Stearate-rich diet and oleate restriction directly inhibit tumor growth via the unfolded protein response

Stearate-rich diet and oleate restriction directly inhibit tumor growth via the unfolded protein response | 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 Stearate-rich diet and oleate restriction directly inhibit tumor growth via the unfolded protein response Yamanoi Koji, Ogura Jumpei, Nakamura Eijiro, Ito Shinji, Nakanishi Yuki, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4198546/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Dec, 2024 Read the published version in Experimental & Molecular Medicine → Version 1 posted 11 You are reading this latest preprint version Abstract Fatty acids are known to have a significant impact on the properties of cancer cells. Therefore, Incorporating them into therapeutic strategies has been reported. However, few studies have examined the effects of individual fatty acids and their interaction in depth. The study analyzed the effects of various fatty acids on cancer cells and found that stearic acid, an abundant saturated fatty acid, had a stronger inhibitory effect on cell growth compared to palmitic acid, which is also an abundant saturated fatty acid, by inducing DNA damage and apoptosis through the unfolded protein response (UPR) pathway. Intriguingly, the negative effects of stearate were reduced by the presence of oleate, a different type of abundant fatty acid. In exploring the dietary impact on tumor growth, we combined a stearate-rich diet with the inhibition of stearoyl-CoA desaturase-1. This approach significantly reduced tumor growth in both ovarian cancer models and patient-derived xenografts (PDXs), including those with chemotherapy-resistant cases, by notably elevating stearate levels while reducing oleate levels within the tumors. Conversely, the negative effects of a stearate-rich diet were mitigated by an oleate-rich diet. The study shows that the dietary stearate can directly inhibit tumor growth through mechanisms involving DNA damage and apoptosis mediated by the UPR pathway. The results suggest that dietary interventions, which increase stearic acid levels while decreasing oleic acid levels, may be a promising therapeutic strategy in cancer treatment. This could lead to the development of new cancer treatment strategies. Biological sciences/Cancer/Cancer metabolism Biological sciences/Cell biology/Cell death/Apoptosis Health sciences/Diseases/Cancer/Cancer metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction A notable correlation exists between various types of cancers and obesity, which is characterized by an excessive accumulation of body fat. Obesity elevates the risk of carcinogenesis 1 . The consumption of a high-fat diet (HFD) augments the malignant potential of cancer cells 2 . Therefore, ingesting an excessive amount of dietary fat is implicated in escalating the risk of cancer development, exacerbating the malignancy of cancer in a tumor-bearing state, and potentially effectuating adverse clinical outcomes 3, 4 . However, fatty acids are not universally detrimental, and their effects on cancer cells vary depending on the type of fatty acid 5 . In biological systems, most fatty acids comprise 16 or more carbon atoms and are classified as long-chain fatty acids 6, 7 . These long-chain fatty acids can be further categorized into saturated fatty acids (SFAs), which possess only single bonds between carbon atoms, and unsaturated fatty acids (UFAs), which are characterized by the presence of double bonds. UFAs may enhance cancer survival, including stemness and ferroptosis resistance, particularly in renal and ovarian cancers 8, 9, 10 , suggesting a potential association between UFAs and adverse clinical outcomes. Conversely, SFAs exert cytotoxic effects on normal cells—especially hepatocytes, endothelial cells, adipocytes, and pancreatic β cells—commonly referred to as lipotoxicity 11, 12, 13 . The potential anti-proliferative effects of SFAs on cancer cells has been documented recently 14, 15, 16 . Caloric restriction markedly elevates the proportion of SFAs in the fatty acid composition of biological systems, which may in turn promote tumor suppression 17 . Inhibiting the activity of stearoyl-CoA desaturase (SCD), which catalyzes the conversion of SFAs to UFAs, induces an increase in SFA levels and subsequently hinders glioblastoma cell proliferation 18 . An SCD inhibition-mediated elevation in the SFA/UFA ratio can enhance antitumor effects in ovarian cancer 16 . Nevertheless, comprehensive studies examining the in vivo effects of SFAs and the subtle differences between them are scarce. Most reports investigating the role of SFAs use palmitate for analysis. Palmitate, comprising 16 carbon atoms, is the most abundant SFA in biological systems, and stearate, comprising 18 carbon atoms, is also quite abundant 6 . They are structurally similar, differing solely by a two-carbon atom variation in chain length. Therefore, it has been thought that there is little difference in the effects of palmitate and stearate on cells. However, the two SFA types can have different effects on cancer cells 19 , but few studies have comprehensively explored their cellular effects; hence, the effects of stearate on cancer cells are largely unknown. Moreover, manipulation of dietary long-chain fatty acid composition and its in vivo effects remain to be evaluated. Therefore, here, we investigated the impact of palmitate, stearate, and oleate on cancer cells and whether dietary changes would have a sufficient clinical impact. To this end, in addition to a usual HFD, a specialized HFD rich in stearate (S-HFD) was employed to study the differential effects of dietary stearate and oleate on cancer in an in vivo setting. Materials and Methods Study Approval The Ethics Committee of the Graduate School and Faculty of Medicine at Kyoto University approved this study, assigning reference numbers G531 and G288, ensuring compliance with the Declaration of Helsinki's principles. The university's Animal Research Committee authorized the animal experiments conducted in this research. Cell Culture The National Institutes of Health's Dr. Melinda Hollingshead provided the human ovarian cancer cell line OVCAR8. Dr. Iwakoshi, Dr. Masashi Kanai, Dr. Shigeo Takaishi, and Dr. Susan K. Murphy donated the human ovarian cancer cell lines ES-2, OVCA5, SKOV3, OVCAR3, the human colon cancer cell lines DLD1 and LoVo, and the human epithelial cell line HOSE 20 , respectively. The American Type Culture Collection (Manassas, VA, USA) supplied the human mammary cancer cell lines MCF-7, MDA-MB-453, MDA-MB-231, Hs578t, the human mammary epithelial cell line MCF10A, the human lung cancer cell lines NCI-H460, NCI-H1299, NCI-H1650, and the human colon cancer cell lines HCT116, HT29, Caco2. The human lung cancer cell line A549 was purchased from the RIKEN BRC cell bank (Tsukuba, Japan). All cell lines, except MCF10A, were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin-streptomycin. MCF10A cells were grown in DMEM/F12 supplemented with 5% FBS, 0.02% Epidermal Growth Factor (EGF), 0.05% insulin, 0.5 µg/mL hydrocortisone, and 1% penicillin-streptomycin. Animal Models BALB/cAJcl-nu/nu mice, aged 4 and 6 weeks, were obtained from CLEA Japan. Five-week-old nonobese diabetic/Shi-scid IL-2RγKO Jic (NOG) mice were acquired from In-Vivo Science Inc. These animals were kept in specific pathogen-free conditions. Initially, the mice were provided a standard solid diet, formula F-2, with 12.0% fat by caloric content, and had unrestricted access to water. Later, as part of the study design, they were assigned to specific dietary regimens. Preparation of Mouse Xenograft Models Utilizing Human Ovarian Cancer Cell Lines To study the effect of dietary conditions and therapeutic interventions on tumor growth, mice were grouped into experimental categories. Xenografts were established using human ovarian cancer cell lines and genetically modified cells, and these were treated with either a vehicle or CAY10566. Before transplantation, mice were acclimatized to specific diets - a sterile regular chow diet (NFD) with 12.0% fat, a high-fat diet with 60% fat (S-HFD), or another high-fat diet with 56.7% fat (O-HFD) for three days, and these diets were maintained throughout the study. Tumor growth was regularly monitored by measuring tumor dimensions, and humane treatment protocols, including euthanasia criteria to prevent excessive tumor growth or ulceration, were strictly followed. Mouse Xenograft Models Harboring PDXs Ovarian cancer patient surgical specimens were obtained with informed consent at Kyoto University Hospital and used to create primary xenograft tumors in NOD SCID mice using Matrigel Matrix Basement Membrane for transplantation. Following tumor establishment, mice were divided into four treatment groups - NFD + vehicle, NFD + CAY10566, S-HFD + CAY10566, and O-HFD + CAY10566 - to assess the effects of dietary conditions and CAY10566 on tumor growth. Tumor volume was periodically measured using methods similar to those employed in ovarian cancer cell line xenograft models, to evaluate treatment outcomes. Detail descriptions of the in vivo and in vitro analyses, mouse work protocols, preparation of reagents and samples, flow cytometry, Enzyme-Linked Immunosorbent Assay (ELISA), RNA sequencing, Western immunoblotting, Immunohistochemistry (IHC), and Liquid Chromatography-Mass Spectrometry (LC-MS) are described in supplementary material and method. The comprehensive list of reagents and antibodies used for Western immunoblotting or IHC, including the corresponding dilutions, primers, short hairpin RNA (shRNA) sequences, and software, along with their sources and research resource identifier numbers, are detailed in the Supplementary Data. Additionally, a detailed composition of the dietary components used in this study are listed in the Supplementary Table. Statistical Analyses At least three independent in vitro experiments and a minimum of two cell lines for in vivo experiments were utilized. Mice in in vivo studies were randomly assigned to experimental groups. Sample sizes were determined to ensure experiment reproducibility, adhering to the principles of replacement, reduction, and refinement in animal ethics. Results are presented as mean ± Standard Error of the Mean (SEM). The Mann–Whitney U test was employed for in vitro proliferation assay data analysis, ELISA group comparisons using in vivo samples, and for assessing tumor volume, weight, and IHC outcomes. The Wilcoxon matched-pairs signed-rank test was used for IC50 analysis, apoptosis assays, and comparisons of in vivo tumor volume and weight between shCtr and shSCD tumors. An unpaired t-test was applied for LC/MS analysis results. All statistical analyses were conducted using Prism 10.0.2 software, setting significance levels at *p < 0.05, **p < 0.01, ***p < 0.001, with 'ns' indicating no significance. Data availability: The RNA-seq data of this study have been deposited in the Gene Expression Omnibus (GEO) under accession no. GSE248408. Results Stearate Inhibits Growth Across Multiple Cancer Cell Lines First, we evaluated the impact of palmitate and stearate on cellular function. Multiple human cancer cell lines (ovarian: OVCAR5, ES-2, SKOV3, OVCAR3, and OVCAR8; lung: H460, A549, H1650, and H1299; breast: Hs578T, MDA-MB0231, MDA-MB-453, and MCF7; and colorectal: Lovo, HCT116, HT290, DLD-1, and Caco-2) were cultured in palmitate- and stearate-supplemented media for 72 h, and their impact on cell proliferation was assessed. Overall, stearate inhibited cell growth to a greater extent than palmitate across all cell lines (p = 0.000252, Wilcoxon matched-pairs test). Furthermore, a k-means clustering analysis revealed that the cells could be segregated into three distinct groups based on their proliferative responses to the fatty acids: stearate ≈ palmitate, stearate > palmitate, and a group where neither fatty acid influenced cellular proliferation (Fig. 1 a). The ovarian cancer cell lines were exclusively categorized under the stearate > palmitate group, suggesting that a stronger inhibitory effect on ovarian cancer cell growth was seen with stearate than with palmitate. Additionally, similar experiments were conducted using the human mammary epithelial cell line MCF10A and the human ovarian surface epithelial cell line HOSE 20 . Despite both being immortalized normal cell lines, the sensitivity to stearate varied significantly between these two cell lines. In HOSE cells, the addition of stearate had a more pronounced impact on cell proliferation than that in MCF10A cells (p = 0.002165). To further investigate the potential anticancer effects of stearate, subsequent experiments primarily focused on the ovarian cancer cell lines classified into the stearate > palmitate group. Conversely, in culture media supplemented with monounsaturated fatty acids (MUFA), palmitoleate and oleate did not markedly inhibit ovarian cancer cell growth. In fact, in certain cases, cell growth was enhanced, suggesting the differential effects of SFAs and MUFAs on the cancer cells (Supplementary Fig. S1 a). To identify which fatty acids significantly affect cell proliferation, we conducted experiments using long-chain fatty acids that are frequently encountered in dietary and cellular contexts 7, 21 . Ovarian cancer cell lines, namely OVCAR5, OVCAR8, SKOV3, ES-2, and OVCAR3, were treated with 50 µM each of palmitate, stearate, and oleate, and the cell viability was measured after 24, 48, and 72 h. Stearate markedly impeded cell growth in all lines from 24 h (Fig. 1 b). Dose-response curves for each long-chain fatty acid revealed substantially lower IC50 values of stearate, at 36.96 ± 3.22 µM for OVCAR5 and 31.04 ± 1.97 µM for OVCAR8, than those of palmitate, at 1469.75 ± 74.61 µM and 74.97 ± 2.7 µM, respectively (Fig. 1 c, Supplementary Fig. S1 b). Stearate Induces Apoptosis and DNA Damage in Ovarian Cancer Cells In Vitro and In Vivo Next, we investigated whether stearate induced apoptosis like palmitate, as reported previously 12, 14, 16 . A flow cytometry analysis of Annexin V-positive cells showed that stearate increased apoptosis of OVCAR5 cells dose-dependently (Figs. 2 a, b). As palmitate-induced apoptosis is related to DNA damage 22, 23 , we examined whether stearate similarly affects OVCAR5 cells. A dose-dependent increase in γH2AX expression was observed following 24 h stearate treatment (Fig. 2 c). The obtained findings were corroborated through comparative experiments utilizing OVCAR8 cells; flow cytometry and western blot assays were conducted to elucidate the influence of stearate on apoptosis and DNA damage, respectively (Supplementary Figs. S3a-c). To gain further insight into our findings, we conducted an in vivo study. We