HGF/c-Met Signaling induces Glutamine Metabolism in Head and Neck Squamous Cell Carcinoma via MAPK/ERK-Dependent Induction of GLS-1

HGF/c-Met Signaling induces Glutamine Metabolism in Head and Neck Squamous Cell Carcinoma via MAPK/ERK-Dependent Induction of GLS-1 | 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 HGF/c-Met Signaling induces Glutamine Metabolism in Head and Neck Squamous Cell Carcinoma via MAPK/ERK-Dependent Induction of GLS-1 Marius Hörner, Florian Mersdorf, Andreas Vollmer, Tobias Renner, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8233600/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Background Head and neck squamous cell carcinoma (HNSCC) is a common malignancy characterized by poor survival due to recurrence, metastasis and therapy resistance. In addition to genetic alterations, metabolic reprogramming is a hallmark of HNSCC and contributes to tumor progression and treatment failure. The hepatocyte growth factor (HGF)/c-Met signaling pathway is frequently activated in head and neck squamous cell carcinoma (HNSCC), where it promotes tumor cell proliferation, invasion, and increased glucose metabolism. However, its contribution to the regulation of glutamine metabolism in HNSCC remains largely unexplored. Methods Human HNSCC cell lines (FaDu, SCC-154, and Detroit562) were stimulated with HGF. Expression of glutaminase 1 (GLS-1) was analyzed by quantitative PCR and Western blotting. The functional relevance of GLS-1 was evaluated by pharmacological inhibition and genetic silencing using siRNA. Cell viability and migratory capacity were assessed by wound-healing assays. Results HGF stimulation induced a pronounced increase in GLS-1 expression in HNSCC cell lines, as confirmed by qPCR and Western blotting. To elucidate the signaling mechanisms underlying this regulation, we next analyzed major c-Met downstream pathways. HGF treatment led to strong ERK1/2 phosphorylation. Pharmacological inhibition of c-Met with Foretinib or blockade of MEK1/2 with U0126 abolished ERK1/2 phosphorylation and prevented the HGF-induced upregulation of GLS-1 protein levels. These results demonstrate that HGF/c-Met–driven GLS-1 expression is mediated through activation of the MAPK/ERK pathway. Moreover, GLS-1 silencing by siRNA significantly impaired wound closure, indicating reduced proliferative and migratory capacity. Conclusion Our findings show that HGF/c-Met signaling activates the MAPK/ERK pathway to induce GLS-1 expression, thereby promoting glutamine metabolism and tumor cell motility in HNSCC. Consequently, targeting glutaminase or ERK signaling may represent a promising therapeutic approach to counteract HGF/c-Met–driven metabolic reprogramming and therapy resistance in HNSCC. HNSCC MAPK/ERK HGF Glutamine metabolism MET-signaling Figures Figure 1 Figure 2 Figure 3 Introduction Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer worldwide, with nearly 900,000 new cases annually and mortality rates approaching 50% (Johnson et al., 2020 , Ferlay et al., 2019 ). The high lethality is primarily due to late-stage diagnosis, loco-regional recurrence, and resistance to current therapeutic regimens. Despite advances in surgery, radiotherapy, chemotherapy, and immunotherapy, treatment options remain limited, particularly for patients with recurrent or metastatic disease. The incidence of oral cancer is closely associated with detrimental oral-related behaviors such as tobacco smoking and excessive alcohol consumption, as well as exposure to human papillomavirus (HPV). Both smoking and alcohol have been linked to altered cellular metabolism and tumorigenesis (Johnson et al., 2020 ). Indeed, metabolic reprogramming is a hallmark of cancer and enables tumor cells to adapt to nutrient stress and sustain uncontrolled proliferation (Qin et al., 2025 ). Classical metabolic adaptations include enhanced glycolytic flux, the so-called Warburg effect, as well as the utilization of alternative energy sources such as glutamine through glutaminolysis (Hartmann et al., 2016 ). Glutamine serves as a critical nutrient for proliferating cancer cells, providing carbon and nitrogen to support anabolic processes including nucleotide, amino acid, and lipid biosynthesis. The rate-limiting enzyme glutaminase 1 (GLS-1) catalyzes the conversion of glutamine to glutamate, fueling the tricarboxylic acid (TCA) cycle and promoting tumor growth. Upregulation of GLS-1 has been reported in several cancers and is associated with aggressive tumor behavior and poor prognosis (Xiang et al., 2019 ). The hepatocyte growth factor (HGF)/c-Met signaling pathway is a receptor tyrosine kinase axis that plays essential roles in embryonic development, tissue regeneration, and wound healing. In cancer, aberrant activation of this pathway promotes cell proliferation, invasion, angiogenesis, and metastasis. Overexpression of c-Met has been reported in more than 80% of HNSCC cases and correlates with poor prognosis (Seiwert et al., 2009 ). Moreover, HGF/c-Met signaling contributes to therapeutic resistance, including resistance to EGFR inhibitors such as Cetuximab (Wheeler et al., 2008 ). While HGF/c-Met signaling has been linked to enhanced glucose metabolism, its impact on glutamine utilization in HNSCC remains poorly understood. Given the central role of glutamine metabolism in tumor progression, elucidating the interaction between HGF/c-Met signaling and GLS-1 dependent metabolic regulation may reveal new therapeutic vulnerabilities. In this study, we investigated whether HGF stimulation modulates GLS-1 expression and glutamine metabolism in HNSCC cells and evaluated the functional and therapeutic implications of GLS-1 inhibition in this context. Material and Methods Cell lines Among the cell lines listed in Table 11 are established, adherent tumor cells derived from squamous cell carcinomas of the head and neck region. SCC-154 and FaDu originate from the respective primary tumors, while Detroit 562 was derived from pleural metastases (ATCC, 2018). The tumor cells were cultured in an incubator at 37°C with 5% CO₂. Cultures were split twice weekly and reseeded in fresh medium (composition shown in Table 12). The cell lines were routinely tested for possible mycoplasma contamination during the experiments. The following steps were performed using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza, Oregon, USA): 2 ml tube of cell culture supernatant were transferred into a 1.5 ml microcentrifuge tube and centrifuged for 5 min at 200 RCF. Subsequently, 100 µl of the supernatant were transferred into a 96-well microtiter plate (655094), followed by the addition of 100 µl MycoAlert PLUS Reagent. After 5 min incubation, luminescence was measured. Then, 100 µl MycoAlert PLUS Substrate was added to each well, and a second luminescence measurement was performed after 10 min. siRNA The cells were cultured in 6-well plates for a period of 24 hours prior to transfection as described before (Nagler et al., 2023 ). This was achieved by the introduction of human GLS-1- specific siRNA (Thermo Fisher Scientific, USA), with non-target siRNA serving as the control (Abcam, UK). Transfection was initiated once the cells had reached 80% confluence. The transfection was conducted using Lipofectamine Transfection LTX Reagent (Thermo Fisher Scientific, USA), in accordance with the instructions provided by the manufacturer. Based on preliminary experiments, sufficient knockdown GLS-1 was observed after 24 hours (Western bots), and all subsequent experiments were conducted at this time point. Western Blot For Western blotting, Detroit 562, FaDu, and SCC-154 cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors, as previously described in (Kelm et al., 2025 ). Protein lysates were homogenized and quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific, Massachusetts, USA). Equal amounts of protein were separated by SDS–PAGE and transferred onto nitrocellulose membranes. Membranes were blocked and incubated overnight at 4°C with the respective primary antibodies (Table 1 ), followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using the SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Massachusetts, USA). Densitometric analysis and normalization to β-actin were performed using ImageLab software (Bio-Rad, Hercules, CA, USA). Table 1 Antibodies, Growth Factors and Inhibitors used in this study. Primary and Secondary Antibodies and Their Dilutions used for Western Blots (WB), in vitro (iv) and Immunofluorescence (IF) Antibody Catalog No. source Application Glutaminase-1 #49363 Cell Signaling Technology, Danvers, USA WB Glutaminase-1/GLS-1 #56750 Cell Signaling Technology, Danvers, USA WB / IF β-Actin #A5316 Sigma Aldrich, St. Louis, USA WB Erk1/2 (9107), Selleckchem Houston, TX, USA WB P-Erk1/2 (4370) Selleckchem Houston, TX, USA WB Secondary Ab (HRP) #7074 Cell Signaling Technology, Danvers, USA WB Alexa Fluor 488 Ab (IF) #4412S Cell Signaling Technology, Danvers, USA IF Foretinib GSK-1363029 Selleckchem (Houston, TX, USA iv FR180204 FR 180204 Sigma-Aldrich, St. Louis, MO, USA iv Table 2 Table 2 lists the cell lines used in this study, including their tissue of origin and supplier. Name Tissue origin supplier SCC-154 Tongue German Collection of Microorganisms and Cell Cultures (DSMZ) FaDu Pharynx Head