FAK Inhibition Demonstrates Dual Effects of Tumor Suppression and Dormancy Induction Through Region-Specific COUP-TF1 Regulation

FAK Inhibition Demonstrates Dual Effects of Tumor Suppression and Dormancy Induction Through Region-Specific COUP-TF1 Regulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article FAK Inhibition Demonstrates Dual Effects of Tumor Suppression and Dormancy Induction Through Region-Specific COUP-TF1 Regulation Chih-Jen Yang, Po-Kai Chuang, Cheng-Hao Chuang, Yen-Yi Zhen, Huei-Yang Hung, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7615921/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Although focal adhesion kinase (FAK) inhibition shows promise in lung cancer therapy, emerging evidence suggests it may promote cellular dormancy and drug resistance through transcriptional regulation. We investigated the therapeutic efficacy and drug resistance inhibition mechanisms of FAK inhibitor VS-4718 in lung cancer. Methods Using an orthotopic syngeneic LLC1 mouse model, we evaluated the effects of VS-4718 (50 mg/kg/day) on tumor progression, survival, and molecular mechanisms. Comprehensive analyses included histological examination, immunohistochemistry, Western blotting, and clinical tissue validation from TKI-treated patients. Results VS-4718 demonstrated significant anti-tumor efficacy, reducing tumor burden by 60%, decreasing surface nodules, and improving overall survival (p < 0.01). Mechanistically, FAK inhibition induced cell cycle arrest through spatially heterogeneous p27 upregulation at tumor margins while suppressing Cyclin A1 expression. Unexpectedly, VS-4718 controlled COUP-TF1/β-catenin interactions, leading to reciprocal protein regulation. Critically, region-specific analysis revealed selective COUP-TF1 upregulation in bronchiolar areas, indicating anatomically-restricted dormancy pathway activation. Clinical validation in TKI-treated patient samples confirmed variable COUP-TF1 expression patterns, supporting its potential as a therapeutic resistance biomarker. Conclusions VS-4718 achieved significant therapeutic benefits through coordinated regulation of cell cycle and transcriptional networks. However, concurrent induction of COUP-TF1-mediated dormancy pathways, particularly in bronchiolar niches, may promote the formation of therapy-resistant cell populations. These findings reveal a fundamental paradox in FAK-targeted therapy and suggest that monotherapy may be insufficient for complete tumor eradication. Our Biological sciences/Cancer/Cancer therapy/Cancer therapeutic resistance Biological sciences/Cell biology/Senescence Biological sciences/Drug discovery/Drug regulation Biological sciences/Drug discovery/Biomarkers/Prognostic markers Biological sciences/Cell biology/Cell signalling/Checkpoint signalling Focal adhesion kinase Lung cancer Cellular dormancy COUP-TF1 Cell cycle arrest Therapy resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Therapy-resistance in cancer treatments lead to detrimental effect that the therapeutic compounds no longer destroy cancer cells [ 1 ]. Confronting the chemical compounds that are applied to kill cancer cells, the cancer cells can adaptively evolve themselves, and dynamically alter tumor microenvironment and signaling pathways in response to inhibitor of chemical targeting kinases [ 2 ]. Oncological literature has documented that there are variety of drug-resistant ways [ 3 ]. Metabolism for the small molecule compound, including drug efflux, drug-metabolism, drug sequestering and altering drug target, is one of ways that cancer cells survive from the administration of anti-cancer drug regimens [ 3 , 4 ]. Alternatively, the tumor cell ceases its dividing program by which the tumor cell is in non-proliferative state can also escape from cytotoxicity of small molecule chemical compound [ 1 , 2 ]. After anti-neoplastic agents’ treatment, it is frequently observed that sporadic tumor cells can survive in the tumor mass that was harvested from tumor resection surgery [ 5 , 6 ]. Even so the tumor cells expose to anti-neoplastic agents, handful tumor cells can alive. Those remnant cells in the tumor biopsies are in non-proliferation. Biologically, the non-proliferative cells are possibly stating in quiescence, senescence, and dormancy [ 7 – 10 ]. Of the three, senescent cells are alive but totally lost their proliferative ability [ 9 , 11 ]. In contrast, quiescent and dormant cells both temporally halt proliferative program but can reenter cell division cycle [ 10 , 12 ]. Biologically, quiescence and dormancy both are transient and reversible non-proliferative state [ 13 – 15 ]. The quiescent and dormant cells in tumor tissue are alive cells who temporary stop cell proliferation cessation [ 14 – 16 ]. Those non-proliferative cells in lung cancer patients were identified in patient-derived organoid-like cell clusters that are the therapy-resistant cells [ 17 ]. Those non-proliferative cells that enriched in tyrosine kinase inhibitors treated lung cancer mass bears an alveolar stem cell signature in parallel [ 18 ]. Commonly, quiescent and dormant cells are transiently in non-proliferative state and have drug-resistance, stemness, plasticity and diapause-like. With respect to dormancy, the chicken ovalbumin upstream promoter-transcriptional factor (Coup-TF1) based transcriptional repertoire programs cellular dormancy and regulates stem cell decision [ 13 , 16 ]. In our previous study, we attempt to manipulate FAK signaling in lung cancer cells to activate apoptosis and to turn down tumor cell proliferation and metastasis [ 7 , 8 ], since FAK pathway in the lung cancer cells derived from type II alveolar epithelial cells significantly regulates cell proliferation, survival, and metastasis. FAK inhibition led to growth cessation in tumor cell culture. The lung cancer cells exposed to FAK inhibitor partly are in senescence and epigenomic alternation in vitro. To evaluate the pharmacological effect of FAK inhibitor in vivo, orthotopic implantation of LLC1 cells in C57BL/6 mice lung was established as the lung cancer mice to test therapeutic efficacy of FAK inhibitor in vivo [ 19 , 20 ]. Intriguingly, cellular senescence in the tumor tissue in the C57BL/6 mice having LLC1 cell implantation is not detected after FAK inhibitor administration to the lung cancer mice, though FAK inhibitor can mitigate tumor cell number in lung cancer tissue. It seemed that those remnant cells in lung cancer tissue do not have proliferative ability. In turn, the antibodies that are used to evaluate non-proliferative cells were employed to immunohistochemical examination for dormant cell or quiescent cell in the lung cancer mass. Our data has revealed that tumor cells can survive under FAK inhibition or TKI-drug administration. This hinted cellular dormancy is a drug-resistant program in clinical cases as well as in the lung cancer animals. 2 Materials and Methods 2.1 Cell Culture LLC1 murine lung carcinoma cells and A549 human lung adenocarcinoma cells were obtained from ATCC (Manassas, VA, USA). LLC1 cells were cultured in DMEM (Gibco, 11965092) while A549 cells were maintained in RPMI-1640 medium (Gibco, 11875093). Both media were supplemented with 10% FBS (Gibco, 16000044) and 1% penicillin-streptomycin (Gibco, 15140122). Cells were incubated at 37°C in a humidified atmosphere with 5% CO2. Subculturing was performed using 0.25% trypsin-EDTA (Gibco, 25200056) when cells reached 80–90% confluency. 2.2 Cell viability assay The antiproliferative effects of VS-4718 were evaluated using MTT assay in LLC1 cell lines. Cells (5×10³/well) were seeded in 96-well plates and treated with VS-4718 (0.1–50 µM) for 72 hours. MTT reagent (100 µL, 0.5 mg/mL) was added and incubated for 4 hours at 37°C. Formazan crystals were dissolved in 100 µL DMSO with gentle agitation. Absorbance was measured at 570 nm using a microplate reader (BioTek Instruments). Cell viability was calculated as percentage of vehicle control (0.01% DMSO). IC₅₀ values were determined using non-linear regression analysis with GraphPad Prism software. Experiments were performed in sextuplicate and repeated three times independently. 2.3 Lung cancer cell growing in lung and FAK inhibitor administration. C57BL/6 mice that applied to establishment of lung cancer mice were purchased from Charles River Breeding Laboratory (Charles River Technology, Bio-LASCO Taiwan Co. Ltd., Taiwan). Animals were housed at room temperature (22 ± 2°C), with a humidity of 50 ± 10%, and automatically controlled cycle of 12 h day and night. Animal experiments, including surgical procedures, drug administration and animal sacrifice, were approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University and permission was issued in 112246 documents. C57BL/6 mice (equal numbers of males and females) aged 10–12 weeks and weighing in the range of 20 to 25 g were subjected to orthotopic implantation for in situ lung tumor growth [ 19 ]. Lung cancer animals were established with the orthotopic implantation by 10 3 mouse Lewis lung carcinoma cells (LLC1) in 50 µl serum free DMEM/matrix injected to upper lobe of left lung. Prior to orthotopic implantation, animals were anesthetized with a mixture of 3.5% isoflurane in 100% oxygen throughout the experimental procedure and the surgical site was disinfected with ethanol-free iodine solution. Then, 5 mm incision was made in the area of the left lateral thorax to make thoracic ribs and intercostal muscle exposed. Following, a 3 mm incision in muscle layer was made and the LLC1 cells in mixture of DMEM and matrix gel were directly injected to upper lobe of left lung with a 1 ml syringe. After implantation, the intercostal muscle layer and skin were swan up with 6 − 0 silk sutures. 2.4 FAK inhibitor administration Four animals were settled in this study, namely sham is the mice were without LLC1 implantation and VS-4718 administration, vehicle are the mice were without LLC1 implantation but had VS-4718 administration, lung cancer group is the mice received LLC1 implantation but did not have VS-4718 administration and FAK inhibitor treated lung cancer group is the mice had LLC1 implantation and VS-4718 administration. At 7th day post-LLC1 cells implantation, FAK inhibitor treated lung cancer mice began with VS-4718 administration and the VS-4718 administration lasted for two weeks. FAK inhibitor administration was performed with daily oral garage of FAK inhibitor with dosage 50 mg/kg/day VS-4718. For FAK inhibitor administration, the VS-4718 was prepared in 0.5% carboxymethyl cellulose and 0.1% Tween 80. The lung cancer mice had daily 0.5% carboxymethyl cellulose and 0.1% Tween 80 without VS-4718 for two weeks. Mice for vehicle group were daily dosed 50 mg/kg VS-4718 for two weeks. 2.5 Histological and immunohistochemical examination Lung and lung cancer tissues were harvested when animals were sacrificed. Human lung cancer tissue samples were obtained from patients under approval from the Institutional Review Board of Kaohsiung Medical University Hospital (IRB No: KMUHIRB-E(I)-20220330). All biopsies were immersed in 4% paraformaldehyde phosphate buffered normal saline for 24 hours. Following fixation, lung and lung cancer tissues were dehydrated and embedded in paraffin wax. Tissue sections in paraffin blocks were sliced at 4 µm with a sliding microtome (SM2125, Leica Biosystem, Nussloch, Germany) and placed on adhesive microscope slides (Pro-01, Matsunami Glass Ind., Ltd., Osaka, Japan). To examine cancer cells in lung tissues, specimens were dewaxed with xylene and rehydrated with serial dilutions of 100%, 90%, 70%, 50% ethanol in water, followed by water. The specimens were subjected to Hematoxylin & Eosin staining (HMM500 and EYB500, ScyTek Lab. Inc., Logan, UT-84323, USA). Antibody against COUP-TF1 was applied for immunohistochemical examination of COUP-TF1 expression in lung cancer tissues. After dewaxing and rehydration, specimens were probed with COUP-TF1 antibody overnight. COUP-TF1 in lung cancer tissues was visualized using a peroxidase-catalyzed Diaminobenzidine (DAB) substrate system (IHC detection kit) and conducted according to the manufacturers' instructions. 