TFE3 and HIF1a regulates the expression of SHMT2 isoforms via alternative promoter utilization in ovarian cancer cells

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Platinum-based chemotherapy constitutes the first-line therapeutic regime. However, primary or acquired resistance seriously affects the survival rate of patients with ovarian cancer. Serine hydroxy methyltransferase (SHMT) catalyzes conversion of serine to glycine and is responsible for production of S-adenosylmethionine (SAM) for methylation. There are cytosolic SHMT1 and mitochondrial SHMT2 in human. Alternative promoter usage is a proteome-expanding mechanism that allows multiple pre-mRNAs to be transcribed from a single gene. The current study demonstrated that cisplatin-sensitive and cisplatin-resistant ovarian cancer cells expressed discrete SHMT2 isoforms, which was ascribed to the selective utilization of SHMT2 alternative promoters. SHMT2 isoforms exerted somewhat paradoxical roles in ovarian cancer cells, with tumor-suppressive role of isoform 1, and tumor-promotive role of isoform 3. In addition, the current study demonstrated that SHMT2 alternative promoter usage mediated by HIF1α and TFE3 might represent adaptive response of ovarian cancer cells to metabolic stress. Collectively, regulation of SHMT2 isoform expression via alternative promoter usage by transcription factors HIF1α and TFE3 provides a novel basis and potential drug targets for the clinical treatment of platin-resistant ovarian cancer. Biological sciences/Cancer/Gynaecological cancer/Ovarian cancer Biological sciences/Drug discovery/Drug regulation Ovarian cancer SHMT2 cisplatin-resistant isoform Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ovarian cancer is one of the most common and lethal gynecological malignancy. Due to the concealment of the incidence and the lack of perfect early diagnosis methods, about 70% of the patients were diagnosed at an advanced stage [ 1 – 3 ] . Although a certain clinical effect has been obtained based on the generally treated by tumor cell reduction surgery combined with platinum-based chemotherapy, ovarian cancer patients still may have a primary or acquired resistance to chemotherapeutic drugs, especially platinum drugs in first-line chemotherapy, and it seriously affects the quality of life and survival rate of patients [ 4 – 6 ] . However, there is no specific drug for clinical treatment of recurrent platinum-resistant ovarian cancer. Therefore, to improve the sensitivity of ovarian cancer chemotherapy and reverse platinum resistance is an urgent problem to be solved in the treatment of ovarian cancer. Glycolysis is abnormally activated in tumor cells and becomes an important source for energy production [ 7 ] . Serine synthesis pathway as a branch of glycolysis, which contributes to nucleotide synthesis, methylation reactions and generation of NADPH for redox homeostasis, has received more and more attention. As a non-essential amino acid, normal cells can rely on endogenous synthesis to satisfy the proliferation needs, while tumor cells also need exogenous intake due to accelerated proliferation and enhanced metabolism [ 7 ] . Enzymes responsible for the serine synthesis pathway are highly expressed in various cancers, including colorectal cancer, breast cancer, and hepatocellular carcinoma. Serine is converted into glycine and a tetrahydrofolate by serine hydroxy methyltransferase (SHMT), SHMT1 mainly exists in cytosolic and SHMT2 mainly exists in mitochondrial [ 8 ] . SHMT2 is a key mitochondrial enzyme in serine catabolism that converts serine to glycine and produces a one-carbon unit that generates S-adenosylmethionine (SAM) for methylation. Recent studies have shown that SHMT2 is significantly overexpressed in a variety of tumors and is associated with poor prognosis [ 9 – 13 ] . Increased serine catabolism supports malignant growth through diverse mechanisms [ 14 ] . However, the functions and underlying mechanisms of SHMT2 in ovarian cancer remain rarely studied. Alternative promoter refers to the variable region corresponding to the initial start site of a specific gene transcript. It was found that about 36% and 40% of protein coding genes in human and mouse genomes respectively have variable promoters [ 15 ] . The selection of alternative promoters greatly increases the diversity of transcriptome, however, the research on the biological function and molecular mechanism of alternative promoters is still very scarce. The current study reported that HIF1α and TFE3 were implicated in the utilization of SHMT2 alternative promoter, resulting in expression of SHMT2 isoforms in cisplatin-resistant ovarian cancer cells under metabolic stress. Our study verified complicated implication of SHMT2 isoforms in ovarian cancer cells. Method and Materials Clinical samples 36 patients with ovarian cancer who underwent surgical resection in Shengjing Hospital of China Medical University from June 2014 to July 2017 were enrolled in this project. None of the patients had received radiotherapy or chemotherapy before the surgery. The patients were divided into platinum-sensitive group (24 cases) and platinum-resistant group (12 cases) according to the recurrence within 6 months after the first cisplatin chemotherapy. The collected tissues were frozen in liquid nitrogen and stored in a refrigerator − 80°C for further analysis. The program was approved by the Institutional Review Board of China Medical University without the informed consent of patients or their families. Clinicopathological characteristics analysis and survival analysis The correlation between SHMT2 expression and clinicopathological characteristics was explored using UALCAN ( http://ualcan.path.uab.edu/ ), a web portal to analyze the relative expression of the desired gene(s) in tumor and normal samples, and association with clinicopathological characteristics of the patients such as cancer stage, tumor grade. We collected survival information of 1440 ovarian cancer patients from GEO, EGA, and TCGA databases and used them to examine the effect of SHMT2 on the prognosis of OV using Kaplan–Meier curve 18 and GEPIA databases. The examination probe ID used for SHMT2 was 227198_at. The log-rank P‐value and hazard ratio (HR) with 95% confidence intervals (CI) were analyzed. Singlecell RNA sequencing analysis The single-cell RNA-sequencing of ovarian cancer were downloaded from Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/ , GSE138794) and analyzed using R package “Seurat 4.1.0”. Method “UMAP” was applied for the visualization of different cell clusters. Cell culture The human ovarian cancer cell lines SKOV3 and A2780, as well as their cisplatin-resistant counterparts SKOV3/DDP