and Neck Cancer Panel (TCP-1012), American Type Culture Collection (ATCC), Manassas, Virginia, USA Detroit 562 Pharyngeal metastases, pleura American Type Culture Collection (ATCC), Manassas, Virginia, USA Scratch assay Detroit 562 cell monolayers were cultured in 6-well plates until reaching confluence. Linear wounds were generated using a 10 µL sterile pipette tip, as previously described in (Hörner et al., 2024 ). All scratches were performed by the same blinded investigator to ensure reproducibility, followed by replacement of the culture medium. Images of the wound area were captured at 0, 24 and 48 post-scratch. Wound closure was quantified using ImageJ software by measuring the remaining wound area relative to the initial (t = 0) area. Cells were treated with HGF (50 ng/mL), Foretinib (100 nM), FR180204 (10 µM), or the respective combinations as indicated. qPCR Total RNA was extracted from Detroit 562 cells using TRIzol™ reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the iScript™ cDNA Synthesis Kit (Bio-Rad, Munich, Germany). Quantitative PCR was performed with MESA GREEN qPCR MasterMix Plus for SYBR® Assay (Eurogentec, Cologne, Germany) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Munich, Germany). Relative gene expression was calculated using the ΔΔCt method, with β-actin as the reference gene. Reactions were run in duplicate at an annealing temperature of 60°C, with primer concentrations of 5 µM. Primer sequences are listed in Table 3 . Table 3 Table 3 lists the qPCR primers. Prime gene Supplier Hs_ACTB_2_SG Qiagen, Venlo, Netherlands; Order No.: 3507814 Hs_GLS_1_SG Qiagen, Venlo, Netherlands Order No.: 3507814 Statistics Statistical analyses were performed using Prism (GraphPad Software, La Jolla, CA, USA). Data are presented as mean ± SEM, and p < 0.05 was considered statistically significant. Normality was assessed using the Shapiro–Wilk test. Two-group comparisons were conducted using paired Student’s t-tests or Mann–Whitney U tests, as appropriate. Multiple-group analyses were performed using one-way ANOVA with Tukey’s or Šídák’s multiple comparisons test or Bonferroni correction; non-parametric data were analyzed using the Kruskal-Wallis test. Results HGF/c-Met signaling induces GLS-1 expression in Met-amplified HNSCC cells Previous studies have demonstrated that stimulation of the c-Met signaling pathway by HGF enhances the expression of key glycolytic enzymes in HNSCC (Boschert et al., 2020a ). However, whether HGF/c-Met signaling also affects glutamine metabolism has remained unclear. We therefore hypothesized that HGF stimulation modulates the glutamine metabolic program of HNSCC cells through c-Met dependent mechanisms. To test this, we analyzed the expression of glutaminase 1 (GLS-1), the rate-limiting enzyme in glutamine metabolism, in three representative HNSCC cell lines with distinct MET status: Detroit 562 (MET-amplified), and FaDu and SCC-154 (both MET wild-type). Cells were treated with recombinant HGF (50 ng/mL) for 24 h, and GLS-1 expression was quantified by qPCR (Fig. 1 A) and Western blot analysis (Fig. 1 B). HGF stimulation markedly increased GLS-1 mRNA levels in MET-amplified Detroit 562 cells (3.41 ± 0.33-fold vs. control), whereas no significant changes were observed in FaDu (0.42 ± 0.18) or SCC-154 cells (0.95 ± 0.35 ;Fig. 1 A). To determine whether this regulation also occurred at the protein level, GLS-1 expression was examined by Western blot analysis (Fig. 1 B). Consistent with the qPCR results, HGF treatment led to a robust increase in GLS-1 protein levels in MET-amplified Detroit 562 cells, showing several-fold higher expression compared to untreated controls (Fig. 1 C). In contrast, FaDu and SCC-154 cells showed no significant changes in GLS-1 expression under identical conditions. To confirm that GLS-1 induction depends on c-Met activity, Detroit 562 cells were treated with the selective c-Met inhibitor Foretinib prior to HGF stimulation (Fig. 1 D). HGF stimulation markedly increased GLS-1 protein levels in Detroit 562 cells, whereas co-treatment with the c-Met inhibitor Foretinib completely abrogated this effect, restoring GLS-1 expression to baseline levels. Treatment with Foretinib alone had no detectable impact on GLS-1 expression (Fig. 1 D-E). Collectively, these findings identify GLS-1 as a downstream effector of HGF/c-Met signaling in MET-amplified HNSCC cells and establish a mechanistic connection between c-Met activation and glutamine metabolism as a key component of tumor-associated metabolic reprogramming. HGF/c-Met signaling induces GLS-1 expression via MAPK/ERK activation To elucidate the signaling mechanisms underlying HGF-induced GLS-1 upregulation, we focused first on the MAPK/ERK pathway. This choice was based on our previous glucose-metabolism studies, in which neither PI3K/AKT nor JAK/STAT signaling contributed to the metabolic switch, making MAPK/ERK the most plausible candidate for further investigation in MET-amplified Detroit 562 cells (Boschert et al., 2020a ). Quantitative PCR analysis showed that HGF stimulation led to a marked increase in GLS-1 mRNA levels, whereas co-treatment with the selective ERK1/2 inhibitor FR180204 completely abolished this effect (Fig. 2 A). Western blot analysis confirmed that HGF induced a strong phosphorylation of ERK1/2 Tyr204 and concomitantly elevated GLS-1 protein levels (Fig. 2 B). Densitometric quantification revealed a significant increase in GLS-1 protein expression and p-ERK/ERK ratios following HGF stimulation, both of which were reduced to baseline upon ERK inhibition (Fig. 2 C). Treatment with the inhibitor alone had no detectable effect on GLS-1 or ERK phosphorylation. Functionally, HGF stimulation significantly enhanced cell motility, as evidenced by accelerated wound closure of 65.42 ± 5.13% after 24 h and 89.67 ± 3.25% after 48 h, compared to 40.28 ± 4.31% and 70.54 ± 6.02% in control cells. In contrast, pharmacological inhibition of ERK1/2 with FR180204 abrogated this pro-migratory effect, reducing wound closure to near baseline levels (Fig. 2 E–F). Collectively, these findings demonstrate that HGF/c-Met signaling activates the MAPK/ERK pathway, which in turn drives GLS-1 expression and promotes a migratory phenotype in MET-amplified HNSCC cells. siRNA-mediated GLS-1 knockdown impairs cell motility in HNSCC cells To evaluate the functional relevance of GLS-1 induction, we next assessed the effects of GLS-1 depletion on cell proliferation and migration. GLS-1 expression was efficiently silenced by siRNA in Detroit 562 cells, as confirmed by Western blot analysis showing a marked reduction of GLS-1 protein levels compared with non-targeting controls (Fig. 3A). Upon HGF stimulation, GLS-1 expression was strongly upregulated in control cells, whereas this effect was completely abolished in GLS-1 siRNA-transfected cells, indicating that HGF-induced GLS-1 elevation depends on intact GLS-1 expression (Fig. 3A–B). Following GLS-1 knockdown, wound-healing assays were performed to determine migratory capacity. GLS-1 depletion significantly delayed wound closure in Detroit 562 cells, indicating impaired migratory behavior (Fig. 3C-D). Graphical abstract The schematic illustration summarizes the proposed mechanism (graphical abstract.): HGF binding to the c-Met receptor activates the MAPK/ERK signaling pathway, resulting in increased GLS-1 expression and enhanced cell motility. Inhibition of c-Met or ERK1/2, or GLS-1 silencing, disrupts this signaling axis and suppresses HGF-driven migration in MET-amplified HNSCC cells. Collectively, these results identify GLS-1 as a critical downstream effector that promotes cell motility in HNSCC, emphasizing its pivotal role in mediating HGF/c-Met–induced tumor progression. Discussion Head and neck squamous cell carcinoma (HNSCC) remains a challenging malignancy with poor prognosis due to frequent recurrence and therapy resistance. Beyond genetic alterations, recent evidence highlights metabolic reprogramming as a key driver of tumor progression and immune escape. Previous reports have shown that HGF enhances glucose metabolism via glycolytic reprogramming in HNSCC (Boschert et al., 2020a ). Our data extend this concept by demonstrating that c-Met activation also drives glutamine metabolism through GLS-1 induction. Such dual control of glucose and glutamine pathways highlights a broader metabolic plasticity of HNSCC cells under HGF stimulation. In line with this, other oncogenic signaling axes such as the PGE₂/PTEN pathway have also been shown to modulate tumor metabolism and growth in HNSCC (Nguyen et al., 2025 ), underscoring the central role of metabolic reprogramming in disease progression. HGF stimulation markedly upregulated GLS-1 expression, most prominently in MET-amplified Detroit 562 cells, as demonstrated by qPCR and Western blot analysis. To elucidate the underlying signaling mechanism, we examined major c-Met downstream pathways. HGF treatment induced robust phosphorylation of ERK1/2 in MET-amplified Detroit 562 cells. Pharmacological inhibition of c-Met with Foretinib or blockade of ERK1/2 with FR180204 abrogated ERK1/2 phosphorylation and prevented the HGF-induced increase in GLS-1 protein levels. Moreover, GLS-1 silencing by siRNA significantly reduced cell motility in wound-healing