ONC201 enhances the cytotoxic effect of cisplatin through ATF3/ATF4/CHOP in HNSCC cells

ONC201 enhances the cytotoxic effect of cisplatin through ATF3/ATF4/CHOP in HNSCC cells | 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 ONC201 enhances the cytotoxic effect of cisplatin through ATF3/ATF4/CHOP in HNSCC cells Ming Huei Chou, Hui-Ching Chuang, Jiin-Haur Chuang, Ya-Ting Hong, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5230558/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Jun, 2025 Read the published version in Oncogenesis → Version 1 posted 9 You are reading this latest preprint version Abstract Head and neck squamous cell carcinoma (HNSCC) remains a prevalent and challenging cancer to treat due to its genetic heterogeneity. Cisplatin resistance is one of important causes in treatment failure of locally advanced HNSCC. This study is aimed to investigate the potential of ONC201 to enhance the cisplatin cytotoxicity in HNSCC cells through the ATF3/ATF4/CHOP pathway. At first, cisplatin-resistant HNSCC cell lines (OC2-CR1) were built up and then, the treatment effectiveness of ONC201 alone and cisplatin in combination on cell viability, DNA damage, reactive oxygen species (ROS) production, and stress response markers were evaluated. Cisplatin-resistant cells showed increased IC50 values compared to parental OC2 cells. Combined treatment with ONC201 and cisplatin significantly increased mitochondrial ROS and intracellular ROS levels, enhancing DNA damage and apoptosis through ATF3/ATF4/CHOP-mediated pathways. In vivo studies by using xenograft models demonstrated that the combination treatment suppressed tumor growth more effectively than either agent alone. ONC201 significantly potentiates the cytotoxic effects of cisplatin in HNSCC by modulating stress response pathways, suggesting a promising therapeutic strategy for cisplatin-resistant HNSCC. Biological sciences/Cancer/Head and neck cancer Biological sciences/Immunology/Cell death and immune response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers globally, often associated with risk factors such as tobacco use, betel quid chewing, alcohol consumption, and human papillomavirus (HPV) infection 3 , 6 , 9 , 12 . HNSCC primarily originates from the mucosal epithelium of the upper aerodigestive tract, including the oral cavity, larynx, oropharynx, and hypopharynx. Due to the lack of enhanced public awareness about the hazards from these substances, many patients are still diagnosed at an advanced stage. Current treatment strategies include surgery, radiotherapy, chemotherapy, target therapy, immunotherapy or combination. Despite of the multidisciplinary approach in the care of patients with HNSCC, there are still many patients who would experience the treatment failure. This is largely due to the remarkable heterogeneity of HNSCC, characterized by various genetic mutations and aberrant gene expressions 1 , 24 . Consequently, there is a need to develop a novel therapeutic agent that is able to provide more effective in the treatment outcomes. One of the current types of research focuses on advancing targeted therapy that exploit specific molecular pathways critical to cancer growth and survival. Cisplatin, a platinum-based agent is widely used as the first-line chemotherapy agent for HNSCC because of its potent anticancer activity. The mechanisms of cisplatin in treating HNSCC involve the formation of platinum-DNA adducts, which induce G2/S cell cycle arrest and subsequent cell death by causing DNA damage in rapidly dividing cancer cells 17 , 19 . Additionally, cisplatin can trigger cellular stress responses through the activation of ATF3 and ATF4, leading to the inhibition of MCL1 expression and inducing the apoptosis in HNSCC cells 4 , 21 . ONC201, a small molecule which has been shown to have anti-tumor efficacy by using a monotherapy or in combination with other treatments 13 , 18 in phase II clinical trials for various solid tumors and hematological malignancies. ONC201 which would activate the integrated stress response (ISR), inhibition of AKT/ERK signaling, and induction of ATF3, ATF4, and CHOP is a DRD2 antagonist and ClpP agonist. These actions culminate in TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in cancer cells. In HNSCC, ATF3 has been identified as a tumor suppressor that promotes apoptosis and inhibits cell growth, regulating various cellular processes in response to stressors such as DNA damage 16 . ATF4, another stress-responsive transcription factor, plays a role in programmed cell death by regulating downstream targets, including MCL1 15 . CHOP, also known as GADD153, is a multifunctional transcription factor that is upregulated in response to various stresses, such as DNA damage, ER stress, and mitochondrial dysfunction. CHOP mediates apoptosis by downregulating anti-apoptotic proteins like BCL2, BCL-XL, and MCL-1, and upregulating pro-apoptotic proteins like BIM, BAK, and BAX 10 . It plays a crucial role in coordinating cellular responses to stress, mediating cell fate decisions, including cell survival or programmed cell death 14 . The role of MCL1 in regulating apoptosis makes it a critical factor in determining cancer cell survival and resistance to therapy in HNSCC 20 . Cisplatin is the major backbone of chemotherapy agent for treatment of HNSCC 5 but the genetic heterogeneity and aberrant mutations could lead to primary cisplatin resistance, causing the treatment failure in locally advanced tumor or recurrent ones 8 . The ONC201 is able to induce mitochondria-mediated apoptosis by downregulating MCL1 and this phenomenon suggests its potential as an effective agent to overcome cisplatin resistance. In this study, we explore the combination of cisplatin and ONC201 to enhance the sensitivity of HNSCC to cisplatin treatment, aiming to demonstrate the synergistic efficacy of this combination as a promising strategy. Results Generation and Characterization of Cisplatin-Resistant HNSCC Cells Cisplatin-resistant HNSCC cells were generated from the OC2 cell line through a progressive selection process involving increasing concentrations of cisplatin (Sigma-Aldrich, UK) over a 6-month period. The selection began with low doses and culminated in continuous exposure to 1 μM cisplatin, resulting into the stable resistant phenotype designated as OC2-CR1 (Fig. 1A). To assess cisplatin sensitivity, OC2 and OC2-CR1 cells were treated with a range of cisplatin concentrations for 24 and 48 hours, and cell viability was evaluated by using the WST1 assay (Fig.1B). OC2 cells displayed sensitivity to cisplatin, while OC2-CR1 cells exhibited marked resistance. At 48 hours, the IC50 values were 2 μM for OC2 cells and 9 μM for OC2-CR1 cells, confirming the significant resistance of OC2-CR1 cells compared to the parental OC2 cells. To investigate the effects of cisplatin on DNA damage and apoptosis, Western blot analysis was performed to detect γH2AX and cleaved Poly (ADP-ribose) polymerase (PARP). Cisplatin treatment led to a dose-dependent increase in γH2AX and cleaved PARP expression in OC2 cells, indicative of DNA damage and activation of apoptotic pathways (Fig. 1C). In contrast, cisplatin-resistant OC2-CR1 cells showed only a modest increase in γH2AX and cleaved PARP expression, suggesting a reduced capacity to respond to cisplatin-induced DNA damage and apoptosis. ONC201 and Cisplatin Induced mtROS in HNSCC cells Excess cellular levels of reactive oxygen species (ROS) cause damage to intracellular lipids, proteins, nucleic acids and organelles, leading to cell death and damaged biological molecules via the endoplasmic reticulum (ER) stress-mediated pathway 7 , 22 . To investigate the role of oxidative stress in the anti-tumor effects of cisplatin and ONC201, mitochondrial ROS levels were assessed using confocal microscopy and flow cytometry. Mitochondrial ROS levels were measured with the MitoSox Red probe following treatment with ONC201, cisplatin, or their combination for 24 and 48 hours (Fig.2A). Flow cytometry was used to analyze both mitochondrial and intracellular ROS levels at 24, 48, and 72 hours by using MitoSOX Red and dihydroethidium (DHE) assays, respectively (Fig.2B, 2C and S1). The results demonstrated that ONC201 and cisplatin treatments, either alone or in combination, led to increased mitochondrial ROS production in OC2 cells at 24, 48, and 72 hours. Similarly, mitochondrial ROS levels were elevated in OC2-CR1 cells at 48 and 72 hours, but not at 24 hours. The DHE assay revealed that cisplatin treatment significantly raised intracellular ROS levels in both OC2 and OC2-CR1 cells after 48 hours. However, the combination of cisplatin and ONC201 at high doses resulted in a marked increase in intracellular ROS production, which was significant only in OC2-CR1 cells. Effects of ONC201 and Cisplatin on ER Stress-Mediated Cell Death Expression in HNSCC Cells Cisplatin and the ERK inhibitor ONC201 are known to induce an ATF-mediated integrated stress response (ISR) that inhibits the growth of various solid tumors. These agents promote cell death by inducing ISR and ER stress-mediated pathways. However, in resistant cancer cells, altered ISR and ER stress can diminish the efficacy of these drugs 23 . We investigated the expression of ER stress-related and cell death markers in OC2 and cisplatin-resistant OC2-CR1 cells following treatment with ONC201, cisplatin, or theses combination (Fig. 3 and S2). Western blot analysis revealed that ONC201 significantly increased ATF4 and CHOP expression at 24 hours but decreased