and A2780/DDP were provided by Shanghai Genechem Co., Ltd. Cells were maintained in RPMI-1640 (Life Technologies, USA) with 10% fetal bovine serum (FBS, Sigma, USA) and penicillin (100 IU/ml, Sigma, USA) and streptomycin (100 µg/ml, Sigma, USA) and the cells were maintained at 37°C with 5% CO2. Western blot and immunoprecipitation Total cellular proteins were extracted using the lysis buffer containing Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100, and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), and the extracted protein was quantified by the BCA protein assay kit. 20µg total protein was separated with 10% SDS-PAGE and transferred to PVDF membrane (Millipore Corporation, Billerica, MA). The immunoprecipitants were treated with various antibodies, incubated overnight in 4°C, washed three times with lysis buffer, and analyzed using Western blot. Knockdown of SHMT2 by CRISPR-Cas9 Lentivirus infection experiments were performed to knockdown SHMT2 stably. Lentivirus was purchased from Nanjing Jikeyin Company. 1×10 5 cells/well were inoculated in 6-well plates and cultured for 24 hours. The virus solution was added into the cell culture medium and cultured for 8h, then the culture medium was replaced with a normal medium containing 1µg/ mL purinomycin for 48h, and the protein was extracted and verified by Western blot. Cell Counting Kit 8 (CCK-8) assay CCK8 (Dojindo Laboratories) assays was performed by exposing cells to different concentrations of cisplatin for 24 hours. 10 µL diluted CCK-8 solution was added to each well. Cells were incubated at 37◦C for 4 hours, and the absorbance at 450nm was determined with a microplate reader. Sphere formation assay Logarithmic growth phase cells were harvested and resuspended with 20mg/ mL EGF (Sigma-Aldrich, St. Louis, MO, USA), 5µg/ mL insulin (Sigma-Aldrich) and 2% B27 (Invitrogen, Carlesbad, CA, USA) in serum-free DMEM/F12 medium. The cells were inoculated in 6-well plates, the medium was changed every 3 days. The cell morphology was observed and the images were taken under the microscope. The number of spheres was counted according to the images. Chromatin immunoprecipitation (ChIP) assay ChIP was performed according to the manual using the ChIP™ assay (Merck Millipore, Billerica, Massachusetts). 1x10 7 cells were fixed with 1% formaldehyde for 10min and quenched with 0.125M glycine for 10 min. Cells were washed and resuspended sequentially in three lysis buffers to isolate chromatin. Chromatin was sonicated and incubated overnight at 4 o C with 10µg of antibody for the indicated proteins and 50µl of Protein G magnetic beads. Magnetic beads were the washed and followed by DNA purification. The ChIP and input DNAs were measured by quantitative PCR qPCR using intergenic regions as negative controls Statistical analysis ANOVA and post hoc Dunnett’s test were used to analyzed the statistical significance in the most experiments, the significant difference was defined as p < 0.05. All experiments were repeated 3 times, and data were expressed as mean ± SD (standard deviation) of representative experiments. Results Distinct expression of SHMT2 isoform 1 and 3 in cisplatin-sensitive and cisplatin-resistant ovarian cancer To clarify the mechanisms underlying cisplatin resistance in ovarian cancer, quantitative proteomics analysis was performed to globally screen the potential molecular targets regulated by cisplatin resistance. We used protein to perform on 9 cisplatin-sensitive and 9 cisplatin-resistant ovarian cancer tissues were used for quantitative proteomics analysis and SHMT2 was identified to be downregulated in the cisplatin-resistant ovarian cancer tissues (Fig. 1 A). Expression of SHMT2 was further analyzed using 24 cisplatin-sensitive and 12 cisplatin-resistant ovarian cancer tissues, and confirmed the downregulation of SHMT2 in cisplatin-resistant ovarian cancer tissues (Fig. 1 B). Two SHMT2 isoforms were identified in ovarian cancer tissues, the smaller isoform was upregulated, even though downregulation of total SHMT2 in cisplatin-resistant tissues (Fig. 1 B). Real-time RT-PCR demonstrated that the total SHMT2 mRNA expression was significantly decreased in cisplatin-resistant compared to the cisplatin-sensitive ovarian cancer tissues (Fig. 1 C). There are multiple SHMT2 variants, the individual variant expression was then studied and found that variant 1 and variant 4 were significantly decreased and increased in the cisplatin-resistant tissues, which encode isoform 1 and isoform 3 of SHMT2 respectively (Fig. 1 C). With regards to other variants, there were no significant difference in the cisplatin-sensitive and cisplatin-resistant ovarian cancer tissues (Fig. 1 C). The online splicing data from TCGA database (tsvdb.com/index.html) also confirmed expression of multiple SHMT2 variants in ovarian cancer tissues (Fig. 1 D). To further determine the potential importance of SHMT2 in clinical settings, we analyzed clinical outcomes from GEPIA OV samples. Compared with normal tissues, the expression level of SHMT2 was significantly increased (Fig. 1 E). In addition, the expression of SHMT2 among Stage II, Stage III and Stage III group were significantly different (Fig. 1 F). Kaplan-Meier curve showed that neither OS (Fig. 1 G) nor PFS (Fig. 1 H) were significantly associated with the mRNA expression level of SHMT2. To better understand the relevance of SHMT2 expression in OV patients, KM plotter was used to evaluate prognostic value based on Affymetrix microarrays. It is worth noting that the expression of SHMT2 was detected by different Affymetrix microarrays, the effect on prognosis was different (Fig. 1 I-J). This might because different probes recognize different SHMT2 variants, and the different effects on prognosis suggest that different SHMT2 variants might have different biological behaviors, which may be related to cisplatin resistance. SHMT2 isoform 3 is expressed in cisplatin-resistant ovarian cancer under metabolic stress To further explore the role of SHMT2 isoforms in the process of cisplatin resistance, SHMT2 expression was studied using paired cisplatin-resistant and parental SKOV3 and A2780 cells. SHMT2 expression did not show significant difference between the cisplatin-resistant SKOV3 (SKOV3/DDP) and A2780 (A2780/DDP) cells compared with their respective parental cells (Fig. 2 A). In addition, only single band representing SHMT2 isoform 1 was observed in ovarian cancer cell lines (Fig. 2 A). Single-cell RNA sequencing analysis showed that SHMT2 was mainly expressed in epithelial and mesenchymal cells, in addition, SHMT2 also expressed in endothelial cells (Fig. 2 B-C). Next, cancer-associated fibroblast (CAF) and ovarian cancer (OC) cells were extracted