assays. These findings demonstrate that HGF/c-Met–driven GLS-1 upregulation is mediated via activation of the MAPK/ERK pathway. The MAPK/ERK cascade represents a central downstream effector of receptor tyrosine kinase signaling, integrating mitogenic and metabolic cues to regulate transcriptional programs that support cell growth and survival (Ngan et al., 2022 ). Through activation of transcription factors such as c-Myc, ERK signaling can promote the expression of metabolic enzymes including GLS-1, thereby coupling oncogenic signaling to glutamine metabolism. Functionally, GLS-1 knockdown by siRNA reduced cell proliferation and significantly impaired wound closure, indicating diminished proliferative and migratory capacity. Our data demonstrate that HGF/c-Met signaling drives glutamine metabolism via GLS-1 and that combined inhibition of GLS-1 and c-Met may have synergistic effects on tumor progression, representing a promising therapeutic strategy particularly in advanced or metastatic HNSCC. Among the molecular pathways implicated in HNSCC progression, the HGF/c-Met axis has attracted particular attention. This pathway promotes proliferation, migration, invasion, and metastasis (Arnold et al., 2017), and clinical data link HGF and c-Met overexpression to reduced survival in HNSCC patients (Szturz et al., 2017 ). HGF stimulation has further been shown to increase PD-L1 expression in renal carcinoma (Balan et al., 2015 ). A similar association was demonstrated HNSCC in previous work from our group (Boschert et al., 2020b). In hepatocellular carcinoma, HGF was also shown to promote glutaminolysis through activation of GLS-1 (Huang et al., 2019 ), suggesting a broader role in metabolic reprogramming. The pronounced upregulation of GLS-1 following HGF stimulation, particularly in MET-amplified Detroit 562 cells, underscores the metabolic relevance of the HGF/c-Met signaling axis in HNSCC. Detroit 562 cells originate from a metastasis of a pharyngeal carcinoma, suggesting that c-Met–driven glutamine metabolism may represent a metabolic adaptation associated with the metastatic phenotype (Boschert et al., 2020a ). Our findings expand this concept by linking c-Met activation to glutamine metabolism via upregulation of GLS-1, the rate-limiting enzyme in glutaminolysis. Interestingly, earlier reports have shown that c-Met activation can also induce PD-L1 expression in metastatic HNSCC, suggesting a broader role of this pathway at the intersection of oncogenic signaling, metabolic plasticity, and immune evasion (Boschert et al., 2020b). This dual regulation of metabolism and immune modulation by c-Met may provide a mechanistic rationale for combined targeting strategies in advanced disease. This finding is consistent with the observation that c-Met overexpression is associated with advanced tumor stages and metastasis (Szturz et al., 2017 , Di Renzo et al., 2000 ). The therapeutic implications of these findings are notable. While single-agent c-Met inhibitors such as Foretinib or Tivantinib have demonstrated limited efficacy in HNSCC (Seiwert et al., 2013 , Kochanny et al., 2020 ), our data suggest that dual targeting of c-Met and GLS-1 may circumvent compensatory signaling and synergistic antitumor effects. Of particular relevance in this context is Amivantamab, a bispecific EGFR/MET antibody currently approved for NSCLC and now being evaluated in HNSCC. Early results from the OrigAMI-4 trial indicate promising activity in patients progressing after checkpoint inhibition and chemotherapy, supporting the concept that combined inhibition of EGFR/MET signaling may enhance therapeutic responsiveness (Harrington et al., 2025 ). Together with our findings, these observations highlight a potential rationale for therapeutic strategies integrating MET blockade with metabolic targeting through GLS-1 inhibition. Next-generation glutaminase inhibitors such as CB-839 have demonstrated activity in several malignancies, including triple-negative breast cancer, renal cell carcinoma, and hematological cancers (Yu et al., 2021 ). Combination strategies targeting both c-Met and GLS-1 therefore represent a promising avenue for translational research in HNSCC. Our study is limited by the use of 2D cell culture models, which cannot fully capture the metabolic heterogeneity and immune interactions of the tumor microenvironment. Future work employing 3D spheroids, patient-derived organoids, and in vivo models will be essential to validate the HGF/c-Met–GLS-1 axis and explore its interplay with antitumor immunity. Moreover, glutamine availability plays a critical role in shaping immune responses, and the intersection between HGF/c-Met signaling, glutamine metabolism, and antitumor immunity warrants further investigation (Shah et al., 2020 , Hartmann et al., 2016 ). In summary, our findings suggest a mechanistic connection between oncogenic c-Met signaling and glutamine metabolism in HNSCC. Through ERK1/2 activation, HGF/c-Met induces GLS-1 expression and cellular motility. The identification of this pathway expands the functional spectrum of c-Met beyond its classical roles in proliferation and migration to include metabolic regulation. Given the established link between c-Met and PD-L1 induction, these results suggest a convergence of metabolic and immune escape mechanisms. Combined inhibition of c-Met and GLS-1, potentially in conjunction with immune checkpoint blockade, may therefore represent a promising therapeutic strategy for advanced or metastatic HNSCC. Declarations Declaration of interests: The authors have no relevant financial or non-financial interests to disclose. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution MH: Designing research studies; conducting experiments; acquiring data; analysing data and writing the manuscript. FM: Conducting experiments; acquiring data; writing the manuscript. AV: interpreting data; providing intellectual support. TR: interpreting data; writing the manuscript JV: interpreting data; providing intellectual support. AK: Providing intellectual support. 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Targeting GLS1 to cancer therapy through glutamine metabolism. Clin Transl Oncol. 2021;23:2253–68. Additional Declarations No competing interests reported. Supplementary Files GA.jpg Graphical abstract: HGF binding to the c-Met receptor activates the MAPK/ ERK1/2 signaling cascade, leading to transcriptional upregulation of GLS-1 and enhanced tumor cell motility. Pharmacological inhibition of c-Met (Foretinib) or ERK1/2 (FR180204) disrupts this pathway and attenuates GLS-1-mediated migration in HNSCC cells. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 10 Feb, 2026 Reviews received at journal 17 Jan, 2026 Reviews received at journal 17 Jan, 2026 Reviews received at journal 15 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviews received at journal 06 Jan, 2026 Reviewers agreed at journal 05 Jan, 2026 Reviewers agreed at journal 05 Jan, 2026 Reviewers agreed at journal 04 Jan, 2026 Reviewers invited by journal 04 Jan, 2026 Editor assigned by journal 28 Nov, 2025 Submission checks completed at journal 28 Nov, 2025 First submitted to journal 28 Nov, 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-8233600","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":569641298,"identity":"fb7edebe-5ece-4925-b5a4-b8bb869e7fe8","order_by":0,"name":"Marius Hörner","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYBACgwMwxmHmA4wNDAwgTKQWy+NsCSRqsT/PY0CkluPNxz4XVNxjMDvM801yZhuDbD8hLfZnjiXPnnGmGKiFd5vkxjYG45mErDG4kWPMzNuWANHycBtD4oYDBLXkf2bm/ZcADDGeZ2At+wlryWFm5m0Aa2GT3AiyhZBfDM4cM2bmOQbSwmZsOfOfhPEMgrYcb37MzFMD1HL+8MObPWdsZPsbCFkDBfVQhRJEqh8Fo2AUjIJRgBcAALYZRZt6x38PAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Oral and Maxillofacial Plastic-head and neck surgery University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Marius","middleName":"","lastName":"Hörner","suffix":""},{"id":569641299,"identity":"af76ca50-1d4d-4ea6-96d3-6d4239d7b433","order_by":1,"name":"Florian Mersdorf","email":"","orcid":"","institution":"Department of Oral and Maxillofacial Plastic-head and neck surgery University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Florian","middleName":"","lastName":"Mersdorf","suffix":""},{"id":569641300,"identity":"c8850d64-ab93-4b1a-8623-1b25352155f9","order_by":2,"name":"Andreas Vollmer","email":"","orcid":"","institution":"Department of Oral and Maxillofacial Plastic-head and neck surgery University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Vollmer","suffix":""},{"id":569641302,"identity":"a0f7b49a-2b6e-477e-a879-478d106fee80","order_by":3,"name":"Tobias Renner","email":"","orcid":"","institution":"Department of Oral and Maxillofacial Plastic-head and neck surgery University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tobias","middleName":"","lastName":"Renner","suffix":""},{"id":569641304,"identity":"eaf4436d-ae49-4618-84cc-40758a15ed59","order_by":4,"name":"Juian Volland","email":"","orcid":"","institution":"Department of Oral and Maxillofacial Plastic-head and neck surgery University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Juian","middleName":"","lastName":"Volland","suffix":""},{"id":569641308,"identity":"aaa08067-f63c-42b8-8214-6e3083e47803","order_by":5,"name":"Alexander