2.6 Immunofluorescence Analysis For protein localization studies, cells were plated onto 12-mm glass coverslips in 24-well dishes and exposed to VS-4718 (10 µM) or DMSO vehicle for 48 hours. Cells were subsequently fixed using 4% formaldehyde solution in phosphate-buffered saline for 15 minutes, followed by membrane permeabilization with 0.1% Triton X-100 for 10 minutes at ambient temperature. Non-specific antibody binding was prevented by incubating cells in 5% normal goat serum blocking solution for 60 minutes. Primary antibody staining was performed using mouse anti-β-catenin (1:200 dilution) and rabbit anti-COUP-TF1 (1:100 dilution) antibodies applied overnight at 4°C. Following triple PBS washes, cells were labeled with species-specific secondary antibodies: goat anti-rabbit Alexa Fluor 488 (1:500, green fluorescence) targeting COUP-TF1 and goat anti-mouse Alexa Fluor 594 (1:500, red fluorescence) targeting β-catenin, incubated for 1 hour in darkness at room temperature. Nuclear staining was achieved using DAPI (1 µg/mL) applied for 5 minutes. Coverslips were sealed with ProLong Gold mounting solution to preserve fluorescence. Image acquisition was performed using a Leica DMi8S fluorescence microscope with various magnification objectives (20×, 40×, 63×). High-resolution confocal microscopy was conducted on an OLYMPUS FV1000 system employing sequential laser excitation to minimize spectral overlap. Protein co-localization was quantified using ImageJ analysis software. 2.7 Western Blot Analysis Cells were harvested and lysed in 1x RIPA buffer (Merck, Darmstadt, Germany) containing protease and phosphatase inhibitors. The protein concentration was determined using a Bio-Rad DC protein assay kit (Bio-Rad, California, USA). For Western blot analysis, 30 µg of total protein was subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% skim milk for 2 h in TBST buffer (20 mM Tris-Cl, 150 mM NaCl, 0.1% Tween 20, pH 7.4). After blocking, the membranes were incubated with primary antibodies overnight at 4°C. Primary antibodies against FAK (1:1000), phospho-FAK (Tyr397) (1:1000), β-catenin (1:1000), COUP-TF1 (1:500), p27 (1:1000), and Cyclin A1 (1:1000) were used for immunoblotting. β-actin (1:5000) served as a loading control. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (ECL, GE Healthcare) and quantified by densitometric analysis using ImageJ software. All experiments were performed in triplicate, and representative blots are shown. 2.8 Statistical analysis The experimental data were digitized and analyzed using GraphPad Prism software (version 9.0). Data are presented as the mean ± standard deviation (SD). One-way ANOVA followed by Tukey's post-hoc test was used to compare digitized data and measurements from independent experiments in multiple groups, and the unpaired Student's t -test was used to compare two independent samples. For survival analysis, Kaplan-Meier curves were constructed and compared using the log-rank test. Statistical significance was defined as p < 0.05, with p < 0.01 and p < 0.001 indicating higher levels of significance. 3 Results 3.1 FAK Inhibitor, VS-4718, Treatment Suppresses Tumor Progression and Improves Survival in LLC1 Lung Cancer Mouse Model To determine the direct anti-tumor effects of VS-4718 on lung cancer cells we assessed its cytotoxic activity in vitro using lung cancer cell lines. VS-4718 treatment significantly reduced cell viability in a dose-dependent manner over 4 days in murine LLC1 cells (Fig. 1 A) or human A549 lung cancer cells line (Figure S1 A). VS-4718 exhibited concentration-dependent growth inhibition in LLC1 cells, with modest effects at 0.1–1 µM and pronounced cytotoxicity at 10–50 µM that nearly eliminated proliferation at the highest concentration tested. Doubling time analysis confirmed these anti-proliferative effects in LLC1 cells (Fig. 1 B) or A549 cells (Figure S1 B). VS-4718 treatment gradually increased doubling times from 1.3 d (control) to 1.5 d (0.1 µM) and 2.3 d (1 µM), with 10 µM causing severe growth damage and 50 µM producing negative doubling times showing cell death. To further evaluate the therapeutic potential of FAK inhibition in lung cancer, we established an orthotopic syngeneic mouse model using Lewis lung carcinoma 1 (LLC1) cells implanted into C57BL/6 mice. Following tumor establishment, mice were randomized into treatment groups and administered VS-4718 (50 mg/kg/day, p.o.) or vehicle control for 14 consecutive days (Fig. 1 C). At the experimental endpoint, VS-4718 treatment significantly reduced body weight at sacrifice compared to vehicle controls (22.5 ± 0.8 g vs. 25.0 ± 0.9 g) (Fig. 1 D), potentially reflecting both metabolic effects of FAK inhibition and reduced tumor burden. More importantly, treatment with VS-4718 led to a significant reduction in total lung weight compared to the untreated tumor-bearing group (Fig. 1 E), directly indicating decreased tumor burden and effective suppression of tumor growth. Additionally, the number of visible surface tumor nodules on the lungs was markedly decreased in VS-4718-treated mice, demonstrating the antitumor activity of the compound (Fig. 1 F). Kaplan-Meier survival analysis over a 21-day observation period revealed a significant improvement in overall survival with VS-4718 treatment (Fig. 1 G). Vehicle control tumor-bearing mice showed progressive mortality beginning around day 15, while VS-4718-treated mice maintained high survival rates throughout the observation period. These survival data support the potential of VS-4718 to not only suppress tumor progression but also to extend survival in this lung cancer model. Collectively, these findings demonstrate that VS-4718 effectively suppresses tumor burden and improves survival in the LLC1 syngeneic lung cancer model, exerting potent anti-tumor effects both in vitro and in vivo. 3.2 Histological Evaluation Reveals Significant Tumor Suppression and Cellular Remodeling Following VS-4718 Treatment To further characterize the anti-tumor effects of VS-4718, we performed comprehensive histological analysis of lung tissues from both treatment groups. Hematoxylin and eosin (H&E) staining revealed distinct morphological differences between vehicle control and VS-4718-treated tumor-bearing mice (Fig. 2 A). Histological examination demonstrated markedly reduced tumor infiltration and preservation of normal alveolar architecture in the VS-4718 group compared to vehicle controls. At 100× and 400× magnifications, untreated lung cancer tissues exhibited dense tumor cell accumulation with disrupted alveolar structures and invasive cellular morphology. In contrast, VS-4718-treated lungs showed significantly decreased cellular density, reduced nuclear atypia, and partial reconstitution of alveolar spaces. Quantitative histomorphometric analysis confirmed significant reduction in tumor area following VS-4718 treatment compared to vehicle controls (Fig. 2 B, n = 7 mice per group; p < 0.001). Sirius Red staining analysis revealed a significantly higher percentage of fibrotic areas in the VS-4718 group compared to controls (Fig. 2 C, p < 0.0001), indicating increased stromal deposition associated with tumor suppression. Nuclear-to-cytoplasm ratio analysis showed drastic reduction in tumor cell density per lung cross-section following VS-4718 treatment (Fig. 2 D, p < 0.001). Therefore, these histological findings conclude that VS-4718-mediated FAK inhibition not only diminishes tumor burden but also induces significant tissue remodeling and restoration of pulmonary architecture in a murine lung cancer model, providing further mechanistic support for the anti-tumor efficacy of FAK-targeted therapy. 3.3 VS-4718 Alters Cell Cycle Regulator Expression by Increasing p27 and Reducing Cyclin A1 Levels in Lung Cancer Models To investigate the molecular mechanisms underlying the anti-tumor effects of VS-4718, we examined the expression of key cell cycle regulatory proteins, p27 and Cyclin A1, in both murine (LLC1) lung cancer models. These proteins play crucial roles in the control of G1/S phase progression and are influenced by FAK signaling pathways. Immunohistochemical analysis of LLC1 tumor tissues revealed region-specific p27 expression patterns following VS-4718 treatment (Fig. 3 A). While p27 levels in tumor interior regions remained unchanged between vehicle control and VS-4718-treated groups (P > 0.05, n.s.), tumor margin areas demonstrated significantly increased p27 expression following VS-4718 treatment (P < 0.01, Fig. 3 B). In contrast, Cyclin A1 expression was markedly reduced throughout the tumor following VS-4718 treatment (P < 0.001, Fig. 3 C), indicating suppression of cell cycle progression. To validate these findings at the protein level, western blot analysis was conducted using lysates from LLC1. VS-4718 treatment resulted in increased p27 expression and a consistent downregulation of Cyclin A1 (Fig. 3 D, E). Notably, these effects were irreversible (Figure S2), as withdrawal of VS-4718 treatment for 24 hours failed to restore phospho-FAK and Cyclin A1 levels, suggesting that FAK inhibition induced sustained molecular changes rather than transient pharmacological effects. These results indicate that VS-4718 exerts its anti-proliferative effects through region-specific modulation of p27 expression and consistent downregulation of Cyclin A1, consistent with the immunohistochemical findings, providing mechanistic insight into how FAK inhibition impairs tumor cell cycle progression. 3.4 VS-4718 Treatment Upregulates COUP-TF1 and Suppresses β-catenin Expression in Lung Cancer Cells To evaluate the downstream signaling mechanisms affected by FAK inhibition and identify potential molecular targets involved in the observed anti-tumor effects, we investigated the interaction between COUP-TF1 (chicken ovalbumin upstream promoter transcription factor 1) and β-catenin, key regulators of the Wnt signaling pathway. We employed dual immunofluorescence co-localization analysis in lung cancer cell lines to assess protein-protein interactions and subcellular distribution patterns, complemented by Western blot analysis to quantify protein expression levels following VS-4718 treatment. Immunofluorescence co-staining analysis in LLC1 lung cancer cells revealed marked changes in COUP-TF1 (green) and β-catenin (red) cellular distribution and co-localization after 48-hour VS-4718 treatment (Fig. 4 A). In control cells, COUP-TF1 and β-catenin showed widespread co-localization in cytoplasm, as demonstrated by strong yellow/orange signals in merged images. VS-4718 treatment triggered significant nuclear redistribution of COUP-TF1 and β-catenin, with both proteins showing enhanced nuclear translocation and decreased spatial separation. Immunofluorescence revealed a transition from co-localization to staining patterns clearly concentrated in specific nuclear regions, suggesting that FAK inhibition promotes nuclear translocation of both proteins while avoiding their normal co-localization in the cytoplasm, potentially enhancing their regulatory functions. To assess the effects of FAK inhibition on COUP-TF1 and β-catenin protein levels, we performed Western blot analysis in LLC1 cell lines following 48-hour VS-4718 treatment (Fig. 4 B). VS-4718 treatment significantly increased COUP-TF1 and β-catenin protein expression compared to vehicle controls in LLC1 cells. These data indicate that VS-4718 regulates key transcriptional and signaling molecules by enhancing COUP-TF1 expression while concurrently concentrating β-catenin. This suggests that FAK inhibition may suppress tumor progression and metastatic potential by manipulating the nuclear localization of COUP-TF1/β-catenin regulatory pathways. 