from fresh ovarian cancer tissues, Western blot demonstrated that CAF expressed SHMT2 isoform 1, while some OC cells expressed SHMT2 isoform 3 (Fig. 2 D-E), suggesting that the expression of SHMT2 isoform 3 might be mainly derived from ovarian tumor epithelial cells. SHMT2 isoform 3 could be detected in some primary OC cells at the beginning, but with continuous cell culture, SHMT2 isoform 3 could not be detected. However, SHMT2 isoform 3 could be detected again under the conditions of hypoglycemia and hypoxia (Fig. 2 F). Real-time RT-PCR showed that variant 1 decreased, while variant 4 decreased in OC#2 and OC#5 after 2–3 passages, culture under hypoglycemic and hypoxic condition recovered the expression of variant 4 (Fig. 2 G). On the other hand, such fluctuation of expression of SHMT2 variants was not observed in OC#3, which did not express SHMT2 variant 4 originally (Fig. 2 G). In the cisplatin-resistant SKOV3 (SKOV3/DDP) and A2780 (A2780/DDP) cells, SHMT2 isoform 3 could be detected in cisplatin-resistant SKOV3/DDP and A2780/DDP cells under hypoglycemic and hypoxic culture, but could not be detected in their respective parental cells (Fig. 2 H). RT-PCR showed that hypoglycemic and hypoxic culture increased total and variant 1 SHMT2 expression in SKOV3 cells, while such induction was not observed in A2780 cells (Fig. 2 I-J). SHMT2 variant 1 was consistently decreased (Fig. 2 J), while SHMT2 variant 4 was consistently increased (Fig. 2 K-L) in cisplatin-resistant cells cultured under hypoglycemic and hypoxic condition. In general, OC can express SHMT2 variant 4 only when metabolic stress occurs, that is, under a condition of low-glucose and hypoxic, thereby expressing the SHMT2 isoform 3. Different effects of SHMT2 knockdown in cisplatin responsiveness in cisplatin-resistant and sensitive ovarian cancer cells In order to explore the role of SHMT2 in cisplatin resistance, SHMT2 was knocked down using CRISP/Cas9 system in SKOV3/DDP and A2780/DDP and their respective parental cells (Fig. 3 A). Knockdown of SHMT2 increased the survival of both SKOV3 and A2780 cells exposed to cisplatin treatment. However, in SKOV3/DDP and A2780/DDP cells knockdown of SHMT2 did not show a significant difference in the viability of the cells after cisplatin exposure (Fig. 3 B). Colony formation assay got a similar result. Knockdown of SHMT2 increased the colony number of cisplatin sensitive cells, while there was no significant change in the resistant cells under cisplatin treatment (Fig. 3 C-D). To further understand the role of SHMT2 isoforms in cisplatin resistance, SHMT2 knockdown cells were restored with SHMT2 isoform 1 and 3, respectively (Fig. 3 E). Restoration of SHMT2 isoform 1, but not isoform 3, significantly decreased the cell viability (Fig. 3 F) and colony formation (Fig. 3 G-H) of SKOV3 and A2780 cells under cisplatin exposure. SHMT2 isoform 3 increased colony formation of SKOV3 cells, but not A2780 cells (Fig. 3 G-H). Neither isoform 1 nor isoform 3 demonstrated any effects on viability and colony formation of SKOV3/DDP and A2780/DDP cells (Fig. 3 F-H). Opposite roles of SHMT2 isoforms in maintenance of stem cell like features of ovarian cancer cells As mentioned above, SHMT2 isoform 3 only expressed under metabolic stress situation, in order to further understand the roles of SHMT2 isoforms, a low-glucose and hypoxic cell culture environment were constructed for the following experiments. SHMT2 isoform 3, but not isoform 1, significantly promoted survival of both cisplatin-sensitive and cisplatin-resistant cells under low-glucose and hypoxic conditions (Fig. 4 A-B). SHMT2 isoform 1 decreased, while isoform 3 increased stem cell like features of ovarian cancer cells, as identified by spheroid formation ability (Fig. 4 C-D), expression of stemness markers CD44 and CD133 (Fig. 4 E), as well as tumor formation rate and stem cell frequency using the tumor-limiting dilution assay (Fig. 4 F). Selective utilization of alternative promoters of SHMT2 in cisplatin-resistant ovarian cancer cells under metabolic stress SHMT2 gene contains three promoters, and different variants will be transcribed due to selective utilization of different promoters (Fig. 5 A-B). The reporters containing − 841/+72, -841/+448, -841/+724 fragments showed highest and similar activities, while no obvious activity of reporter containing + 82/+448 or + 520/+724 fragments was observed in both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells under normal condition (Fig. 5 C), indicating that ovarian cancer cells preferentially utilized promoter 1, while promoter 2 and promoter 3 were rarely activated under normal culture condition. The activity of reporter containing − 841/+72 fragment significantly decreased, while the activity of reporter containing + 82/+448 fragment significantly increased in SKOV3/DDP and A2780/DDP under hypoglycemic and hypoxic condition (Fig. 5 C). The enrichment of RNA Pol II pSer2 and Pol II pSer5 of SKOV3/DDP and A2780/DDP to the promoter 1 region was significantly decreased under the low-glucose and hypoxic condition, while there was no significant difference in SKOV3 and A2780 cells (Fig. 5 D). Selective utilization of SHMT2 promoter 2 by HIF1α and TFE3 complex To understand the regulation of SHMT2α by transcription factors, the specific transcription factor binding sites around promoter 2 which were associated with metabolic stress were surveyed and XBP1, HIF1α and TFE3 were screened out. Knockdown of TFE3 and HIFα in low-glucose and hypoxic condition decreased the expression of SHMT2 isoform 3, while XBP1 knockdown demonstrated no obvious effect (Fig. 6 A). ChIP confirmed significant enrichment of HIF1α and TFE3 to the promoter 2 region in cisplatin-resistant cells under low-glucose and hypoxic culture condition (Fig. 6 B). Knockdown of TFE3 and HIFα decreased the activity of promoter 2, and knockdown of XBP1 had no significant difference to the activity of promoter 2 (Fig. 6 C). To further study whether there was a regulation between TFE3 and HIFα, ChIP showed that TFE3 knockdown inhibited the recruitment of HIF1α in both SKOV3/DDP and A2780/DDP cells (Fig. 6 D). HIF1α knockdown decreased recruitment of TFE3 in A2780/DDP, while had no effect in SKOV3/DDP (Fig. 6 D). Molecular docking model predicted direct interaction of HIF1α and TFE3 (Fig. 6 E). DuoLink confirmed interaction of TFE3 and HIF1α in cisplatin-resistant ovarian cancer cells under the low-glucose and hypoxic condition (Fig. 6 E). Discussion In 1955 Thomlinson first proposed the concept of tumor hypoxia, more than 60 years of clinical and experimental evidence have shown that hypoxia is a common feature of many types of solid tumors [ 16 ] . As the tumor continues