first used murine models to determine the impact of a HFD rich in oleate (O-HFD) on the growth of tumors derived from subcutaneously inoculated cancer cell lines (Supplementary Fig. S1 c). Consistent with previous studies, tumor proliferation increased in mice harboring tumors derived from OVCAR5 cells that were fed an O-HFD (Supplementary Figs. S1d–g) 2, 4, 24 . Given the absence of notable disparities in body weight or blood insulin levels 25 resulting from dietary variations, the findings prompted the hypothesis that the influence on tumor proliferation stemmed from the fatty acids themselves rather than alterations in the physiological condition of the mice. Initially, the fat in the O-HFD was mainly composed of oleate (64.3%) with a low stearate content (7.5%; Table S1 ). Next, we investigated the effects of a S-HFD with a significantly higher per-calorie stearate content (33.35%; Table S1 ). Importantly, the S-HFD group exhibited a significant reduction in tumor growth compared with the normal-fat diet (NFD) group (Figs. 2 d, e). Flow cytometry and IHC (immunohistochemistry) results revealed a higher degree of tumor apoptosis in the S-HFD group than in the NFD group (Annexin V-positive rate: 26.6% vs. 30.27%, cleaved caspase-3-positive area: 0.56% vs. 1.0%; Figs. 2 f, g, Supplementary Figs. S2c, d). To evaluate DNA damage, IHC was performed for γH2AX, and the results showed a significant increase in the proportion of γH2AX-positive cells in the S-HFD group compared with that in the NFD group (H score: 38.2 vs. 57.5; Supplementary Figs. S2c, e). These findings were validated using SKOV3 cells (Supplementary Figs. S2f–h). Furthermore, no significant differences were observed in body weight and vital organs, including the liver and kidneys, between the S-HFD and NFD groups (Supplementary Figs. S2a, b). These data collectively demonstrate that stearate induces cytotoxicity, DNA damage, and apoptosis in ovarian cancer cells. Oleate Mitigates Stearate-Induced Cytotoxicity Long-chain fatty acids can be converted to other long-chain fatty acids in biological systems 26 . Therefore, we altered dietary long-chain fatty acid composition to generate S-HFD and evaluated whether these changes were reflected in the tumor tissue. The S-HFD group had significantly higher tumor stearate content compared to that in the NFD group (107.3 vs. 164.9 pmol/mg, p = 0.012094). Notably, stearate levels were elevated even in the O-HFD group, almost matching those in the S-HFD group (O-HFD vs S-HFD: 164.9 vs. 165.7 pmol/mg, p = 0.957080). However, oleate levels were higher in the O-HFD group than in the S-HFD group (O-HFD vs. S-HFD: 172.5 vs. 86.62 pmol/mg, p = 0.007269; Fig. 2 h). This led us to propose that oleate may mitigate the tumor-suppressive effects of stearate. Oleate ameliorates palmitate-induced ER stress and DNA damage 17, 23, 27, 28, 29 , and therefore, we next examined whether similar phenomena occurred in our study. We first investigated the effect of oleate on tumor apoptosis. Stearate-induced apoptosis was significantly reduced following 50 µM oleate addition to OVCAR5 and OVCAR8 cells (Figs. 3 i, j, Supplementary Figs. S3d, e). Moreover, the addition of 50 µM oleate almost abrogated stearate-induced elevated γH2AX expression in OVCAR5 and OVCAR8 cells (Figs. 3 k, Supplementary Fig. S3 f), resulting in reduced cytotoxicity. We treated OVCAR5, OVCAR8, SKOV3, ES-2, and OVCAR3 cells with varying concentrations of stearate under 50 µM oleate treatment (Fig. 2 l). The addition of 50 µM oleate significantly ameliorated the stearate-induced decrease in cell viability. We next explored the effects of oleate on stearate-induced cell death. Oleate rescued the cells incubated with 100 µM stearate from cell death in a concentration-dependent manner (Supplementary Fig. S4 a). In OVCAR5 cells, the addition of 25 µM oleate rescued cell proliferation to levels almost comparable to those achieved with the addition of 100 µM oleate. Overall, oleate attenuates stearate-induced cytotoxic effects, including DNA damage and apoptosis, in ovarian cancer cells. Inhibition of Unsaturation Increases Stearate Toxicity In biological systems, stearate is converted to oleate by stearoyl-CoA desaturase 1 30 (SCD1; Fig. 3 a). SCD1 overexpression has been documented in various cancers, including ovarian cancer 31, 32, 33 . Li et al.8 argued that the malignancy of high-grade serous ovarian cancer (HGSC) is significantly influenced by endogenous oleate produced by SCD1. Therefore, we explored the involvement of endogenous and exogenous oleate in cancer pathogenesis. To inhibit endogenous oleate synthesis, we transduced shRNA sequences targeting SCD1 (shSCD-1 and shSCD-2) or control shRNA (shCtr) into OVCAR-5 and OVCAR-8 cells (Supplementary Figs. S4b–e). The inhibition of SCD1 led to a marked increase in cellular sensitivity to stearate (Figs. 3 b, c). Conversely, the addition of oleate substantially ameliorated stearate-induced cytotoxicity (Fig. 3 d, Supplementary Figs. S4f, g). Incubation with 1 µM CAY10566, an SCD inhibitor 34, 35 , did not inhibit cell proliferation; however, concentrations of stearate and oleate in OVCAR5 cells were significantly altered (Supplementary Figs. S5a–c). Similar to SCD knockdown (SCD-KD), the addition of 1 µM CAY10566 increased cellular sensitivity to stearate (Figs. 3 f, g), while the growth-inhibitory effect was significantly mitigated by the addition of oleate (Fig. 3 e). These trends were consistent across other cell lines, including SKOV3, ES2, and OVCAR3 (Supplementary Figs. S5d–f). Although SCD activity inhibition, using CAY10566 or SCD expression knockdown, decreased cell viability by increasing stearate concentration and decreasing oleate concentration, exogenous oleate abrogated these detrimental effects. Stearate Induces Cytotoxicity via Endoplasmic Reticulum (ER) Stress and CHOP Activation Next, we sought to elucidate the mechanisms underlying stearate-mediated cytotoxicity. We treated OVCAR5 cells with (i) DMSO, (ii) CAY10566 1 µM, (iii) stearate 50 µM + DMSO, (iv) stearate 50 µM + CAY10566 1 µM, (v) oleate 50 µM + DMSO, and (vi) oleate 50 µM + CAY10566 1 µM and performed RNA sequencing analysis. Principal component analysis (PCA) revealed that the presence or absence of stearate strongly contributed to PC1, whereas the presence or absence of oleate influenced PC2. However, 1 µM CAY10566 had limited effects (Fig. 4 a). Gene Ontology (GO) analysis identified 643 differentially expressed genes (DEGs), of which 401 were upregulated and 242 were downregulated between 50 µM stearate-treated and control OVCAR5 cells (false discovery rate [FDR] 1.25; Supplementary Figs. S6a, b). The top 10 significantly upregulated GO categories were enriched in pathways associated with the unfolded protein response (UPR) and ER stress in 50 µM stearate-treated OVCAR5 cells compared with control cells (Fig. 4 b; FDR < 0.05). UPR signaling involves ATF6, IRE1α, and PERK pathways 36 . Our western blot analysis confirmed that stearate induced the concentration-dependent activation of UPR-related proteins, including ATF6, XBP-1 as a downstream transcription factor of IRE1α, and ATF4 as a downstream transcription factor of PERK. Moreover, the expression of pro-apoptotic transcription factor CHOP 37 and apoptotic markers cleaved caspase-3 and γH2AX was upregulated (Figs. 4 c, d). We further examined whether the addition of oleate mitigated the activation of ER stress response pathways. Activation of ER stress response pathways was negated by the addition of 100 µM oleate to OVCAR5 and OVCAR8 cells (Figs. 4 c, d). Furthermore, the addition of 1 µM CAY10566 enhanced the stearate-dependent activation of UPR-related proteins, CHOP, cleaved caspase-3, and γH2AX; however, this activation of the UPR pathway was almost abrogated by exogenous oleate (Figs. 4 c, d). Long-term exposure to mild ER stress or short-term exposure to severe ER stress induces CHOP-mediated apoptosis 13, 38 . To explore whether stearate induced apoptosis via CHOP, we generated CHOP-knockdown OVCAR5 and OVCAR8 cell lines via lentiviral infection of CHOP shRNA (Supplementary Fig. S6c, d). Following inhibition of CHOP expression, the expression of cleaved caspase-3 and γH2AX, which was increased in a concentration-dependent manner by stearate treatment, was significantly reduced (Figs. 4 e, f, Supplementary Figs. S6e, f). Moreover, CHOP knockdown significantly enhanced the resistance to stearate-induced cytotoxicity (Figs. 4 g, h), indicating that stearate-induced cytotoxicity was mediated through ER stress and CHOP activation. Overall, exogenous stearate activated ER stress response pathways, induced DNA damage, and inhibited the proliferation of ovarian cancer cells. Consistently, the addition of exogenous oleate attenuated the ER stress response pathway activated by stearate, reducing its toxicity in ovarian cancer. Additionally, we have confirmed that the sensitivity to stearate and palmitate varies among cell lines (Fig. 1 a). We investigated whether these differences were due to variations in ER stress response pathways. Regarding MCF10A cells (stearate-nonresponsive cells), we observed minimal CHOP induction by stearate, which differs significantly from the findings for HOSE and OVCAR5 cells (stearate-responsive cells) (Supplementary Figs. S7a, b). In the case of H1299 cells (stearate-nonresponsive cells), we found constant CHOP expression regardless of the addition of stearate, which was not decreased by oleate. These findings also significantly differed from those of HOSE and OVCAR5 (Supplementary Figs. S7a, b). We then investigated the responses of OVCAR5 and OVCAR8 cells to stearate and palmitate. Notably, palmitate induced CHOP expression in these cells, but to a lesser extent than stearate. Additionally, the activation of Cleaved Caspase3 by palmitate was less pronounced than that induced by stearate (Supplementary Figs. S7c, d). These findings highlight that the varying sensitivities to palmitate and stearate in different cell lines are primarily a result of their unique responses to the activation of the ER stress pathway. Inhibition of Unsaturation Along with Dietary Supplementation of Stearate Hinders Tumor Growth, Which is Reversed by Oleate Supply To validate our results obtained so far in vivo, we fed mice an S-HFD, O-HFD, or NFD (Supplementary Figs. S8a–c). In the S-HFD group subcutaneously injected with SCD1-knockdown (SCD1-KD) OVCAR5 cells, tumor growth was significantly inhibited compared with that in the NFD group (SCD1-KD & S-HFD vs. SCD1-KD & NFD; 0.125 g vs. 0.240 g, p = 0.006494; Figs. 5 b, c). Conversely, the O-HFD group displayed significantly greater tumor growth than did the S-HFD and NFD groups (Fig. 5 c). In experiments using sh-control cell lines, the S-HFD group exhibited stronger growth suppression than the NFD and O-HFD groups, although this trend was less pronounced than that observed in experiments using SCD1-KD cells (sh-control & S-HFD vs. sh-control & O-HFD; 0.2633 g vs. 0.4017 g, p = 0.006494; Fig. 5 a). Furthermore, no significant differences in tumor growth were observed between sh-control and SCD1-KD cells in the O-HFD group (Fig. 5 c). In the S-HFD group subcutaneously injected with SCD1-KD OVCAR8 cells, the greatest tumor growth suppression was noted, with a significant difference compared with that in the O-HFD group (SCD1-KD & S-HFD vs. SCD1-KD & O-HFD; 0.02667 g vs. 0.0733 g, p = 0.019481; Figs. 5 d–f). The same trend was observed when animals were injected with the sh-control cell line; however, no significant differences were observed between the S-HFD and O-HFD groups (sh-control & S-HFD vs. sh-control & O-HFD: 0.0433 g vs. 0.0533 g, p = 0.4848). Additionally, no significant differences were observed in tumor growth between mice injected with sh-control and SCD1-KD cells and fed on the O-HFD, as observed with OVCAR5 cells (Fig. 5 f). Next, we examined whether the UPR pathway, DNA damage, or apoptosis were modulated in vivo. IHC of OVCAR5 cell-derived tumors revealed marked upregulation of CHOP expression in the S-HFD group and the most significant upregulation in SCD1-KD cells (Figs. 5 g–j). Conversely, CHOP expression was almost abrogated in the O-HFD group, regardless of whether the sh-control or SCD1-KD cells were used. We also assessed γH2AX and cleaved caspase-3 expression and observed trends consistent with those of CHOP expression. Similar results were obtained using OVCAR8 cells (Supplementary Figs. S8d–g). We conducted further experiments using CAY10566 (Supplementary Fig. S9a). In mice injected with OVCAR5 and OVCAR8 cells, the CAY10566-treated group exhibited the most significant tumor growth suppression when fed the S-HFD compared with the NFD and O-HFD groups (Supplementary Figs. S9b–f, S10a–c). Tumor growth in the vehicle-treated group, those fed on the S-HFD, was the lowest, but this trend was less pronounced than that in the CAY10566 group. Moreover, no significant differences were observed between the vehicle and CAY10566 groups when fed on the O-HFD. The expression levels of γH2AX and cleaved caspase-3 were most significantly upregulated in the CAY10566 + S-HFD group, whereas almost no expression was observed in the O-HFD groups, irrespective of whether they were in the vehicle or CAY10566 group (Supplementary Fig. S9g–j, S10d–g). Assessments of stearate and oleate concentrations within tumor tissues demonstrated a stearate increment of 1.5- to 2-fold in the S-HFD-, O-HFD-, or CAY10566-administered group compared with that in the NFD + vehicle group. Despite the administration of CAY10566, O-HFD elevated oleate levels by approximately 1.5-fold, correlating with an actual proliferation enhancement in the tumors. Conversely, S-HFD in combination with CAY10566 administration resulted in a significant elevation of stearate to 185 pmol/mg while maintaining oleate levels at 50 pmol/mg, which was lower than that in the NFD-vehicle group, thus exhibiting a pronounced inhibitory effect on tumor growth (Supplementary Fig. S10h). Overall, robust tumor-suppressive effects were achieved in vivo by increasing tumor stearate levels via S-HFD feeding, coupled with oleate inhibition mediated via SCD inhibition. Additionally, excessive intake of oleate through the O-HFD significantly diminished this effect. Supply of Stearate along with Inhibition of Unsaturation Shows Significant Anti-proliferative Effects on Ovarian Cancer Patient-derived Xenograft (PDX) Models To evaluate the applicability of our findings in the clinical setting, we next conducted experiments using PDXs. Conducting large-scale interventions to assess the effects of dietary changes is challenging; however, drug responses in PDXs have been suggested to correlate with patient clinical outcomes 39 . Therefore, we utilized two PDXs from distinct