3.5 VS-4718 treatment reduces FAK expression in vivo and correlates with clinical COUP-TF1 expression patterns. To validate target engagement and assess the clinical relevance of our preclinical findings, we performed comprehensive immunohistochemical analysis to examine COUP-TF1 expression in VS-4718-treated LLC1 tumor tissues and evaluated COUP-TF1 expression patterns in human lung cancer specimens from patients receiving tyrosine kinase inhibitor (TKI) therapy. This dual approach aimed to confirm direct pharmacological effects of VS-4718 on its primary target while establishing potential clinical correlations for the identified molecular pathways. To address the effects of FAK inhibition on COUP-TF1 expression and assess the clinical relevance of our preclinical findings, we performed comprehensive immunohistochemical analysis of COUP-TF1 protein expression in LLC1 tumor tissues revealed region-specific responses to VS-4718 treatment (Fig. 5 A). Representative images demonstrated distinct differences in COUP-TF1 staining patterns between vehicle control and VS-4718-treated tumors across anatomically different regions. In non-defined tumor areas, quantitative analysis using DAB staining area measurements showed no significant difference between control and VS-4718-treated groups (Fig. 5 B, P > 0.05, n.s.). However, analysis of bronchiole areas revealed a markedly different response pattern. Control tumors in bronchiole regions exhibited moderate COUP-TF1 expression, while VS-4718 treatment resulted in significantly increased COUP-TF1 expression (Fig. 5 C, P < 0.05). This region-specific upregulation of COUP-TF1 in bronchiole areas supports our in vitro findings and suggests that FAK inhibition preferentially affects COUP-TF1 expression in specific anatomical contexts. To evaluate the clinical relevance of COUP-TF1 regulation, we analyzed COUP-TF1 expression in human lung cancer tissues obtained from patients before and after TKI therapy (Fig. 5 D). We focused our analysis on bronchiole areas, as these regions demonstrated the most pronounced responses in our preclinical studies. Analysis of patient samples revealed variable COUP-TF1 expression patterns between patients and treatment timepoints. In Subject 1, weak COUP-TF1 staining was observed in bronchiole areas prior to TKI therapy, while enhanced expression and improved tissue organization were evident following treatment. Post-treatment samples demonstrated increased numbers of COUP-TF1-positive cells and more organized bronchiolar architecture. Subject 2 displayed distinct baseline COUP-TF1 expression patterns, with marked changes in cellular distribution following TKI therapy. Post-treatment samples exhibited altered COUP-TF1 localization, characterized by increased nuclear staining in bronchiolar epithelial cells and adjacent tissue regions. These data demonstrate that VS-4718-mediated FAK inhibition specifically enhances COUP-TF1 expression in bronchiole regions of tumor tissues, establishing COUP-TF1 as a downstream target of FAK signaling in tissue-specific contexts. The regional specificity of this response indicates that FAK-COUP-TF1 pathway interactions may play a critical role in bronchiole-associated tumor development. The changes in COUP-TF1 expression patterns observed after TKI therapy indicate the potential clinical value of COUP-TF1 as both a biomarker for treatment response and a therapeutic target. 3.6 Therapeutic Promise of VS-4718 and COUP-TF1-Mediated Resistance Mechanisms This study demonstrates that FAK inhibition with VS-4718 produces significant anti-tumor effects in the LLC1 lung cancer model through multiple mechanisms. VS-4718 treatment reduced tumor burden, improved survival, and restored normal pulmonary architecture with increased fibrotic remodeling. At the molecular regulatory level (Fig. 6 ), VS-4718 induced cell cycle arrest through selective p27 upregulation at tumor margins and reduced Cyclin A1 expression. Additionally, FAK inhibition induced COUP-TF1/β-catenin co-expression level, indicating modulatory signaling pathways. The region-specific upregulation of COUP-TF1 in bronchiole areas suggests tissue-dependent regulation of these pathways. Clinical analysis of COUP-TF1 expression in TKI-treated patients revealed variable expression patterns, supporting its potential utility as a biomarker for treatment monitoring and patient stratification. In conclusion, VS-4718 represents a promising therapeutic approach for lung cancer through simultaneous targeting of cell cycle regulation and COUP-TF1/β-catenin signaling pathways. These findings support further clinical development of FAK inhibitors and suggest COUP-TF1 expression monitoring may provide valuable insights for personalized lung cancer therapy. 4 Discussion This study provides comprehensive evidence that pharmacological inhibition of FAK using VS-4718 exerts potent anti-tumor effects in lung cancer through coordinated modulation of cell cycle progression and transcriptional regulatory networks. Our findings demonstrate that FAK inhibition not only reduces tumor burden and improves survival but also induces significant molecular changes that may contribute to therapy resistance through cellular dormancy mechanisms. The therapeutic efficacy of VS-4718 in the LLC1 syngeneic mouse model is consistent with previous reports demonstrating the critical role of FAK in lung cancer progression [ 21 , 22 ]. The significant reduction in tumor burden, decreased surface nodules, and improved survival outcomes align with studies showing that FAK inhibition disrupts key oncogenic processes including cell proliferation, survival, and metastasis [ 23 , 24 ]. The preservation of normal pulmonary architecture following VS-4718 treatment suggests that FAK inhibition may offer therapeutic benefits beyond simple growth suppression, potentially facilitating tissue repair and regeneration processes [ 7 ]. Our molecular analysis reveals that VS-4718 induces cell cycle arrest through region-specific modulation of key checkpoint proteins. The selective upregulation of p27 at tumor margins, while maintaining unchanged levels in tumor interior regions, indicates spatial heterogeneity in treatment response [ 25 , 26 ]. This pattern suggests that FAK inhibition preferentially affects actively proliferating cells at the tumor-host interface, which may be more vulnerable to cell cycle disruption. The concurrent reduction in Cyclin A1 expression provides additional evidence for G1/S checkpoint arrest, consistent with the established role of these proteins in cell cycle regulation [ 27 , 28 ]. The disruption of COUP-TF1/β-catenin interactions represent a novel mechanism by which FAK inhibition affects cellular behavior. COUP-TF1 has been increasingly recognized as a master regulator of cellular dormancy and stemness in cancer cells [ 13 , 16 ]. The reciprocal regulation of COUP-TF1 upregulation and β-catenin downregulation following VS-4718 treatment suggests that FAK signaling normally suppresses dormancy-associated transcriptional programs while promoting Wnt pathway activity [ 17 , 29 ]. This finding is particularly significant given that cellular dormancy represents a major mechanism of therapy resistance in cancer treatment [ 30 , 31 ]. The region-specific upregulation of COUP-TF1 in bronchiole areas following VS-4718 treatment provides important insights into tissue-dependent regulation of dormancy pathways. Bronchiolar structures in the lung have been identified as niches that harbor stem-like cells and may serve as reservoirs for therapy-resistant cancer cells [ 18 , 32 ]. The preferential induction of COUP-TF1 in these anatomical regions suggests that FAK inhibition may inadvertently promote cellular dormancy in specific microenvironmental contexts, potentially contributing to the survival of residual cancer cells observed in our histological analysis. The clinical analysis of COUP-TF1 expression in TKI-treated patients provides translational validation of our preclinical findings while highlighting the complexity of therapy resistance mechanisms in clinical settings. The variable expression patterns observed between patients reflect the heterogeneous nature of treatment responses and underscore the need for personalized therapeutic approaches [ 1 , 4 ]. The enhanced COUP-TF1 expression following TKI therapy supports the hypothesis that targeted therapies may inadvertently select for dormant, therapy-resistant cell populations [ 17 ]. Our findings have important implications for the clinical development of FAK inhibitors in lung cancer therapy. While VS-4718 demonstrates clear anti-tumor efficacy, the induction of COUP-TF1-mediated dormancy pathways may contribute to the emergence of therapy-resistant cell populations. This dual effect suggests that FAK inhibitor monotherapy may be insufficient for complete tumor eradication and supports the development of combination therapeutic strategies that simultaneously target both proliferating and dormant cancer cell populations [ 11 , 33 ]. Several limitations of this study should be acknowledged. The use of a single mouse model may not fully recapitulate the heterogeneity of human lung cancer, and longer follow-up studies are needed to assess the potential for tumor recurrence from dormant cell populations. Additionally, the molecular mechanisms linking FAK signaling to COUP-TF1 regulation require further investigation to identify potential therapeutic targets for preventing dormancy induction. In summary, our study demonstrates that FAK inhibition with VS-4718 produces significant anti-tumor effects through coordinated modulation of cell cycle progression and transcriptional regulatory networks. However, the concurrent induction of COUP-TF1-mediated dormancy pathways highlights the complexity of therapy resistance mechanisms and underscores the need for combination therapeutic approaches in lung cancer treatment. 