to proliferate, low-glucose that often occur at the same time as hypoxia is also a common change in the tumor microenvironment (TME). This is because cancer cells are characterized by a high proliferation rate and active metabolism, and they need to consume a lot of energy to support their increased proliferation and growth rate compared to normal cells [ 17 ] . When the oxygen and glucose required by cell metabolism cannot be satisfied by organism, it will change TME into a hypoxia and low-glucose situation [ 18 ] , and it might cause a series of metabolic disorders [ 19 ] . SHMT2 is a key mitochondrial enzyme in serine catabolism that converts serine to glycine [ 20 , 21 ] . Studies have shown that high expression of SHMT2 may promote tumor proliferation in bladder cancer, rhabdomyosarcoma, esophageal cancer and diffuse large B-cell lymphoma [ 14 , 22 – 24 ] . SHMT2 induces stemness of head and neck cancer, Burkitt lymphoma [ 25 , 26 ] . On the contrary, SHMT2 functions as a tumor suppressor and negatively regulates proliferation and metastasis in prostate cancer [ 27 ] . These data indicate that SHMT2 might function as both tumor-promoting and tumor-suppressive functions in a cell-dependent context. The current study demonstrated that SHMT2 isoforms might exert paradoxical functions in ovarian cancer cells. SHMT2 isoform 1 might function as tumor suppressive function, as it suppressed cancer stem cell (CSC)-like features including increase of cisplatin responsiveness of parent ovarian cancer cells, suppression of spheroid formation, downregulation of CSC markers. On the contrary, SHMT2 isoforms 3 might function as tumor promoting function to promote CSC-like features. However, exact mechanisms underlying discrete roles of SHMT2 isoforms in ovarian cancer require further investigation. Anyway, the current study suggested that discrete isoform expression might represent another layer of paradoxical function of SHMT2 in different cancer. In order to survive under metabolic stress, cancer cells regulate metabolism, protein synthesis and cell cycle processes by the adjustment of transcription factors [ 28 , 29 ] . Hypoxia is a common characteristic of the tumor microenvironment found in most solid tumors, including ovarian cancer [ 30 ] . Cancer cells under hypoxic stress regulate protein translation through reprogramming gene expression, leading to microenvironmental changes [ 31 ] . HIF1-α is an important regulatory transcription factor in the hypoxia and low-glucose situation. Accumulating data indicate complex implication of SHMT2 in hypoxia. Co-induction of SHMT2 by HIF1α and Myc maintains cell growth by balancing the NADPH/NADP + ratios in neuroblastoma tumors upon lack of oxygen [ 32 ] . Hypoxia stabilizes SHMT2 lactylation, thereby promoting glycolysis and stemness of esophageal cancer cells [ 24 ] . Furthermore, SHMT2 regulates HIF1α expression through both enzymatic and non-enzymatic functions in gastric cancer [ 33 ] . Identification of selective utilization of SHMT2 alternative promoter by the combination of HIFα and TFE3 in cisplatin-resistant ovarian cancer cells in the current study further strengthen the complicated interlinkage between hypoxia and SHMT2. Many genes have multiple promoters, which may even lead to different functions of the same gene. The utilization of alternative promoter produces different pre-mRNAs being transcribed from variable transcription start sites (TSSs) within a gene locus. The patterns of alternative promoter selection result in the regulation of diverse cell types, tissue types and complex developmental genes. The abnormal usage of alternative promoter plays an important role in the occurrence and development of various diseases. As a general mechanism, alternative promoter is diverse and flexible in gene expression regulation. In the current study, we reported that under hypoxia and hypoglycemia, the combination of HIFα and TFE3 leads to a reduction in the utilization of promoter 1, while the increase in the selection of promoter 2, resulting in transcriptional dysregulation and an increase in the transcription of variant 4, followed by increase of SHMT2 isoform 3 under metabolic stress conditions. In general, the high expression of SHMT2α and the low expression of SHMT2 in the hypoxia and low-glucose situation may be one of the causes of cisplatin resistance in ovarian cancer patients. However, further studies are needed to dissect, how SHMT2 isoforms exerted paradoxical functions in ovarian cancer. Collectively, the current study demonstrated that ovarian cancer cells expressed multiple SHMT2 isoforms, which exerted complicated functions. Selective utilization of SHMT2 alternative promoter by HIF1α and TFE3 resulted in SHMT2 isoform expression shift, which might be implicated in adaptation of ovarian cancer cells under metabolic stress. Abbreviations SHMT: Serine hydroxy methyltransferase SAM: S-adenosylmethionine HR: hazard ratio CI: confidence intervals CAF: cancer-associated fibroblast OC: ovarian cancer Declarations Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Huaqin Wang and Chuan Liu designed this study. Siqi Wang and Ning Liu performed all the experiments and analyzed the data. Baiqiang Li provided clinical samples and conducted pathological evaluation. Fuying Zhao and Chao Li provided the technical support. Siqi Wang and Ning Liu conceptualized and initiated the project and wrote the manuscript. All authors have read and approved the final manuscript. References Juarez-Mendez, S., et al., Splice variants of zinc finger protein 695 mRNA associated to ovarian cancer. J Ovarian Res, 2013. 6 (1): p. 61. 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Clark, R.A., et al., Induction of serine hydroxymethyltransferase 2 promotes tumorigenesis and metastasis in neuroblastoma. Oncotarget, 2022. 13 : p. 32-45. Wang, W., et al., SHMT2 Promotes Gastric Cancer Development through Regulation of HIF1alpha/VEGF/STAT3 Signaling. Int J Mol Sci, 2023. 