Kübler","email":"","orcid":"","institution":"Department of Oral and Maxillofacial Plastic-head and neck surgery University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Kübler","suffix":""},{"id":569641309,"identity":"52a43050-9285-47af-ba0c-5fb256a472d2","order_by":6,"name":"Nicolas Schlegel","email":"","orcid":"","institution":"Department of General, Visceral, Transplant, Vascular and Pediatric Surgery, University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Schlegel","suffix":""},{"id":569641310,"identity":"402973c0-e530-4fbd-9a3e-417e2f74d912","order_by":7,"name":"Stefan Hartmann","email":"","orcid":"","institution":"Department of Oral and Maxillofacial Plastic-head and neck surgery University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Hartmann","suffix":""}],"badges":[],"createdAt":"2025-11-29 00:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8233600/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8233600/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99793552,"identity":"37fbf46c-0361-4d2e-b1ad-b87cd354f92f","added_by":"auto","created_at":"2026-01-08 13:31:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":936509,"visible":true,"origin":"","legend":"","description":"","filename":"finalHGFcMetHNSCC.docx","url":"https://assets-eu.researchsquare.com/files/rs-8233600/v1/71182f96b69707655c3279c2.docx"},{"id":99616758,"identity":"8cb73143-8eed-49a6-bbf0-4d40050fa201","added_by":"auto","created_at":"2026-01-06 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13:34:11","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":83412,"visible":true,"origin":"","legend":"","description":"","filename":"d7d31815f5e344cb95d22f086936411f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8233600/v1/28b38804bf758e434ea308ae.xml"},{"id":99616772,"identity":"74edc32d-cb6c-4420-a0b2-0906591a6155","added_by":"auto","created_at":"2026-01-06 13:34:11","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91118,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8233600/v1/52565beeb5c093a7b0bec305.html"},{"id":99793648,"identity":"61648ed0-33fa-404a-8c9f-441b81f5fc97","added_by":"auto","created_at":"2026-01-08 13:32:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":148533,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHGF induces GLS-1 expression in MET-amplified HNSCC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e(A) Quantitative RT–PCR analysis of GLS-1 mRNA expression in HNSCC cell lines (Detroit 562, FaDu, SCC-154) following HGF stimulation (50 ng/mL, 24 h). Data are presented as mean ± SEM from three independent experiments. (B) Representative Western blot analysis showing GLS-1 protein expression in Detroit562, FaDu and SCC-154 cell lines after HGF treatment, β-actin served as a loading control. (C) Quantification of GLS-1 protein expression relative to β-actin normalized to untreated control. (D) Representative Western blot analysis of GLS-1 protein levels in Detroit 562 cells treated with HGF (50 ng/mL, 24 h) in the presence or absence of the c-Met inhibitor Foretinib (100 nM, 2 h pre-treatment). (E) Quantification of GLS-1 optical density (OD) values normalized to β-actin and expressed as fold change relative to control. HGF-induced GLS-1 upregulation was abrogated by c-Met inhibition with Foretinib. Data represent mean ± SEM of at least three independent experiments. Data are shown as mean ± SEM and were analysed by one-way ANOVA followed by Tukey’s post hoc test; *p ≤ 0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8233600/v1/ea557f2b61122719d83a0618.jpg"},{"id":99793357,"identity":"1ebf62fe-2d50-42b8-a020-68ca9fd66d30","added_by":"auto","created_at":"2026-01-08 13:31:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":271780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMAPK/ERK signaling mediates HGF-induced GLS-1 upregulation and cell migration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e(A) Quantitative RT–PCR analysis of GLS-1 mRNA expression in Detroit 562 cells treated with HGF (50 ng/mL, 24 h) in the presence or absence of the selective ERK1/2 inhibitor FR180204 (10 µM, 2 h pre-treatment). Data are shown as mean ± SEM from three independent experiments. (B) Representative Western blot analysis of GLS-1, phosphorylated ERK1/2 (p-ERK), and total ERK protein levels following HGF stimulation and ERK inhibition. β-actin served as a loading control. (C) Densitometric quantification of GLS-1 protein expression relative to β-actin normalized to control. (D) Quantification of ERK activation shown as p-ERK/ERK ratio relative to control. (E) Representative wound-healing assay images showing enhanced migration after HGF stimulation and suppression of this effect by ERK inhibition at 24 h and 48 h. (F) Quantification of wound closure expressed as percentage of the initial wound area. Data are shown as mean ± SEM and were analysed by one-way ANOVA followed by Tukey’s post hoc test; *p ≤ 0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8233600/v1/37c69e910c77cf0385f43277.jpg"},{"id":99793917,"identity":"c5ee5655-4d6a-4826-b493-90a6274d0720","added_by":"auto","created_at":"2026-01-08 13:33:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGLS-1 knockdown suppresses HGF-driven GLS-1 expression and cell motility in Detroit 562 cells\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e(A) Representative Western blot analysis showing GLS-1 protein expression in Detroit 562 cells following HGF stimulation (50 ng/mL, 24 h) with or without GLS-1 knockdown by siRNA. β-actin served as a loading control. (B) Densitometric quantification of GLS-1 expression normalized to β-actin and presented as fold change relative to control. HGF treatment markedly increased GLS-1 protein levels, whereas GLS-1 siRNA silencing effectively suppressed both basal and HGF-induced GLS-1 expression. (C) Representative images of wound-healing assays at 24 h and 48 h post-scratch in control and GLS-1 siRNA-transfected Detroit 562 cells. (D) Quantification of wound closure relative to the initial scratch area (t = 0). GLS-1 knockdown significantly impaired wound healing, indicating reduced migratory capacity. Data are shown as mean ± SEM and were analysed by one-way ANOVA followed by Tukey’s post hoc test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8233600/v1/413cc179a006ed586c090657.jpg"},{"id":100356332,"identity":"be30498e-a037-4b23-93f2-3b4841829159","added_by":"auto","created_at":"2026-01-16 07:03:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1303542,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8233600/v1/7990809f-4bad-4ef7-8bc5-4c329583433b.pdf"},{"id":99616756,"identity":"35766260-0dd0-4c85-ab84-000a743dedee","added_by":"auto","created_at":"2026-01-06 13:34:11","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":95062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGraphical abstract:\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHGF binding to the c-Met receptor activates the MAPK/ ERK1/2 signaling cascade, leading to transcriptional upregulation of GLS-1 and enhanced tumor cell motility. Pharmacological inhibition of c-Met (Foretinib) or ERK1/2 (FR180204) disrupts this pathway and attenuates GLS-1-mediated migration in HNSCC cells.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"GA.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8233600/v1/e464643c00ee4c92df3730c5.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"HGF/c-Met Signaling induces Glutamine Metabolism in Head and Neck Squamous Cell Carcinoma via MAPK/ERK-Dependent Induction of GLS-1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHead and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer worldwide, with nearly 900,000 new cases annually and mortality rates approaching 50% (Johnson et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Ferlay et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The high lethality is primarily due to late-stage diagnosis, loco-regional recurrence, and resistance to current therapeutic regimens. Despite advances in surgery, radiotherapy, chemotherapy, and immunotherapy, treatment options remain limited, particularly for patients with recurrent or metastatic disease.