5. Conclusion VS-4718 exhibits significant anti-tumor efficacy in lung cancer models by inducing cell cycle arrest through p27 upregulation, modulating COUP-TF1/β-catenin signaling pathways, and restoring normal pulmonary architecture. It demonstrates promising therapeutic potential with region-specific molecular effects and measurable survival benefits. However, the concurrent induction of COUP-TF1-mediated cellular dormancy pathways, particularly in bronchiole regions, may contribute to therapy resistance mechanisms. Therefore, this study focused on developing combination therapeutic regimens that simultaneously target FAK-mediated proliferation and COUP-TF1-induced dormancy mechanisms to prevent therapy resistance. Declarations Acknowledgement: Not applicable. Funding Statement: This work was supported by grants from the National Science and Technology Council (NSTC), Taiwan (NSTC 113-2314-B-037-104-MY3 and NSTC 114-2314-B-110-006), Kaohsiung Medical University Hospital (KMUH112-2R16), the NYCU-KMU Joint Research Program (NYCUKMU-113-I1003), and the NSYSU-KMU Joint Research Project (NSYSU-KMU-113-P36). Author Contributions: Conceptualization: Po-Kai Chuang, Chih-Jen Yang; Methodology: Chih-Jen Yang, Cheng-Hao Chuang, Yen-Yi Zhen, Ching-Tang Huang, Pei-Hui Wang; Investigation: Yen-Yi Zhen, Ching-Tang Huang, Pei-Hui Wang; Formal Analysis: Po-Kai Chuang, Chih-Jen Yang, Cheng-Hao Chuang, Yen-Yi Zhen, Ching-Tang Huang, Pei-Hui Wang, Ying-Ray Lee; Resources: Cheng-Hao Chuang, Huei-Yang Hung, Michael Hsiao, Ping-Lun Jiang; Visualization: Po-Kai Chuang, Cheng-Hao Chuang, Yen-Yi Zhen, Ching-Tang Huang, Pei-Hui Wang, Yung-Kuo Lee, Chih-Yang Wang; Writing—Original Draft: Po-Kai Chuang, Yen-Yi Zhen; Writing—Review & Editing: Po-Kai Chuang, Chih-Jen Yang; Project Administration: Po-Kai Chuang, Chih-Jen Yang, Ming-Shyan Huang; Funding Acquisition: Po-Kai Chuang, Chih-Jen Yang, Cheng-Hao Chuang; Validation: Ching-Tang Huang, Pei-Hui Wang. All authors reviewed and approved the final manuscript. Availability of Data and Materials: Not applicable. Ethics Approval: All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University (approval number: 112246) and conducted in accordance with institutional guidelines for animal welfare. Human lung cancer tissue samples were obtained under approval from the Institutional Review Board of Kaohsiung Medical University Hospital (IRB No: KMUHIRB-E(I)-20220330). All procedures involving human subjects were performed in accordance with the Declaration of Helsinki and relevant ethical guidelines. Conflicts of Interest: The authors declare no conflicts of interest to report regarding the present study. Supplementary Materials: The supplementary material is available online. References Konieczkowski DJ, Johannessen CM, Garraway LA. A convergence-based framework for cancer drug resistance. Cancer Cell. 2018;33(5):801-815. Skarkova A, Bizzarri M, Janostiak R, Masek J, Rosel D, Brabek J. 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Proteins secreted by lung cancer cells induce the onset of proteinuria via focal adhesion kinase signaling in mice. Lab Invest. 2023;103:100156. Roles and inhibitors of FAK in cancer: current advances and future directions. Front Pharmacol. 2024;15:1274209. Focal adhesion kinase (FAK): emerging target for drug-resistant malignant tumors. PMC. 2025. FAK in cancer: from mechanisms to therapeutic strategies. Biomolecules. 2023;14:446. Development of novel focal adhesion kinase (FAK) inhibitors for targeting cancer: structural insights and therapeutic potential. Eur J Med Chem. 2024;276:116743. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999;13(12):1501-1512. Chu IM, Hengst L, Slingerland JM. The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer. 2008;8(4):253-267. Malumbres M, Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer. 2009;9(3):153-166. Besson A, Dowdy SF, Roberts JM. CDK inhibitors: cell cycle regulators and beyond. Dev Cell. 2008;14(2):159-169. Pereira B, Chin SF, Rueda OM, Vollan HK, Provenzano E, Bardwell HA, et al. The somatic mutation profiles of 2,433 breast cancers refine their genomic and transcriptomic landscapes. Nat Commun. 2016;7(1):11479. Wnt/β-catenin signaling pathway in carcinogenesis and cancer therapy. J Hematol Oncol. 2024;17:44. Chronic WNT/β-catenin signaling induces cellular senescence in lung epithelial cells. Cell Mol Life Sci. 2020;87:1425-1439. WNT/beta-catenin signalling interrupts a senescence-induction cascade in human mesenchymal stem cells. Cell Mol Life Sci. 2022;79:88. Rock JR, Onaitis MW, Rawlins EL, Lu Y, Clark CP, Xue Y, et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci USA. 2009;106(31):12771-12775. Additional Declarations (Not answered) Supplementary Files SupplementarydataWB.docx FAK Inhibition Demonstrates Dual Effects of Tumor Suppression and Dormancy Induction Through Region-Specific COUP-TF1 Regulation FigureS1ands2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Data are expressed as mean ± standard deviation (SD). (B) Doubling time analysis showing dose-dependent effects of VS-4718 treatment in LLC1 cells. *p \u0026lt; 0.05, **p \u0026lt; 0.01 compared to control group. (C) Schematic diagram of experimental design. LLC1 cells were implanted into C57BL/6 mice, followed by daily oral gavage with VS-4718 (50 mg/kg/day) or vehicle control for 14 days. Serum was collected and mice were sacrificed for analysis. (D) Body weight at sacrifice. Mice were weighed at the end of treatment period. Data are expressed as mean ± standard deviation (SD). n = 3-7 mice per group. **p \u0026lt; 0.01 compared to vehicle control group. (E) Total lung weight measurement. Lungs were harvested and weighed immediately after sacrifice. Data are expressed as mean ± SD. **p \u0026lt; 0.01 compared to lung cancer control group. (F) Quantification of lung surface tumor nodules. Macroscopic tumor nodules on lung surface were counted under stereomicroscope. Each data point represents individual mouse. **p \u0026lt; 0.01 compared to lung cancer control group. (G) Survival analysis using Kaplan-Meier method. Mice were monitored daily for 21 days post-implantation. Survival curves were compared using log-rank test. **p \u0026lt; 0.01 compared to lung cancer control group.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7615921/v1/275328489cb42faaf44810f7.jpg"},{"id":93729007,"identity":"3848bcdd-c223-4c3e-806b-7294e8714df5","added_by":"auto","created_at":"2025-10-17 02:13:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68703,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological analysis reveals reduced tumor burden and collagen deposition following VS-4718 treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative cross-sectional images of lung tissues from tumor-bearing mice with or without VS-4718 treatment. Upper panels show lung cancer control group, lower panels show lung cancer treated with VS-4718. Left panels display overview images (scale bar = 1 mm), middle panels show 100× magnification, and right panels show 400× magnification of selected areas (indicated by blue rectangles). (B) Tissues were stained with hematoxylin and eosin (H\u0026amp;E) for analysis of cell number per unit area. \u0026nbsp;(C) Quantitative analysis of tumor area and collagen content. (D) Nuclear-cytoplasm ratio analysis comparing control and VS-4718 treated groups. Data are expressed as mean ± standard deviation (SD). n = 7 mice per group. Lower right graph shows percentage of necrotic/collagen area quantification. ***p \u0026lt; 0.001 compared to lung cancer control group using unpaired Student's t-test.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7615921/v1/2f298ff521e0f13974081b2b.jpg"},{"id":93729008,"identity":"4b0b8495-9349-489b-a8f5-4aa136faae87","added_by":"auto","created_at":"2025-10-17 02:13:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":133417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVS-4718 treatment modulates cell cycle regulatory proteins p27 and Cyclin A1 expression in lung cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunohistochemical staining of p27 protein in lung tumor tissues. Representative images showing p27 expression in LLC1 tumor-bearing mice without (left) or with (right) VS-4718 treatment. Scale bar = 20 μm. (B) Quantitative analysis of p27 expression levels measured separately in tumor interior regions and tumor margin areas. Data are expressed as mean ± standard deviation (SD). n = 5-6 mice per group. Statistical comparisons between treatment groups were performed using unpaired Student's t-test. n.s., not significant for p27 interior expression; **p \u0026lt; 0.01 for p27 margin expression compared to lung cancer control group. (C) Immunohistochemical staining of Cyclin A1 protein in lung tumor tissues. Representative images showing Cyclin A1 expression in LLC1 tumor-bearing mice without (left) or with (right) VS-4718 treatment. Scale bar = 20 μm. (D) Quantitative analysis of Cyclin A1 expression levels in tumor tissues. Data are expressed as mean ± SD. n = 5-6 mice per group. ***p \u0026lt; 0.001 compared to lung cancer control group using unpaired Student's t-test. (E) Western blot analysis of p27 and Cyclin A1 protein expression in LLC1 lung cancer cell lines. Cells were treated with VS-4718 or vehicle control for 48 hours. Protein expression was normalized to β-actin loading control. Representative blots are shown with quantitative densitometric analysis. VS-4718 treatment increased p27 expression and decreased Cyclin A1 expression in both cell lines, indicating cell cycle arrest at the G1/S checkpoint.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7615921/v1/fbc46c5d02b291aa7638e972.jpg"},{"id":93729027,"identity":"1f90c3f9-9a7c-4b76-a691-cc2448c771c7","added_by":"auto","created_at":"2025-10-17 02:13:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":125088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAK inhibitor, VS-4718, treatment through interaction of β-catenin and COUP-TF1 in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunofluorescence analysis at higher magnification showing detailed subcellular distribution of COUP-TF1 (green) and β-catenin (red) proteins in LLC1 lung cancer cells. Control cells (upper row) display extensive co-localization in both cytoplasmic and nuclear compartments, while VS-4718-treated cells (lower row) show disrupted co-localization patterns with altered protein distribution. Merged images demonstrate the spatial relationship between these proteins, and DAPI staining reveals nuclear morphology. Scale bar = 200 μm \u003cstrong\u003e(Ｂ)\u003c/strong\u003eWestern blot analysis of COUP-TF1 and β-catenin protein expression levels in LLC1 lung cancer cell lines. Cells were treated with VS-4718 or vehicle control for 48 hours. (C-F) Protein expression was normalized to β-actin loading control. Representative blots are shown with corresponding densitometric quantification. VS-4718 treatment significantly increased COUP-TF1 expression while decreasing β-catenin levels in LLC1 cell lines.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7615921/v1/b9c1ec7fbf2accf3a567e0ab.jpg"},{"id":93729015,"identity":"f93e5762-7035-490a-895d-213f665abeef","added_by":"auto","created_at":"2025-10-17 02:13:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":216974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCOUP-TF1 expression patterns in preclinical and clinical lung cancer tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Immunohistochemical analysis of COUP-TF1 protein expression in LLC1 tumor tissues. Representative images showing COUP-TF1 staining intensity in non-defined tumor areas (upper panels) and bronchiole areas (lower panels) from untreated LLC1 tumors (left column) and VS-4718-treated tumors (right column). Scale bar = 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B and C)\u003c/strong\u003e Quantitative analysis of COUP-TF1 expression measuring DAB (3,3'-diaminobenzidine) staining area in arbitrary units. Data are expressed as mean ± standard deviation (SD). Individual data points represent measurements from different tumor regions. n.s., not significant for non-defined areas; **p \u0026lt; 0.01 for bronchiole areas compared to lung cancer control group using unpaired Student's t-test. VS-4718 treatment significantly increased COUP-TF1 expression specifically in bronchiole regions. \u003cstrong\u003e(D)\u003c/strong\u003e Immunohistochemical analysis of COUP-TF1 protein expression in human lung cancer tissues from patients prior to and post tyrosine kinase inhibitor (TKI) administration. Representative images from two different subjects (Subject 1 and Subject 2) showing COUP-TF1 staining patterns in bronchiole areas before tyrosine kinase inhibitor (TKI) treatment (upper panels) and after TKI treatment (lower panels). Scale bar = 20 μm. The analysis demonstrates variable COUP-TF1 expression patterns between different patients and treatment timepoints, with enhanced COUP-TF1 expression observed in bronchiolar structures following TKI therapy.