24 (8). Additional Declarations (Not answered) Supplementary Files WB.pdf original and repeat experiments of WB Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2025 Read the published version in Cell Death & Disease → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-4981006","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":347982394,"identity":"dc80a18c-771c-4300-83b8-ba23d7f5f79a","order_by":0,"name":"Siqi Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApklEQVRIiWNgGAWjYPACGx5+/gbStKTJSM44QJqWwzYGDQlEquWfdsZM8kfZeR4DhgOMHz7mEKFF4naOmTTPuds85swNzJIztxGhxUA6x+w2Y9ttHsuGA2zMvMRqufmz7RyPwYEEErTc4G07QIIWidtp5b95ziXzSM442EycX/hnJ282/FFmZ8/P33zww0ditEAAG4hgbCBaPUzLKBgFo2AUjAIcAACM9TKPBTyE1QAAAABJRU5ErkJggg==","orcid":"","institution":"China Medical University","correspondingAuthor":true,"prefix":"","firstName":"Siqi","middleName":"","lastName":"Wang","suffix":""},{"id":347982395,"identity":"dcb87aa8-c911-4c0c-865a-9c740784fc79","order_by":1,"name":"Ning Liu","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Liu","suffix":""},{"id":347982396,"identity":"c700064a-2427-4823-9cc9-d5e77ebbb830","order_by":2,"name":"Baiqiang Li","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Baiqiang","middleName":"","lastName":"Li","suffix":""},{"id":347982397,"identity":"27e3b0da-f01f-4e26-a926-4e78be87d154","order_by":3,"name":"Fuying Zhao","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fuying","middleName":"","lastName":"Zhao","suffix":""},{"id":347982398,"identity":"a072c168-736a-4675-bcf6-8ceee67e6797","order_by":4,"name":"Chao Li","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Li","suffix":""},{"id":347982399,"identity":"ed21d7f9-ea6d-4cb8-827b-4b0dbf9772db","order_by":5,"name":"Huaqin Wang","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huaqin","middleName":"","lastName":"Wang","suffix":""},{"id":347982400,"identity":"7131fc41-c853-44bc-a3d3-756b76fd3adc","order_by":6,"name":"chuan liu","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University;","correspondingAuthor":false,"prefix":"","firstName":"chuan","middleName":"","lastName":"liu","suffix":""}],"badges":[],"createdAt":"2024-08-27 03:00:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4981006/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4981006/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-07445-y","type":"published","date":"2025-03-17T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66930690,"identity":"02d014e7-50d7-4d85-beb9-d2a9817ce9bb","added_by":"auto","created_at":"2024-10-18 06:58:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2270405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCisplatin-sensitive and cisplatin-resistant ovarian cancer mainly express SHMT2 isoform 1 and 3, respectively.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Top 20 differentially expressed proteins between cisplatin-sensitive and resistant pathological tissue in ovarian cancer. B：Wetern Blot showed the presence of SHMT2 and SHM2a isoforms in ovarian cancer. C：RT-PCR showed that mRNA expression in real-time in different variants expression between cisplatin sensitive and resistant ovarian cancer tissue. D: Different variants expression between cisplatin sensitive and resistant ovarian cancer in bulk database. E-H: Different expression of SHMT2 in GEPIA database. I-J: Kaplan–Meier curves revealing overall survival of SHMT2s’ expression according to different Affymetrix microarrays detected.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4981006/v1/c2080de758777417fb33b8cd.png"},{"id":66930899,"identity":"b68a8e90-d4cf-44ce-95af-03575679661d","added_by":"auto","created_at":"2024-10-18 07:06:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2473963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHMT2 isoform 3 is expressed in cisplatin-resistant ovarian cancer under metabolic stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Western blotting of SHMT2 expression in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells. B-C: Single-cell RNA sequencing of the expression of SHMT2 among different stages visualizing UMAP cell clusters. D: Western blotting of SHMT2 expression in CAF extracted from patients with ovarian cancer. E: Western blotting of SHMT2 expression in ovarian cancer cells extracted from patients. F: Western blotting of SHMT2 expression in ovarian cancer cells extracted from patients under different situations. G: RT-PCR showed the difference among different SHMT2 isomers under different situations. H: Western blotting of SHMT2 expression in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells under different situations. I-K: RT-PCR showed SHMT2, SHMT2 V1 and SHMT2 V4 in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells under different situations. L: Agarose gel electrophoresis showed the expression of SHMT2 V4 in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells under different situations.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4981006/v1/f444d5ffdb7dfeb9f2744ff6.png"},{"id":66930898,"identity":"3896ac18-448d-4552-bba1-023ef8dd19d8","added_by":"auto","created_at":"2024-10-18 07:06:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5239502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHMT2 knockdown had discrete effects on cisplatin responsiveness of cisplatin-resistant and sensitive ovarian cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Western blot analysis of SHMT2 protein expression upon SHMT2 knockdown in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells. B: The quantification of cell viability of cisplatin-sensitive SKOV3 and A2780 cells treated with the indicated concentrations of cisplatin for 24 h, using CCK8 assays. C: The quantification of cell viability of cisplatin- resistant (DDP) SKOV3 and A2780 cells were treated with the indicated concentrations of cisplatin for 24 h, cell viability was assessed using CCK8 assays. D-E: Colony number shown upon SHMT2 knockdown in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells. F: Western blot analysis of SHMT2 and SHMT2a protein expression in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells. G: The quantification of cell viability of cisplatin- resistant (DDP) SKOV3 and A2780 cells transduced with SHMT2 and SHMT2α overexpression sequences, which were treated with the indicated concentrations of cisplatin for 24 h, H-I: Colony number shown upon SHMT2 and SHMT2α overexpression in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4981006/v1/61955e940e80869687d38fd9.png"},{"id":66930692,"identity":"48cfc50e-2322-439e-b54d-dcd365754ed4","added_by":"auto","created_at":"2024-10-18 06:58:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3651548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOpposite roles of SHMT2 isoforms in maintenance of stem cell like features of ovarian cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B: Colony number shown upon SHMT2 and SHMT2α overexpression in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells under a low-glucose and hypoxic environment. C-D: The quantification of tumor sphere formation of cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells transduced upon SHMT2 and SHMT2α overexpression sequences under a low-glucose and hypoxic environment. E: Western blot analysis of CD44 and CD133 protein expression upon SHMT2 and SHMT2α overexpression sequences under a low-glucose and hypoxic environment. F:Image of the xenograft tumors dissected from nude mice （n=3 in each group） was shown, and the ultimate weight and volume of the tumors were evaluated.