\u003c/p\u003e \u003cp\u003eThe incidence of oral cancer is closely associated with detrimental oral-related behaviors such as tobacco smoking and excessive alcohol consumption, as well as exposure to human papillomavirus (HPV). Both smoking and alcohol have been linked to altered cellular metabolism and tumorigenesis (Johnson et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Indeed, metabolic reprogramming is a hallmark of cancer and enables tumor cells to adapt to nutrient stress and sustain uncontrolled proliferation (Qin et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Classical metabolic adaptations include enhanced glycolytic flux, the so-called Warburg effect, as well as the utilization of alternative energy sources such as glutamine through glutaminolysis (Hartmann et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Glutamine serves as a critical nutrient for proliferating cancer cells, providing carbon and nitrogen to support anabolic processes including nucleotide, amino acid, and lipid biosynthesis. The rate-limiting enzyme glutaminase 1 (GLS-1) catalyzes the conversion of glutamine to glutamate, fueling the tricarboxylic acid (TCA) cycle and promoting tumor growth. Upregulation of GLS-1 has been reported in several cancers and is associated with aggressive tumor behavior and poor prognosis (Xiang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe hepatocyte growth factor (HGF)/c-Met signaling pathway is a receptor tyrosine kinase axis that plays essential roles in embryonic development, tissue regeneration, and wound healing. In cancer, aberrant activation of this pathway promotes cell proliferation, invasion, angiogenesis, and metastasis. Overexpression of c-Met has been reported in more than 80% of HNSCC cases and correlates with poor prognosis (Seiwert et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Moreover, HGF/c-Met signaling contributes to therapeutic resistance, including resistance to EGFR inhibitors such as Cetuximab (Wheeler et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile HGF/c-Met signaling has been linked to enhanced glucose metabolism, its impact on glutamine utilization in HNSCC remains poorly understood. Given the central role of glutamine metabolism in tumor progression, elucidating the interaction between HGF/c-Met signaling and GLS-1 dependent metabolic regulation may reveal new therapeutic vulnerabilities. In this study, we investigated whether HGF stimulation modulates GLS-1 expression and glutamine metabolism in HNSCC cells and evaluated the functional and therapeutic implications of GLS-1 inhibition in this context.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eAmong the cell lines listed in Table\u0026nbsp;11 are established, adherent tumor cells derived from squamous cell carcinomas of the head and neck region. SCC-154 and FaDu originate from the respective primary tumors, while Detroit 562 was derived from pleural metastases (ATCC, 2018). The tumor cells were cultured in an incubator at 37\u0026deg;C with 5% CO₂. Cultures were split twice weekly and reseeded in fresh medium (composition shown in Table\u0026nbsp;12). The cell lines were routinely tested for possible mycoplasma contamination during the experiments. The following steps were performed using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza, Oregon, USA): 2 ml tube of cell culture supernatant were transferred into a 1.5 ml microcentrifuge tube and centrifuged for 5 min at 200 RCF. Subsequently, 100 \u0026micro;l of the supernatant were transferred into a 96-well microtiter plate (655094), followed by the addition of 100 \u0026micro;l MycoAlert PLUS Reagent. After 5 min incubation, luminescence was measured. Then, 100 \u0026micro;l MycoAlert PLUS Substrate was added to each well, and a second luminescence measurement was performed after 10 min.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003esiRNA\u003c/h3\u003e\n\u003cp\u003eThe cells were cultured in 6-well plates for a period of 24 hours prior to transfection as described before (Nagler et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This was achieved by the introduction of human GLS-1- specific siRNA (Thermo Fisher Scientific, USA), with non-target siRNA serving as the control (Abcam, UK). Transfection was initiated once the cells had reached 80% confluence. The transfection was conducted using Lipofectamine Transfection LTX Reagent (Thermo Fisher Scientific, USA), in accordance with the instructions provided by the manufacturer. Based on preliminary experiments, sufficient knockdown GLS-1 was observed after 24 hours (Western bots), and all subsequent experiments were conducted at this time point.\u003c/p\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cp\u003eFor Western blotting, Detroit 562, FaDu, and SCC-154 cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors, as previously described in (Kelm et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Protein lysates were homogenized and quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific, Massachusetts, USA). Equal amounts of protein were separated by SDS\u0026ndash;PAGE and transferred onto nitrocellulose membranes. Membranes were blocked and incubated overnight at 4\u0026deg;C with the respective primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using the SuperSignal\u0026trade; West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Massachusetts, USA). Densitometric analysis and normalization to β-actin were performed using ImageLab software (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibodies, Growth Factors and Inhibitors used in this study. Primary and Secondary Antibodies and Their Dilutions used for Western Blots (WB), \u003cem\u003ein vitro\u003c/em\u003e (iv) and Immunofluorescence (IF)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCatalog No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003esource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApplication\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutaminase-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#49363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology, Danvers, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutaminase-1/GLS-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#56750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology, Danvers, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWB / IF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-Actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#A5316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSigma Aldrich, St. Louis, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eErk1/2 (9107),\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSelleckchem Houston, TX, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-Erk1/2 (4370)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSelleckchem Houston, TX, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSecondary Ab (HRP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#7074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology, Danvers, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlexa Fluor 488 Ab (IF)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#4412S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology, Danvers, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForetinib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGSK-1363029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSelleckchem (Houston, TX, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eiv\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFR180204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFR 180204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSigma-Aldrich, St. Louis, MO, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eiv\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e lists the cell lines used in this study, including their tissue of origin and supplier.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTissue origin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003esupplier\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSCC-154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTongue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGerman Collection of Microorganisms and Cell Cultures (DSMZ)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFaDu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePharynx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHead and Neck Cancer Panel (TCP-1012), American Type Culture Collection (ATCC), Manassas, Virginia, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetroit 562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePharyngeal metastases, pleura\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmerican Type Culture Collection (ATCC), Manassas, Virginia, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eScratch assay\u003c/h3\u003e\n\u003cp\u003eDetroit 562 cell monolayers were cultured in 6-well plates until reaching confluence. Linear wounds were generated using a 10 \u0026micro;L sterile pipette tip, as previously described in (H\u0026ouml;rner et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). All scratches were performed by the same blinded investigator to ensure reproducibility, followed by replacement of the culture medium. Images of the wound area were captured at 0, 24 and 48 post-scratch. Wound closure was quantified using ImageJ software by measuring the remaining wound area relative to the initial (t\u0026thinsp;=\u0026thinsp;0) area. Cells were treated with HGF (50 ng/mL), Foretinib (100 nM), FR180204 (10 \u0026micro;M), or the respective combinations as indicated.\u003c/p\u003e\n\u003ch3\u003eqPCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from Detroit 562 cells using TRIzol\u0026trade; reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s instructions. Complementary DNA (cDNA) was synthesized from 1 \u0026micro;g of total RNA using the iScript\u0026trade; cDNA Synthesis Kit (Bio-Rad, Munich, Germany). Quantitative PCR was performed with MESA GREEN qPCR MasterMix Plus for SYBR\u0026reg; Assay (Eurogentec, Cologne, Germany) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Munich, Germany). Relative gene expression was calculated using the ΔΔCt method, with β-actin as the reference gene. Reactions were run in duplicate at an annealing temperature of 60\u0026deg;C, with primer concentrations of 5 \u0026micro;M. Primer sequences are listed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e lists the qPCR primers.