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7615921/v1/75a19a7427534d6bac769e4e.jpg"},{"id":93732306,"identity":"9ebe20f7-347f-4f60-a833-943b019d60ef","added_by":"auto","created_at":"2025-10-17 02:29:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":95446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanism of VS-4718-mediated FAK inhibition in lung cancer therapy and its dual effects on tumor suppression and cellular dormancy induction.\u003c/strong\u003e \u003cbr\u003e\nVS-4718, a small molecule FAK inhibitor, demonstrates reduced tumor burden with increased necrosis in the representative lung tumor microenvironment following treatment. Cell cycle analysis reveals cell cycle arrest with upregulated p27 and downregulated Cyclin A1. The disrupted β-catenin/COUP-TF1 signaling pathway exhibits decreased β-catenin expression and increased COUP-TF1 levels, particularly in bronchiolar regions. Extracellular matrix remodeling shows enhanced collagen deposition and stromal reorganization, with disorganized tumor tissue transforming into organized fibrous remodeling. Kaplan-Meier survival curves demonstrate improved outcomes in the VS-4718-treated group compared to controls. VS-4718 treatment results in coordinated effects including tissue remodeling, cell cycle arrest, and transcriptional reprogramming, supporting FAK inhibition as a therapeutic strategy and COUP-TF1 as a spatial biomarker in lung cancer.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7615921/v1/133c2a7492f66880d131d962.jpg"},{"id":96249120,"identity":"f3d52d80-6185-44a7-95cf-ba2c71df75af","added_by":"auto","created_at":"2025-11-19 07:30:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1953854,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7615921/v1/4da3027e-83dc-4b72-a8be-5ab836707d62.pdf"},{"id":93729037,"identity":"bf607e23-8093-49d4-ab1d-e4ddfb9d3b45","added_by":"auto","created_at":"2025-10-17 02:13:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37077034,"visible":true,"origin":"","legend":"FAK Inhibition Demonstrates Dual Effects of Tumor Suppression and Dormancy Induction Through Region-Specific COUP-TF1 Regulation","description":"","filename":"SupplementarydataWB.docx","url":"https://assets-eu.researchsquare.com/files/rs-7615921/v1/c0831edd38ee78c61db8f911.docx"},{"id":93729011,"identity":"8a215de2-c442-46d9-8b88-0491d129c8a7","added_by":"auto","created_at":"2025-10-17 02:13:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":140932,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1ands2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7615921/v1/c8f396799b4b91810d75352e.docx"}],"financialInterests":"(Not answered)","formattedTitle":"FAK Inhibition Demonstrates Dual Effects of Tumor Suppression and Dormancy Induction Through Region-Specific COUP-TF1 Regulation","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eTherapy-resistance in cancer treatments lead to detrimental effect that the therapeutic compounds no longer destroy cancer cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Confronting the chemical compounds that are applied to kill cancer cells, the cancer cells can adaptively evolve themselves, and dynamically alter tumor microenvironment and signaling pathways in response to inhibitor of chemical targeting kinases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Oncological literature has documented that there are variety of drug-resistant ways [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Metabolism for the small molecule compound, including drug efflux, drug-metabolism, drug sequestering and altering drug target, is one of ways that cancer cells survive from the administration of anti-cancer drug regimens [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Alternatively, the tumor cell ceases its dividing program by which the tumor cell is in non-proliferative state can also escape from cytotoxicity of small molecule chemical compound [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAfter anti-neoplastic agents\u0026rsquo; treatment, it is frequently observed that sporadic tumor cells can survive in the tumor mass that was harvested from tumor resection surgery [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Even so the tumor cells expose to anti-neoplastic agents, handful tumor cells can alive. Those remnant cells in the tumor biopsies are in non-proliferation. Biologically, the non-proliferative cells are possibly stating in quiescence, senescence, and dormancy [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Of the three, senescent cells are alive but totally lost their proliferative ability [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast, quiescent and dormant cells both temporally halt proliferative program but can reenter cell division cycle [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBiologically, quiescence and dormancy both are transient and reversible non-proliferative state [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The quiescent and dormant cells in tumor tissue are alive cells who temporary stop cell proliferation cessation [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Those non-proliferative cells in lung cancer patients were identified in patient-derived organoid-like cell clusters that are the therapy-resistant cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Those non-proliferative cells that enriched in tyrosine kinase inhibitors treated lung cancer mass bears an alveolar stem cell signature in parallel [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Commonly, quiescent and dormant cells are transiently in non-proliferative state and have drug-resistance, stemness, plasticity and diapause-like. With respect to dormancy, the chicken ovalbumin upstream promoter-transcriptional factor (Coup-TF1) based transcriptional repertoire programs cellular dormancy and regulates stem cell decision [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our previous study, we attempt to manipulate FAK signaling in lung cancer cells to activate apoptosis and to turn down tumor cell proliferation and metastasis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], since FAK pathway in the lung cancer cells derived from type II alveolar epithelial cells significantly regulates cell proliferation, survival, and metastasis. FAK inhibition led to growth cessation in tumor cell culture. The lung cancer cells exposed to FAK inhibitor partly are in senescence and epigenomic alternation in vitro. To evaluate the pharmacological effect of FAK inhibitor in vivo, orthotopic implantation of LLC1 cells in C57BL/6 mice lung was established as the lung cancer mice to test therapeutic efficacy of FAK inhibitor in vivo [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Intriguingly, cellular senescence in the tumor tissue in the C57BL/6 mice having LLC1 cell implantation is not detected after FAK inhibitor administration to the lung cancer mice, though FAK inhibitor can mitigate tumor cell number in lung cancer tissue. It seemed that those remnant cells in lung cancer tissue do not have proliferative ability. In turn, the antibodies that are used to evaluate non-proliferative cells were employed to immunohistochemical examination for dormant cell or quiescent cell in the lung cancer mass. Our data has revealed that tumor cells can survive under FAK inhibition or TKI-drug administration. This hinted cellular dormancy is a drug-resistant program in clinical cases as well as in the lung cancer animals.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Cell Culture\u003c/h2\u003e\u003cp\u003eLLC1 murine lung carcinoma cells and A549 human lung adenocarcinoma cells were obtained from ATCC (Manassas, VA, USA). LLC1 cells were cultured in DMEM (Gibco, 11965092) while A549 cells were maintained in RPMI-1640 medium (Gibco, 11875093). Both media were supplemented with 10% FBS (Gibco, 16000044) and 1% penicillin-streptomycin (Gibco, 15140122). Cells were incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO2. Subculturing was performed using 0.25% trypsin-EDTA (Gibco, 25200056) when cells reached 80\u0026ndash;90% confluency.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell viability assay\u003c/h2\u003e\u003cp\u003eThe antiproliferative effects of VS-4718 were evaluated using MTT assay in LLC1 cell lines. Cells (5\u0026times;10\u0026sup3;/well) were seeded in 96-well plates and treated with VS-4718 (0.1\u0026ndash;50 \u0026micro;M) for 72 hours. MTT reagent (100 \u0026micro;L, 0.5 mg/mL) was added and incubated for 4 hours at 37\u0026deg;C. Formazan crystals were dissolved in 100 \u0026micro;L DMSO with gentle agitation. Absorbance was measured at 570 nm using a microplate reader (BioTek Instruments). Cell viability was calculated as percentage of vehicle control (0.01% DMSO). IC₅₀ values were determined using non-linear regression analysis with GraphPad Prism software. Experiments were performed in sextuplicate and repeated three times independently.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Lung cancer cell growing in lung and FAK inhibitor administration.\u003c/h2\u003e\u003cp\u003eC57BL/6 mice that applied to establishment of lung cancer mice were purchased from Charles River Breeding Laboratory (Charles River Technology, Bio-LASCO Taiwan Co. Ltd., Taiwan). Animals were housed at room temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C), with a humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, and automatically controlled cycle of 12 h day and night. Animal experiments, including surgical procedures, drug administration and animal sacrifice, were approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University and permission was issued in 112246 documents.\u003c/p\u003e\u003cp\u003eC57BL/6 mice (equal numbers of males and females) aged 10\u0026ndash;12 weeks and weighing in the range of 20 to 25 g were subjected to orthotopic implantation for in situ lung tumor growth [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Lung cancer animals were established with the orthotopic implantation by 10\u003csup\u003e3\u003c/sup\u003e mouse Lewis lung carcinoma cells (LLC1) in 50 \u0026micro;l serum free DMEM/matrix injected to upper lobe of left lung. Prior to orthotopic implantation, animals were anesthetized with a mixture of 3.5% isoflurane in 100% oxygen throughout the experimental procedure and the surgical site was disinfected with ethanol-free iodine solution. Then, 5 mm incision was made in the area of the left lateral thorax to make thoracic ribs and intercostal muscle exposed. Following, a 3 mm incision in muscle layer was made and the LLC1 cells in mixture of DMEM and matrix gel were directly injected to upper lobe of left lung with a 1 ml syringe. After implantation, the intercostal muscle layer and skin were swan up with 6\u0026thinsp;\u0026minus;\u0026thinsp;0 silk sutures.