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4981006/v1/715fdc56f7e1dca7869b390a.png"},{"id":66930694,"identity":"b5646a64-6f39-42ce-a4bd-e98e31ed5ae8","added_by":"auto","created_at":"2024-10-18 06:58:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":982983,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelective utilization of alternative promoters of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSHMT2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in cisplatin-resistant ovarian cancer cells under metabolic stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B：Diagram of SHMT2 isoform formation. C: Luciferase assay different promoter activity in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells under different cell culture. D: RT-PCR shown enrichment of different RNA polymerase in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells under different cell culture.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4981006/v1/44bed4a0e796a8f4136ce2af.png"},{"id":66930696,"identity":"bc30ca42-0e59-40f8-8565-64f7eeaeba55","added_by":"auto","created_at":"2024-10-18 06:58:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2871991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelective utilization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSHMT2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromoter 2 by HIF1α and TFE3 complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B: Western blot analysis of SHMT2, SHMT2α, XBP1, TFE3, HIF1α protein expression upon XBP1, TFE3, HIF1α knockdown respectively in cisplatin-sensitive (parental) and cisplatin-resistant (DDP) SKOV3 or A2780 cells under different cell culture. C: Luciferase assay different promoter activity upon XBP1, TFE3, HIF1α knockdown respectively cisplatin-resistant (DDP) SKOV3 or A2780 cells under low-glucose and hypoxic environment. D: Co-IP experiment testing the interaction between TFE3, HIF1α in cisplatin-resistant (DDP) SKOV3 or A2780 cells under low-glucose and hypoxic environment. E: Binding pose of TFE3 with HIF1α in 3D. F: Co-IF staining showing the distribution of TFE3 with HIF1α in cisplatin-resistant (DDP) SKOV3 or A2780 cells under different cell culture.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4981006/v1/dac2b8303b7b4389e5181e97.png"},{"id":78729603,"identity":"92943a4c-d317-4b6d-85ef-fde8a2055575","added_by":"auto","created_at":"2025-03-18 07:10:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19602817,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4981006/v1/7ca7dbcc-2545-45d3-89cb-7412eda6b9b8.pdf"},{"id":66930695,"identity":"e721535a-3767-4aa0-961f-55ed45f6801a","added_by":"auto","created_at":"2024-10-18 06:58:06","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":26780348,"visible":true,"origin":"","legend":"original and repeat experiments of WB","description":"","filename":"WB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4981006/v1/8ee3b5859ce2a44e529ea4ed.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"TFE3 and HIF1a regulates the expression of SHMT2 isoforms via alternative promoter utilization in ovarian cancer cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer is one of the most common and lethal gynecological malignancy. Due to the concealment of the incidence and the lack of perfect early diagnosis methods, about 70% of the patients were diagnosed at an advanced stage\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Although a certain clinical effect has been obtained based on the generally treated by tumor cell reduction surgery combined with platinum-based chemotherapy, ovarian cancer patients still may have a primary or acquired resistance to chemotherapeutic drugs, especially platinum drugs in first-line chemotherapy, and it seriously affects the quality of life and survival rate of patients\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, there is no specific drug for clinical treatment of recurrent platinum-resistant ovarian cancer. Therefore, to improve the sensitivity of ovarian cancer chemotherapy and reverse platinum resistance is an urgent problem to be solved in the treatment of ovarian cancer.\u003c/p\u003e \u003cp\u003eGlycolysis is abnormally activated in tumor cells and becomes an important source for energy production \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Serine synthesis pathway as a branch of glycolysis, which contributes to nucleotide synthesis, methylation reactions and generation of NADPH for redox homeostasis, has received more and more attention. As a non-essential amino acid, normal cells can rely on endogenous synthesis to satisfy the proliferation needs, while tumor cells also need exogenous intake due to accelerated proliferation and enhanced metabolism\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Enzymes responsible for the serine synthesis pathway are highly expressed in various cancers, including colorectal cancer, breast cancer, and hepatocellular carcinoma. Serine is converted into glycine and a tetrahydrofolate by serine hydroxy methyltransferase (SHMT), SHMT1 mainly exists in cytosolic and SHMT2 mainly exists in mitochondrial \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSHMT2 is a key mitochondrial enzyme in serine catabolism that converts serine to glycine and produces a one-carbon unit that generates S-adenosylmethionine (SAM) for methylation. Recent studies have shown that SHMT2 is significantly overexpressed in a variety of tumors and is associated with poor prognosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Increased serine catabolism supports malignant growth through diverse mechanisms\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. However, the functions and underlying mechanisms of SHMT2 in ovarian cancer remain rarely studied.\u003c/p\u003e \u003cp\u003eAlternative promoter refers to the variable region corresponding to the initial start site of a specific gene transcript. It was found that about 36% and 40% of protein coding genes in human and mouse genomes respectively have variable promoters\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The selection of alternative promoters greatly increases the diversity of transcriptome, however, the research on the biological function and molecular mechanism of alternative promoters is still very scarce. The current study reported that HIF1α and TFE3 were implicated in the utilization of \u003cem\u003eSHMT2\u003c/em\u003e alternative promoter, resulting in expression of SHMT2 isoforms in cisplatin-resistant ovarian cancer cells under metabolic stress. Our study verified complicated implication of SHMT2 isoforms in ovarian cancer cells.