clinical backgrounds (PDX72 and PDX82; Supplementary Texts) that were established from patients treated at our institution. PDX82 was sourced from a 38-year-old female patient with stage IIIC HGSC harboring a BRCA2 mutation. This patient was sensitive to platinum-based chemotherapy and maintained no long-term evidence of disease under poly (ADP-ribose) polymerase inhibitor (PARPi) 40, 41 treatment (Figs. 6 a–c, Supplementary Figs. S11a–c). In PDX82 experiments, while treatment with CAY10566 alone showed limited effectiveness, tumor growth was significantly inhibited when these mice were fed S-HFD (NFD-CAY10566: 2685 mg vs. S-HFD-CAY10566: 970 mg, p = 0.0285; Figs. 6 d–f). However, feeding mice on O-HFD led to significantly larger tumor sizes compared to the S-HFD-fed group, even with CAY10566 administration (S-HFD-CAY10566: 970 mg vs. O-HFD-CAY10566: 970 mg, p = 0.0285). Another PDX, PDX72, was sourced from a 43-year-old female who developed platinum-resistant recurrent HGSC. The tumor was collected during secondary debulking surgery (SDS). Despite surgery, the patient relapsed quickly, and neither platinum-based chemotherapy nor anti-VEGF antibodies 42 were effective, resulting in a poor prognosis (Figs. 6 g–i, Supplementary Figs. S12a–c). Studies using PDX72 revealed that CAY10566 administration alone inhibited tumor growth, and this effect was further enhanced by feeding the S-HFD to mice (NFD-vehicle: 678.3 mg vs. NFD-CAY10566: 245.0 mg vs. S-HFD-CAY10566: 150 mg, p = 0.0021, 0.0043, respectively; Figs. 6 j–l). However, despite CAY10566 treatment, mice fed on the O-HFD developed significantly larger tumors than those fed on the S-HFD (S-HFD-CAY10566: 150 mg vs. O-HFD-CAY10566: 798.3 mg, p = 0.0021). IHC analysis results of these two PDX models regarding UPR, DNA damage, and apoptosis markers were consistent; the highest expression levels of CHOP, γH2AX, and cleaved caspase-3 were observed in the CAY10566 + S-HFD group, whereas these markers were significantly inhibited in the O-HFD group (Supplementary Figs. S11d–g, S12d–g). Overall, combined administration of CAY10566 and S-HFD significantly suppressed tumor growth in two distinct PDX models with different clinical backgrounds and outcomes. Furthermore, even in cases sensitive to CAY10566 alone, tumor proliferation was enhanced when the O-HFD was consumed, suggesting that the antitumor effect of CAY10566 can be compromised by an O-HFD. Discussion In this study, we extensively explored the varied effects of long-chain fatty acids on cancer cell proliferation. Studies using multiple organ-derived cancer cells have revealed that SFAs, known for their lipotoxicity in normal cells 43 —specifically palmitate and stearate— impart inhibitory effects on the growth of cancer cell lines. Notably, stearate exhibited an anti-proliferative effect on broader range of cancer cells in comparison to palmitate. Specifically, there were several cell lines in where palmitate showed limited efficacy, whereas stearate was more potent, with all six ovarian cancer cell lines included in this study falling into this category. Furthermore, the normal human ovarian surface epithelial cell line (HOSE) was also strongly affected by stearate, a result that differed significantly from that of the normal human mammary epithelial cell line (MCF10A). Given the variable influence of long-chain fatty acids across different tissue types 5 ; this finding suggests that ovarian tissues might possess heightened susceptibility to the cytotoxic effects of stearate, and, this sensitivity could potentially be extended to ovarian cancers, although the detailed mechanisms remain unclarified. Our findings demonstrated that stearate induced DNA damage and apoptosis through dose-dependent activation of the UPR pathway. This phenomenon was directly mitigated by oleate, and impeding the conversion of stearate to oleate amplified the cytotoxic effects of stearate. Wieder et al. 5 segregated long-chain fatty acid-elicited cellular damage into two major pathways, UPR and ROS generation, and showed that the detrimental effects associated with UPR could be rescued by oleate treatment. Our results corroborate the aforementioned findings. Moreover, our findings established that S-HFD and SCD1-rich diets exert the most potent anti-proliferative effects and reduced oleate levels in mice harboring xenografts derived from various cancer cell lines. These findings provide evidence that dietary modifications can induce the accumulation of excess stearate and limit oleate contents in tumors, inhibiting tumor growth. To our knowledge, this is the first study to specify the therapeutic role of excess dietary intake of stearate and limited intake of oleate in cancers. In vivo studies on ovarian cancer with palmitate treatment did not portray the marked changes observed with stearate treatment 16 . As previously stated, most ovarian cancer cell lines were more sensitive to stearate than to palmitate. Additionally, the UPR pathway response was more strongly induced by stearate compared to palmitate. This explains the difference between the results of our study and previous in vivo studies16 Our experiments suggest that stearate should produce stronger antitumor effects compared to palmitate, although they have similar structures. HGSC is the predominant histological subtype of ovarian cancer 44 and is often diagnosed at advanced stages accompanied by peritoneal dissemination 45, 46 . Despite the promising outcomes achieved through the administration of targeted therapies against aberrant DNA repair mechanisms, including PARPis 40, 41 , HGSC eventually becomes therapy-resistant and worsens prognosis in numerous patients 47 . Notably, the development of drug resistance in ovarian cancers also induces limited genetic alterations 48 , requiring the implementation of alternative treatment strategies. Our results hold significant clinical potential, as similar anticancer effects of dietary modulations were observed using mice harboring PDXs derived from drug-resistant tumors. Wieder et al. 5 proposed that the UPR is a promising therapeutic target for various states of HGSC, and targeting UPR along with a dietary intervention to steer stearate accumulation and limit oleate content in tumors may offer a novel therapeutic approach for refractory HGSC. There are still some limitations to this study. The detail mechanism of why stearate and palmitate have different effects remains unclear. Therefore, it is difficult to identify a population for which the activation of UPR with stearate is more effective. The effects on the immune system have not yet been investigated, and the details of their effects on normal organs are still unknown. Additionally, the dietary conditions employed here may lack direct applicability in clinical settings. Nonetheless, the implications of our study are noteworthy. Although dietary interventions are garnering increased attention in clinical research on cancer treatment 49 , it is generally regarded as a complementary therapy. Our findings suggest that dietary modifications can exert direct antitumor effects, broadening the scope for dietary interventions in cancer treatment. Based on this study, we would like to build a more solid evidence-based dietary intervention for cancer treatment. Declarations Grants: This work was supported by the JST SPRING, Grant Number JPMJSP2110, and the MEXT/JSPS KAKENHI, Grant Numbers JP20K18166 and JP23K15834. Acknowledgments We would like to thank Editage (www.editage.com) for English language editing. We are also grateful to Junko Satoh and Atsuko Nakao for their assistance with the measurement of fatty acids using LC/MS. Conflict of interests: Eijiro Nakamura has received research funding from Sumitomo Pharma CO., Ltd. The other authors declare that they have no competing interests. This work was supported by the JST SPRING, Grant Number JPMJSP2110 (to JO), and the MEXT/JSPS KAKENHI, Grant Numbers JP20K18166 and JP23K15834 (to YK). End Notes ： Author contributions: J.O. designed and performed the experiments and wrote the manuscript. K.Y., M.T., R.M., and J.H. all contributed to designing the experiments and editing the manuscript. Y.H., S.I., J.S., A.N., and E.N. were involved in performing the experiments. Y.N. and K.K. provided samples and assisted in editing the manuscript. M.M. designed the experiments, provided funding, and edited the manuscript. Supplementary information accompanies the manuscript on the Experimental & Molecular Medicine’s website (http://www.nature.com/emm/). References Lauby-Secretan B, Scoccianti C, Loomis D, Grosse Y, Bianchini F, Straif K. Body Fatness and Cancer–Viewpoint of the IARC Working Group. N Engl J Med 375 , 794–798 (2016). Yang J, et al. High-Fat Diet Promotes Colorectal Tumorigenesis Through Modulating Gut Microbiota and Metabolites. Gastroenterology 162 , 135–149.e132 (2022). Beyaz S, et al. High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. Nature 531 , 53–58 (2016). Labbé DP, et al. High-fat diet fuels prostate cancer progression by rewiring the metabolome and amplifying the MYC program. Nature Communications 10 , (2019). Wieder N, et al. FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity. Cell Metab 35 , 887–905.e811 (2023). Fraser DA, Thoen J, Rustan AC, Førre O, Kjeldsen-Kragh J. Changes in plasma free fatty acid concentrations in rheumatoid arthritis patients during fasting and their effects upon T-lymphocyte proliferation. Rheumatology (Oxford) 38 , 948–952 (1999). Puri P, et al. A lipidomic analysis of nonalcoholic fatty liver disease. Hepatology 46 , 1081–1090 (2007). Li J, et al. Lipid Desaturation Is a Metabolic Marker and Therapeutic Target of Ovarian Cancer Stem Cells. Cell Stem Cell 20 , 303–314 e305 (2017). Magtanong L, et al. Exogenous Monounsaturated Fatty Acids Promote a Ferroptosis-Resistant Cell State. Cell Chem Biol 26 , 420–432 e429 (2019). Yang Y, et al. Palmitoylation stabilizes PD-L1 to promote breast tumor growth. Cell Res 29 , 83–86 (2019). Harvey KA, et al. Oleic acid inhibits stearic acid-induced inhibition of cell growth and pro-inflammatory responses in human aortic endothelial cells. J Lipid Res 51 , 3470–3480 (2010). Nemcova-Furstova V, Balusikova K, Halada P, Pavlikova N, Sramek J, Kovar J. Stearate-Induced Apoptosis in Human Pancreatic beta-Cells is Associated with Changes in Membrane Protein Expression and These Changes are Inhibited by Oleate. Proteomics Clin Appl 13 , e1800104 (2019). Cao J, et al. Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway. Mol Cell Biochem 364 , 115–129 (2012). Evans LM, Cowey SL, Siegal GP, Hardy RW. Stearate Preferentially Induces Apoptosis in Human Breast Cancer Cells. Nutr Cancer 61 , 746–753 (2009). Lin L, et al. Functional lipidomics: Palmitic acid impairs hepatocellular carcinoma development by modulating membrane fluidity and glucose metabolism. Hepatology 66 , 432–448 (2017). Zhao G, et al. Ovarian cancer cell fate regulation by the dynamics between saturated and unsaturated fatty acids. Proc Natl Acad Sci U S A 119 , e2203480119 (2022). Lien EC, et al. Low glycaemic diets alter lipid metabolism to influence tumour growth. Nature 599 , 302–307 (2021). Eyme KM, et al. Targeting de novo lipid synthesis induces lipotoxicity and impairs DNA damage repair in glioblastoma mouse models. Sci Transl Med 15 , eabq6288 (2023). Zhang L, Lv J, Chen C, Wang X. Roles of acyl-CoA synthetase long-chain family member 5 and colony stimulating factor 2 in inhibition of palmitic or stearic acids in lung cancer cell proliferation and metabolism. Cell Biol Toxicol 37 , 15–34 (2021). Nagayoshi Y, et al. Characterization of 17beta-hydroxysteroid dehydrogenase type 4 in human ovarian surface epithelial cells. Mol Hum Reprod 11 , 615–621 (2005). de Almeida IT, Cortez-Pinto H, Fidalgo G, Rodrigues D, Camilo ME. Plasma total and free fatty acids composition in human non-alcoholic steatohepatitis. Clin Nutr 21 , 219–223 (2002). Yuzefovych L, Wilson G, Rachek L. Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress. Am J Physiol Endocrinol Metab 299 , E1096-1105 (2010). Ishaq A, Tchkonia T, Kirkland JL, Siervo M, Saretzki G. Palmitate induces DNA damage and senescence in human adipocytes in vitro that can be alleviated by oleic acid but not inorganic nitrate. Exp Gerontol 163 , 111798 (2022). Hayashi T, et al. High-Fat Diet-Induced Inflammation Accelerates Prostate Cancer Growth via IL6 Signaling. Clin Cancer Res 24 , 4309–4318 (2018). Straus DS. Growth-stimulatory actions of insulin in vitro and in vivo. Endocr Rev 5 , 356–369 (1984). Luo D, et al. Covalent inhibition of endoplasmic reticulum chaperone GRP78 disconnects the transduction of ER stress signals to inflammation and lipid accumulation in diet-induced obese mice. eLife 11 , e72182 (2022). Oshima M, et al. Stearoyl CoA desaturase is a gatekeeper that protects human beta cells against lipotoxicity and maintains their identity. Diabetologia 63 , 395–409 (2020). Salvado L, et al. Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism. Diabetologia 56 , 1372–1382 (2013). Zeng X, et al. Oleic acid ameliorates palmitic acid induced hepatocellular lipotoxicity by inhibition of ER stress and pyroptosis. Nutrition & Metabolism 17 , (2020). Ntambi JM. The regulation of stearoyl-CoA desaturase (SCD). Prog Lipid Res 34 , 139–150 (1995). Qiang L, et al. Hepatic SirT1-dependent gain of function of stearoyl-CoA desaturase-1 conveys dysmetabolic and tumor progression functions. Cell Rep 11 , 1797–1808 (2015). Sen U, Coleman C, Sen T. Stearoyl coenzyme A desaturase-1: multitasker in cancer, metabolism, and ferroptosis. Trends in Cancer 9 , 480–489 (2023). Vivas-García Y, et al. Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity. Mol Cell 77 , 120–137. e129 (2020). Kamphorst JJ, et al. Hypoxic and Ras-transformed cells support growth by scavenging unsaturated fatty acids from lysophospholipids. Proceedings of the National Academy of Sciences 110 , 8882–8887 (2013). Liu G, et al. Discovery of potent, selective, orally bioavailable stearoyl-CoA desaturase 1 inhibitors. J Med Chem 50 , 3086–3100 (2007). Hetz C, Papa FR. The Unfolded Protein Response and Cell Fate Control. Mol Cell 69 , 169–181 (2018). Xu Z, Bu Y, Chitnis N, Koumenis C, Fuchs SY, Diehl JA. miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis. Nat Commun 7 , 11422 (2016). Kim I, Xu W, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nature Reviews Drug Discovery 7 , 1013–1030 (2008). Izumchenko E, et al. Patient-derived xenografts effectively capture responses to oncology therapy in a heterogeneous cohort of patients with solid tumors. Ann Oncol 28 , 2595–2605 (2017). Baert T, et al. The systemic treatment of recurrent ovarian cancer revisited. Ann Oncol 32 , 710–725 (2021). Mirza MR, et al. The forefront of ovarian cancer therapy: update on PARP inhibitors. Ann Oncol 31 , 1148–1159 (2020). Burger RA. Experience with bevacizumab in the management of epithelial ovarian cancer. J Clin Oncol 25 , 2902–2908 (2007). Masuda M, et al. Saturated phosphatidic acids mediate saturated fatty acid-induced vascular calcification and lipotoxicity. J Clin Invest 125 , 4544–4558 (2015). Matulonis UA, Sood AK, Fallowfield L, Howitt BE, Sehouli J, Karlan BY. Ovarian cancer. Nat Rev Dis Primers 2 , 16061 (2016). Nieman KM, et al. Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nat Med 17 , 1498–1503 (2011). Ladanyi A, et al. Adipocyte-induced CD36 expression drives ovarian cancer progression and metastasis. Oncogene 37 , 2285–2301 (2018). Christie EL, Bowtell DDL. Acquired chemotherapy resistance in ovarian cancer. Ann Oncol 28 , viii13-viii15 (2017). Smith P, et al. The copy number and mutational landscape of recurrent ovarian high-grade serous carcinoma. Nature Communications 14 , (2023). Vernieri C, Ligorio F, Zattarin E, Rivoltini L, De Braud F. Fasting-mimicking diet plus chemotherapy in breast cancer treatment. Nature Communications 11 , (2020). Additional Declarations There is a conflict of interest Eijiro Nakamura has received research funding from Sumitomo Pharma CO., Ltd. The other authors declare that they have no competing interests. Supplementary Files Supplementarytext.docx Supplementary text SupplementaryInformation.docx Supplementary Information. Cite Share Download PDF Status: Published Journal Publication published 02 Dec, 2024 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 14 May, 2024 Review # 3 received at journal 12 May, 2024 Review # 1 received at journal 03 May, 2024 Reviewer # 3 agreed at journal 29 Apr, 2024 Reviewer # 2 agreed at journal 22 Apr, 2024 Reviewer # 1 agreed at journal 19 Apr, 2024 Reviewers invited by journal 12 Apr, 2024 Submission checks completed at journal 11 Apr, 2024 First submitted to journal 11 Apr, 2024 Unknown event 01 Apr, 2024 Editor assigned by journal 01 Apr, 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-4198546","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":290651984,"identity":"d229e44d-72b1-4e25-bac7-945d7270ced8","order_by":0,"name":"Yamanoi 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06:25:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4198546/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4198546/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-024-01356-2","type":"published","date":"2024-12-02T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55005361,"identity":"3a7b219b-1045-4df7-b8fa-74ea727f736c","added_by":"auto","created_at":"2024-04-19 18:48:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":946072,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent anti-proliferative effects of stearate and palmitate across various cancer cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eProliferation assay clustering. Data of MTT proliferation assays for samples, including lung, breast, ovarian, and colon cancer cell lines, were subjected to k-means clustering based on absorbance. Cells were cultured for 72 h and treated with 50 µM stearate or palmitate, followed by an MTT assay. The results were normalized to those of the fatty acid-free control. The ovarian cancer cell lines are highlighted in red (n=6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. \u003c/strong\u003eMTT assay of ovarian cancer cell lines. Ovarian cancer cell lines (OVCAR5, OVCAR8, SKOV3, ES-2, and OVCAR3) were treated with 50 µM free fatty acids (palmitate, palmitoleate, stearate, and oleate) for 72 h and then subjected to an MTT assay. Proliferation was assessed every 24 h. Data represent mean ± SEM (n=4; *p \u0026lt; 0.05, Mann–Whitney test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003eDose-response and IC50 values for various fatty acids using different ovarian cancer cell lines. Representative dose-response curves for stearate, palmitate, and oleate are shown, based on quadruplicate data. Scatter plots display absolute IC50 values from independent experiments across multiple cell lines, including OVCAR5, OVCAR8, SKOV3, ES-2, and OVCAR3.\u003c/p\u003e","description":"","filename":"EMMfigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4198546/v1/089d097458906b18f0fbb16d.png"},{"id":55005362,"identity":"b5fad116-d868-4baf-b3ab-5a39e23aed39","added_by":"auto","created_at":"2024-04-19 18:48:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1347597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStearate induces apoptosis and DNA damage in ovarian cancer cells in vitro and in vivo while oleate mitigates stearate-induced cytotoxicity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, b.\u003c/strong\u003e Flow cytometry image of stearate-induced apoptosis with Annexin V/PI and bar graphs. Apoptosis in ovarian cancer cells, OVCAR5, treated with varying stearate concentrations was analyzed using Annexin V/PI staining. Bar graphs (b) depict the percentage of Annexin V-positive cells compared to that in untreated controls, as determined using flow cytometry (a). Data represent mean ± SEM (n=4; *p \u0026lt; 0.05, Wilcoxon test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003e γH2AX expression analysis using western blot. The OVCAR5 cell lines were treated with the indicated stearate concentrations for 24 h and γH2AX expression was determined. α-tubulin served as the loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed-g.\u003c/strong\u003e Xenograft mice models harboring OVCAR5.\u003c/p\u003e\n\u003cp\u003ed, e. Tumor growth curves (d) and tumor weights at collection (e). Data represent mean ± SEM (n=6; *p \u0026lt; 0.05, **p \u0026lt; 0.01, Mann–Whitney test). f, g. Apoptosis assay of tumor tissue using flow cytometry (f) and histograms (g) present the percentages of apoptotic cells (n=4; *p \u0026lt; 0.05, **p \u0026lt; 0.01, Mann–Whitney test).\u003cbr\u003e\nh\u003cstrong\u003e.\u003c/strong\u003e Fatty acid levels in OVCAR5 xenografts assessed via LC/MS. Fatty acid concentrations were quantified in tumors from mice on NFD, S-HFD, or O-HFD (identical to those in Figs. S9a, d–f).\u003c/p\u003e\n\u003cp\u003ei\u003cstrong\u003e, j.\u003c/strong\u003e Flow cytometry of stearate-induced \u0026nbsp;apoptosis with Annexin V/PI (i). Bar graph (j) illustrates the ratio of Annexin V-positive cells to control cells. Mean ± SEM (n=6, **p \u0026lt; 0.01, ns; not significant, Wilcoxon matched-pairs test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ek.\u003c/strong\u003e γH2AX expression analysis using western blotting. OVCAR5 cells were cultured for 24 h with the indicated concentrations of oleate and stearate, and γH2AX expression was analyzed. α-tubulin served as a loading control.\u003cbr\u003e\n \u003cstrong\u003el\u003c/strong\u003e. Viability of various cell lines exposed to varying concentrations of stearate and oleate. Cells were treated with indicated stearate concentrations and co-incubated with 50 µM oleate for 72 h. Cell viability, relative to that of the control cells, was assessed using an MTT assay (n=4; *p \u0026lt; 0.05, Mann–Whitney test).\u003c/p\u003e","description":"","filename":"EMMfigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4198546/v1/bbef8e272342a0b57071eccd.png"},{"id":55005364,"identity":"1386e736-03db-494a-8f6d-59a47e5d1c3f","added_by":"auto","created_at":"2024-04-19 18:48:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":928298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of unsaturation increases stearate toxicity, and exogenous oleate mitigates it.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eProposed functional mechanism of SCD in stearate metabolism. shRNA knockdown and CAY10566 were used to inhibit SCD, the enzyme converting stearate to oleate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003eIC50 of stearate in shSCD-treated OVCAR5 cells. SCD knockdown lowered the IC50 (n=6; *p \u0026lt; 0.05, **p \u0026lt; 0.01, Mann–Whitney test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003eAnalogous IC50 findings in shSCD-treated OVCAR8 cells (n=6; *p \u0026lt; 0.05, **p \u0026lt; 0.01, Mann–Whitney test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed.\u003c/strong\u003eEffects of oleate on stearate-treated shSCD-transfected OVCAR5 cell viability. Cells were treated with indicated stearate concentrations and co-incubated with 50 µM oleate for 72 h; viability was measured via an MTT assay (n=6; *p \u0026lt; 0.05, **p \u0026lt; 0.01, Mann–Whitney test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee.\u003c/strong\u003eCell viability in response to treatment with stearate and oleate under 1 μM of CAY10566. Cells were treated with indicated stearate concentrations and co-incubated with 50 µM oleate for 72 h; viability was assessed after 72 h via an MTT assay (n=6; *p \u0026lt; 0.05, Mann–Whitney test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef, g.\u003c/strong\u003eIC50 values of stearate with 1 μM CAY10566 and DMSO control using OVCAR5 (f) and OVCAR8 (g) cells (n=6; **p \u0026lt; 0.01, Mann–Whitney test).\u003c/p\u003e","description":"","filename":"EMMfigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4198546/v1/6506253a2009cf86fe473b7c.png"},{"id":55005363,"identity":"f4e7543a-9783-4eed-8757-8937301eeebc","added_by":"auto","created_at":"2024-04-19 18:48:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1506896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStearate induces cytotoxicity via ER stress and CHOP activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e RNA-Seq in OVCAR5 cells subjected to various treatments. Cells were treated and cultured for 24 h before RNA-seq. Principal component analysis revealed distinct gene expression profiles without treatment-based separation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Top 10 functionally enriched terms. Biological processes induced by stearate compared with DMSO are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec, d.\u003c/strong\u003e Representative western blot analysis of proteins involved in the unfolded protein response (UPR), apoptosis, and DNA damage in OVCAR5 and OVCAR8 cells treated with stearate and oleate at the indicated concentrations. GAPDH was used as an internal control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee, f.\u003c/strong\u003e Representative western blot analysis of protein expression following knockdown of CHOP (shCHOP) in OVCAR5 and OVCAR8 cells. a-tubulin was used as an internal control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg, h.\u003c/strong\u003e IC50 values of stearate in shCHOP-transfected OVCAR5 and OVCAR8 cells (n=6; *p \u0026lt; 0.05, **p \u0026lt; 0.01, Mann–Whitney test).\u003c/p\u003e","description":"","filename":"EMMfigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4198546/v1/0226e3330631a0c513d0e742.png"},{"id":55006019,"identity":"330e8372-1e30-42b5-8b88-79c44b4e260a","added_by":"auto","created_at":"2024-04-19 18:56:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2023243,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of unsaturation along with dietary supplementation of stearate hinders tumor growth, which is reversed by oleate supply.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, b.\u003c/strong\u003eTumor growth in mice following subcutaneous injection of control (shCtr) (a) or SCD knockdown (shSCD) (b) OVCAR5 cells. Mice were divided into three dietary groups: normal-fat diet (NFD), O-HFD, and S-HFD from 3 days before injection until the end of the study (n=6). Data are presented as the mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ns stands for not significant, Mann–Whitney test applied between shCtr samples and Wilcoxon matched-pairs signed rank test between shCtr and shSCD-1 pairs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003eWeight of the tumors at the end of the experimental period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed–f.\u003c/strong\u003e Result validation using OVCAR8 cells (n=6). Data are presented as the mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ns stands for not significant, Mann–Whitney test applied between shCtr samples and Wilcoxon matched-pairs signed rank test between shCtr and shSCD-1 pairs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg.\u003c/strong\u003eRepresentative immunohistochemical images of tumor tissues derived from shCtr-OVCAR5 cells or shSCD-1-OVCAR5 cells depicting the expression of cleaved caspase-3, γH2AX, and CHOP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh–j.\u003c/strong\u003e Quantitative analysis of cleaved caspase-3, γH2AX, and CHOP expression in tissue. (n=30; ***p \u0026lt; 0.001, ns: no significance, Mann–Whitney test).\u003c/p\u003e","description":"","filename":"EMMfigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4198546/v1/b8e3e0a23b7a8f8d37dae714.png"},{"id":55005365,"identity":"0bbae337-16b7-4fa2-aa12-360bde8baef4","added_by":"auto","created_at":"2024-04-19 18:48:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3335623,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupply of stearate along with inhibition of unsaturation shows significant anti-proliferative effects in ovarian cancer patient-derived xenograft (PDX) models.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eLongitudinal assessment of serum CA-125 levels during therapeutic intervention in a patient with high-grade serous ovarian carcinoma \u0026nbsp;(source of PDX82).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Magnetic resonance imaging (MRI) of a 38-year-old female (source of PDX82 ). Sagittal T2-weighted MRI highlights the tumor mass; the white arrow indicates the tumor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003e Diagnostic laparoscopy depicts frozen pelvis phenomenon due to significant tumor occupation in the pelvic cavity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed.\u003c/strong\u003e PDX82 proliferation in a mouse model under various nutritional and CAY10566 treatment conditions. (n=4; *p \u0026lt; 0.05, Mann–Whitney test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee.\u003c/strong\u003e End-point tumor mass in mice harboring PDX82 (n=4; *p \u0026lt; 0.05, **p \u0026lt; 0.01, Mann–Whitney test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef.\u003c/strong\u003e PDX82 tumor specimens. Representative images from each condition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg.\u003c/strong\u003e Longitudinal assessment of serum CA-125 levels during therapeutic intervention in a patient with high-grade serous ovarian carcinoma (source of PDX72).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh.\u003c/strong\u003e MRI of a platinum-resistant 43-year-old female (source of PDX72): coronal T2-weighted MRI highlights the tumor mass; white arrow indicates the tumor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei.\u003c/strong\u003e Intraoperative abdominal imaging: captured during hepatic metastatic tumor resections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej.\u003c/strong\u003e PDX72 tumor proliferation in mouse model under various nutritional and CAY10566 treatment conditions (n=6; *p \u0026lt; 0.05, **p \u0026lt; 0.01, Mann–Whitney test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ek.\u003c/strong\u003e End-point tumor mass in PDX72 model (n=6; **p \u0026lt; 0.01, Mann–Whitney test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003el.\u003c/strong\u003e PDX72 tumor specimens: images from each experimental group .\u003c/p\u003e","description":"","filename":"EMMfigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4198546/v1/4bba06510da20bdca4b3da8e.png"},{"id":70328513,"identity":"9ac67d33-6cc2-489e-866d-8c2a80c5ed2b","added_by":"auto","created_at":"2024-12-02 08:05:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14259886,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4198546/v1/a81fe1c7-8d73-45b3-80ac-16aac426c6fc.pdf"},{"id":55005366,"identity":"88f43c56-8cd6-4e19-ab0f-130570a5495a","added_by":"auto","created_at":"2024-04-19 18:48:49","extension":"docx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":33313,"visible":true,"origin":"","legend":"Supplementary text","description":"","filename":"Supplementarytext.docx","url":"https://assets-eu.researchsquare.com/files/rs-4198546/v1/89d0cf9243e1ba46bce0425b.docx"},{"id":55005370,"identity":"c03cae80-7427-457e-8b89-59300b689a77","added_by":"auto","created_at":"2024-04-19 18:48:55","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":107432357,"visible":true,"origin":"","legend":"Supplementary Information.","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4198546/v1/1bdec96f989f83fe3557a60b.docx"}],"financialInterests":"There is a conflict of interest\nEijiro Nakamura has received research funding from Sumitomo Pharma CO., Ltd. The other authors declare that they have no competing interests.","formattedTitle":"Stearate-rich diet and oleate restriction directly inhibit tumor growth via the unfolded protein response","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA notable correlation exists between various types of cancers and obesity, which is characterized by an excessive accumulation of body fat. Obesity elevates the risk of carcinogenesis\u003csup\u003e1\u003c/sup\u003e. The consumption of a high-fat diet (HFD) augments the malignant potential of cancer cells\u003csup\u003e2\u003c/sup\u003e. Therefore, ingesting an excessive amount of dietary fat is implicated in escalating the risk of cancer development, exacerbating the malignancy of cancer in a tumor-bearing state, and potentially effectuating adverse clinical outcomes\u003csup\u003e3, 4\u003c/sup\u003e. However, fatty acids are not universally detrimental, and their effects on cancer cells vary depending on the type of fatty acid\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn biological systems, most fatty acids comprise 16 or more carbon atoms and are classified as long-chain fatty acids\u003csup\u003e6, 7\u003c/sup\u003e. These long-chain fatty acids can be further categorized into saturated fatty acids (SFAs), which possess only single bonds between carbon atoms, and unsaturated fatty acids (UFAs), which are characterized by the presence of double bonds. UFAs may enhance cancer survival, including stemness and ferroptosis resistance, particularly in renal and ovarian cancers\u003csup\u003e8, 9, 10\u003c/sup\u003e, suggesting a potential association between UFAs and adverse clinical outcomes. Conversely, SFAs exert cytotoxic effects on normal cells\u0026mdash;especially hepatocytes, endothelial cells, adipocytes, and pancreatic β cells\u0026mdash;commonly referred to as lipotoxicity\u003csup\u003e11, 12, 13\u003c/sup\u003e. The potential anti-proliferative effects of SFAs on cancer cells has been documented recently\u003csup\u003e14, 15, 16\u003c/sup\u003e. Caloric restriction markedly elevates the proportion of SFAs in the fatty acid composition of biological systems, which may in turn promote tumor suppression\u003csup\u003e17\u003c/sup\u003e. Inhibiting the activity of stearoyl-CoA desaturase (SCD), which catalyzes the conversion of SFAs to UFAs, induces an increase in SFA levels and subsequently hinders glioblastoma cell proliferation\u003csup\u003e18\u003c/sup\u003e. An SCD inhibition-mediated elevation in the SFA/UFA ratio can enhance antitumor effects in ovarian cancer\u003csup\u003e16\u003c/sup\u003e. Nevertheless, comprehensive studies examining the in vivo effects of SFAs and the subtle differences between them are scarce.\u003c/p\u003e \u003cp\u003eMost reports investigating the role of SFAs use palmitate for analysis. Palmitate, comprising 16 carbon atoms, is the most abundant SFA in biological systems, and stearate, comprising 18 carbon atoms, is also quite abundant\u003csup\u003e6\u003c/sup\u003e. They are structurally similar, differing solely by a two-carbon atom variation in chain length. Therefore, it has been thought that there is little difference in the effects of palmitate and stearate on cells. However, the two SFA types can have different effects on cancer cells\u003csup\u003e19\u003c/sup\u003e, but few studies have comprehensively explored their cellular effects; hence, the effects of stearate on cancer cells are largely unknown. Moreover, manipulation of dietary long-chain fatty acid composition and its in vivo effects remain to be evaluated.\u003c/p\u003e \u003cp\u003eTherefore, here, we investigated the impact of palmitate, stearate, and oleate on cancer cells and whether dietary changes would have a sufficient clinical impact. To this end, in addition to a usual HFD, a specialized HFD rich in stearate (S-HFD) was employed to study the differential effects of dietary stearate and oleate on cancer in an in vivo setting.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Approval\u003c/h2\u003e \u003cp\u003e The Ethics Committee of the Graduate School and Faculty of Medicine at Kyoto University approved this study, assigning reference numbers G531 and G288, ensuring compliance with the Declaration of Helsinki's principles. The university's Animal Research Committee authorized the animal experiments conducted in this research.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eThe National Institutes of Health's Dr. Melinda Hollingshead provided the human ovarian cancer cell line OVCAR8. Dr. Iwakoshi, Dr. Masashi Kanai, Dr. Shigeo Takaishi, and Dr. Susan K. Murphy donated the human ovarian cancer cell lines ES-2, OVCA5, SKOV3, OVCAR3, the human colon cancer cell lines DLD1 and LoVo, and the human epithelial cell line HOSE\u003csup\u003e20\u003c/sup\u003e, respectively. The American Type Culture Collection (Manassas, VA, USA) supplied the human mammary cancer cell lines MCF-7, MDA-MB-453, MDA-MB-231, Hs578t, the human mammary epithelial cell line MCF10A, the human lung cancer cell lines NCI-H460, NCI-H1299, NCI-H1650, and the human colon cancer cell lines HCT116, HT29, Caco2. The human lung cancer cell line A549 was purchased from the RIKEN BRC cell bank (Tsukuba, Japan). All cell lines, except MCF10A, were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin-streptomycin. MCF10A cells were grown in DMEM/F12 supplemented with 5% FBS, 0.02% Epidermal Growth Factor (EGF), 0.05% insulin, 0.5 \u0026micro;g/mL hydrocortisone, and 1% penicillin-streptomycin.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Models\u003c/h2\u003e \u003cp\u003eBALB/cAJcl-nu/nu mice, aged 4 and 6 weeks, were obtained from CLEA Japan. Five-week-old nonobese diabetic/Shi-scid IL-2RγKO Jic (NOG) mice were acquired from In-Vivo Science Inc. These animals were kept in specific pathogen-free conditions. Initially, the mice were provided a standard solid diet, formula F-2, with 12.0% fat by caloric content, and had unrestricted access to water. Later, as part of the study design, they were assigned to specific dietary regimens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Mouse Xenograft Models Utilizing Human Ovarian Cancer Cell Lines\u003c/h2\u003e \u003cp\u003eTo study the effect of dietary conditions and therapeutic interventions on tumor growth, mice were grouped into experimental categories. Xenografts were established using human ovarian cancer cell lines and genetically modified cells, and these were treated with either a vehicle or CAY10566. Before transplantation, mice were acclimatized to specific diets - a sterile regular chow diet (NFD) with 12.0% fat, a high-fat diet with 60% fat (S-HFD), or another high-fat diet with 56.7% fat (O-HFD) for three days, and these diets were maintained throughout the study. Tumor growth was regularly monitored by measuring tumor dimensions, and humane treatment protocols, including euthanasia criteria to prevent excessive tumor growth or ulceration, were strictly followed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMouse Xenograft Models Harboring PDXs\u003c/h2\u003e \u003cp\u003eOvarian cancer patient surgical specimens were obtained with informed consent at Kyoto University Hospital and used to create primary xenograft tumors in NOD SCID mice using Matrigel Matrix Basement Membrane for transplantation. Following tumor establishment, mice were divided into four treatment groups - NFD\u0026thinsp;+\u0026thinsp;vehicle, NFD\u0026thinsp;+\u0026thinsp;CAY10566, S-HFD\u0026thinsp;+\u0026thinsp;CAY10566, and O-HFD\u0026thinsp;+\u0026thinsp;CAY10566 - to assess the effects of dietary conditions and CAY10566 on tumor growth. Tumor volume was periodically measured using methods similar to those employed in ovarian cancer cell line xenograft models, to evaluate treatment outcomes.\u003c/p\u003e \u003cp\u003eDetail descriptions of the in vivo and in vitro analyses, mouse work protocols, preparation of reagents and samples, flow cytometry, Enzyme-Linked Immunosorbent Assay (ELISA), RNA sequencing, Western immunoblotting, Immunohistochemistry (IHC), and Liquid Chromatography-Mass Spectrometry (LC-MS) are described in supplementary material and method.\u003c/p\u003e \u003cp\u003eThe comprehensive list of reagents and antibodies used for Western immunoblotting or IHC, including the corresponding dilutions, primers, short hairpin RNA (shRNA) sequences, and software, along with their sources and research resource identifier numbers, are detailed in the Supplementary Data.\u003c/p\u003e \u003cp\u003eAdditionally, a detailed composition of the dietary components used in this study are listed in the Supplementary Table.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eAt least three independent in vitro experiments and a minimum of two cell lines for in vivo experiments were utilized. Mice in in vivo studies were randomly assigned to experimental groups. Sample sizes were determined to ensure experiment reproducibility, adhering to the principles of replacement, reduction, and refinement in animal ethics. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Error of the Mean (SEM). The Mann\u0026ndash;Whitney U test was employed for in vitro proliferation assay data analysis, ELISA group comparisons using in vivo samples, and for assessing tumor volume, weight, and IHC outcomes. The Wilcoxon matched-pairs signed-rank test was used for IC50 analysis, apoptosis assays, and comparisons of in vivo tumor volume and weight between shCtr and shSCD tumors. An unpaired t-test was applied for LC/MS analysis results. All statistical analyses were conducted using Prism 10.0.2 software, setting significance levels at *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, with 'ns' indicating no significance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData availability:\u003c/h2\u003e \u003cp\u003eThe RNA-seq data of this study have been deposited in the Gene Expression Omnibus (GEO) under accession no. GSE248408.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStearate Inhibits Growth Across Multiple Cancer Cell Lines\u003c/h2\u003e \u003cp\u003eFirst, we evaluated the impact of palmitate and stearate on cellular function. Multiple human cancer cell lines (ovarian: OVCAR5, ES-2, SKOV3, OVCAR3, and OVCAR8; lung: H460, A549, H1650, and H1299; breast: Hs578T, MDA-MB0231, MDA-MB-453, and MCF7; and colorectal: Lovo, HCT116, HT290, DLD-1, and Caco-2) were cultured in palmitate- and stearate-supplemented media for 72 h, and their impact on cell proliferation was assessed. Overall, stearate inhibited cell growth to a greater extent than palmitate across all cell lines (p\u0026thinsp;=\u0026thinsp;0.000252, Wilcoxon matched-pairs test). Furthermore, a k-means clustering analysis revealed that the cells could be segregated into three distinct groups based on their proliferative responses to the fatty acids: stearate\u0026thinsp;\u0026asymp;\u0026thinsp;palmitate, stearate\u0026thinsp;\u0026gt;\u0026thinsp;palmitate, and a group where neither fatty acid influenced cellular proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe ovarian cancer cell lines were exclusively categorized under the stearate\u0026thinsp;\u0026gt;\u0026thinsp;palmitate group, suggesting that a stronger inhibitory effect on ovarian cancer cell growth was seen with stearate than with palmitate. Additionally, similar experiments were conducted using the human mammary epithelial cell line MCF10A and the human ovarian surface epithelial cell line HOSE\u003csup\u003e20\u003c/sup\u003e. Despite both being immortalized normal cell lines, the sensitivity to stearate varied significantly between these two cell lines. In HOSE cells, the addition of stearate had a more pronounced impact on cell proliferation than that in MCF10A cells (p\u0026thinsp;=\u0026thinsp;0.002165). To further investigate the potential anticancer effects of stearate, subsequent experiments primarily focused on the ovarian cancer cell lines classified into the stearate\u0026thinsp;\u0026gt;\u0026thinsp;palmitate group.\u003c/p\u003e \u003cp\u003eConversely, in culture media supplemented with monounsaturated fatty acids (MUFA), palmitoleate and oleate did not markedly inhibit ovarian cancer cell growth. In fact, in certain cases, cell growth was enhanced, suggesting the differential effects of SFAs and MUFAs on the cancer cells (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eTo identify which fatty acids significantly affect cell proliferation, we conducted experiments using long-chain fatty acids that are frequently encountered in dietary and cellular contexts\u003csup\u003e7, 21\u003c/sup\u003e. Ovarian cancer cell lines, namely OVCAR5, OVCAR8, SKOV3, ES-2, and OVCAR3, were treated with 50 \u0026micro;M each of palmitate, stearate, and oleate, and the cell viability was measured after 24, 48, and 72 h. Stearate markedly impeded cell growth in all lines from 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Dose-response curves for each long-chain fatty acid revealed substantially lower IC50 values of stearate, at 36.96\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22 \u0026micro;M for OVCAR5 and 31.