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 FAK inhibitor administration\u003c/h2\u003e\u003cp\u003eFour animals were settled in this study, namely sham is the mice were without LLC1 implantation and VS-4718 administration, vehicle are the mice were without LLC1 implantation but had VS-4718 administration, lung cancer group is the mice received LLC1 implantation but did not have VS-4718 administration and FAK inhibitor treated lung cancer group is the mice had LLC1 implantation and VS-4718 administration. At 7th day post-LLC1 cells implantation, FAK inhibitor treated lung cancer mice began with VS-4718 administration and the VS-4718 administration lasted for two weeks. FAK inhibitor administration was performed with daily oral garage of FAK inhibitor with dosage 50 mg/kg/day VS-4718. For FAK inhibitor administration, the VS-4718 was prepared in 0.5% carboxymethyl cellulose and 0.1% Tween 80. The lung cancer mice had daily 0.5% carboxymethyl cellulose and 0.1% Tween 80 without VS-4718 for two weeks. Mice for vehicle group were daily dosed 50 mg/kg VS-4718 for two weeks.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Histological and immunohistochemical examination\u003c/h2\u003e\u003cp\u003eLung and lung cancer tissues were harvested when animals were sacrificed. Human lung cancer tissue samples were obtained from patients under approval from the Institutional Review Board of Kaohsiung Medical University Hospital (IRB No: KMUHIRB-E(I)-20220330). All biopsies were immersed in 4% paraformaldehyde phosphate buffered normal saline for 24 hours. Following fixation, lung and lung cancer tissues were dehydrated and embedded in paraffin wax. Tissue sections in paraffin blocks were sliced at 4 \u0026micro;m with a sliding microtome (SM2125, Leica Biosystem, Nussloch, Germany) and placed on adhesive microscope slides (Pro-01, Matsunami Glass Ind., Ltd., Osaka, Japan). To examine cancer cells in lung tissues, specimens were dewaxed with xylene and rehydrated with serial dilutions of 100%, 90%, 70%, 50% ethanol in water, followed by water. The specimens were subjected to Hematoxylin \u0026amp; Eosin staining (HMM500 and EYB500, ScyTek Lab. Inc., Logan, UT-84323, USA). Antibody against COUP-TF1 was applied for immunohistochemical examination of COUP-TF1 expression in lung cancer tissues. After dewaxing and rehydration, specimens were probed with COUP-TF1 antibody overnight. COUP-TF1 in lung cancer tissues was visualized using a peroxidase-catalyzed Diaminobenzidine (DAB) substrate system (IHC detection kit) and conducted according to the manufacturers' instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Immunofluorescence Analysis\u003c/h2\u003e\u003cp\u003eFor protein localization studies, cells were plated onto 12-mm glass coverslips in 24-well dishes and exposed to VS-4718 (10 \u0026micro;M) or DMSO vehicle for 48 hours. Cells were subsequently fixed using 4% formaldehyde solution in phosphate-buffered saline for 15 minutes, followed by membrane permeabilization with 0.1% Triton X-100 for 10 minutes at ambient temperature. Non-specific antibody binding was prevented by incubating cells in 5% normal goat serum blocking solution for 60 minutes. Primary antibody staining was performed using mouse anti-β-catenin (1:200 dilution) and rabbit anti-COUP-TF1 (1:100 dilution) antibodies applied overnight at 4\u0026deg;C. Following triple PBS washes, cells were labeled with species-specific secondary antibodies: goat anti-rabbit Alexa Fluor 488 (1:500, green fluorescence) targeting COUP-TF1 and goat anti-mouse Alexa Fluor 594 (1:500, red fluorescence) targeting β-catenin, incubated for 1 hour in darkness at room temperature. Nuclear staining was achieved using DAPI (1 \u0026micro;g/mL) applied for 5 minutes. Coverslips were sealed with ProLong Gold mounting solution to preserve fluorescence. Image acquisition was performed using a Leica DMi8S fluorescence microscope with various magnification objectives (20\u0026times;, 40\u0026times;, 63\u0026times;). High-resolution confocal microscopy was conducted on an OLYMPUS FV1000 system employing sequential laser excitation to minimize spectral overlap. Protein co-localization was quantified using ImageJ analysis software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Western Blot Analysis\u003c/h2\u003e\u003cp\u003eCells were harvested and lysed in 1x RIPA buffer (Merck, Darmstadt, Germany) containing protease and phosphatase inhibitors. The protein concentration was determined using a Bio-Rad DC protein assay kit (Bio-Rad, California, USA). For Western blot analysis, 30 \u0026micro;g of total protein was subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% skim milk for 2 h in TBST buffer (20 mM Tris-Cl, 150 mM NaCl, 0.1% Tween 20, pH 7.4). After blocking, the membranes were incubated with primary antibodies overnight at 4\u0026deg;C. Primary antibodies against FAK (1:1000), phospho-FAK (Tyr397) (1:1000), β-catenin (1:1000), COUP-TF1 (1:500), p27 (1:1000), and Cyclin A1 (1:1000) were used for immunoblotting. β-actin (1:5000) served as a loading control. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (ECL, GE Healthcare) and quantified by densitometric analysis using ImageJ software. All experiments were performed in triplicate, and representative blots are shown.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe experimental data were digitized and analyzed using GraphPad Prism software (version 9.0). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). One-way ANOVA followed by Tukey's post-hoc test was used to compare digitized data and measurements from independent experiments in multiple groups, and the unpaired Student's \u003cem\u003et\u003c/em\u003e-test was used to compare two independent samples. For survival analysis, Kaplan-Meier curves were constructed and compared using the log-rank test. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 indicating higher levels of significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003e\u003cb\u003e3.1 FAK Inhibitor, VS-4718, Treatment Suppresses Tumor Progression and Improves Survival in LLC1 Lung Cancer Mouse Model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the direct anti-tumor effects of VS-4718 on lung cancer cells we assessed its cytotoxic activity in vitro using lung cancer cell lines. VS-4718 treatment significantly reduced cell viability in a dose-dependent manner over 4 days in murine LLC1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) or human A549 lung cancer cells line (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). VS-4718 exhibited concentration-dependent growth inhibition in LLC1 cells, with modest effects at 0.1\u0026ndash;1 \u0026micro;M and pronounced cytotoxicity at 10\u0026ndash;50 \u0026micro;M that nearly eliminated proliferation at the highest concentration tested. Doubling time analysis confirmed these anti-proliferative effects in LLC1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) or A549 cells (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). VS-4718 treatment gradually increased doubling times from 1.3 d (control) to 1.5 d (0.1 \u0026micro;M) and 2.3 d (1 \u0026micro;M), with 10 \u0026micro;M causing severe growth damage and 50 \u0026micro;M producing negative doubling times showing cell death.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further evaluate the therapeutic potential of FAK inhibition in lung cancer, we established an orthotopic syngeneic mouse model using Lewis lung carcinoma 1 (LLC1) cells implanted into C57BL/6 mice. Following tumor establishment, mice were randomized into treatment groups and administered VS-4718 (50 mg/kg/day, p.o.) or vehicle control for 14 consecutive days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). At the experimental endpoint, VS-4718 treatment significantly reduced body weight at sacrifice compared to vehicle controls (22.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 g vs. 25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 g) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), potentially reflecting both metabolic effects of FAK inhibition and reduced tumor burden. More importantly, treatment with VS-4718 led to a significant reduction in total lung weight compared to the untreated tumor-bearing group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), directly indicating decreased tumor burden and effective suppression of tumor growth. Additionally, the number of visible surface tumor nodules on the lungs was markedly decreased in VS-4718-treated mice, demonstrating the antitumor activity of the compound (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Kaplan-Meier survival analysis over a 21-day observation period revealed a significant improvement in overall survival with VS-4718 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Vehicle control tumor-bearing mice showed progressive mortality beginning around day 15, while VS-4718-treated mice maintained high survival rates throughout the observation period. These survival data support the potential of VS-4718 to not only suppress tumor progression but also to extend survival in this lung cancer model. Collectively, these findings demonstrate that VS-4718 effectively suppresses tumor burden and improves survival in the LLC1 syngeneic lung cancer model, exerting potent anti-tumor effects both in vitro and in vivo.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Histological Evaluation Reveals Significant Tumor Suppression and Cellular Remodeling Following VS-4718 Treatment\u003c/h2\u003e\u003cp\u003eTo further characterize the anti-tumor effects of VS-4718, we performed comprehensive histological analysis of lung tissues from both treatment groups. Hematoxylin and eosin (H\u0026amp;E) staining revealed distinct morphological differences between vehicle control and VS-4718-treated tumor-bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHistological examination demonstrated markedly reduced tumor infiltration and preservation of normal alveolar architecture in the VS-4718 group compared to vehicle controls. At 100\u0026times; and 400\u0026times; magnifications, untreated lung cancer tissues exhibited dense tumor cell accumulation with disrupted alveolar structures and invasive cellular morphology. In contrast, VS-4718-treated lungs showed significantly decreased cellular density, reduced nuclear atypia, and partial reconstitution of alveolar spaces.\u003c/p\u003e\u003cp\u003eQuantitative histomorphometric analysis confirmed significant reduction in tumor area following VS-4718 treatment compared to vehicle controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, n\u0026thinsp;=\u0026thinsp;7 mice per group; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Sirius Red staining analysis revealed a significantly higher percentage of fibrotic areas in the VS-4718 group compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating increased stromal deposition associated with tumor suppression. Nuclear-to-cytoplasm ratio analysis showed drastic reduction in tumor cell density per lung cross-section following VS-4718 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eTherefore, these histological findings conclude that VS-4718-mediated FAK inhibition not only diminishes tumor burden but also induces significant tissue remodeling and restoration of pulmonary architecture in a murine lung cancer model, providing further mechanistic support for the anti-tumor efficacy of FAK-targeted therapy.