\u003c/p\u003e"},{"header":"Method and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical samples\u003c/h2\u003e \u003cp\u003e36 patients with ovarian cancer who underwent surgical resection in Shengjing Hospital of China Medical University from June 2014 to July 2017 were enrolled in this project. None of the patients had received radiotherapy or chemotherapy before the surgery. The patients were divided into platinum-sensitive group (24 cases) and platinum-resistant group (12 cases) according to the recurrence within 6 months after the first cisplatin chemotherapy. The collected tissues were frozen in liquid nitrogen and stored in a refrigerator \u0026minus;\u0026thinsp;80\u0026deg;C for further analysis. The program was approved by the Institutional Review Board of China Medical University without the informed consent of patients or their families.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eClinicopathological characteristics analysis and survival analysis\u003c/h2\u003e \u003cp\u003eThe correlation between SHMT2 expression and clinicopathological characteristics was explored using UALCAN (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ualcan.path.uab.edu/\u003c/span\u003e\u003cspan address=\"http://ualcan.path.uab.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a web portal to analyze the relative expression of the desired gene(s) in tumor and normal samples, and association with clinicopathological characteristics of the patients such as cancer stage, tumor grade. We collected survival information of 1440 ovarian cancer patients from GEO, EGA, and TCGA databases and used them to examine the effect of SHMT2 on the prognosis of OV using Kaplan\u0026ndash;Meier curve 18 and GEPIA databases. The examination probe ID used for SHMT2 was 227198_at. The log-rank P‐value and hazard ratio (HR) with 95% confidence intervals (CI) were analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSinglecell RNA sequencing analysis\u003c/h2\u003e \u003cp\u003eThe single-cell RNA-sequencing of ovarian cancer were downloaded from Gene Expression Omnibus (GEO, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, GSE138794) and analyzed using R package \u0026ldquo;Seurat 4.1.0\u0026rdquo;. Method \u0026ldquo;UMAP\u0026rdquo; was applied for the visualization of different cell clusters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe human ovarian cancer cell lines SKOV3 and A2780, as well as their cisplatin-resistant counterparts SKOV3/DDP and A2780/DDP were provided by Shanghai Genechem Co., Ltd. Cells were maintained in RPMI-1640 (Life Technologies, USA) with 10% fetal bovine serum (FBS, Sigma, USA) and penicillin (100 IU/ml, Sigma, USA) and streptomycin (100 \u0026micro;g/ml, Sigma, USA) and the cells were maintained at 37\u0026deg;C with 5% CO2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot and immunoprecipitation\u003c/h2\u003e \u003cp\u003eTotal cellular proteins were extracted using the lysis buffer containing Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100, and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), and the extracted protein was quantified by the BCA protein assay kit. 20\u0026micro;g total protein was separated with 10% SDS-PAGE and transferred to PVDF membrane (Millipore Corporation, Billerica, MA). The immunoprecipitants were treated with various antibodies, incubated overnight in 4\u0026deg;C, washed three times with lysis buffer, and analyzed using Western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of SHMT2 by CRISPR-Cas9\u003c/h2\u003e \u003cp\u003eLentivirus infection experiments were performed to knockdown SHMT2 stably. Lentivirus was purchased from Nanjing Jikeyin Company. 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well were inoculated in 6-well plates and cultured for 24 hours. The virus solution was added into the cell culture medium and cultured for 8h, then the culture medium was replaced with a normal medium containing 1\u0026micro;g/ mL purinomycin for 48h, and the protein was extracted and verified by Western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell Counting Kit 8 (CCK-8) assay\u003c/h2\u003e \u003cp\u003eCCK8 (Dojindo Laboratories) assays was performed by exposing cells to different concentrations of cisplatin for 24 hours. 10 \u0026micro;L diluted CCK-8 solution was added to each well. Cells were incubated at 37◦C for 4 hours, and the absorbance at 450nm was determined with a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSphere formation assay\u003c/h2\u003e \u003cp\u003eLogarithmic growth phase cells were harvested and resuspended with 20mg/ mL EGF (Sigma-Aldrich, St. Louis, MO, USA), 5\u0026micro;g/ mL insulin (Sigma-Aldrich) and 2% B27 (Invitrogen, Carlesbad, CA, USA) in serum-free DMEM/F12 medium. The cells were inoculated in 6-well plates, the medium was changed every 3 days. The cell morphology was observed and the images were taken under the microscope. The number of spheres was counted according to the images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP) assay\u003c/h2\u003e \u003cp\u003eChIP was performed according to the manual using the ChIP\u0026trade; assay (Merck Millipore, Billerica, Massachusetts). 1x10\u003csup\u003e7\u003c/sup\u003e cells were fixed with 1% formaldehyde for 10min and quenched with 0.125M glycine for 10 min. Cells were washed and resuspended sequentially in three lysis buffers to isolate chromatin. Chromatin was sonicated and incubated overnight at 4\u003csup\u003eo\u003c/sup\u003eC with 10\u0026micro;g of antibody for the indicated proteins and 50\u0026micro;l of Protein G magnetic beads. Magnetic beads were the washed and followed by DNA purification. The ChIP and input DNAs were measured by quantitative PCR qPCR using intergenic regions as negative controls\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eANOVA and post hoc Dunnett\u0026rsquo;s test were used to analyzed the statistical significance in the most experiments, the significant difference was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All experiments were repeated 3 times, and data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (standard deviation) of representative experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDistinct expression of SHMT2 isoform 1 and 3 in cisplatin-sensitive and cisplatin-resistant ovarian cancer\u003c/h2\u003e \u003cp\u003eTo clarify the mechanisms underlying cisplatin resistance in ovarian cancer, quantitative proteomics analysis was performed to globally screen the potential molecular targets regulated by cisplatin resistance. We used protein to perform on 9 cisplatin-sensitive and 9 cisplatin-resistant