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97 \u0026micro;M for OVCAR8, than those of palmitate, at 1469.75\u0026thinsp;\u0026plusmn;\u0026thinsp;74.61 \u0026micro;M and 74.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStearate Induces Apoptosis and DNA Damage in Ovarian Cancer Cells In Vitro and In Vivo\u003c/h2\u003e \u003cp\u003eNext, we investigated whether stearate induced apoptosis like palmitate, as reported previously\u003csup\u003e12, 14, 16\u003c/sup\u003e. A flow cytometry analysis of Annexin V-positive cells showed that stearate increased apoptosis of OVCAR5 cells dose-dependently (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). As palmitate-induced apoptosis is related to DNA damage\u003csup\u003e22, 23\u003c/sup\u003e, we examined whether stearate similarly affects OVCAR5 cells. A dose-dependent increase in γH2AX expression was observed following 24 h stearate treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The obtained findings were corroborated through comparative experiments utilizing OVCAR8 cells; flow cytometry and western blot assays were conducted to elucidate the influence of stearate on apoptosis and DNA damage, respectively (Supplementary Figs. S3a-c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo gain further insight into our findings, we conducted an in vivo study. We first used murine models to determine the impact of a HFD rich in oleate (O-HFD) on the growth of tumors derived from subcutaneously inoculated cancer cell lines (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec). Consistent with previous studies, tumor proliferation increased in mice harboring tumors derived from OVCAR5 cells that were fed an O-HFD (Supplementary Figs. S1d\u0026ndash;g)\u003csup\u003e2, 4, 24\u003c/sup\u003e. Given the absence of notable disparities in body weight or blood insulin levels\u003csup\u003e25\u003c/sup\u003e resulting from dietary variations, the findings prompted the hypothesis that the influence on tumor proliferation stemmed from the fatty acids themselves rather than alterations in the physiological condition of the mice. Initially, the fat in the O-HFD was mainly composed of oleate (64.3%) with a low stearate content (7.5%; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNext, we investigated the effects of a S-HFD with a significantly higher per-calorie stearate content (33.35%; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Importantly, the S-HFD group exhibited a significant reduction in tumor growth compared with the normal-fat diet (NFD) group (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). Flow cytometry and IHC (immunohistochemistry) results revealed a higher degree of tumor apoptosis in the S-HFD group than in the NFD group (Annexin V-positive rate: 26.6% vs. 30.27%, cleaved caspase-3-positive area: 0.56% vs. 1.0%; Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g, Supplementary Figs. S2c, d). To evaluate DNA damage, IHC was performed for γH2AX, and the results showed a significant increase in the proportion of γH2AX-positive cells in the S-HFD group compared with that in the NFD group (H score: 38.2 vs. 57.5; Supplementary Figs. S2c, e). These findings were validated using SKOV3 cells (Supplementary Figs. S2f\u0026ndash;h). Furthermore, no significant differences were observed in body weight and vital organs, including the liver and kidneys, between the S-HFD and NFD groups (Supplementary Figs. S2a, b). These data collectively demonstrate that stearate induces cytotoxicity, DNA damage, and apoptosis in ovarian cancer cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOleate Mitigates Stearate-Induced Cytotoxicity\u003c/h2\u003e \u003cp\u003eLong-chain fatty acids can be converted to other long-chain fatty acids in biological systems\u003csup\u003e26\u003c/sup\u003e. Therefore, we altered dietary long-chain fatty acid composition to generate S-HFD and evaluated whether these changes were reflected in the tumor tissue. The S-HFD group had significantly higher tumor stearate content compared to that in the NFD group (107.3 vs. 164.9 pmol/mg, p\u0026thinsp;=\u0026thinsp;0.012094). Notably, stearate levels were elevated even in the O-HFD group, almost matching those in the S-HFD group (O-HFD vs S-HFD: 164.9 vs. 165.7 pmol/mg, p\u0026thinsp;=\u0026thinsp;0.957080). However, oleate levels were higher in the O-HFD group than in the S-HFD group (O-HFD vs. S-HFD: 172.5 vs. 86.62 pmol/mg, p\u0026thinsp;=\u0026thinsp;0.007269; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). This led us to propose that oleate may mitigate the tumor-suppressive effects of stearate. Oleate ameliorates palmitate-induced ER stress and DNA damage\u003csup\u003e17, 23, 27, 28, 29\u003c/sup\u003e, and therefore, we next examined whether similar phenomena occurred in our study.\u003c/p\u003e \u003cp\u003eWe first investigated the effect of oleate on tumor apoptosis. Stearate-induced apoptosis was significantly reduced following 50 \u0026micro;M oleate addition to OVCAR5 and OVCAR8 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei, j, Supplementary Figs. S3d, e). Moreover, the addition of 50 \u0026micro;M oleate almost abrogated stearate-induced elevated γH2AX expression in OVCAR5 and OVCAR8 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek, Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ef), resulting in reduced cytotoxicity. We treated OVCAR5, OVCAR8, SKOV3, ES-2, and OVCAR3 cells with varying concentrations of stearate under 50 \u0026micro;M oleate treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el). The addition of 50 \u0026micro;M oleate significantly ameliorated the stearate-induced decrease in cell viability. We next explored the effects of oleate on stearate-induced cell death. Oleate rescued the cells incubated with 100 \u0026micro;M stearate from cell death in a concentration-dependent manner (Supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003ea). In OVCAR5 cells, the addition of 25 \u0026micro;M oleate rescued cell proliferation to levels almost comparable to those achieved with the addition of 100 \u0026micro;M oleate. Overall, oleate attenuates stearate-induced cytotoxic effects, including DNA damage and apoptosis, in ovarian cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of Unsaturation Increases Stearate Toxicity\u003c/h2\u003e \u003cp\u003eIn biological systems, stearate is converted to oleate by stearoyl-CoA desaturase 1\u003csup\u003e30\u003c/sup\u003e (SCD1; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). SCD1 overexpression has been documented in various cancers, including ovarian cancer\u003csup\u003e31, 32, 33\u003c/sup\u003e. Li et al.8 argued that the malignancy of high-grade serous ovarian cancer (HGSC) is significantly influenced by endogenous oleate produced by SCD1. Therefore, we explored the involvement of endogenous and exogenous oleate in cancer pathogenesis. To inhibit endogenous oleate synthesis, we transduced shRNA sequences targeting SCD1 (shSCD-1 and shSCD-2) or control shRNA (shCtr) into OVCAR-5 and OVCAR-8 cells (Supplementary Figs. S4b\u0026ndash;e).\u003c/p\u003e \u003cp\u003eThe inhibition of SCD1 led to a marked increase in cellular sensitivity to stearate (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c). Conversely, the addition of oleate substantially ameliorated stearate-induced cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, Supplementary Figs. S4f, g). Incubation with 1 \u0026micro;M CAY10566, an SCD inhibitor\u003csup\u003e34, 35\u003c/sup\u003e, did not inhibit cell proliferation; however, concentrations of stearate and oleate in OVCAR5 cells were significantly altered (Supplementary Figs. S5a\u0026ndash;c). Similar to SCD knockdown (SCD-KD), the addition of 1 \u0026micro;M CAY10566 increased cellular sensitivity to stearate (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g), while the growth-inhibitory effect was significantly mitigated by the addition of oleate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). These trends were consistent across other cell lines, including SKOV3, ES2, and OVCAR3 (Supplementary Figs. S5d\u0026ndash;f).\u003c/p\u003e \u003cp\u003eAlthough SCD activity inhibition, using CAY10566 or SCD expression knockdown, decreased cell viability by increasing stearate concentration and decreasing oleate concentration, exogenous oleate abrogated these detrimental effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStearate Induces Cytotoxicity via Endoplasmic Reticulum (ER) Stress and CHOP Activation\u003c/h2\u003e \u003cp\u003eNext, we sought to elucidate the mechanisms underlying stearate-mediated cytotoxicity. We treated OVCAR5 cells with (i) DMSO, (ii) CAY10566 1 \u0026micro;M, (iii) stearate 50 \u0026micro;M\u0026thinsp;+\u0026thinsp;DMSO, (iv) stearate 50 \u0026micro;M\u0026thinsp;+\u0026thinsp;CAY10566 1 \u0026micro;M, (v) oleate 50 \u0026micro;M\u0026thinsp;+\u0026thinsp;DMSO, and (vi) oleate 50 \u0026micro;M\u0026thinsp;+\u0026thinsp;CAY10566 1 \u0026micro;M and performed RNA sequencing analysis. Principal component analysis (PCA) revealed that the presence or absence of stearate strongly contributed to PC1, whereas the presence or absence of oleate influenced PC2. However, 1 \u0026micro;M CAY10566 had limited effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGene Ontology (GO) analysis identified 643 differentially expressed genes (DEGs), of which 401 were upregulated and 242 were downregulated between 50 \u0026micro;M stearate-treated and control OVCAR5 cells (false discovery rate [FDR]\u0026thinsp;\u0026lt;\u0026thinsp;0.05, minimum fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.25; Supplementary Figs. S6a, b). The top 10 significantly upregulated GO categories were enriched in pathways associated with the unfolded protein response (UPR) and ER stress in 50 \u0026micro;M stearate-treated OVCAR5 cells compared with control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb; FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eUPR signaling involves ATF6, IRE1α, and PERK pathways\u003csup\u003e36\u003c/sup\u003e. Our western blot analysis confirmed that stearate induced the concentration-dependent activation of UPR-related proteins, including ATF6, XBP-1 as a downstream transcription factor of IRE1α, and ATF4 as a downstream transcription factor of PERK. Moreover, the expression of pro-apoptotic transcription factor CHOP\u003csup\u003e37\u003c/sup\u003e and apoptotic markers cleaved caspase-3 and γH2AX was upregulated (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003eWe further examined whether the addition of oleate mitigated the activation of ER stress response pathways. Activation of ER stress response pathways was negated by the addition of 100 \u0026micro;M oleate to OVCAR5 and OVCAR8 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). Furthermore, the addition of 1 \u0026micro;M CAY10566 enhanced the stearate-dependent activation of UPR-related proteins, CHOP, cleaved caspase-3, and γH2AX; however, this activation of the UPR pathway was almost abrogated by exogenous oleate (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003eLong-term exposure to mild ER stress or short-term exposure to severe ER stress induces CHOP-mediated apoptosis\u003csup\u003e13, 38\u003c/sup\u003e. To explore whether stearate induced apoptosis via CHOP, we generated CHOP-knockdown OVCAR5 and OVCAR8 cell lines via lentiviral infection of CHOP shRNA (Supplementary Fig. S6c, d).\u003c/p\u003e \u003cp\u003eFollowing inhibition of CHOP expression, the expression of cleaved caspase-3 and γH2AX, which was increased in a concentration-dependent manner by stearate treatment, was significantly reduced (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f, Supplementary Figs. S6e, f). Moreover, CHOP knockdown significantly enhanced the resistance to stearate-induced cytotoxicity (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, h), indicating that stearate-induced cytotoxicity was mediated through ER stress and CHOP activation.\u003c/p\u003e \u003cp\u003eOverall, exogenous stearate activated ER stress response pathways, induced DNA damage, and inhibited the proliferation of ovarian cancer cells. Consistently, the addition of exogenous oleate attenuated the ER stress response pathway activated by stearate, reducing its toxicity in ovarian cancer.