it by 72 hours in OC2 cells. ATF3 expression was not elevated until 72 hours post-treatment. Conversely, cisplatin did not induce ATF4 expression at any time point (24, 48, or 72 hours) but did lead to high levels of ATF3 and CHOP expression. In the combination treatment, ATF3 levels were consistently higher at 24, 48, and 72 hours, while CHOP expression peaked at 24 and 48 hours before declining at 72 hours, potentially dependent on ATF4 expression. In OC2-CR1 cells, ONC201 and cisplatin treatment similarly induced ATF3 and CHOP at 24 and 48 hours, with a decline by 72 hours. This suggests that ONC201 and cisplatin induce CHOP through distinct mechanisms: ATF3/ATF4 in OC2 cells and primarily ATF3 in OC2-CR1 cells (Fig. 3A). Further analysis of ATF3/ATF4/CHOP-mediated cell death showed that ONC201 did not induce γH2AX, a marker of DNA damage, but did upregulate MCL1 at 24 hours. Both MCL1 and cleaved-PARP levels decreased at 48 and 72 hours in both cell lines. Cisplatin treatment in OC2 cells significantly increased γH2AX and cleaved-PARP expression while decreasing MCL1 in a dose-dependent manner. In OC2-CR1 cells, similar trends were observed, though the increases in γH2AX and cleaved-PARP were less pronounced. Combined treatment with ONC201 and cisplatin induced γH2AX and cleaved-PARP similarly to cisplatin alone at 24 and 48 hours in both cell lines, but significantly reduced cleaved-PARP and MCL1 levels by 72 hours (Fig. 3B). These findings suggest that ONC201 and cisplatin effectively inhibit MCL1 through distinct ER stress-mediated pathways, involving ATF4/CHOP and ATF3/CHOP in OC2 and OC2-CR1 cells, respectively. ONC201 and Cisplatin Inhibit Cell Proliferation and Migration in HNSCC Cells To assess the effects of cisplatin and ONC201 on colony formation in HNSCC cells, equal numbers of OC2 and cisplatin-resistant OC2-CR1 cells (100 cells per well) were treated with varying concentrations of cisplatin (0.25–1 µM) or ONC201 (0.25–1 µM) and allowed to grow for 9 days. Colony formation in both OC2 and OC2-CR1 cells was inhibited in a dose-dependent manner by both agents (Fig. 4A). When cells were treated with a combination of 1 µM cisplatin and 0.25–1 µM ONC201, there was a significant synergistic inhibition of colony formation. The combined treatment led to a more pronounced reduction in colony formation in both OC2 and OC2-CR1 cells compared to treatment with 1 µM cisplatin alone. Notably, OC2 cells exhibited a significantly greater decrease in colony formation when treated with 1 µM cisplatin, 1 µM ONC201, or the combination, compared to the more resistant OC2-CR1 cells. In addition to colony formation, we evaluated the effects of cisplatin and ONC201 on cell migration using a two-dimensional Oris™ cell migration assay. OC2 and OC2-CR1 cells were treated with 1 µM ONC201, 10 µM or 20 µM cisplatin, or a combination of ONC201 and cisplatin for 48 hours. The results demonstrated that both cisplatin and ONC201 significantly inhibited cell migration in HNSCC cells (Fig. 4B). The combination of cisplatin and ONC201 was more effective at inhibiting migration in both OC2 and OC2-CR1 cells than either agent alone. Interestingly, the combined treatment was more effective in inhibiting the migration of OC2-CR1 cells compared to OC2 cells, suggesting a heightened sensitivity of the resistant cells to the combination therapy. These findings indicate that ONC201 and cisplatin, particularly when used in combination, effectively inhibit both the proliferation and migration of HNSCC cells, with differential effects observed between the sensitive OC2 and resistant OC2-CR1 cell lines. Significant Anti-Proliferative Effects of Cisplatin and ONC201 in an Orthotopic Graft Model of HNSCC To evaluate the efficacy of cisplatin and ONC201 on tumor growth, we assessed these in vivo antitumor effects by using a xenograft model derived from OC2 and cisplatin-resistant OC2-CR1 cells in NU/NU mice. Tumor volumes were monitored twice a week with a digital vernier caliper, and visible dorsal tumors developed 7 days post-injection. Starting 7 days after tumor inoculation, mice received intraperitoneal injections of cisplatin (3 mg/kg) and ONC201 (50 mg/kg), either alone or in combination, twice a week for 4 weeks. Control groups underwent injections of saline (for cisplatin) or a vehicle solution (5% DMSO, 5% Tween 80, 40% PEG300, 50% saline) for ONC201 (Fig. 5A). In the OC2 xenograft model, both cisplatin and ONC201 significantly suppressed tumor growth compared to the control group. The combined treatment with cisplatin and ONC201 led to even more pronounced tumor suppression, with no significant differences observed in the body weights of the mice among the different treatment groups (Fig. 5B-C). However, in the cisplatin-resistant OC2-CR1 xenograft model, neither cisplatin nor ONC201 alone effectively inhibited tumor growth when compared to the control group. Remarkably, only the combination of cisplatin and ONC201 significantly suppressed the growth of OC2-CR1-derived tumors. These in vivo findings suggest that the combined treatment of cisplatin and ONC201 holds potential as a therapeutic strategy for overcoming cisplatin resistance in HNSCC, offering a promising approach to improve outcomes in resistant cases. Discussion The present study provides significant insights into the therapeutic potential of combining ONC201 with cisplatin to overcome the issue of resistance in HNSCC. Through comprehensive in vitro and in vivo investigations, we elucidated the mechanisms underlying the enhanced anti-cancer effects of this combination, particularly focusing on the modulation of the ATF3/ATF4/CHOP pathway (Fig.6). Our findings also underscore the challenges posed by the genetic heterogeneity of HNSCC and the need for innovative strategies to address the cisplatin resistance. The development of cisplatin resistance in HNSCC is a great challenge in the treatment of locally advanced tumor or recurrent metastasis tumor and it often leads to therapeutic failure and dismal clinical outcomes 1 , 11 . In this study, the cisplatin-resistant OC2-CR1 cells exhibited significantly higher IC50 values compared to the parental OC2 cells, confirming their resistant phenotype (Fig. 1). This marked difference in sensitivity highlights the difficulty in treating cisplatin-resistant HNSCC and address the importance of combination therapies to restore drug sensitivity. While cisplatin alone was able to induce DNA damage and apoptosis, its efficacy was notably reduced in the resistant OC2-CR1 cells, likely due to the upregulation of stress-responsive transcription factors like ATF3 (Fig. 3). This aligns with previous studies which had implicated ATF3 in the promotion of cell survival and resistance to radiation therapy 25 or chemotherapy 2 through the induction of anti-apoptotic proteins such as MCL1. ONC201, known for its role as a DRD2 antagonist and ClpP agonist, has demonstrated potent anti-tumor activity across various cancer types. In this study, ONC201 was shown to significantly sensitize OC2-CR1 cells to cisplatin, primarily by exacerbating oxidative stress and modulating ER stress pathways. The increase in mitochondrial and intracellular ROS levels following treatment with ONC201, particularly when combined with cisplatin, suggests that the generation of oxidative stress is a critical mechanism by which ONC201 enhances the cytotoxic effects of cisplatin. This increase in ROS levels was associated with heightened DNA damage and apoptosis, particularly in the cisplatin-resistant OC2-CR1 cells, thereby highlighting the potential of ONC201 to overcome chemoresistance (Fig. 2). The differential induction of stress response markers, such as ATF3, ATF4, and CHOP, between OC2 and OC2-CR1 cells demonstrates the distinct cellular mechanisms employed by these cell lines to cope with therapeutic stress (Fig.6). In both OC2 and cisplatin-resistant OC2-CR1 cells, cisplatin treatment primarily induced CHOP expression through ATF3. However, ONC201 induced CHOP expression primarily through ATF4 in OC2 cells, while in the resistant OC2-CR1 cells, CHOP induction was mediated predominantly by ATF3 (Fig. 3). This differential regulation of CHOP, a key pro-apoptotic factor, suggests that the combination of ONC201 and cisplatin effectively targets multiple pathways to induce apoptosis, thereby overcoming the resistance mechanisms presented in OC2-CR1 cells. Additionally, the downregulation of MCL1, an anti-apoptotic protein, by the combined treatment in both cell lines further supports the role of this pathway in sensitizing cells to apoptosis. The combination of ONC201 and cisplatin also demonstrated a significant inhibitory effect on colony formation and cell migration in both OC2 and OC2-CR1 cells, with a more pronounced effect observed in the resistant OC2-CR1 cells (Fig. 4). These findings suggest that the combination therapy not only enhances the cytotoxic effects of cisplatin but also effectively inhibits key processes involved in tumor progression and metastasis. The in vivo xenograft experiments provided strong evidence for the therapeutic potential of the ONC201 and cisplatin combination (Fig. 5). The combined treatment significantly suppressed tumor growth in both OC2 and OC2-CR1 derived xenograft models, demonstrating its efficacy in overcoming resistant mechanisms. Importantly, this treatment regimen did not result in significant weight loss in the mice, showing a less toxicity profile. These findings suggest that ONC201, when used in combination with cisplatin