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrime gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSupplier\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHs_ACTB_2_SG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQiagen, Venlo, Netherlands;\u003c/p\u003e \u003cp\u003eOrder No.: 3507814\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHs_GLS_1_SG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQiagen, Venlo, Netherlands\u003c/p\u003e \u003cp\u003eOrder No.: 3507814\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Prism (GraphPad Software, La Jolla, CA, USA). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Normality was assessed using the Shapiro\u0026ndash;Wilk test. Two-group comparisons were conducted using paired Student\u0026rsquo;s t-tests or Mann\u0026ndash;Whitney U tests, as appropriate. Multiple-group analyses were performed using one-way ANOVA with Tukey\u0026rsquo;s or Š\u0026iacute;d\u0026aacute;k\u0026rsquo;s multiple comparisons test or Bonferroni correction; non-parametric data were analyzed using the Kruskal-Wallis test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eHGF/c-Met signaling induces GLS-1 expression in Met-amplified HNSCC cells\u003c/h2\u003e\n\u003cp\u003ePrevious studies have demonstrated that stimulation of the c-Met signaling pathway by HGF enhances the expression of key glycolytic enzymes in HNSCC (Boschert et al., \u003cspan class=\"CitationRef\"\u003e2020a\u003c/span\u003e). However, whether HGF/c-Met signaling also affects glutamine metabolism has remained unclear. We therefore hypothesized that HGF stimulation modulates the glutamine metabolic program of HNSCC cells through c-Met dependent mechanisms.\u003c/p\u003e\n\u003cp\u003eTo test this, we analyzed the expression of glutaminase 1 (GLS-1), the rate-limiting enzyme in glutamine metabolism, in three representative HNSCC cell lines with distinct MET status: Detroit 562 (MET-amplified), and FaDu and SCC-154 (both MET wild-type). Cells were treated with recombinant HGF (50 ng/mL) for 24 h, and GLS-1 expression was quantified by qPCR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) and Western blot analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eHGF stimulation markedly increased GLS-1 mRNA levels in MET-amplified Detroit 562 cells (3.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33-fold vs. control), whereas no significant changes were observed in FaDu (0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18) or SCC-154 cells (0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 ;Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eTo determine whether this regulation also occurred at the protein level, GLS-1 expression was examined by Western blot analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistent with the qPCR results, HGF treatment led to a robust increase in GLS-1 protein levels in MET-amplified Detroit 562 cells, showing several-fold higher expression compared to untreated controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). In contrast, FaDu and SCC-154 cells showed no significant changes in GLS-1 expression under identical conditions. To confirm that GLS-1 induction depends on c-Met activity, Detroit 562 cells were treated with the selective c-Met inhibitor Foretinib prior to HGF stimulation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003eHGF stimulation markedly increased GLS-1 protein levels in Detroit 562 cells, whereas co-treatment with the c-Met inhibitor Foretinib completely abrogated this effect, restoring GLS-1 expression to baseline levels. Treatment with Foretinib alone had no detectable impact on GLS-1 expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). Collectively, these findings identify GLS-1 as a downstream effector of HGF/c-Met signaling in MET-amplified HNSCC cells and establish a mechanistic connection between c-Met activation and glutamine metabolism as a key component of tumor-associated metabolic reprogramming.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eHGF/c-Met signaling induces GLS-1 expression via MAPK/ERK activation\u003c/h2\u003e\n\u003cp\u003eTo elucidate the signaling mechanisms underlying HGF-induced GLS-1 upregulation, we focused first on the MAPK/ERK pathway. This choice was based on our previous glucose-metabolism studies, in which neither PI3K/AKT nor JAK/STAT signaling contributed to the metabolic switch, making MAPK/ERK the most plausible candidate for further investigation in MET-amplified Detroit 562 cells (Boschert et al., \u003cspan class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Quantitative PCR analysis showed that HGF stimulation led to a marked increase in GLS-1 mRNA levels, whereas co-treatment with the selective ERK1/2 inhibitor FR180204 completely abolished this effect (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eWestern blot analysis confirmed that HGF induced a strong phosphorylation of ERK1/2\u003csup\u003eTyr204\u003c/sup\u003e and concomitantly elevated GLS-1 protein levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Densitometric quantification revealed a significant increase in GLS-1 protein expression and p-ERK/ERK ratios following HGF stimulation, both of which were reduced to baseline upon ERK inhibition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Treatment with the inhibitor alone had no detectable effect on GLS-1 or ERK phosphorylation.\u003c/p\u003e\n\u003cp\u003eFunctionally, HGF stimulation significantly enhanced cell motility, as evidenced by accelerated wound closure of 65.42\u0026thinsp;\u0026plusmn;\u0026thinsp;5.13% after 24 h and 89.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25% after 48 h, compared to 40.28\u0026thinsp;\u0026plusmn;\u0026thinsp;4.31% and 70.54\u0026thinsp;\u0026plusmn;\u0026thinsp;6.02% in control cells. In contrast, pharmacological inhibition of ERK1/2 with FR180204 abrogated this pro-migratory effect, reducing wound closure to near baseline levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE\u0026ndash;F). Collectively, these findings demonstrate that HGF/c-Met signaling activates the MAPK/ERK pathway, which in turn drives GLS-1 expression and promotes a migratory phenotype in MET-amplified HNSCC cells.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003esiRNA-mediated GLS-1 knockdown impairs cell motility in HNSCC cells\u003c/h2\u003e\n\u003cp\u003eTo evaluate the functional relevance of GLS-1 induction, we next assessed the effects of GLS-1 depletion on cell proliferation and migration. GLS-1 expression was efficiently silenced by siRNA in Detroit 562 cells, as confirmed by Western blot analysis showing a marked reduction of GLS-1 protein levels compared with non-targeting controls (Fig.\u0026nbsp;3A). Upon HGF stimulation, GLS-1 expression was strongly upregulated in control cells, whereas this effect was completely abolished in GLS-1 siRNA-transfected cells, indicating that HGF-induced GLS-1 elevation depends on intact GLS-1 expression (Fig.\u0026nbsp;3A\u0026ndash;B).\u003c/p\u003e\n\u003cp\u003eFollowing GLS-1 knockdown, wound-healing assays were performed to determine migratory capacity. GLS-1 depletion significantly delayed wound closure in Detroit 562 cells, indicating impaired migratory behavior (Fig.\u0026nbsp;3C-D).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe schematic illustration summarizes the proposed mechanism (graphical abstract.): HGF binding to the c-Met receptor activates the MAPK/ERK signaling pathway, resulting in increased GLS-1 expression and enhanced cell motility. Inhibition of c-Met or ERK1/2, or GLS-1 silencing, disrupts this signaling axis and suppresses HGF-driven migration in MET-amplified HNSCC cells. Collectively, these results identify GLS-1 as a critical downstream effector that promotes cell motility in HNSCC, emphasizing its pivotal role in mediating HGF/c-Met\u0026ndash;induced tumor progression.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHead and neck squamous cell carcinoma (HNSCC) remains a challenging malignancy with poor prognosis due to frequent recurrence and therapy resistance. Beyond genetic alterations, recent evidence highlights metabolic reprogramming as a key driver of tumor progression and immune escape. Previous reports have shown that HGF enhances glucose metabolism via glycolytic reprogramming in HNSCC (Boschert et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Our data extend this concept by demonstrating that c-Met activation also drives glutamine metabolism through GLS-1 induction. Such dual control of glucose and glutamine pathways highlights a broader metabolic plasticity of HNSCC cells under HGF stimulation. In line with this, other oncogenic signaling axes such as the PGE₂/PTEN pathway have also been shown to modulate tumor metabolism and growth in HNSCC (Nguyen et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), underscoring the central role of metabolic reprogramming in disease progression.