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.3 VS-4718 Alters Cell Cycle Regulator Expression by Increasing p27 and Reducing Cyclin A1 Levels in Lung Cancer Models\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the molecular mechanisms underlying the anti-tumor effects of VS-4718, we examined the expression of key cell cycle regulatory proteins, p27 and Cyclin A1, in both murine (LLC1) lung cancer models. These proteins play crucial roles in the control of G1/S phase progression and are influenced by FAK signaling pathways. Immunohistochemical analysis of LLC1 tumor tissues revealed \u003cb\u003eregion-specific\u003c/b\u003e p27 expression patterns following VS-4718 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). While p27 levels in tumor interior regions remained unchanged between vehicle control and VS-4718-treated groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n.s.), tumor margin areas demonstrated significantly increased p27 expression following VS-4718 treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, Cyclin A1 expression was markedly reduced throughout the tumor following VS-4718 treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicating suppression of cell cycle progression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo validate these findings at the protein level, western blot analysis was conducted using lysates from LLC1. VS-4718 treatment resulted in increased p27 expression and a consistent downregulation of Cyclin A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). Notably, these effects were irreversible (Figure S2), as withdrawal of VS-4718 treatment for 24 hours failed to restore phospho-FAK and Cyclin A1 levels, suggesting that FAK inhibition induced sustained molecular changes rather than transient pharmacological effects. These results indicate that VS-4718 exerts its anti-proliferative effects through region-specific modulation of p27 expression and consistent downregulation of Cyclin A1, consistent with the immunohistochemical findings, providing mechanistic insight into how FAK inhibition impairs tumor cell cycle progression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 VS-4718 Treatment Upregulates COUP-TF1 and Suppresses β-catenin Expression in Lung Cancer Cells\u003c/h2\u003e\u003cp\u003eTo evaluate the downstream signaling mechanisms affected by FAK inhibition and identify potential molecular targets involved in the observed anti-tumor effects, we investigated the interaction between COUP-TF1 (chicken ovalbumin upstream promoter transcription factor 1) and β-catenin, key regulators of the Wnt signaling pathway. We employed dual immunofluorescence co-localization analysis in lung cancer cell lines to assess protein-protein interactions and subcellular distribution patterns, complemented by Western blot analysis to quantify protein expression levels following VS-4718 treatment.\u003c/p\u003e\u003cp\u003e Immunofluorescence co-staining analysis in LLC1 lung cancer cells revealed marked changes in COUP-TF1 (green) and β-catenin (red) cellular distribution and co-localization after 48-hour VS-4718 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In control cells, COUP-TF1 and β-catenin showed widespread co-localization in cytoplasm, as demonstrated by strong yellow/orange signals in merged images. VS-4718 treatment triggered significant nuclear redistribution of COUP-TF1 and β-catenin, with both proteins showing enhanced nuclear translocation and decreased spatial separation. Immunofluorescence revealed a transition from co-localization to staining patterns clearly concentrated in specific nuclear regions, suggesting that FAK inhibition promotes nuclear translocation of both proteins while avoiding their normal co-localization in the cytoplasm, potentially enhancing their regulatory functions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess the effects of FAK inhibition on COUP-TF1 and β-catenin protein levels, we performed Western blot analysis in LLC1 cell lines following 48-hour VS-4718 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). VS-4718 treatment significantly increased COUP-TF1 and β-catenin protein expression compared to vehicle controls in LLC1 cells.\u003c/p\u003e\u003cp\u003eThese data indicate that VS-4718 regulates key transcriptional and signaling molecules by enhancing COUP-TF1 expression while concurrently concentrating β-catenin. This suggests that FAK inhibition may suppress tumor progression and metastatic potential by manipulating the nuclear localization of COUP-TF1/β-catenin regulatory pathways.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 VS-4718 treatment reduces FAK expression in vivo and correlates with clinical COUP-TF1 expression patterns.\u003c/h2\u003e\u003cp\u003eTo validate target engagement and assess the clinical relevance of our preclinical findings, we performed comprehensive immunohistochemical analysis to examine COUP-TF1 expression in VS-4718-treated LLC1 tumor tissues and evaluated COUP-TF1 expression patterns in human lung cancer specimens from patients receiving tyrosine kinase inhibitor (TKI) therapy. This dual approach aimed to confirm direct pharmacological effects of VS-4718 on its primary target while establishing potential clinical correlations for the identified molecular pathways.\u003c/p\u003e\u003cp\u003eTo address the effects of FAK inhibition on COUP-TF1 expression and assess the clinical relevance of our preclinical findings, we performed comprehensive immunohistochemical analysis of COUP-TF1 protein expression in LLC1 tumor tissues revealed region-specific responses to VS-4718 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Representative images demonstrated distinct differences in COUP-TF1 staining patterns between vehicle control and VS-4718-treated tumors across anatomically different regions. In non-defined tumor areas, quantitative analysis using DAB staining area measurements showed no significant difference between control and VS-4718-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n.s.). However, analysis of bronchiole areas revealed a markedly different response pattern. Control tumors in bronchiole regions exhibited moderate COUP-TF1 expression, while VS-4718 treatment resulted in significantly increased COUP-TF1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This region-specific upregulation of COUP-TF1 in bronchiole areas supports our in vitro findings and suggests that FAK inhibition preferentially affects COUP-TF1 expression in specific anatomical contexts. To evaluate the clinical relevance of COUP-TF1 regulation, we analyzed COUP-TF1 expression in human lung cancer tissues obtained from patients before and after TKI therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). We focused our analysis on bronchiole areas, as these regions demonstrated the most pronounced responses in our preclinical studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of patient samples revealed variable COUP-TF1 expression patterns between patients and treatment timepoints. In Subject 1, weak COUP-TF1 staining was observed in bronchiole areas prior to TKI therapy, while enhanced expression and improved tissue organization were evident following treatment. Post-treatment samples demonstrated increased numbers of COUP-TF1-positive cells and more organized bronchiolar architecture. Subject 2 displayed distinct baseline COUP-TF1 expression patterns, with marked changes in cellular distribution following TKI therapy. Post-treatment samples exhibited altered COUP-TF1 localization, characterized by increased nuclear staining in bronchiolar epithelial cells and adjacent tissue regions.\u003c/p\u003e\u003cp\u003eThese data demonstrate that VS-4718-mediated FAK inhibition specifically enhances COUP-TF1 expression in bronchiole regions of tumor tissues, establishing COUP-TF1 as a downstream target of FAK signaling in tissue-specific contexts. The regional specificity of this response indicates that FAK-COUP-TF1 pathway interactions may play a critical role in bronchiole-associated tumor development. The changes in COUP-TF1 expression patterns observed after TKI therapy indicate the potential clinical value of COUP-TF1 as both a biomarker for treatment response and a therapeutic target.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Therapeutic Promise of VS-4718 and COUP-TF1-Mediated Resistance Mechanisms\u003c/h2\u003e\u003cp\u003eThis study demonstrates that FAK inhibition with VS-4718 produces significant anti-tumor effects in the LLC1 lung cancer model through multiple mechanisms. VS-4718 treatment reduced tumor burden, improved survival, and restored normal pulmonary architecture with increased fibrotic remodeling. At the molecular regulatory level (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e), VS-4718 induced cell cycle arrest through selective p27 upregulation at tumor margins and reduced Cyclin A1 expression. Additionally, FAK inhibition induced COUP-TF1/β-catenin co-expression level, indicating modulatory signaling pathways. The region-specific upregulation of COUP-TF1 in bronchiole areas suggests tissue-dependent regulation of these pathways. Clinical analysis of COUP-TF1 expression in TKI-treated patients revealed variable expression patterns, supporting its potential utility as a biomarker for treatment monitoring and patient stratification. In conclusion, VS-4718 represents a promising therapeutic approach for lung cancer through simultaneous targeting of cell cycle regulation and COUP-TF1/β-catenin signaling pathways. These findings support further clinical development of FAK inhibitors and suggest COUP-TF1 expression monitoring may provide valuable insights for personalized lung cancer therapy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThis study provides comprehensive evidence that pharmacological inhibition of FAK using VS-4718 exerts potent anti-tumor effects in lung cancer through coordinated modulation of cell cycle progression and transcriptional regulatory networks. Our findings demonstrate that FAK inhibition not only reduces tumor burden and improves survival but also induces significant molecular changes that may contribute to therapy resistance through cellular dormancy mechanisms.