is a novel strategy to treat HNSCC, particularly in cases where resistance to cisplatin has evolved. Additionally, combination with other therapeutic agents to ONC201 such as chemotherapy agents, check point inhibitors, target therapy agents to treat HNSCC may provide other possible options to improve the clinical outcomes although more studies are necessary to prove this assumption. In conclusion, this study highlights the potential of ONC201 to augment the cytotoxic effects of cisplatin in HNSCC through the modulation of the ATF3/ATF4/CHOP pathway, particularly in overcoming the problem of cisplatin resistance. The differential responses between OC2 and OC2-CR1 cells demonstrate the importance of targeting multiple pathways to effectively treat against the resistant cancer phenotypes. The therapeutic value of combining ONC201 with cisplatin shows its ability to target multiple resistant mechanisms simultaneously. By inducing oxidative stress, modulating ER stress pathways, and promoting apoptosis through different molecular routes, this combination addresses the genetic heterogeneity and adaptive responses of HNSCC cells. This multi-faceted approach is helpful and promising in coping with the complexity of cancer resistance from cisplatin and improving the clinical outcomes in HNSCC. Materials/Subjects and Methods Induction of Cisplatin Resistance in OC2 Cells and Cell Culture Cisplatin-resistant OC2 cells (designated OC2-CR1) were established by exposing the parental OC2 cell line to progressively increasing concentrations of cisplatin (Sigma-Aldrich, UK) over a 6-month period. The selection process began with a low dose and culminated in continuous exposure to 1 μM cisplatin, resulting in the stable resistant phenotype. To validate resistance, IC50 values were determined by measuring the cells' response to cisplatin. Both OC2 and OC2-CR1 cells were maintained in RPMI 1640 medium (Invitrogen), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 μg/mL Amphotericin B solution (Invitrogen). Cells were seeded at a density of 5×10 6 cells per 10 cm culture dish and treated with varying concentrations of cisplatin, ONC201 (Selleckchem), or a combination of both. The cells were harvested at specific time points for subsequent in vitro analyses. Cell Viability Analysis (WST-1 Assay) OC2 and OC2-CR1 cells were seeded at a density of 10 4 cells per well in 96-well plates and treated with varying concentrations of cisplatin, ONC201, or their combination for 24 and 48 hours. Following the treatment period, 10 μL of WST-1 reagent (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene sulfonate) (Roche Diagnostics) was added to each well. The plates were then incubated at 37°C for an additional 1-4 hours to allow the formation of formazan dye. Absorbance was measured using a microplate reader, with the test wavelength set to 450 nm and the reference wavelength at 630 nm to correct for any background signal. The absorbance values were used to quantify cell viability, reflecting the metabolic activity of the cells post-treatment. Detection of Mitochondrial and Intracellular Reactive Oxygen Species (ROS) Mitochondrial and intracellular ROS levels in OC2 and OC2-CR1 cells were assessed using MitoSOX™ Red (Invitrogen) and Dihydroethidium (DHE, Merck/Sigma-Aldrich) fluorescent probes, respectively. Cells were first washed twice with PBS to remove any residual media. For mitochondrial ROS detection, cells were incubated with 5 μM MitoSOX™ Red, while intracellular ROS was detected by staining with 5 μM DHE. Both staining procedures were carried out for 15 minutes at 37°C. Following the staining, the cells were collected and washed twice with PBS to remove excess dye. The cells were then resuspended in 1 mL of PBS and transferred into flow cytometry tubes. The fluorescence intensity, indicative of ROS levels, was measured using a BD LSRII flow cytometer (BD Biosciences). Immunofluorescence Staining of Mitochondrial ROS Cells cultured on glass slides were washed twice with PBS and then incubated with 5 μM MitoSOX™ Red (Invitrogen) for 2 hours at 37°C to detect mitochondrial ROS. After incubation, the slides were fixed using an appropriate fixative (4% paraformaldehyde) and blocked to minimize non-specific binding. The slides were then co-stained with DAPI (4′,6-diamidino-2-phenylindole; Molecular Probes) to visualize the nuclei. Following staining, the slides were mounted using a fluorescent mounting medium (Dako Cytomation) and sealed with coverslips. Images were captured using an Olympus FluoView® confocal microscope, ensuring that exposure gains and rates were consistent across all samples to allow for accurate comparison. Fluorescence intensities were quantified separately for each color channel. Western Blot Analysis OC2 and OC2-CR1 cells were lysed in PRO-PREP protein extraction solution (iNtRON Bio) for 30 minutes on ice. The lysates were centrifuged at 13,000 rpm for 5 minutes at 4°C, and the resulting supernatants were collected. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad). Equal amounts of protein (20-50 μg) from each sample were mixed with loading buffer, boiled for 10 minutes, and then separated by SDS-PAGE on a 10% polyacrylamide gel. The proteins were transferred onto nitrocellulose membranes. Following transfer, the membranes were blocked in 5% non-fat milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies against ATF3, γ-H2AX (Abcam), ATF4, CHOP, c-PARP, MCL1 (Cell Signaling Technology), and β-actin (Sigma-Aldrich) according to the manufacturers’ protocols. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using a chemiluminescent substrate (Santa Cruz Biotechnology) and detected by exposure to X-ray film. Densitometric analysis was performed to quantify the intensity of the protein bands relative to β-actin as a loading control. Clonogenic Assay OC2 and OC2-CR1 cells were seeded at a density of 200 cells per well in 6-well plates and allowed to adhere for 48 hours. The cells were then treated with various concentrations of cisplatin, ONC201, or a combination of both, and cultured for an additional 10 days under standard conditions. After the treatment period, the cells were fixed with 4% paraformaldehyde and stained with 0.5% Crystal Violet for 30 minutes at room temperature. Excess stain was washed away with distilled water, and the plates were air-dried. Images of the stained colonies were captured using a digital camera. The number of colonies in each plate was counted manually or using imaging software. Relative plating efficiency (RPE) was calculated using the following formula: RPE = [total colonies of three well (test) / total colonies of three well (control)] x 100%. The results were expressed as a percentage of the control, and statistical analysis was performed to compare the clonogenic survival between different treatment groups. Oris™ Cell Migration Assay The migration of OC2 and OC2-CR1 cells was assessed using the Oris™ Cell Migration Assembly Kit (Platypus Technologies) following the manufacturer’s protocol. Cells were seeded at a density of 5×10^4 cells per well in Oris™ 96-well plates, each equipped with stoppers that create a central cell-free detection zone. The cells were allowed to adhere and grow overnight. After incubation, the Oris™ stoppers were gently removed to initiate the migration process. Tetramethylrhodamine ethyl ester perchlorate (TMRE, Invitrogen) was added to each well to fluorescently label the cells. The plates were then incubated for an additional 48 hours to allow for cell migration into the detection zones. At designated time points, the fluorescence intensity in the detection zones, which correlates with cell migration, was measured using a fluorescence plate reader. The fluorescence signals were converted to cell numbers, and the data were used to generate real-time migration curves. Animal Care and Xenograft Model Male NU/NU nude mice, 6 weeks of age, were obtained from the National Laboratory Animal Center and maintained in a specific pathogen-free (SPF) facility. The mice were provided with irradiated chow and autoclaved reverse-osmosis water ad libitum. All animal procedures were conducted following protocols approved by the Institutional Animal Care and Usage Committee. For tumor inoculation, the mice were anesthetized with Zoletil (15 mg/kg body weight), and 1×10^7 OC2 or OC2-CR1 cells suspended in 0.1 mL of sterile saline were subcutaneously injected into the right flank using a 26-gauge needle. Tumor development was monitored, and visible dorsal tumors were observed approximately 14 days post-injection. Starting from day 14 post-inoculation, the mice were treated with cisplatin (3 mg/kg), ONC201 (30 mg/kg), or a combination of both, administered intraperitoneally twice a week for 4 weeks. Control groups received either saline or vehicle (10% DMSO, 40% PEG, 5% Tween 80, and 45% saline). Tumor growth was measured twice weekly using a caliper, with tumor size calculated as the product of the longest dimension and its perpendicular. Mice were monitored every other day for tumor progression and signs of weight loss. The study was concluded 7 days after the final treatment, and mice were humanely euthanized by CO2 inhalation if they lost more than 25% of their pre-injection body weight. Statistical Analysis All data were expressed as the mean ± SEM for animal studies and cell migration analyses, and as the mean ± SD for in vitro experiments, with each experiment performed at least three times independently. Statistical comparisons between experimental