\u003c/p\u003e \u003cp\u003eAdditionally, we have confirmed that the sensitivity to stearate and palmitate varies among cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We investigated whether these differences were due to variations in ER stress response pathways. Regarding MCF10A cells (stearate-nonresponsive cells), we observed minimal CHOP induction by stearate, which differs significantly from the findings for HOSE and OVCAR5 cells (stearate-responsive cells) (Supplementary Figs. S7a, b). In the case of H1299 cells (stearate-nonresponsive cells), we found constant CHOP expression regardless of the addition of stearate, which was not decreased by oleate. These findings also significantly differed from those of HOSE and OVCAR5 (Supplementary Figs. S7a, b). We then investigated the responses of OVCAR5 and OVCAR8 cells to stearate and palmitate. Notably, palmitate induced CHOP expression in these cells, but to a lesser extent than stearate. Additionally, the activation of Cleaved Caspase3 by palmitate was less pronounced than that induced by stearate (Supplementary Figs. S7c, d). These findings highlight that the varying sensitivities to palmitate and stearate in different cell lines are primarily a result of their unique responses to the activation of the ER stress pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibition of Unsaturation Along with Dietary Supplementation of Stearate Hinders Tumor Growth, Which is Reversed by Oleate Supply\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo validate our results obtained so far in vivo, we fed mice an S-HFD, O-HFD, or NFD (Supplementary Figs. S8a\u0026ndash;c). In the S-HFD group subcutaneously injected with SCD1-knockdown (SCD1-KD) OVCAR5 cells, tumor growth was significantly inhibited compared with that in the NFD group (SCD1-KD \u0026amp; S-HFD vs. SCD1-KD \u0026amp; NFD; 0.125 g vs. 0.240 g, p\u0026thinsp;=\u0026thinsp;0.006494; Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c). Conversely, the O-HFD group displayed significantly greater tumor growth than did the S-HFD and NFD groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In experiments using sh-control cell lines, the S-HFD group exhibited stronger growth suppression than the NFD and O-HFD groups, although this trend was less pronounced than that observed in experiments using SCD1-KD cells (sh-control \u0026amp; S-HFD vs. sh-control \u0026amp; O-HFD; 0.2633 g vs. 0.4017 g, p\u0026thinsp;=\u0026thinsp;0.006494; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Furthermore, no significant differences in tumor growth were observed between sh-control and SCD1-KD cells in the O-HFD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the S-HFD group subcutaneously injected with SCD1-KD OVCAR8 cells, the greatest tumor growth suppression was noted, with a significant difference compared with that in the O-HFD group (SCD1-KD \u0026amp; S-HFD vs. SCD1-KD \u0026amp; O-HFD; 0.02667 g vs. 0.0733 g, p\u0026thinsp;=\u0026thinsp;0.019481; Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u0026ndash;f). The same trend was observed when animals were injected with the sh-control cell line; however, no significant differences were observed between the S-HFD and O-HFD groups (sh-control \u0026amp; S-HFD vs. sh-control \u0026amp; O-HFD: 0.0433 g vs. 0.0533 g, p\u0026thinsp;=\u0026thinsp;0.4848). Additionally, no significant differences were observed in tumor growth between mice injected with sh-control and SCD1-KD cells and fed on the O-HFD, as observed with OVCAR5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eNext, we examined whether the UPR pathway, DNA damage, or apoptosis were modulated in vivo. IHC of OVCAR5 cell-derived tumors revealed marked upregulation of CHOP expression in the S-HFD group and the most significant upregulation in SCD1-KD cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg\u0026ndash;j). Conversely, CHOP expression was almost abrogated in the O-HFD group, regardless of whether the sh-control or SCD1-KD cells were used. We also assessed γH2AX and cleaved caspase-3 expression and observed trends consistent with those of CHOP expression. Similar results were obtained using OVCAR8 cells (Supplementary Figs. S8d\u0026ndash;g).\u003c/p\u003e \u003cp\u003eWe conducted further experiments using CAY10566 (Supplementary Fig. S9a). In mice injected with OVCAR5 and OVCAR8 cells, the CAY10566-treated group exhibited the most significant tumor growth suppression when fed the S-HFD compared with the NFD and O-HFD groups (Supplementary Figs. S9b\u0026ndash;f, S10a\u0026ndash;c). Tumor growth in the vehicle-treated group, those fed on the S-HFD, was the lowest, but this trend was less pronounced than that in the CAY10566 group. Moreover, no significant differences were observed between the vehicle and CAY10566 groups when fed on the O-HFD. The expression levels of γH2AX and cleaved caspase-3 were most significantly upregulated in the CAY10566\u0026thinsp;+\u0026thinsp;S-HFD group, whereas almost no expression was observed in the O-HFD groups, irrespective of whether they were in the vehicle or CAY10566 group (Supplementary Fig. S9g\u0026ndash;j, S10d\u0026ndash;g). Assessments of stearate and oleate concentrations within tumor tissues demonstrated a stearate increment of 1.5- to 2-fold in the S-HFD-, O-HFD-, or CAY10566-administered group compared with that in the NFD\u0026thinsp;+\u0026thinsp;vehicle group. Despite the administration of CAY10566, O-HFD elevated oleate levels by approximately 1.5-fold, correlating with an actual proliferation enhancement in the tumors. Conversely, S-HFD in combination with CAY10566 administration resulted in a significant elevation of stearate to 185 pmol/mg while maintaining oleate levels at 50 pmol/mg, which was lower than that in the NFD-vehicle group, thus exhibiting a pronounced inhibitory effect on tumor growth (Supplementary Fig. S10h).\u003c/p\u003e \u003cp\u003eOverall, robust tumor-suppressive effects were achieved in vivo by increasing tumor stearate levels via S-HFD feeding, coupled with oleate inhibition mediated via SCD inhibition. Additionally, excessive intake of oleate through the O-HFD significantly diminished this effect.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSupply of Stearate along with Inhibition of Unsaturation Shows Significant Anti-proliferative Effects on Ovarian Cancer Patient-derived Xenograft (PDX) Models\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the applicability of our findings in the clinical setting, we next conducted experiments using PDXs. Conducting large-scale interventions to assess the effects of dietary changes is challenging; however, drug responses in PDXs have been suggested to correlate with patient clinical outcomes\u003csup\u003e39\u003c/sup\u003e. Therefore, we utilized two PDXs from distinct clinical backgrounds (PDX72 and PDX82; Supplementary Texts) that were established from patients treated at our institution. PDX82 was sourced from a 38-year-old female patient with stage IIIC HGSC harboring a \u003cem\u003eBRCA2\u003c/em\u003e mutation. This patient was sensitive to platinum-based chemotherapy and maintained no long-term evidence of disease under poly (ADP-ribose) polymerase inhibitor (PARPi)\u003csup\u003e40, 41\u003c/sup\u003e treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u0026ndash;c, Supplementary Figs. S11a\u0026ndash;c). In PDX82 experiments, while treatment with CAY10566 alone showed limited effectiveness, tumor growth was significantly inhibited when these mice were fed S-HFD (NFD-CAY10566: 2685 mg vs. S-HFD-CAY10566: 970 mg, p\u0026thinsp;=\u0026thinsp;0.0285; Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed\u0026ndash;f). However, feeding mice on O-HFD led to significantly larger tumor sizes compared to the S-HFD-fed group, even with CAY10566 administration (S-HFD-CAY10566: 970 mg vs. O-HFD-CAY10566: 970 mg, p\u0026thinsp;=\u0026thinsp;0.0285).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnother PDX, PDX72, was sourced from a 43-year-old female who developed platinum-resistant recurrent HGSC. The tumor was collected during secondary debulking surgery (SDS). Despite surgery, the patient relapsed quickly, and neither platinum-based chemotherapy nor anti-VEGF antibodies\u003csup\u003e42\u003c/sup\u003e were effective, resulting in a poor prognosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg\u0026ndash;i, Supplementary Figs. S12a\u0026ndash;c). Studies using PDX72 revealed that CAY10566 administration alone inhibited tumor growth, and this effect was further enhanced by feeding the S-HFD to mice (NFD-vehicle: 678.3 mg vs. NFD-CAY10566: 245.0 mg vs. S-HFD-CAY10566: 150 mg, p\u0026thinsp;=\u0026thinsp;0.0021, 0.0043, respectively; Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej\u0026ndash;l). However, despite CAY10566 treatment, mice fed on the O-HFD developed significantly larger tumors than those fed on the S-HFD (S-HFD-CAY10566: 150 mg vs. O-HFD-CAY10566: 798.3 mg, p\u0026thinsp;=\u0026thinsp;0.0021).\u003c/p\u003e \u003cp\u003eIHC analysis results of these two PDX models regarding UPR, DNA damage, and apoptosis markers were consistent; the highest expression levels of CHOP, γH2AX, and cleaved caspase-3 were observed in the CAY10566\u0026thinsp;+\u0026thinsp;S-HFD group, whereas these markers were significantly inhibited in the O-HFD group (Supplementary Figs. S11d\u0026ndash;g, S12d\u0026ndash;g).\u003c/p\u003e \u003cp\u003eOverall, combined administration of CAY10566 and S-HFD significantly suppressed tumor growth in two distinct PDX models with different clinical backgrounds and outcomes. Furthermore, even in cases sensitive to CAY10566 alone, tumor proliferation was enhanced when the O-HFD was consumed, suggesting that the antitumor effect of CAY10566 can be compromised by an O-HFD.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we extensively explored the varied effects of long-chain fatty acids on cancer cell proliferation. Studies using multiple organ-derived cancer cells have revealed that SFAs, known for their lipotoxicity in normal cells\u003csup\u003e43\u003c/sup\u003e\u0026mdash;specifically palmitate and stearate\u0026mdash; impart inhibitory effects on the growth of cancer cell lines. Notably, stearate exhibited an anti-proliferative effect on broader range of cancer cells in comparison to palmitate. Specifically, there were several cell lines in where palmitate showed limited efficacy, whereas stearate was more potent, with all six ovarian cancer cell lines included in this study falling into this category. Furthermore, the normal human ovarian surface epithelial cell line (HOSE) was also strongly affected by stearate, a result that differed significantly from that of the normal human mammary epithelial cell line (MCF10A). Given the variable influence of long-chain fatty acids across different tissue types\u003csup\u003e5\u003c/sup\u003e; this finding suggests that ovarian tissues might possess heightened susceptibility to the cytotoxic effects of stearate, and, this sensitivity could potentially be extended to ovarian cancers, although the detailed mechanisms remain unclarified.\u003c/p\u003e \u003cp\u003eOur findings demonstrated that stearate induced DNA damage and apoptosis through dose-dependent activation of the UPR pathway. This phenomenon was directly mitigated by oleate, and impeding the conversion of stearate to oleate amplified the cytotoxic effects of stearate. Wieder et al.\u003csup\u003e5\u003c/sup\u003e segregated long-chain fatty acid-elicited cellular damage into two major pathways, UPR and ROS generation, and showed that the detrimental effects associated with UPR could be rescued by oleate treatment. Our results corroborate the aforementioned findings. Moreover, our findings established that S-HFD and SCD1-rich diets exert the most potent anti-proliferative effects and reduced oleate levels in mice harboring xenografts derived from various cancer cell lines. These findings provide evidence that dietary modifications can induce the accumulation of excess stearate and limit oleate contents in tumors, inhibiting tumor growth. To our knowledge, this is the first study to specify the therapeutic role of excess dietary intake of stearate and limited intake of oleate in cancers. In vivo studies on ovarian cancer with palmitate treatment did not portray the marked changes observed with stearate treatment\u003csup\u003e16\u003c/sup\u003e. As previously stated, most ovarian cancer cell lines were more sensitive to stearate than to palmitate. Additionally, the UPR pathway response was more strongly induced by stearate compared to palmitate. This explains the difference between the results of our study and previous in vivo studies16 Our experiments suggest that stearate should produce stronger antitumor effects compared to palmitate, although they have similar structures.\u003c/p\u003e \u003cp\u003eHGSC is the predominant histological subtype of ovarian cancer\u003csup\u003e44\u003c/sup\u003e and is often diagnosed at advanced stages accompanied by peritoneal dissemination\u003csup\u003e45, 46\u003c/sup\u003e. Despite the promising outcomes achieved through the administration of targeted therapies against aberrant DNA repair mechanisms, including PARPis\u003csup\u003e40, 41\u003c/sup\u003e, HGSC eventually becomes therapy-resistant and worsens prognosis in numerous patients\u003csup\u003e47\u003c/sup\u003e. Notably, the development of drug resistance in ovarian cancers also induces limited genetic alterations\u003csup\u003e48\u003c/sup\u003e, requiring the implementation of alternative treatment strategies. Our results hold significant clinical potential, as similar anticancer effects of dietary modulations were observed using mice harboring PDXs derived from drug-resistant tumors. Wieder et al.\u003csup\u003e5\u003c/sup\u003e proposed that the UPR is a promising therapeutic target for various states of HGSC, and targeting UPR along with a dietary intervention to steer stearate accumulation and limit oleate content in tumors may offer a novel therapeutic approach for refractory HGSC.\u003c/p\u003e \u003cp\u003eThere are still some limitations to this study. The detail mechanism of why stearate and palmitate have different effects remains unclear. Therefore, it is difficult to identify a population for which the activation of UPR with stearate is more effective. The effects on the immune system have not yet been investigated, and the details of their effects on normal organs are still unknown. Additionally, the dietary conditions employed here may lack direct applicability in clinical settings. Nonetheless, the implications of our study are noteworthy. Although dietary interventions are garnering increased attention in clinical research on cancer treatment\u003csup\u003e49\u003c/sup\u003e, it is generally regarded as a complementary therapy. Our findings suggest that dietary modifications can exert direct antitumor effects, broadening the scope for dietary interventions in cancer treatment. Based on this study, we would like to build a more solid evidence-based dietary intervention for cancer treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eGrants:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the JST SPRING, Grant Number JPMJSP2110, and the MEXT/JSPS KAKENHI, Grant Numbers JP20K18166 and JP23K15834.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Editage (www.editage.com) for English language editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe are also grateful to Junko Satoh and Atsuko Nakao for their assistance with the measurement of fatty acids using LC/MS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEijiro Nakamura has received research funding from Sumitomo Pharma CO., Ltd.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe other authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eThis work was supported by the JST SPRING, Grant Number JPMJSP2110 (to JO), and the MEXT/JSPS KAKENHI, Grant Numbers JP20K18166 and JP23K15834 (to YK).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnd Notes\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.O. designed and performed the experiments and wrote the manuscript. K.Y., M.T., R.M., and J.H. all contributed to designing the experiments and editing the manuscript. Y.H., S.I., J.S., A.N., and E.N. were involved in performing the experiments. Y.N. and K.K. provided samples and assisted in editing the manuscript. M.M. designed the experiments, provided funding, and edited the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e accompanies the manuscript on the Experimental \u0026amp; Molecular Medicine\u0026rsquo;s website (http://www.nature.com/emm/).