\u003c/p\u003e\u003cp\u003eThe therapeutic efficacy of VS-4718 in the LLC1 syngeneic mouse model is consistent with previous reports demonstrating the critical role of FAK in lung cancer progression [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The significant reduction in tumor burden, decreased surface nodules, and improved survival outcomes align with studies showing that FAK inhibition disrupts key oncogenic processes including cell proliferation, survival, and metastasis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The preservation of normal pulmonary architecture following VS-4718 treatment suggests that FAK inhibition may offer therapeutic benefits beyond simple growth suppression, potentially facilitating tissue repair and regeneration processes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur molecular analysis reveals that VS-4718 induces cell cycle arrest through region-specific modulation of key checkpoint proteins. The selective upregulation of p27 at tumor margins, while maintaining unchanged levels in tumor interior regions, indicates spatial heterogeneity in treatment response [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This pattern suggests that FAK inhibition preferentially affects actively proliferating cells at the tumor-host interface, which may be more vulnerable to cell cycle disruption. The concurrent reduction in Cyclin A1 expression provides additional evidence for G1/S checkpoint arrest, consistent with the established role of these proteins in cell cycle regulation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe disruption of COUP-TF1/β-catenin interactions represent a novel mechanism by which FAK inhibition affects cellular behavior. COUP-TF1 has been increasingly recognized as a master regulator of cellular dormancy and stemness in cancer cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The reciprocal regulation of COUP-TF1 upregulation and β-catenin downregulation following VS-4718 treatment suggests that FAK signaling normally suppresses dormancy-associated transcriptional programs while promoting Wnt pathway activity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This finding is particularly significant given that cellular dormancy represents a major mechanism of therapy resistance in cancer treatment [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe region-specific upregulation of COUP-TF1 in bronchiole areas following VS-4718 treatment provides important insights into tissue-dependent regulation of dormancy pathways. Bronchiolar structures in the lung have been identified as niches that harbor stem-like cells and may serve as reservoirs for therapy-resistant cancer cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The preferential induction of COUP-TF1 in these anatomical regions suggests that FAK inhibition may inadvertently promote cellular dormancy in specific microenvironmental contexts, potentially contributing to the survival of residual cancer cells observed in our histological analysis.\u003c/p\u003e\u003cp\u003eThe clinical analysis of COUP-TF1 expression in TKI-treated patients provides translational validation of our preclinical findings while highlighting the complexity of therapy resistance mechanisms in clinical settings. The variable expression patterns observed between patients reflect the heterogeneous nature of treatment responses and underscore the need for personalized therapeutic approaches [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The enhanced COUP-TF1 expression following TKI therapy supports the hypothesis that targeted therapies may inadvertently select for dormant, therapy-resistant cell populations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur findings have important implications for the clinical development of FAK inhibitors in lung cancer therapy. While VS-4718 demonstrates clear anti-tumor efficacy, the induction of COUP-TF1-mediated dormancy pathways may contribute to the emergence of therapy-resistant cell populations. This dual effect suggests that FAK inhibitor monotherapy may be insufficient for complete tumor eradication and supports the development of combination therapeutic strategies that simultaneously target both proliferating and dormant cancer cell populations [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSeveral limitations of this study should be acknowledged. The use of a single mouse model may not fully recapitulate the heterogeneity of human lung cancer, and longer follow-up studies are needed to assess the potential for tumor recurrence from dormant cell populations. Additionally, the molecular mechanisms linking FAK signaling to COUP-TF1 regulation require further investigation to identify potential therapeutic targets for preventing dormancy induction.\u003c/p\u003e\u003cp\u003eIn summary, our study demonstrates that FAK inhibition with VS-4718 produces significant anti-tumor effects through coordinated modulation of cell cycle progression and transcriptional regulatory networks. However, the concurrent induction of COUP-TF1-mediated dormancy pathways highlights the complexity of therapy resistance mechanisms and underscores the need for combination therapeutic approaches in lung cancer treatment.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eVS-4718 exhibits significant anti-tumor efficacy in lung cancer models by inducing cell cycle arrest through p27 upregulation, modulating COUP-TF1/β-catenin signaling pathways, and restoring normal pulmonary architecture. It demonstrates promising therapeutic potential with region-specific molecular effects and measurable survival benefits. However, the concurrent induction of COUP-TF1-mediated cellular dormancy pathways, particularly in bronchiole regions, may contribute to therapy resistance mechanisms. Therefore, this study focused on developing combination therapeutic regimens that simultaneously target FAK-mediated proliferation and COUP-TF1-induced dormancy mechanisms to prevent therapy resistance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eFunding Statement:\u0026nbsp;\u003c/strong\u003eThis work was supported by grants from the National Science and Technology Council (NSTC), Taiwan (NSTC 113-2314-B-037-104-MY3 and NSTC 114-2314-B-110-006), Kaohsiung Medical University Hospital (KMUH112-2R16), the NYCU-KMU Joint Research Program (NYCUKMU-113-I1003), and the NSYSU-KMU Joint Research Project (NSYSU-KMU-113-P36).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization: Po-Kai Chuang, Chih-Jen Yang; Methodology: Chih-Jen Yang, Cheng-Hao Chuang, Yen-Yi Zhen, Ching-Tang Huang, Pei-Hui Wang; Investigation: Yen-Yi Zhen, Ching-Tang Huang, Pei-Hui Wang; Formal Analysis: Po-Kai Chuang, Chih-Jen Yang, Cheng-Hao Chuang, Yen-Yi Zhen, Ching-Tang Huang, Pei-Hui Wang, Ying-Ray Lee; Resources: Cheng-Hao Chuang, Huei-Yang Hung, Michael Hsiao, Ping-Lun Jiang; Visualization: Po-Kai Chuang, Cheng-Hao Chuang, Yen-Yi Zhen, Ching-Tang Huang, Pei-Hui Wang, Yung-Kuo Lee, Chih-Yang Wang; Writing\u0026mdash;Original Draft: Po-Kai Chuang, Yen-Yi Zhen; Writing\u0026mdash;Review \u0026amp; Editing: Po-Kai Chuang, Chih-Jen Yang; Project Administration: Po-Kai Chuang, Chih-Jen Yang, Ming-Shyan Huang; Funding Acquisition: Po-Kai Chuang, Chih-Jen Yang, Cheng-Hao Chuang; Validation: Ching-Tang Huang, Pei-Hui Wang. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials:\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval:\u0026nbsp;\u003c/strong\u003eAll animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University (approval number: 112246) and conducted in accordance with institutional guidelines for animal welfare. Human lung cancer tissue samples were obtained under approval from the Institutional Review Board of Kaohsiung Medical University Hospital (IRB No: KMUHIRB-E(I)-20220330). All procedures involving human subjects were performed in accordance with the Declaration of Helsinki and relevant ethical guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest to report regarding the present study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u0026nbsp;\u003c/strong\u003eThe supplementary material is available online.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKonieczkowski DJ, Johannessen CM, Garraway LA. A convergence-based framework for cancer drug resistance. Cancer Cell. 2018;33(5):801-815.\u003c/li\u003e\n\u003cli\u003eSkarkova A, Bizzarri M, Janostiak R, Masek J, Rosel D, Brabek J. Educate, not kill: treating cancer without triggering its defenses. Trends Mol Med. 2024;30:673-685.\u003c/li\u003e\n\u003cli\u003eIngham J, Ruan JL, Coelho MA. 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The somatic mutation profiles of 2,433 breast cancers refine their genomic and transcriptomic landscapes. Nat Commun. 2016;7(1):11479.\u003c/li\u003e\n\u003cli\u003eWnt/\u0026beta;-catenin signaling pathway in carcinogenesis and cancer therapy. J Hematol Oncol. 2024;17:44.\u003c/li\u003e\n\u003cli\u003eChronic WNT/\u0026beta;-catenin signaling induces cellular senescence in lung epithelial cells. Cell Mol Life Sci. 2020;87:1425-1439.\u003c/li\u003e\n\u003cli\u003eWNT/beta-catenin signalling interrupts a senescence-induction cascade in human mesenchymal stem cells. Cell Mol Life Sci. 2022;79:88.\u003c/li\u003e\n\u003cli\u003eRock JR, Onaitis MW, Rawlins EL, Lu Y, Clark CP, Xue Y, et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci USA. 2009;106(31):12771-12775.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Focal adhesion kinase, Lung cancer, Cellular dormancy, COUP-TF1, Cell cycle arrest, Therapy resistance","lastPublishedDoi":"10.21203/rs.3.rs-7615921/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7615921/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAlthough focal adhesion kinase (FAK) inhibition shows promise in lung cancer therapy, emerging evidence suggests it may promote cellular dormancy and drug resistance through transcriptional regulation. We investigated the therapeutic efficacy and drug resistance inhibition mechanisms of FAK inhibitor VS-4718 in lung cancer.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eUsing an orthotopic syngeneic LLC1 mouse model, we evaluated the effects of VS-4718 (50 mg/kg/day) on tumor progression, survival, and molecular mechanisms. Comprehensive analyses included histological examination, immunohistochemistry, Western blotting, and clinical tissue validation from TKI-treated patients.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eVS-4718 demonstrated significant anti-tumor efficacy, reducing tumor burden by 60%, decreasing surface nodules, and improving overall survival (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Mechanistically, FAK inhibition induced cell cycle arrest through spatially heterogeneous p27 upregulation at tumor margins while suppressing Cyclin A1 expression. Unexpectedly, VS-4718 controlled COUP-TF1/β-catenin interactions, leading to reciprocal protein regulation. Critically, region-specific analysis revealed selective COUP-TF1 upregulation in bronchiolar areas, indicating anatomically-restricted dormancy pathway activation. Clinical validation in TKI-treated patient samples confirmed variable COUP-TF1 expression patterns, supporting its potential as a therapeutic resistance biomarker.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eVS-4718 achieved significant therapeutic benefits through coordinated regulation of cell cycle and transcriptional networks. However, concurrent induction of COUP-TF1-mediated dormancy pathways, particularly in bronchiolar niches, may promote the formation of therapy-resistant cell populations. These findings reveal a fundamental paradox in FAK-targeted therapy and suggest that monotherapy may be insufficient for complete tumor eradication. Our\u003c/p\u003e","manuscriptTitle":"FAK Inhibition Demonstrates Dual Effects of Tumor Suppression and Dormancy Induction Through Region-Specific COUP-TF1 Regulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 02:13:41","doi":"10.21203/rs.3.rs-7615921/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9b3b59c8-f584-4b86-93d4-b93cf19b2d16","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55729854,"name":"Biological sciences/Cancer/Cancer therapy/Cancer therapeutic resistance"},{"id":55729855,"name":"Biological sciences/Cell biology/Senescence"},{"id":55729856,"name":"Biological sciences/Drug discovery/Drug regulation"},{"id":55729857,"name":"Biological sciences/Drug discovery/Biomarkers/Prognostic markers"},{"id":55729858,"name":"Biological sciences/Cell biology/Cell signalling/Checkpoint signalling"}],"tags":[],"updatedAt":"2025-11-17T19:13:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-17 02:13:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7615921","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7615921","identity":"rs-7615921","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}