groups and continuous variables were made by using Student’s t-test (unpaired, two-tailed). For comparisons involving multiple groups, a one-way ANOVA followed by a post-hoc test was employed to determine significant differences. Statistical significance was defined as P < 0.05. Declarations Acknowledgements: This study was supported by grant number CRRPG8K0081-3, CRRPG8N0041-3 from the Chang Gung Memorial Hospital and MOST 111-2314-B-182A-099, MOST 111-2314-B-182A-079 -MY3 and MOST 108-2314-B-182A-112-MY3 from the Ministry of Science and Technology (National Science and Technology Council). We thank Yu-Li Liu, Rui-An Lin, Shin-Yan Lee, Yu-An Li and Chih-Kuan Chao for discussions and technical assistance. Author Contributions: HCC was responsible for drafting the article, acquisition of data and final approval of this manuscript; JHC was responsible for critical review of this article and final approval of this manuscript; YTH was responsible for analysis and interpretation of data and final approval of this manuscript; MHC and CYC was responsible for conception and design and final approval of this manuscript. All authors have read and agreed to the published version of the manuscript. Conflict of Interest: The authors declare no conflict of interest. Availability of Data and Materials: All the raw data of this study are available from the corresponding author upon reasonable. References Alsahafi E, Begg K, Amelio I, Raulf N, Lucarelli P, Sauter T et al . Clinical update on head and neck cancer: molecular biology and ongoing challenges. Cell Death Dis 2019; 10: 540. Böpple K, Oren Y, Henry WS, Dong M, Weller S, Thiel J et al . 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Characterization of cancer genomic heterogeneity by next-generation sequencing advances precision medicine in cancer treatment. Precis Clin Med 2018; 1: 29-48. Zhao W, Sun M, Li S, Chen Z, Geng D. Transcription factor ATF3 mediates the radioresistance of breast cancer. J Cell Mol Med 2018; 22: 4664-4675. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files supplementaryfiguresONC201andcisplatininHNSCC.pdf Cite Share Download PDF Status: Published Journal Publication published 18 Jun, 2025 Read the published version in Oncogenesis → Version 1 posted Editorial decision: revise 13 Jan, 2025 Review # 1 received at journal 12 Jan, 2025 Review # 2 received at journal 17 Nov, 2024 Reviewer # 2 agreed at journal 13 Nov, 2024 Reviewer # 1 agreed at journal 03 Nov, 2024 Reviewers invited by journal 28 Oct, 2024 Submission checks completed at journal 10 Oct, 2024 Editor assigned by journal 09 Oct, 2024 First submitted to journal 09 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5230558","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":364519240,"identity":"b64d7792-53d6-420e-9cda-714bdbff6345","order_by":0,"name":"Ming Huei Chou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIie3NsQrCMBCA4UigXQ6yRhT6CidCVSjoowiFTAU7iYNDxcFXCPgU4gsoQrp0FQp1EATnjg4Oxg7iVOMmmJ+EW+7jCLHZfjGu/xjnwPSk5qSMVbuZvMgnqUlDljTAnSlh66VqATrQTdPjNibBxBsxxWuPnJTQpA1+Fk0LScRgs6NOLUEe+dUVPwdRADlgJ6FObkAodOU3pCk1Qe6qinjkA+G5CPslKuBZ5BSAApHS3q2OMBnu8/F9PmSr9FrALEBvtbhgHXkL9OLzHQz3de65Gl5iTmw2m+0/egBFiT94uraEswAAAABJRU5ErkJggg==","orcid":"","institution":"Chang Gung University College of Medicine, Kaohsiung, Taiwan","correspondingAuthor":true,"prefix":"","firstName":"Ming","middleName":"Huei","lastName":"Chou","suffix":""},{"id":364519241,"identity":"5d6a8fc9-4530-4959-a2b4-d6a10c099341","order_by":1,"name":"Hui-Ching Chuang","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hui-Ching","middleName":"","lastName":"Chuang","suffix":""},{"id":364519242,"identity":"ce2894a5-5b46-4d16-89e3-91914426f4a2","order_by":2,"name":"Jiin-Haur Chuang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiin-Haur","middleName":"","lastName":"Chuang","suffix":""},{"id":364519243,"identity":"d0ed3048-a9cf-4570-8a38-5aa136a2ee1a","order_by":3,"name":"Ya-Ting Hong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ya-Ting","middleName":"","lastName":"Hong","suffix":""},{"id":364519244,"identity":"5ff55316-0c8a-4968-9806-c63458dfba14","order_by":4,"name":"Chih-Yen Chien","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chih-Yen","middleName":"","lastName":"Chien","suffix":""}],"badges":[],"createdAt":"2024-10-09 08:21:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5230558/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5230558/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41389-025-00563-4","type":"published","date":"2025-06-18T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68434448,"identity":"48360b51-ddf2-4184-b25a-9e331f52790e","added_by":"auto","created_at":"2024-11-07 08:34:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":709800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional characterization of OC2 and cisplatin-resistant OC2-CR1 cells. (A) \u003c/strong\u003eTimeline for the generation of cisplatin-resistant HNSCC.\u003cstrong\u003e (B)\u003c/strong\u003e Cell viability of OC2 and OC2-CR1 cells treated with cisplatin at concentrations ranging from 1 to 20 μM for 24 h or 48 h, determined by WST-1 assay. Data represent the mean ± SD from six independent experiments. \u003cstrong\u003e(C)\u003c/strong\u003eWestern blot analysis of γH2AX and cleaved PARP levels in OC2 and OC2-CR1 cells following cisplatin treatment for 4, 24, 36, and 48 h. Densitometric analysis results are shown as histograms. β-actin was used as a loading control. Quantitative data are presented as the mean ± SEM of three independent experiments. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5230558/v1/0e143e2a8a054e419d58bed0.jpg"},{"id":68434275,"identity":"f189e4df-e510-4f37-aa05-9a4bf24e001d","added_by":"auto","created_at":"2024-11-07 08:26:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1071895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of ONC201 and cisplatin on reactive oxygen species (ROS) levels in OC2 and OC2-CR1 cells.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(A)\u003c/strong\u003e Mitochondrial ROS levels in OC2 and OC2-CR1 cells after 24 h and 48 h treatment with ONC201, cisplatin, or their combination, determined by confocal microscopy. Cells were stained with MitoSOX™ Red (5 μM) for 15 min at 37°C. Images were acquired at 600× magnification.\u003cstrong\u003e (B,C) \u003c/strong\u003eFlow cytometry analysis of ROS levels: (B) mitochondrial ROS and (C) intracellular ROS after treatment with ONC201, cisplatin, or their combination for 24 h, 48 h, and 72 h. Cells were stained with 5 μM MitoSOX™ Red (mitochondrial ROS) or 5 μM DHE (intracellular ROS) for 15 min at 37°C. Histogram data represent the mean ± SD of six independent experiments. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5230558/v1/5027208e59576077f596e1a2.jpg"},{"id":68434449,"identity":"2a5588d3-7593-4cc4-84cc-b66b45dadec0","added_by":"auto","created_at":"2024-11-07 08:34:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1021146,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot analysis of ER stress-mediated cell death regulators in treated HNSCC cells.\u003c/strong\u003e\u003cbr\u003e\nRepresentative Western blot analysis of \u003cstrong\u003e(A)\u003c/strong\u003e ATF3, ATF4, CHOP, and \u003cstrong\u003e(B)\u003c/strong\u003eγH2AX, cleaved PARP (cPARP), and MCL1 expression levels in OC2 and OC2-CR1 cells after 24 h, 48 h, and 72 h of treatment with ONC201, cisplatin, or their combination. β-actin was used as a loading control. Densitometric analysis results are presented as histograms. Data are expressed as the mean ± SEM from three independent experiments. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5230558/v1/d3f6dae838129f2d06730f0f.jpg"},{"id":68434273,"identity":"e80ad77f-ee75-4064-a021-af0e8cb8b3a9","added_by":"auto","created_at":"2024-11-07 08:26:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1437079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination therapy synergistically inhibits cell proliferation and migration in HNSCC cells.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(A) \u003c/strong\u003eColony formation assays of OC2 and OC2-CR1 cells treated with various concentrations of cisplatin, ONC201, or their combination for 10 days, stained with crystal violet, and quantified using ImageJ software. Data are presented as the mean ± SD (n = 6). *p \u0026lt; 0.05 versus control. \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative images and quantification of cell migration assays. OC2 and OC2-CR1 cells were treated with various concentrations of cisplatin, ONC201, or their combination and assessed using the Oris™ Cell Migration Assay. Real-time migration curves were generated, and data are expressed as the mean ± SEM (n = 6). *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5230558/v1/fbf0d7ed1383ab5576bfa292.jpg"},{"id":68434279,"identity":"ceca0b1a-4c5f-407d-9778-94d3111bc208","added_by":"auto","created_at":"2024-11-07 08:26:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":534754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of ONC201 with cisplatin improves tumor response in cisplatin-resistant HNSCC xenograft models.