent a novel therapeutic approach to overcome chemoresistance and metabolic adaptations in esophageal cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conceived and designed with contributions from all authors. Investigation and data curation were carried out by Aprajita. Conceptualization, investigation, and supervision were provided by Dr. Rinu Sharma. The initial manuscript draft was written by Aprajita, with all authors reviewing and commenting on previous versions. Dr. N.R. Dash served as the clinician, providing tissue samples and performing the surgical procedures. The final manuscript was read and approved by all authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData generated in the current study will be made available on request by contacting \u0026nbsp;the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study ethicaly cleared from the Institutional Research Ethics Committee of the A.I.I.M.S, New Delhi (Ref. No.: IEC-545/06.08.2021), as well as from the Institutional Research Ethics Committee of GGSIP University, Dwarka, New Delhi (Ref. No.: GGSIPU/IEC/2021-A1). All procedures where human participants were involved were conducted in compliance with the ethical guidelines of both committees, 1964 Declaration of Helsinki \u0026nbsp;principles and its subsequent amendments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided written informed consent prior to enrollment in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no conflicts of interest related to the content of this manuscript. They further confirm that no financial or personal relationships exist that influenced the work presented here.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement :\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding: Financial support for this study was provided by Guru Gobind Singh Indraprastha University (GGSIPU; Grant No. GGSIPU/DRC/FRGS/2021/594/17) and SERB, DST (Grant No. CRG/2022/003194).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHerskovic A, Russell W, et al. Esophageal carcinoma: advances in treatment results for locally advanced disease. \u003cem\u003eAnn Oncol.\u003c/em\u003e 2012;23(5):1095-1103. doi:10.1093/annonc/mdr602\u003c/li\u003e\n \u003cli\u003eAjani JA, D\u0026rsquo;Amico TA, et al. 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O-GlcNAc modification mediates aquaporin 3 to coordinate endometrial cell glycolysis and affects embryo implantation. \u003cem\u003eJ Adv Res.\u003c/em\u003e 2023;47:153-166. doi:10.1016/j.jare.2022.11.008\u003c/li\u003e\n\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-biochemistry-and-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbbi","sideBox":"Learn more about [Cell Biochemistry and Biophysics](http://link.springer.com/journal/12013)","snPcode":"12013","submissionUrl":"https://submission.nature.com/new-submission/12013/3","title":"Cell Biochemistry and Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"FGFRL1, esophageal cancer, chemoresistance, Notch signaling, glucose metabolism, neoadjuvant chemotherapy","lastPublishedDoi":"10.21203/rs.3.rs-7525805/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7525805/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChemoresistance remains a significant barrier to the treatment of esophageal cancer (EC), regulated by metabolic and signaling adaptations. Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) is a key regulator of cancer progression; however, its involvement in driving chemoresistance remains poorly understood. This study investigates the functional significance of FGFRL1 in chemo-resistant EC cells and its association with response to chemotherapy. FGFRL1 expression was analyzed in cisplatin-resistant EC cells using real-time PCR and Western blotting. FGFRL1 protein levels were examined in clinical specimens from EC patients post-neoadjuvant chemotherapy (NACT) to evaluate their correlation with treatment response using immunohistochemistry. Significantly decreased expression of FGFRL1 was observed in cisplatin-resistant EC cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Interestingly, overexpression of FGFRL1 suppressed proliferation, migration, and clonogenic potential (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while activating Notch signaling via JAG1, DLL1, DLL4, NOTCH1, NOTCH2, and HES1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in cisplatin-resistant EC cells. FGFRL1 overexpression also shifted glucose metabolism toward glycogen synthesis, involving regulators GFPT2, AQP3, and GALT5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In patient specimens, FGFRL1 upregulation was significantly associated with chemotherapy response, observed in 80% of complete responders versus 36.4% of non-responders (p\u0026thinsp;=\u0026thinsp;0.000, OR\u0026thinsp;=\u0026thinsp;8.61). We report for the first time that FGFRL1 regulates metabolic and signaling pathways in chemo-resistant EC, suggesting its potential as a drug target to counter resistance.\u003c/p\u003e","manuscriptTitle":"FGFRL1 Modulates Notch Signaling and Glucose-Glycogen Homeostasis to Suppress Chemoresistance in Esophageal Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 04:53:56","doi":"10.21203/rs.3.rs-7525805/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-19T16:35:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-13T14:49:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-13T12:55:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60434904613972960867453735206381488415","date":"2025-09-09T05:44:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240977762301728322166084015255984100957","date":"2025-09-08T09:30:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39016970522630899588237167619026921849","date":"2025-09-05T22:34:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-05T17:09:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-05T06:40:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-05T06:40:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Biochemistry and Biophysics","date":"2025-09-03T10:08:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-biochemistry-and-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbbi","sideBox":"Learn more about [Cell Biochemistry and Biophysics](http://link.springer.com/journal/12013)","snPcode":"12013","submissionUrl":"https://submission.nature.com/new-submission/12013/3","title":"Cell Biochemistry and Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4b2cf868-0de5-4a6f-adc0-8073d6fdace5","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:04:32+00:00","versionOfRecord":{"articleIdentity":"rs-7525805","link":"https://doi.org/10.1007/s12013-026-02065-5","journal":{"identity":"cell-biochemistry-and-biophysics","isVorOnly":false,"title":"Cell Biochemistry and Biophysics"},"publishedOn":"2026-04-07 15:58:16","publishedOnDateReadable":"April 7th, 2026"},"versionCreatedAt":"2025-09-17 04:53:56","video":"","vorDoi":"10.1007/s12013-026-02065-5","vorDoiUrl":"https://doi.org/10.1007/s12013-026-02065-5","workflowStages":[]},"version":"v1","identity":"rs-7525805","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7525805","identity":"rs-7525805","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}