\u003c/p\u003e \u003cp\u003eHGF stimulation markedly upregulated GLS-1 expression, most prominently in MET-amplified Detroit 562 cells, as demonstrated by qPCR and Western blot analysis. To elucidate the underlying signaling mechanism, we examined major c-Met downstream pathways. HGF treatment induced robust phosphorylation of ERK1/2 in MET-amplified Detroit 562 cells. Pharmacological inhibition of c-Met with Foretinib or blockade of ERK1/2 with FR180204 abrogated ERK1/2 phosphorylation and prevented the HGF-induced increase in GLS-1 protein levels. Moreover, GLS-1 silencing by siRNA significantly reduced cell motility in wound-healing assays.\u003c/p\u003e \u003cp\u003eThese findings demonstrate that HGF/c-Met\u0026ndash;driven GLS-1 upregulation is mediated via activation of the MAPK/ERK pathway. The MAPK/ERK cascade represents a central downstream effector of receptor tyrosine kinase signaling, integrating mitogenic and metabolic cues to regulate transcriptional programs that support cell growth and survival (Ngan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Through activation of transcription factors such as c-Myc, ERK signaling can promote the expression of metabolic enzymes including GLS-1, thereby coupling oncogenic signaling to glutamine metabolism. Functionally, GLS-1 knockdown by siRNA reduced cell proliferation and significantly impaired wound closure, indicating diminished proliferative and migratory capacity.\u003c/p\u003e \u003cp\u003eOur data demonstrate that HGF/c-Met signaling drives glutamine metabolism via GLS-1 and that combined inhibition of GLS-1 and c-Met may have synergistic effects on tumor progression, representing a promising therapeutic strategy particularly in advanced or metastatic HNSCC.\u003c/p\u003e \u003cp\u003eAmong the molecular pathways implicated in HNSCC progression, the HGF/c-Met axis has attracted particular attention. This pathway promotes proliferation, migration, invasion, and metastasis (Arnold et al., 2017), and clinical data link HGF and c-Met overexpression to reduced survival in HNSCC patients (Szturz et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). HGF stimulation has further been shown to increase PD-L1 expression in renal carcinoma (Balan et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). A similar association was demonstrated HNSCC in previous work from our group (Boschert et al., 2020b).\u003c/p\u003e \u003cp\u003eIn hepatocellular carcinoma, HGF was also shown to promote glutaminolysis through activation of GLS-1 (Huang et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), suggesting a broader role in metabolic reprogramming.\u003c/p\u003e \u003cp\u003eThe pronounced upregulation of GLS-1 following HGF stimulation, particularly in MET-amplified Detroit 562 cells, underscores the metabolic relevance of the HGF/c-Met signaling axis in HNSCC. Detroit 562 cells originate from a metastasis of a pharyngeal carcinoma, suggesting that c-Met\u0026ndash;driven glutamine metabolism may represent a metabolic adaptation associated with the metastatic phenotype (Boschert et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Our findings expand this concept by linking c-Met activation to glutamine metabolism via upregulation of GLS-1, the rate-limiting enzyme in glutaminolysis. Interestingly, earlier reports have shown that c-Met activation can also induce PD-L1 expression in metastatic HNSCC, suggesting a broader role of this pathway at the intersection of oncogenic signaling, metabolic plasticity, and immune evasion (Boschert et al., 2020b). This dual regulation of metabolism and immune modulation by c-Met may provide a mechanistic rationale for combined targeting strategies in advanced disease.\u003c/p\u003e \u003cp\u003eThis finding is consistent with the observation that c-Met overexpression is associated with advanced tumor stages and metastasis (Szturz et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Di Renzo et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe therapeutic implications of these findings are notable. While single-agent c-Met inhibitors such as Foretinib or Tivantinib have demonstrated limited efficacy in HNSCC (Seiwert et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Kochanny et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), our data suggest that dual targeting of c-Met and GLS-1 may circumvent compensatory signaling and synergistic antitumor effects. Of particular relevance in this context is Amivantamab, a bispecific EGFR/MET antibody currently approved for NSCLC and now being evaluated in HNSCC. Early results from the OrigAMI-4 trial indicate promising activity in patients progressing after checkpoint inhibition and chemotherapy, supporting the concept that combined inhibition of EGFR/MET signaling may enhance therapeutic responsiveness (Harrington et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Together with our findings, these observations highlight a potential rationale for therapeutic strategies integrating MET blockade with metabolic targeting through GLS-1 inhibition. Next-generation glutaminase inhibitors such as CB-839 have demonstrated activity in several malignancies, including triple-negative breast cancer, renal cell carcinoma, and hematological cancers (Yu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Combination strategies targeting both c-Met and GLS-1 therefore represent a promising avenue for translational research in HNSCC.\u003c/p\u003e \u003cp\u003eOur study is limited by the use of 2D cell culture models, which cannot fully capture the metabolic heterogeneity and immune interactions of the tumor microenvironment. Future work employing 3D spheroids, patient-derived organoids, and \u003cem\u003ein vivo\u003c/em\u003e models will be essential to validate the HGF/c-Met\u0026ndash;GLS-1 axis and explore its interplay with antitumor immunity.\u003c/p\u003e \u003cp\u003eMoreover, glutamine availability plays a critical role in shaping immune responses, and the intersection between HGF/c-Met signaling, glutamine metabolism, and antitumor immunity warrants further investigation (Shah et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Hartmann et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn summary, our findings suggest a mechanistic connection between oncogenic c-Met signaling and glutamine metabolism in HNSCC. Through ERK1/2 activation, HGF/c-Met induces GLS-1 expression and cellular motility. The identification of this pathway expands the functional spectrum of c-Met beyond its classical roles in proliferation and migration to include metabolic regulation. Given the established link between c-Met and PD-L1 induction, these results suggest a convergence of metabolic and immune escape mechanisms. Combined inhibition of c-Met and GLS-1, potentially in conjunction with immune checkpoint blockade, may therefore represent a promising therapeutic strategy for advanced or metastatic HNSCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of interests:\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMH: Designing research studies; conducting experiments; acquiring data; analysing data and writing the manuscript. FM: Conducting experiments; acquiring data; writing the manuscript. AV: interpreting data; providing intellectual support. TR: interpreting data; writing the manuscript JV: interpreting data; providing intellectual support. AK: Providing intellectual support. NS: interpreting data;providing intellectual support. SH:designing and supervision of all studies; analysing and interpreting data; writing the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank Olga Frank for skillful technical assistance.\u003c/p\u003e\u003ch2\u003eData availability statement:\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author, M.H., upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eARNOLD L, ENDERS, J., THOMAS SM. 2017. Activated HGF-c-Met Axis in Head and Neck Cancer. Cancers (Basel), 9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBALAN M, MIER Y TERAN E, WAAGA-GASSER AM, GASSER M, FREEMAN CHOUEIRITK, G., PAL S. Novel roles of c-Met in the survival of renal cancer cells through the regulation of HO-1 and PD-L1 expression. J Biol Chem. 2015;290:8110\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBOSCHERT V, JANAKI RAMAN KLENKNABTA, FISCHER S, LINZ MBRANDSRCSEHERA, M\u0026Uuml;LLER-RICHTER C, BISCHLER UDA. T. \u0026amp; HARTMANN, S. 2020a. The Influence of Met Receptor Level on HGF-Induced Glycolytic Reprogramming in Head and Neck Squamous Cell Carcinoma. \u003cem\u003eInt J Mol Sci\u003c/em\u003e, 21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBOSCHERT V, ALJASEM TEUSCHJ, SCHMUCKER A, KLENK P, BITTRICH NSTRAUBA, SEHER M, LINZ A, M\u0026Uuml;LLER-RICHTER C, U. D. A., HARTMANN S. 2020b. HGF-Induced PD-L1 Expression in Head and Neck Cancer: Preclinical and Clinical Findings. Int J Mol Sci, 21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDI RENZO MF, OLIVERO M, MARTONE T, MAFFE A, MAGGIORA P, STEFANI AD, GIORDANO VALENTEG, CORTESINA S, G., COMOGLIO PM. Somatic mutations of the MET oncogene are selected during metastatic spread of human HNSC carcinomas. Oncogene. 2000;19:1547\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFERLAY J, COLOMBET M, SOERJOMATARAM I, MATHERS C, PARKIN DM, PI\u0026Ntilde;EROS M, ZNAOR A, BRAY F. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144:1941\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHARRINGTON KJ, YANG ROSENBERGAJ, OLIVA MHGEIGERJL, AHN M, LIM MJ, INCE SM, KEAM WBHATIAASHETHS, TOYOIZUMI BMETCALFRCURTINJC, WADE K, YILMAZ M, KIM E, SHAH PVERHEIJENRB, BAIG S, M., SWIECICKI PL. Subcutaneous amivantamab in recurrent/metastatic head and neck squamous cell cancer after disease progression on checkpoint inhibitor and chemotherapy: Preliminary results from the phase 1b/2 OrigAMI-4 study. Oral Oncol. 2025;171:107791.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHARTMANN S, BHOLA NE, GRANDIS JR. HGF/Met Signaling in Head and Neck Cancer: Impact on the Tumor Microenvironment. Clin Cancer Res. 2016;22:4005\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH\u0026Ouml;RNER M, LEIST BURKARDNKELMM, SELIG A, MEIR TKOLLMANNC, M., OTTO, C., GERMER, C. T., KRETZSCHMAR, K., FLEMMING, S., SCHLEGEL N. 2024. Glial cell line derived neurotrophic factor (GDNF) induces mucosal healing via intestinal stem cell niche activation. \u003cem\u003eCell Prolif\u003c/em\u003e, e13758.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHUANG X, WANG GANG, XU X, T., XIE W. The HGF-MET axis coordinates liver cancer metabolism and autophagy for chemotherapeutic resistance. Autophagy. 2019;15:1258\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJOHNSON DE, BURTNESS B, LEEMANS, C. R., LUI, V. W. Y., BAUMAN, J. E., GRANDIS JR. Head and neck squamous cell carcinoma. Nat Rev Dis Primers. 2020;6:92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKELM M, KOLLMANN BURKARDNH\u0026Ouml;RNERM, SARAVI COTTOC, SEUBERT B, FORCHEL AC, HOLLFELDER FKOHLERTNPENNERM, SCHMIDT F, KAMPMEIER SGERULLBSPRINGERR, RAUSCHENBERGER S, ARAMPATZI VDEOGHARENGERMERCT, KUGELMANN P, GARC\u0026Iacute;A-PONCE D, FLEMMING AWASCHKEJ, KRETZSCHMAR S, K., SCHLEGEL N. 2025. Junctional epithelial Plakoglobin facilitates intestinal inflammation by p38MAPK-dependent activation of the inflammasome. \u003cem\u003eMucosal Immunol\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKOCHANNY SE, WORDEN FP, ADKINS DR, LIM DW, WAGNER BAUMANJE, BRISSON SA, STADLER RJKARRISONTG, W. M., VOKES, E. E., SEIWERT TY. A randomized phase 2 network trial of tivantinib plus cetuximab versus cetuximab in patients with recurrent/metastatic head and neck squamous cell carcinoma. Cancer. 2020;126:2146\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNAGLER S, GHOREISHI Y, KOLLMANN C, KELM M, BURKARD GERULLBWASCHKEJ, N., SCHLEGEL N. Plakophilin 2 regulates intestinal barrier function by modulating protein kinase C activity in vitro. Tissue Barriers. 2023;11:2138061.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNGAN HL, CHOI LAWCH, Y. C. Y., CHAN, J. Y., LUI VWY. Precision drugging of the MAPK pathway in head and neck cancer. NPJ Genom Med. 2022;7:20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNGUYEN JP, NA'ARA S, HOERNER WOERNERLCVANLANDINGHAMNK, BLUM MSANTURAYRT, KIM K, M. O., JOHNSON, D. E., GRANDIS JR. Blockade of the PGE2 Pathway Inhibits the Growth of PTEN-Deficient HNSCC Tumors. Mol Cancer Ther. 2025;24:931\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQIN X, WU H, SHI PANJKANGK, Y., BU S. Immune-metabolic crosstalk in HNSCC: mechanisms and therapeutic opportunities. Front Oncol. 2025;15:1553284.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSEIWERT T, KALLENDER SARANTOPOULOSJ, MCCALLUM H, KEER S, H. N., BLUMENSCHEIN G, JR. Phase II trial of single-agent foretinib (GSK1363089) in patients with recurrent or metastatic squamous cell carcinoma of the head and neck. Invest New Drugs. 2013;31:417\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSEIWERT TY, JAGADEESWARAN R, FAORO L, JANAMANCHI V, NALLASURA V, EL DINALI M, COHEN YALASKANTETIR, MARTIN EELINGENMW, KRISHNASWAMY L, KLEIN-SZANTO S, VOKES ACHRISTENSENJG, E. E., SALGIA R. The MET receptor tyrosine kinase is a potential novel therapeutic target for head and neck squamous cell carcinoma. Cancer Res. 2009;69:3021\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSHAH AM, WANG Z, MA J. 2020. Glutamine Metabolism and Its Role in Immunity, a Comprehensive Review. Anim (Basel), 10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSZTURZ P, RAYMOND E, ABITBOL C, DE GRAMONT ALBERTS, A., FAIVRE S. Understanding c-MET signalling in squamous cell carcinoma of the head \u0026amp; neck. Crit Rev Oncol Hematol. 2017;111:39\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHEELER DL, HUANG S, KRUSER TJ, NECHREBECKI MM, BENAVENTE ARMSTRONGEA, GONDI S, V., HSU, K. T., HARARI PM. Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members. Oncogene. 2008;27:3944\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXIANG L, SHAO MOUJ, WEI B, LIANG Y, TAKANO H, N., SEMENZA, G. L., XIE G. Glutaminase 1 expression in colorectal cancer cells is induced by hypoxia and required for tumor growth, invasion, and metastatic colonization. Cell Death Dis. 2019;10:40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYU W, YANG X, YUAN ZHANGQSUNL, S., XIN Y. Targeting GLS1 to cancer therapy through glutamine metabolism. Clin Transl Oncol. 2021;23:2253\u0026ndash;68.\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"HNSCC, MAPK/ERK, HGF, Glutamine metabolism, MET-signaling","lastPublishedDoi":"10.21203/rs.3.rs-8233600/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8233600/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHead and neck squamous cell carcinoma (HNSCC) is a common malignancy characterized by poor survival due to recurrence, metastasis and therapy resistance. In addition to genetic alterations, metabolic reprogramming is a hallmark of HNSCC and contributes to tumor progression and treatment failure. The hepatocyte growth factor (HGF)/c-Met signaling pathway is frequently activated in head and neck squamous cell carcinoma (HNSCC), where it promotes tumor cell proliferation, invasion, and increased glucose metabolism. However, its contribution to the regulation of glutamine metabolism in HNSCC remains largely unexplored.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHuman HNSCC cell lines (FaDu, SCC-154, and Detroit562) were stimulated with HGF. Expression of glutaminase 1 (GLS-1) was analyzed by quantitative PCR and Western blotting. The functional relevance of GLS-1 was evaluated by pharmacological inhibition and genetic silencing using siRNA. Cell viability and migratory capacity were assessed by wound-healing assays.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHGF stimulation induced a pronounced increase in GLS-1 expression in HNSCC cell lines, as confirmed by qPCR and Western blotting. To elucidate the signaling mechanisms underlying this regulation, we next analyzed major c-Met downstream pathways. HGF treatment led to strong ERK1/2 phosphorylation. Pharmacological inhibition of c-Met with Foretinib or blockade of MEK1/2 with U0126 abolished ERK1/2 phosphorylation and prevented the HGF-induced upregulation of GLS-1 protein levels. These results demonstrate that HGF/c-Met\u0026ndash;driven GLS-1 expression is mediated through activation of the MAPK/ERK pathway. Moreover, GLS-1 silencing by siRNA significantly impaired wound closure, indicating reduced proliferative and migratory capacity.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings show that HGF/c-Met signaling activates the MAPK/ERK pathway to induce GLS-1 expression, thereby promoting glutamine metabolism and tumor cell motility in HNSCC. Consequently, targeting glutaminase or ERK signaling may represent a promising therapeutic approach to counteract HGF/c-Met\u0026ndash;driven metabolic reprogramming and therapy resistance in HNSCC.\u003c/p\u003e","manuscriptTitle":"HGF/c-Met Signaling induces Glutamine Metabolism in Head and Neck Squamous Cell Carcinoma via MAPK/ERK-Dependent Induction of GLS-1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-06 13:34:07","doi":"10.21203/rs.3.rs-8233600/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-10T12:43:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-17T16:14:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-17T13:08:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-16T03:40:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169049923542276781061406279130911367718","date":"2026-01-08T10:19:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T05:22:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266526707416208393979250601565305114506","date":"2026-01-05T14:41:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212329946903157362915774744584285873710","date":"2026-01-05T05:42:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"241475080730241273585796134400254346119","date":"2026-01-05T04:24:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-05T03:58:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-29T03:19:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-29T03:18:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Oncology","date":"2025-11-29T00:08:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"93959f83-e5cc-4164-b378-eb54d0594aba","owner":[],"postedDate":"January 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T17:23:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-06 13:34:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8233600","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8233600","identity":"rs-8233600","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}