\u003c/strong\u003e\u003cbr\u003e\nOC2 or OC2-CR1 cells were subcutaneously injected into the dorsal flank of athymic mice. After 14 days of tumor growth, mice were divided into four groups (n ≥ 4 per group): control, cisplatin (3 mg/kg), ONC201 (30 mg/kg), or a combination of both, administered intraperitoneally twice a week for 4 weeks. \u003cstrong\u003e(A)\u003c/strong\u003eIn vivo experimental schedule (B.I.W = twice a week). \u003cstrong\u003e(B)\u003c/strong\u003e Body weights of mice in each treatment group over time, shown as mean ± SD at pre-specified time points. \u003cstrong\u003e(C)\u003c/strong\u003e Tumor photographs and volumes at the study endpoint for each treatment group, presented as mean ± SD. *p ≤ 0.05.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5230558/v1/14aa947259317cde23950f02.jpg"},{"id":68434276,"identity":"d4cddce9-461a-48ab-9f45-8d51e7c1cfcf","added_by":"auto","created_at":"2024-11-07 08:26:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":463845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential induction of stress response markers in OC2 and OC2-CR1 cells under therapeutic stress.\u003c/strong\u003e The distinct cellular mechanisms of stress response between OC2 and OC2-CR1 cells are highlighted by the differential induction of ATF3, ATF4, and CHOP. In both OC2 and cisplatin-resistant OC2-CR1 cells, cisplatin treatment primarily induced CHOP expression through ATF3. However, ONC201 treatment induced CHOP via ATF4 in OC2 cells, whereas in OC2-CR1 cells, CHOP induction was predominantly mediated by ATF3. This differential regulation of CHOP, a key pro-apoptotic factor, indicates that the combination of ONC201 and cisplatin targets multiple pathways to effectively induce apoptosis and overcome the resistance mechanisms in OC2-CR1 cells. Additionally, the combination treatment led to the downregulation of MCL1, an anti-apoptotic protein, in both cell lines, further enhancing cellular sensitivity to apoptosis.\u003c/p\u003e","description":"","filename":"16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5230558/v1/bf06bd021478022d08948973.jpg"},{"id":84950207,"identity":"a5de70a8-ee8a-49a5-ad5b-7d8343676654","added_by":"auto","created_at":"2025-06-19 07:07:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6166545,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5230558/v1/e89f0140-7388-4abf-a8e4-da35f1bb789e.pdf"},{"id":68434278,"identity":"713a3f5d-c289-450b-b838-bced07661947","added_by":"auto","created_at":"2024-11-07 08:26:41","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":953979,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfiguresONC201andcisplatininHNSCC.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5230558/v1/2d84ddb951ce95b43b2a3387.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"ONC201 enhances the cytotoxic effect of cisplatin through ATF3/ATF4/CHOP in HNSCC cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHead and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers globally, often associated with risk factors such as tobacco use, betel quid chewing, alcohol consumption, and human papillomavirus (HPV) infection\u003csup\u003e3\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e9\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e12\u003c/sup\u003e. HNSCC primarily originates from the mucosal epithelium of the upper aerodigestive tract, including the oral cavity, larynx, oropharynx, and hypopharynx. Due to the lack of enhanced public awareness about the hazards from these substances, many patients are still diagnosed at an advanced stage. Current treatment strategies include surgery, radiotherapy, chemotherapy, target therapy, immunotherapy or combination. Despite of the multidisciplinary approach in the care of patients with HNSCC, there are still many patients who would experience the treatment failure. This is largely due to the remarkable heterogeneity of HNSCC, characterized by various genetic mutations and aberrant gene expressions\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e24\u003c/sup\u003e. Consequently, there is a need to develop a novel therapeutic agent that is able to provide more effective in the treatment outcomes. One of the current types of research focuses on advancing targeted therapy that exploit specific molecular pathways critical to cancer growth and survival.\u003c/p\u003e\n\u003cp\u003eCisplatin, a platinum-based agent is widely used as the first-line chemotherapy agent for HNSCC because of its potent anticancer activity. The mechanisms of cisplatin in treating HNSCC involve the formation of platinum-DNA adducts, which induce G2/S cell cycle arrest and subsequent cell death by causing DNA damage in rapidly dividing cancer cells\u003csup\u003e17\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e19\u003c/sup\u003e. Additionally, cisplatin can trigger cellular stress responses through the activation of ATF3 and ATF4, leading to the inhibition of MCL1 expression and inducing the apoptosis in HNSCC cells \u003csup\u003e4\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eONC201, a small molecule which has been shown to have anti-tumor efficacy by using a monotherapy or in combination with other treatments\u003csup\u003e13\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e18\u003c/sup\u003e in phase II clinical trials for various solid tumors and hematological malignancies. ONC201 which would activate the integrated stress response (ISR), inhibition of AKT/ERK signaling, and induction of ATF3, ATF4, and CHOP is a DRD2 antagonist and ClpP agonist. These actions culminate in TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in cancer cells.\u003c/p\u003e\n\u003cp\u003eIn HNSCC, ATF3 has been identified as a tumor suppressor that promotes apoptosis and inhibits cell growth, regulating various cellular processes in response to stressors such as DNA damage\u003csup\u003e16\u003c/sup\u003e. ATF4, another stress-responsive transcription factor, plays a role in programmed cell death by regulating downstream targets, including MCL1\u003csup\u003e15\u003c/sup\u003e. CHOP, also known as GADD153, is a multifunctional transcription factor that is upregulated in response to various stresses, such as DNA damage, ER stress, and mitochondrial dysfunction. CHOP mediates apoptosis by downregulating anti-apoptotic proteins like BCL2, BCL-XL, and MCL-1, and upregulating pro-apoptotic proteins like BIM, BAK, and BAX\u003csup\u003e10\u003c/sup\u003e. It plays a crucial role in coordinating cellular responses to stress, mediating cell fate decisions, including cell survival or programmed cell death\u003csup\u003e14\u003c/sup\u003e. The role of MCL1 in regulating apoptosis makes it a critical factor in determining cancer cell survival and resistance to therapy in HNSCC \u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCisplatin is the major backbone of chemotherapy agent for treatment of HNSCC\u003csup\u003e5\u003c/sup\u003e but the genetic heterogeneity and aberrant mutations could lead to primary cisplatin resistance, causing the treatment failure in locally advanced tumor or recurrent ones \u003csup\u003e8\u003c/sup\u003e. The ONC201 is able to induce mitochondria-mediated apoptosis by downregulating MCL1 and this phenomenon suggests its potential as an effective agent to overcome cisplatin resistance. In this study, we explore the combination of cisplatin and ONC201 to enhance the sensitivity of HNSCC to cisplatin treatment, aiming to demonstrate the synergistic efficacy of this combination as a promising strategy.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGeneration and Characterization of Cisplatin-Resistant HNSCC Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCisplatin-resistant HNSCC cells were generated from the OC2 cell line through a progressive selection process involving increasing concentrations of cisplatin (Sigma-Aldrich, UK) over a 6-month period. The selection began with low doses and culminated in continuous exposure to 1 \u0026mu;M cisplatin, resulting into the stable resistant phenotype designated as OC2-CR1 (Fig. 1A). To assess cisplatin sensitivity, OC2 and OC2-CR1 cells were treated with a range of cisplatin concentrations for 24 and 48 hours, and cell viability was evaluated by using the WST1 assay (Fig.1B). OC2 cells displayed sensitivity to cisplatin, while OC2-CR1 cells exhibited marked resistance. At 48 hours, the IC50 values were 2 \u0026mu;M for OC2 cells and 9 \u0026mu;M for OC2-CR1 cells, confirming the significant resistance of OC2-CR1 cells compared to the parental OC2 cells.