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Lauby-Secretan B, Scoccianti C, Loomis D, Grosse Y, Bianchini F, Straif K. Body Fatness and Cancer\u0026ndash;Viewpoint of the IARC Working Group. N Engl J Med \u003cb\u003e375\u003c/b\u003e, 794\u0026ndash;798 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Yang J, et al. High-Fat Diet Promotes Colorectal Tumorigenesis Through Modulating Gut Microbiota and Metabolites. Gastroenterology \u003cb\u003e162\u003c/b\u003e, 135\u0026ndash;149.e132 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Beyaz S, et al. High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. Nature \u003cb\u003e531\u003c/b\u003e, 53\u0026ndash;58 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Labb\u0026eacute; DP, et al. High-fat diet fuels prostate cancer progression by rewiring the metabolome and amplifying the MYC program. Nature Communications \u003cb\u003e10\u003c/b\u003e, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wieder N, et al. FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity. Cell Metab \u003cb\u003e35\u003c/b\u003e, 887\u0026ndash;905.e811 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Fraser DA, Thoen J, Rustan AC, F\u0026oslash;rre O, Kjeldsen-Kragh J. Changes in plasma free fatty acid concentrations in rheumatoid arthritis patients during fasting and their effects upon T-lymphocyte proliferation. Rheumatology (Oxford) \u003cb\u003e38\u003c/b\u003e, 948\u0026ndash;952 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Puri P, et al. A lipidomic analysis of nonalcoholic fatty liver disease. Hepatology \u003cb\u003e46\u003c/b\u003e, 1081\u0026ndash;1090 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Li J, et al. Lipid Desaturation Is a Metabolic Marker and Therapeutic Target of Ovarian Cancer Stem Cells. Cell Stem Cell \u003cb\u003e20\u003c/b\u003e, 303\u0026ndash;314 e305 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Magtanong L, et al. Exogenous Monounsaturated Fatty Acids Promote a Ferroptosis-Resistant Cell State. Cell Chem Biol \u003cb\u003e26\u003c/b\u003e, 420\u0026ndash;432 e429 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Yang Y, et al. Palmitoylation stabilizes PD-L1 to promote breast tumor growth. Cell Res \u003cb\u003e29\u003c/b\u003e, 83\u0026ndash;86 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Harvey KA, et al. Oleic acid inhibits stearic acid-induced inhibition of cell growth and pro-inflammatory responses in human aortic endothelial cells. J Lipid Res \u003cb\u003e51\u003c/b\u003e, 3470\u0026ndash;3480 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Nemcova-Furstova V, Balusikova K, Halada P, Pavlikova N, Sramek J, Kovar J. Stearate-Induced Apoptosis in Human Pancreatic beta-Cells is Associated with Changes in Membrane Protein Expression and These Changes are Inhibited by Oleate. Proteomics Clin Appl \u003cb\u003e13\u003c/b\u003e, e1800104 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Cao J, et al. Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway. Mol Cell Biochem \u003cb\u003e364\u003c/b\u003e, 115\u0026ndash;129 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Evans LM, Cowey SL, Siegal GP, Hardy RW. Stearate Preferentially Induces Apoptosis in Human Breast Cancer Cells. Nutr Cancer \u003cb\u003e61\u003c/b\u003e, 746\u0026ndash;753 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Lin L, et al. Functional lipidomics: Palmitic acid impairs hepatocellular carcinoma development by modulating membrane fluidity and glucose metabolism. Hepatology \u003cb\u003e66\u003c/b\u003e, 432\u0026ndash;448 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Zhao G, et al. Ovarian cancer cell fate regulation by the dynamics between saturated and unsaturated fatty acids. Proc Natl Acad Sci U S A \u003cb\u003e119\u003c/b\u003e, e2203480119 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Lien EC, et al. Low glycaemic diets alter lipid metabolism to influence tumour growth. Nature \u003cb\u003e599\u003c/b\u003e, 302\u0026ndash;307 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Eyme KM, et al. Targeting de novo lipid synthesis induces lipotoxicity and impairs DNA damage repair in glioblastoma mouse models. Sci Transl Med \u003cb\u003e15\u003c/b\u003e, eabq6288 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Zhang L, Lv J, Chen C, Wang X. Roles of acyl-CoA synthetase long-chain family member 5 and colony stimulating factor 2 in inhibition of palmitic or stearic acids in lung cancer cell proliferation and metabolism. Cell Biol Toxicol \u003cb\u003e37\u003c/b\u003e, 15\u0026ndash;34 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Nagayoshi Y, et al. Characterization of 17beta-hydroxysteroid dehydrogenase type 4 in human ovarian surface epithelial cells. Mol Hum Reprod \u003cb\u003e11\u003c/b\u003e, 615\u0026ndash;621 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e de Almeida IT, Cortez-Pinto H, Fidalgo G, Rodrigues D, Camilo ME. Plasma total and free fatty acids composition in human non-alcoholic steatohepatitis. Clin Nutr \u003cb\u003e21\u003c/b\u003e, 219\u0026ndash;223 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Yuzefovych L, Wilson G, Rachek L. Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress. Am J Physiol Endocrinol Metab \u003cb\u003e299\u003c/b\u003e, E1096-1105 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Ishaq A, Tchkonia T, Kirkland JL, Siervo M, Saretzki G. Palmitate induces DNA damage and senescence in human adipocytes in vitro that can be alleviated by oleic acid but not inorganic nitrate. Exp Gerontol \u003cb\u003e163\u003c/b\u003e, 111798 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Hayashi T, et al. High-Fat Diet-Induced Inflammation Accelerates Prostate Cancer Growth via IL6 Signaling. Clin Cancer Res \u003cb\u003e24\u003c/b\u003e, 4309\u0026ndash;4318 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Straus DS. Growth-stimulatory actions of insulin in vitro and in vivo. Endocr Rev \u003cb\u003e5\u003c/b\u003e, 356\u0026ndash;369 (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Luo D, et al. Covalent inhibition of endoplasmic reticulum chaperone GRP78 disconnects the transduction of ER stress signals to inflammation and lipid accumulation in diet-induced obese mice. eLife \u003cb\u003e11\u003c/b\u003e, e72182 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Oshima M, et al. Stearoyl CoA desaturase is a gatekeeper that protects human beta cells against lipotoxicity and maintains their identity. Diabetologia \u003cb\u003e63\u003c/b\u003e, 395\u0026ndash;409 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Salvado L, et al. Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism. Diabetologia \u003cb\u003e56\u003c/b\u003e, 1372\u0026ndash;1382 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Zeng X, et al. Oleic acid ameliorates palmitic acid induced hepatocellular lipotoxicity by inhibition of ER stress and pyroptosis. Nutrition \u0026amp; Metabolism \u003cb\u003e17\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Ntambi JM. The regulation of stearoyl-CoA desaturase (SCD). Prog Lipid Res \u003cb\u003e34\u003c/b\u003e, 139\u0026ndash;150 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Qiang L, et al. Hepatic SirT1-dependent gain of function of stearoyl-CoA desaturase-1 conveys dysmetabolic and tumor progression functions. Cell Rep \u003cb\u003e11\u003c/b\u003e, 1797\u0026ndash;1808 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sen U, Coleman C, Sen T. Stearoyl coenzyme A desaturase-1: multitasker in cancer, metabolism, and ferroptosis. Trends in Cancer \u003cb\u003e9\u003c/b\u003e, 480\u0026ndash;489 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Vivas-Garc\u0026iacute;a Y, et al. Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity. Mol Cell \u003cb\u003e77\u003c/b\u003e, 120\u0026ndash;137. e129 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Kamphorst JJ, et al. Hypoxic and Ras-transformed cells support growth by scavenging unsaturated fatty acids from lysophospholipids. Proceedings of the National Academy of Sciences \u003cb\u003e110\u003c/b\u003e, 8882\u0026ndash;8887 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Liu G, et al. Discovery of potent, selective, orally bioavailable stearoyl-CoA desaturase 1 inhibitors. J Med Chem \u003cb\u003e50\u003c/b\u003e, 3086\u0026ndash;3100 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Hetz C, Papa FR. The Unfolded Protein Response and Cell Fate Control. Mol Cell \u003cb\u003e69\u003c/b\u003e, 169\u0026ndash;181 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Xu Z, Bu Y, Chitnis N, Koumenis C, Fuchs SY, Diehl JA. miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis. Nat Commun \u003cb\u003e7\u003c/b\u003e, 11422 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Kim I, Xu W, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nature Reviews Drug Discovery \u003cb\u003e7\u003c/b\u003e, 1013\u0026ndash;1030 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Izumchenko E, et al. Patient-derived xenografts effectively capture responses to oncology therapy in a heterogeneous cohort of patients with solid tumors. Ann Oncol \u003cb\u003e28\u003c/b\u003e, 2595\u0026ndash;2605 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Baert T, et al. The systemic treatment of recurrent ovarian cancer revisited. Ann Oncol \u003cb\u003e32\u003c/b\u003e, 710\u0026ndash;725 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Mirza MR, et al. The forefront of ovarian cancer therapy: update on PARP inhibitors. Ann Oncol \u003cb\u003e31\u003c/b\u003e, 1148\u0026ndash;1159 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Burger RA. Experience with bevacizumab in the management of epithelial ovarian cancer. J Clin Oncol \u003cb\u003e25\u003c/b\u003e, 2902\u0026ndash;2908 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Masuda M, et al. Saturated phosphatidic acids mediate saturated fatty acid-induced vascular calcification and lipotoxicity. J Clin Invest \u003cb\u003e125\u003c/b\u003e, 4544\u0026ndash;4558 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Matulonis UA, Sood AK, Fallowfield L, Howitt BE, Sehouli J, Karlan BY. Ovarian cancer. Nat Rev Dis Primers \u003cb\u003e2\u003c/b\u003e, 16061 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Nieman KM, et al. Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nat Med \u003cb\u003e17\u003c/b\u003e, 1498\u0026ndash;1503 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Ladanyi A, et al. Adipocyte-induced CD36 expression drives ovarian cancer progression and metastasis. Oncogene \u003cb\u003e37\u003c/b\u003e, 2285\u0026ndash;2301 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Christie EL, Bowtell DDL. Acquired chemotherapy resistance in ovarian cancer. Ann Oncol \u003cb\u003e28\u003c/b\u003e, viii13-viii15 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Smith P, et al. The copy number and mutational landscape of recurrent ovarian high-grade serous carcinoma. Nature Communications \u003cb\u003e14\u003c/b\u003e, (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Vernieri C, Ligorio F, Zattarin E, Rivoltini L, De Braud F. Fasting-mimicking diet plus chemotherapy in breast cancer treatment. Nature Communications \u003cb\u003e11\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4198546/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4198546/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFatty acids are known to have a significant impact on the properties of cancer cells. Therefore, Incorporating them into therapeutic strategies has been reported. However, few studies have examined the effects of individual fatty acids and their interaction in depth. The study analyzed the effects of various fatty acids on cancer cells and found that stearic acid, an abundant saturated fatty acid, had a stronger inhibitory effect on cell growth compared to palmitic acid, which is also an abundant saturated fatty acid, by inducing DNA damage and apoptosis through the unfolded protein response (UPR) pathway. Intriguingly, the negative effects of stearate were reduced by the presence of oleate, a different type of abundant fatty acid. In exploring the dietary impact on tumor growth, we combined a stearate-rich diet with the inhibition of stearoyl-CoA desaturase-1. This approach significantly reduced tumor growth in both ovarian cancer models and patient-derived xenografts (PDXs), including those with chemotherapy-resistant cases, by notably elevating stearate levels while reducing oleate levels within the tumors. Conversely, the negative effects of a stearate-rich diet were mitigated by an oleate-rich diet. The study shows that the dietary stearate can directly inhibit tumor growth through mechanisms involving DNA damage and apoptosis mediated by the UPR pathway. The results suggest that dietary interventions, which increase stearic acid levels while decreasing oleic acid levels, may be a promising therapeutic strategy in cancer treatment. This could lead to the development of new cancer treatment strategies.\u003c/p\u003e","manuscriptTitle":"Stearate-rich diet and oleate restriction directly inhibit tumor growth via the unfolded protein response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-19 18:48:44","doi":"10.21203/rs.3.rs-4198546/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-05-14T04:58:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-05-13T02:44:04+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-05-03T04:12:47+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-04-29T05:36:47+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-04-22T07:40:53+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-04-19T21:33:21+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-04-12T18:36:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-11T23:19:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2024-04-11T09:44:07+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-04-02T00:31:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-01T06:23:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2853c48b-202e-481a-8348-ec484bdb9b4c","owner":[],"postedDate":"April 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30620131,"name":"Biological sciences/Cancer/Cancer metabolism"},{"id":30620132,"name":"Biological sciences/Cell biology/Cell death/Apoptosis"},{"id":30620133,"name":"Health sciences/Diseases/Cancer/Cancer metabolism"}],"tags":[],"updatedAt":"2024-12-02T08:05:26+00:00","versionOfRecord":{"articleIdentity":"rs-4198546","link":"https://doi.org/10.1038/s12276-024-01356-2","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2024-12-02 05:00:00","publishedOnDateReadable":"December 2nd, 2024"},"versionCreatedAt":"2024-04-19 18:48:44","video":"","vorDoi":"10.1038/s12276-024-01356-2","vorDoiUrl":"https://doi.org/10.1038/s12276-024-01356-2","workflowStages":[]},"version":"v1","identity":"rs-4198546","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4198546","identity":"rs-4198546","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}