\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of cisplatin on DNA damage and apoptosis, Western blot analysis was performed to detect \u0026gamma;H2AX and cleaved Poly (ADP-ribose) polymerase (PARP). Cisplatin treatment led to a dose-dependent increase in \u0026gamma;H2AX and cleaved PARP expression in OC2 cells, indicative of DNA damage and activation of apoptotic pathways (Fig. 1C). In contrast, cisplatin-resistant OC2-CR1 cells showed only a modest increase in \u0026gamma;H2AX and cleaved PARP expression, suggesting a reduced capacity to respond to cisplatin-induced DNA damage and apoptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eONC201 and Cisplatin Induced mtROS in HNSCC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExcess cellular levels of reactive oxygen species (ROS) cause damage to intracellular lipids, proteins, nucleic acids and organelles, leading to cell death and damaged biological molecules via the endoplasmic reticulum (ER) stress-mediated pathway\u003csup\u003e7\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e22\u003c/sup\u003e. To investigate the role of oxidative stress in the anti-tumor effects of cisplatin and ONC201, mitochondrial ROS levels were assessed using confocal microscopy and flow cytometry. Mitochondrial ROS levels were measured with the MitoSox Red probe following treatment with ONC201, cisplatin, or their combination for 24 and 48 hours (Fig.2A). Flow cytometry was used to analyze both mitochondrial and intracellular ROS levels at 24, 48, and 72 hours by using MitoSOX Red and dihydroethidium (DHE) assays, respectively (Fig.2B, 2C and S1). \u003c/p\u003e\n\u003cp\u003eThe results demonstrated that ONC201 and cisplatin treatments, either alone or in combination, led to increased mitochondrial ROS production in OC2 cells at 24, 48, and 72 hours. Similarly, mitochondrial ROS levels were elevated in OC2-CR1 cells at 48 and 72 hours, but not at 24 hours. The DHE assay revealed that cisplatin treatment significantly raised intracellular ROS levels in both OC2 and OC2-CR1 cells after 48 hours. However, the combination of cisplatin and ONC201 at high doses resulted in a marked increase in intracellular ROS production, which was significant only in OC2-CR1 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of ONC201 and Cisplatin on \u003c/strong\u003e\u003cstrong\u003eER Stress-Mediated Cell Death\u003c/strong\u003e\u003cstrong\u003e Expression in HNSCC Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCisplatin and the ERK inhibitor ONC201 are known to induce an ATF-mediated integrated stress response (ISR) that inhibits the growth of various solid tumors. These agents promote cell death by inducing ISR and ER stress-mediated pathways. However, in resistant cancer cells, altered ISR and ER stress can diminish the efficacy of these drugs\u003csup\u003e23\u003c/sup\u003e. We investigated the expression of ER stress-related and cell death markers in OC2 and cisplatin-resistant OC2-CR1 cells following treatment with ONC201, cisplatin, or theses combination (Fig. 3 and S2). Western blot analysis revealed that ONC201 significantly increased ATF4 and CHOP expression at 24 hours but decreased it by 72 hours in OC2 cells. ATF3 expression was not elevated until 72 hours post-treatment. Conversely, cisplatin did not induce ATF4 expression at any time point (24, 48, or 72 hours) but did lead to high levels of ATF3 and CHOP expression. In the combination treatment, ATF3 levels were consistently higher at 24, 48, and 72 hours, while CHOP expression peaked at 24 and 48 hours before declining at 72 hours, potentially dependent on ATF4 expression. In OC2-CR1 cells, ONC201 and cisplatin treatment similarly induced ATF3 and CHOP at 24 and 48 hours, with a decline by 72 hours. This suggests that ONC201 and cisplatin induce CHOP through distinct mechanisms: ATF3/ATF4 in OC2 cells and primarily ATF3 in OC2-CR1 cells (Fig. 3A).\u003c/p\u003e\n\u003cp\u003eFurther analysis of ATF3/ATF4/CHOP-mediated cell death showed that ONC201 did not induce \u0026gamma;H2AX, a marker of DNA damage, but did upregulate MCL1 at 24 hours. Both MCL1 and cleaved-PARP levels decreased at 48 and 72 hours in both cell lines. Cisplatin treatment in OC2 cells significantly increased \u0026gamma;H2AX and cleaved-PARP expression while decreasing MCL1 in a dose-dependent manner. In OC2-CR1 cells, similar trends were observed, though the increases in \u0026gamma;H2AX and cleaved-PARP were less pronounced. Combined treatment with ONC201 and cisplatin induced \u0026gamma;H2AX and cleaved-PARP similarly to cisplatin alone at 24 and 48 hours in both cell lines, but significantly reduced cleaved-PARP and MCL1 levels by 72 hours (Fig. 3B). These findings suggest that ONC201 and cisplatin effectively inhibit MCL1 through distinct ER stress-mediated pathways, involving ATF4/CHOP and ATF3/CHOP in OC2 and OC2-CR1 cells, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eONC201 and Cisplatin Inhibit Cell Proliferation and Migration in HNSCC Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the effects of cisplatin and ONC201 on colony formation in HNSCC cells, equal numbers of OC2 and cisplatin-resistant OC2-CR1 cells (100 cells per well) were treated with varying concentrations of cisplatin (0.25\u0026ndash;1 \u0026micro;M) or ONC201 (0.25\u0026ndash;1 \u0026micro;M) and allowed to grow for 9 days. Colony formation in both OC2 and OC2-CR1 cells was inhibited in a dose-dependent manner by both agents (Fig. 4A). When cells were treated with a combination of 1 \u0026micro;M cisplatin and 0.25\u0026ndash;1 \u0026micro;M ONC201, there was a significant synergistic inhibition of colony formation. The combined treatment led to a more pronounced reduction in colony formation in both OC2 and OC2-CR1 cells compared to treatment with 1 \u0026micro;M cisplatin alone. Notably, OC2 cells exhibited a significantly greater decrease in colony formation when treated with 1 \u0026micro;M cisplatin, 1 \u0026micro;M ONC201, or the combination, compared to the more resistant OC2-CR1 cells.\u003c/p\u003e\n\u003cp\u003eIn addition to colony formation, we evaluated the effects of cisplatin and ONC201 on cell migration using a two-dimensional Oris\u0026trade; cell migration assay. OC2 and OC2-CR1 cells were treated with 1 \u0026micro;M ONC201, 10 \u0026micro;M or 20 \u0026micro;M cisplatin, or a combination of ONC201 and cisplatin for 48 hours. The results demonstrated that both cisplatin and ONC201 significantly inhibited cell migration in HNSCC cells (Fig. 4B). The combination of cisplatin and ONC201 was more effective at inhibiting migration in both OC2 and OC2-CR1 cells than either agent alone. Interestingly, the combined treatment was more effective in inhibiting the migration of OC2-CR1 cells compared to OC2 cells, suggesting a heightened sensitivity of the resistant cells to the combination therapy. These findings indicate that ONC201 and cisplatin, particularly when used in combination, effectively inhibit both the proliferation and migration of HNSCC cells, with differential effects observed between the sensitive OC2 and resistant OC2-CR1 cell lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSignificant Anti-Proliferative Effects of Cisplatin and ONC201 in an Orthotopic Graft Model of HNSCC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the efficacy of cisplatin and ONC201 on tumor growth, we assessed these in vivo antitumor effects by using a xenograft model derived from OC2 and cisplatin-resistant OC2-CR1 cells in NU/NU mice. Tumor volumes were monitored twice a week with a digital vernier caliper, and visible dorsal tumors developed 7 days post-injection. Starting 7 days after tumor inoculation, mice received intraperitoneal injections of cisplatin (3 mg/kg) and ONC201 (50 mg/kg), either alone or in combination, twice a week for 4 weeks. Control groups underwent injections of saline (for cisplatin) or a vehicle solution (5% DMSO, 5% Tween 80, 40% PEG300, 50% saline) for ONC201 (Fig. 5A).\u003c/p\u003e\n\u003cp\u003eIn the OC2 xenograft model, both cisplatin and ONC201 significantly suppressed tumor growth compared to the control group. The combined treatment with cisplatin and ONC201 led to even more pronounced tumor suppression, with no significant differences observed in the body weights of the mice among the different treatment groups (Fig. 5B-C). However, in the cisplatin-resistant OC2-CR1 xenograft model, neither cisplatin nor ONC201 alone effectively inhibited tumor growth when compared to the control group. Remarkably, only the combination of cisplatin and ONC201 significantly suppressed the growth of OC2-CR1-derived tumors. These in vivo findings suggest that the combined treatment of cisplatin and ONC201 holds potential as a therapeutic strategy for overcoming cisplatin resistance in HNSCC, offering a promising approach to improve outcomes in resistant cases.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study provides significant insights into the therapeutic potential of combining ONC201 with cisplatin to overcome the issue of resistance in HNSCC. Through comprehensive in vitro and in vivo investigations, we elucidated the mechanisms underlying the enhanced anti-cancer effects of this combination, particularly focusing on the modulation of the ATF3/ATF4/CHOP pathway (Fig.6). Our findings also underscore the challenges posed by the genetic heterogeneity of HNSCC and the need for innovative strategies to address the cisplatin resistance.\u003c/p\u003e\n\u003cp\u003eThe development of cisplatin resistance in HNSCC is a great challenge in the treatment of locally advanced tumor or recurrent metastasis tumor and it often leads to therapeutic failure and dismal clinical outcomes\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e11\u003c/sup\u003e. In this study, the cisplatin-resistant OC2-CR1 cells exhibited significantly higher IC50 values compared to the parental OC2 cells, confirming their resistant phenotype (Fig. 1). This marked difference in sensitivity highlights the difficulty in treating cisplatin-resistant HNSCC and address the importance of combination therapies to restore drug sensitivity. While cisplatin alone was able to induce DNA damage and apoptosis, its efficacy was notably reduced in the resistant OC2-CR1 cells, likely due to the upregulation of stress-responsive transcription factors like ATF3 (Fig. 3). This aligns with previous studies which had implicated ATF3 in the promotion of cell survival and resistance to radiation therapy\u003csup\u003e25\u003c/sup\u003e or chemotherapy\u003csup\u003e2\u003c/sup\u003e through the induction of anti-apoptotic proteins such as MCL1.\u003c/p\u003e\n\u003cp\u003eONC201, known for its role as a DRD2 antagonist and ClpP agonist, has demonstrated potent anti-tumor activity across various cancer types. In this study, ONC201 was shown to significantly sensitize OC2-CR1 cells to cisplatin, primarily by exacerbating oxidative stress and modulating ER stress pathways. The increase in mitochondrial and intracellular ROS levels following treatment with ONC201, particularly when combined with cisplatin, suggests that the generation of oxidative stress is a critical mechanism by which ONC201 enhances the cytotoxic effects of cisplatin. This increase in ROS levels was associated with heightened DNA damage and apoptosis, particularly in the cisplatin-resistant OC2-CR1 cells, thereby highlighting the potential of ONC201 to overcome chemoresistance (Fig. 2).\u003c/p\u003e\n\u003cp\u003eThe differential induction of stress response markers, such as ATF3, ATF4, and CHOP, between OC2 and OC2-CR1 cells demonstrates the distinct cellular mechanisms employed by these cell lines to cope with therapeutic stress (Fig.6). In both OC2 and cisplatin-resistant OC2-CR1 cells, cisplatin treatment primarily induced CHOP expression through ATF3. However, ONC201 induced CHOP expression primarily through ATF4 in OC2 cells, while in the resistant OC2-CR1 cells, CHOP induction was mediated predominantly by ATF3 (Fig. 3). This differential regulation of CHOP, a key pro-apoptotic factor, suggests that the combination of ONC201 and cisplatin effectively targets multiple pathways to induce apoptosis, thereby overcoming the resistance mechanisms presented in OC2-CR1 cells. Additionally, the downregulation of MCL1, an anti-apoptotic protein, by the combined treatment in both cell lines further supports the role of this pathway in sensitizing cells to apoptosis.\u003c/p\u003e\n\u003cp\u003eThe combination of ONC201 and cisplatin also demonstrated a significant inhibitory effect on colony formation and cell migration in both OC2 and OC2-CR1 cells, with a more pronounced effect observed in the resistant OC2-CR1 cells (Fig. 4). These findings suggest that the combination therapy not only enhances the cytotoxic effects of cisplatin but also effectively inhibits key processes involved in tumor progression and metastasis. The in vivo xenograft experiments provided strong evidence for the therapeutic potential of the ONC201 and cisplatin combination (Fig. 5). The combined treatment significantly suppressed tumor growth in both OC2 and OC2-CR1 derived xenograft models, demonstrating its efficacy in overcoming resistant mechanisms. Importantly, this treatment regimen did not result in significant weight loss in the mice, showing a less toxicity profile. These findings suggest that ONC201, when used in combination with cisplatin is a novel strategy to treat HNSCC, particularly in cases where resistance to cisplatin has evolved. Additionally, combination with other therapeutic agents to ONC201 such as chemotherapy agents, check point inhibitors, target therapy agents to treat HNSCC may provide other possible options to improve the clinical outcomes although more studies are necessary to prove this assumption.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study highlights the potential of ONC201 to augment the cytotoxic effects of cisplatin in HNSCC through the modulation of the ATF3/ATF4/CHOP pathway, particularly in overcoming the problem of cisplatin resistance. The differential responses between OC2 and OC2-CR1 cells demonstrate the importance of targeting multiple pathways to effectively treat against the resistant cancer phenotypes. The therapeutic value of combining ONC201 with cisplatin shows its ability to target multiple resistant mechanisms simultaneously. By inducing oxidative stress, modulating ER stress pathways, and promoting apoptosis through different molecular routes, this combination addresses the genetic heterogeneity and adaptive responses of HNSCC cells. This multi-faceted approach is helpful and promising in coping with the complexity of cancer resistance from cisplatin and improving the clinical outcomes in HNSCC. \u003c/p\u003e"},{"header":"Materials/Subjects and Methods","content":"\u003cp\u003e\u003cstrong\u003eInduction of Cisplatin Resistance in OC2 Cells and Cell Culture\u003c/strong\u003e\u003cbr\u003eCisplatin-resistant OC2 cells (designated OC2-CR1) were established by exposing the parental OC2 cell line to progressively increasing concentrations of cisplatin (Sigma-Aldrich, UK) over a 6-month period. The selection process began with a low dose and culminated in continuous exposure to 1 \u0026mu;M cisplatin, resulting in the stable resistant phenotype. To validate resistance, IC50 values were determined by measuring the cells\u0026apos; response to cisplatin. Both OC2 and OC2-CR1 cells were maintained in RPMI 1640 medium (Invitrogen), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 \u0026mu;g/mL streptomycin, and 250 \u0026mu;g/mL Amphotericin B solution (Invitrogen). Cells were seeded at a density of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells per 10 cm culture dish and treated with varying concentrations of cisplatin, ONC201 (Selleckchem), or a combination of both. The cells were harvested at specific time points for subsequent in vitro analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Viability Analysis (WST-1 Assay)\u003c/strong\u003e\u003cbr\u003eOC2 and OC2-CR1 cells were seeded at a density of 10\u003csup\u003e4\u003c/sup\u003e cells per well in 96-well plates and treated with varying concentrations of cisplatin, ONC201, or their combination for 24 and 48 hours. Following the treatment period, 10 \u0026mu;L of WST-1 reagent (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene sulfonate) (Roche Diagnostics) was added to each well. The plates were then incubated at 37\u0026deg;C for an additional 1-4 hours to allow the formation of formazan dye. Absorbance was measured using a microplate reader, with the test wavelength set to 450 nm and the reference wavelength at 630 nm to correct for any background signal. The absorbance values were used to quantify cell viability, reflecting the metabolic activity of the cells post-treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Mitochondrial and Intracellular Reactive Oxygen Species (ROS)\u003c/strong\u003e\u003cbr\u003e\u0026nbsp; \u0026nbsp;Mitochondrial and intracellular ROS levels in OC2 and OC2-CR1 cells were assessed using MitoSOX\u0026trade; Red (Invitrogen) and Dihydroethidium (DHE, Merck/Sigma-Aldrich) fluorescent probes, respectively. Cells were first washed twice with PBS to remove any residual media. For mitochondrial ROS detection, cells were incubated with 5 \u0026mu;M MitoSOX\u0026trade; Red, while intracellular ROS was detected by staining with 5 \u0026mu;M DHE. Both staining procedures were carried out for 15 minutes at 37\u0026deg;C. Following the staining, the cells were collected and washed twice with PBS to remove excess dye. The cells were then resuspended in 1 mL of PBS and transferred into flow cytometry tubes. The fluorescence intensity, indicative of ROS levels, was measured using a BD LSRII flow cytometer (BD Biosciences).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence Staining of Mitochondrial ROS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; Cells cultured on glass slides were washed twice with PBS and then incubated with 5 \u0026mu;M MitoSOX\u0026trade; Red (Invitrogen) for 2 hours at 37\u0026deg;C to detect mitochondrial ROS. After incubation, the slides were fixed using an appropriate fixative (4% paraformaldehyde) and blocked to minimize non-specific binding. The slides were then co-stained with DAPI (4\u0026prime;,6-diamidino-2-phenylindole; Molecular Probes) to visualize the nuclei. Following staining, the slides were mounted using a fluorescent mounting medium (Dako Cytomation) and sealed with coverslips. Images were captured using an Olympus FluoView\u0026reg; confocal microscope, ensuring that exposure gains and rates were consistent across all samples to allow for accurate comparison. Fluorescence intensities were quantified separately for each color channel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; OC2 and OC2-CR1 cells were lysed in PRO-PREP protein extraction solution (iNtRON Bio) for 30 minutes on ice. The lysates were centrifuged at 13,000 rpm for 5 minutes at 4\u0026deg;C, and the resulting supernatants were collected. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad). Equal amounts of protein (20-50 \u0026mu;g) from each sample were mixed with loading buffer, boiled for 10 minutes, and then separated by SDS-PAGE on a 10% polyacrylamide gel. The proteins were transferred onto nitrocellulose membranes. Following transfer, the membranes were blocked in 5% non-fat milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature. The membranes were then incubated overnight at 4\u0026deg;C with primary antibodies against ATF3, \u0026gamma;-H2AX (Abcam), ATF4, CHOP, c-PARP, MCL1 (Cell Signaling Technology), and \u0026beta;-actin (Sigma-Aldrich) according to the manufacturers\u0026rsquo; protocols. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using a chemiluminescent substrate (Santa Cruz Biotechnology) and detected by exposure to X-ray film. Densitometric analysis was performed to quantify the intensity of the protein bands relative to \u0026beta;-actin as a loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClonogenic Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; OC2 and OC2-CR1 cells were seeded at a density of 200 cells per well in 6-well plates and allowed to adhere for 48 hours. The cells were then treated with various concentrations of cisplatin, ONC201, or a combination of both, and cultured for an additional 10 days under standard conditions. After the treatment period, the cells were fixed with 4% paraformaldehyde and stained with 0.5% Crystal Violet for 30 minutes at room temperature. Excess stain was washed away with distilled water, and the plates were air-dried. Images of the stained colonies were captured using a digital camera. The number of colonies in each plate was counted manually or using imaging software. Relative plating efficiency (RPE) was calculated using the following formula: RPE = [total colonies of three well (test) / total colonies of three well (control)] x 100%. The results were expressed as a percentage of the control, and statistical analysis was performed to compare the clonogenic survival between different treatment groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOris\u0026trade; Cell Migration Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; The migration of OC2 and OC2-CR1 cells was assessed using the Oris\u0026trade; Cell Migration Assembly Kit (Platypus Technologies) following the manufacturer\u0026rsquo;s protocol. Cells were seeded at a density of 5\u0026times;10^4 cells per well in Oris\u0026trade; 96-well plates, each equipped with stoppers that create a central cell-free detection zone. The cells were allowed to adhere and grow overnight. After incubation, the Oris\u0026trade; stoppers were gently removed to initiate the migration process. Tetramethylrhodamine ethyl ester perchlorate (TMRE, Invitrogen) was added to each well to fluorescently label the cells. The plates were then incubated for an additional 48 hours to allow for cell migration into the detection zones. At designated time points, the fluorescence intensity in the detection zones, which correlates with cell migration, was measured using a fluorescence plate reader. The fluorescence signals were converted to cell numbers, and the data were used to generate real-time migration curves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Care and Xenograft Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; Male NU/NU nude mice, 6 weeks of age, were obtained from the National Laboratory Animal Center and maintained in a specific pathogen-free (SPF) facility. The mice were provided with irradiated chow and autoclaved reverse-osmosis water ad libitum. All animal procedures were conducted following protocols approved by the Institutional Animal Care and Usage Committee. For tumor inoculation, the mice were anesthetized with Zoletil (15 mg/kg body weight), and 1\u0026times;10^7 OC2 or OC2-CR1 cells suspended in 0.1 mL of sterile saline were subcutaneously injected into the right flank using a 26-gauge needle. Tumor development was monitored, and visible dorsal tumors were observed approximately 14 days post-injection. Starting from day 14 post-inoculation, the mice were treated with cisplatin (3 mg/kg), ONC201 (30 mg/kg), or a combination of both, administered intraperitoneally twice a week for 4 weeks. Control groups received either saline or vehicle (10% DMSO, 40% PEG, 5% Tween 80, and 45% saline). Tumor growth was measured twice weekly using a caliper, with tumor size calculated as the product of the longest dimension and its perpendicular. Mice were monitored every other day for tumor progression and signs of weight loss. The study was concluded 7 days after the final treatment, and mice were humanely euthanized by CO2 inhalation if they lost more than 25% of their pre-injection body weight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; All data were expressed as the mean \u0026plusmn; SEM for animal studies and cell migration analyses, and as the mean \u0026plusmn; SD for in vitro experiments, with each experiment performed at least three times independently. Statistical comparisons between experimental groups and continuous variables were made by using Student\u0026rsquo;s t-test (unpaired, two-tailed). For comparisons involving multiple groups, a one-way ANOVA followed by a post-hoc test was employed to determine significant differences. Statistical significance was defined as P \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by grant number CRRPG8K0081-3, CRRPG8N0041-3 from the Chang Gung Memorial Hospital\u0026nbsp;and MOST 111-2314-B-182A-099,\u0026nbsp;MOST\u0026nbsp;111-2314-B-182A-079 -MY3\u0026nbsp;and\u0026nbsp;MOST 108-2314-B-182A-112-MY3\u0026nbsp;from the Ministry of Science and Technology\u0026nbsp;(National Science and Technology Council). We thank Yu-Li Liu, Rui-An Lin, Shin-Yan Lee, Yu-An Li and Chih-Kuan Chao for discussions and technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e HCC\u0026nbsp;was responsible for drafting the article, acquisition of data and final approval of this manuscript; JHC was responsible for critical review of this article and final approval of this manuscript; YTH was responsible for analysis and interpretation of data and final approval of this manuscript; MHC and CYC was responsible for conception and design and final approval of this manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials:\u003c/strong\u003e All the raw data of this study are available from the corresponding author upon reasonable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlsahafi E, Begg K, Amelio I, Raulf N, Lucarelli P, Sauter T\u003cem\u003e et al\u003c/em\u003e. Clinical update on head and neck cancer: molecular biology and ongoing challenges. Cell Death Dis 2019; 10: 540.\u003c/li\u003e\n\u003cli\u003eB\u0026ouml;pple K, Oren Y, Henry WS, Dong M, Weller S, Thiel J\u003cem\u003e et al\u003c/em\u003e. ATF3 characterizes aggressive drug-tolerant persister cells in HGSOC. 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J Cell Mol Med 2018; 22: 4664-4675.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogenesis","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"oncsis","sideBox":"Learn more about [Oncogenesis](http://www.nature.com/oncsis/)","snPcode":"41389","submissionUrl":"https://mts-oncsis.nature.com/cgi-bin/main.plex","title":"Oncogenesis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5230558/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5230558/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Head and neck squamous cell carcinoma (HNSCC) remains a prevalent and challenging cancer to treat due to its genetic heterogeneity. Cisplatin resistance is one of important causes in treatment failure of locally advanced HNSCC. This study is aimed to investigate the potential of ONC201 to enhance the cisplatin cytotoxicity in HNSCC cells through the ATF3/ATF4/CHOP pathway. At first, cisplatin-resistant HNSCC cell lines (OC2-CR1) were built up and then, the treatment effectiveness of ONC201 alone and cisplatin in combination on cell viability, DNA damage, reactive oxygen species (ROS) production, and stress response markers were evaluated. Cisplatin-resistant cells showed increased IC50 values compared to parental OC2 cells. Combined treatment with ONC201 and cisplatin significantly increased mitochondrial ROS and intracellular ROS levels, enhancing DNA damage and apoptosis through ATF3/ATF4/CHOP-mediated pathways. In vivo studies by using xenograft models demonstrated that the combination treatment suppressed tumor growth more effectively than either agent alone. ONC201 significantly potentiates the cytotoxic effects of cisplatin in HNSCC by modulating stress response pathways, suggesting a promising therapeutic strategy for cisplatin-resistant HNSCC.","manuscriptTitle":"ONC201 enhances the cytotoxic effect of cisplatin through ATF3/ATF4/CHOP in HNSCC cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-07 08:26:36","doi":"10.21203/rs.3.rs-5230558/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-01-13T16:50:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-01-12T16:31:24+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-11-17T22:53:24+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-11-13T19:44:25+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-11-03T13:24:46+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-10-28T16:02:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-10T10:57:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-09T08:19:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogenesis","date":"2024-10-09T08:19:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogenesis","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"oncsis","sideBox":"Learn more about [Oncogenesis](http://www.nature.com/oncsis/)","snPcode":"41389","submissionUrl":"https://mts-oncsis.nature.com/cgi-bin/main.plex","title":"Oncogenesis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d82fd85-22c3-40bc-bcd6-a35114573667","owner":[],"postedDate":"November 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38782301,"name":"Biological sciences/Cancer/Head and neck cancer"},{"id":38782302,"name":"Biological sciences/Immunology/Cell death and immune response"}],"tags":[],"updatedAt":"2025-06-19T07:07:16+00:00","versionOfRecord":{"articleIdentity":"rs-5230558","link":"https://doi.org/10.1038/s41389-025-00563-4","journal":{"identity":"oncogenesis","isVorOnly":false,"title":"Oncogenesis"},"publishedOn":"2025-06-18 04:00:00","publishedOnDateReadable":"June 18th, 2025"},"versionCreatedAt":"2024-11-07 08:26:36","video":"","vorDoi":"10.1038/s41389-025-00563-4","vorDoiUrl":"https://doi.org/10.1038/s41389-025-00563-4","workflowStages":[]},"version":"v1","identity":"rs-5230558","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5230558","identity":"rs-5230558","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}