ovarian cancer tissues were used for quantitative proteomics analysis and SHMT2 was identified to be downregulated in the cisplatin-resistant ovarian cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Expression of SHMT2 was further analyzed using 24 cisplatin-sensitive and 12 cisplatin-resistant ovarian cancer tissues, and confirmed the downregulation of SHMT2 in cisplatin-resistant ovarian cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Two SHMT2 isoforms were identified in ovarian cancer tissues, the smaller isoform was upregulated, even though downregulation of total SHMT2 in cisplatin-resistant tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Real-time RT-PCR demonstrated that the total SHMT2 mRNA expression was significantly decreased in cisplatin-resistant compared to the cisplatin-sensitive ovarian cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). There are multiple SHMT2 variants, the individual variant expression was then studied and found that variant 1 and variant 4 were significantly decreased and increased in the cisplatin-resistant tissues, which encode isoform 1 and isoform 3 of SHMT2 respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). With regards to other variants, there were no significant difference in the cisplatin-sensitive and cisplatin-resistant ovarian cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The online splicing data from TCGA database (tsvdb.com/index.html) also confirmed expression of multiple SHMT2 variants in ovarian cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To further determine the potential importance of SHMT2 in clinical settings, we analyzed clinical outcomes from GEPIA OV samples. Compared with normal tissues, the expression level of SHMT2 was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In addition, the expression of SHMT2 among Stage II, Stage III and Stage III group were significantly different (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Kaplan-Meier curve showed that neither OS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) nor PFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) were significantly associated with the mRNA expression level of SHMT2. To better understand the relevance of SHMT2 expression in OV patients, KM plotter was used to evaluate prognostic value based on Affymetrix microarrays. It is worth noting that the expression of SHMT2 was detected by different Affymetrix microarrays, the effect on prognosis was different (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). This might because different probes recognize different SHMT2 variants, and the different effects on prognosis suggest that different SHMT2 variants might have different biological behaviors, which may be related to cisplatin resistance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSHMT2 isoform 3 is expressed in cisplatin-resistant ovarian cancer under metabolic stress\u003c/h2\u003e \u003cp\u003eTo further explore the role of SHMT2 isoforms in the process of cisplatin resistance, SHMT2 expression was studied using paired cisplatin-resistant and parental SKOV3 and A2780 cells. SHMT2 expression did not show significant difference between the cisplatin-resistant SKOV3 (SKOV3/DDP) and A2780 (A2780/DDP) cells compared with their respective parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In addition, only single band representing SHMT2 isoform 1 was observed in ovarian cancer cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Single-cell RNA sequencing analysis showed that SHMT2 was mainly expressed in epithelial and mesenchymal cells, in addition, SHMT2 also expressed in endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Next, cancer-associated fibroblast (CAF) and ovarian cancer (OC) cells were extracted from fresh ovarian cancer tissues, Western blot demonstrated that CAF expressed SHMT2 isoform 1, while some OC cells expressed SHMT2 isoform 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E), suggesting that the expression of SHMT2 isoform 3 might be mainly derived from ovarian tumor epithelial cells. SHMT2 isoform 3 could be detected in some primary OC cells at the beginning, but with continuous cell culture, SHMT2 isoform 3 could not be detected. However, SHMT2 isoform 3 could be detected again under the conditions of hypoglycemia and hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Real-time RT-PCR showed that variant 1 decreased, while variant 4 decreased in OC#2 and OC#5 after 2\u0026ndash;3 passages, culture under hypoglycemic and hypoxic condition recovered the expression of variant 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). On the other hand, such fluctuation of expression of SHMT2 variants was not observed in OC#3, which did not express SHMT2 variant 4 originally (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). In the cisplatin-resistant SKOV3 (SKOV3/DDP) and A2780 (A2780/DDP) cells, SHMT2 isoform 3 could be detected in cisplatin-resistant SKOV3/DDP and A2780/DDP cells under hypoglycemic and hypoxic culture, but could not be detected in their respective parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). RT-PCR showed that hypoglycemic and hypoxic culture increased total and variant 1 SHMT2 expression in SKOV3 cells, while such induction was not observed in A2780 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-J). SHMT2 variant 1 was consistently decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ), while SHMT2 variant 4 was consistently increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-L) in cisplatin-resistant cells cultured under hypoglycemic and hypoxic condition. In general, OC can express SHMT2 variant 4 only when metabolic stress occurs, that is, under a condition of low-glucose and hypoxic, thereby expressing the SHMT2 isoform 3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDifferent effects of SHMT2 knockdown in cisplatin responsiveness in cisplatin-resistant and sensitive ovarian cancer cells\u003c/h2\u003e \u003cp\u003eIn order to explore the role of SHMT2 in cisplatin resistance, SHMT2 was knocked down using CRISP/Cas9 system in SKOV3/DDP and A2780/DDP and their respective parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Knockdown of SHMT2 increased the survival of both SKOV3 and A2780 cells exposed to cisplatin treatment. However, in SKOV3/DDP and A2780/DDP cells knockdown of SHMT2 did not show a significant difference in the viability of the cells after cisplatin exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Colony formation assay got a similar result. Knockdown of SHMT2 increased the colony number of cisplatin sensitive cells, while there was no significant change in the resistant cells under cisplatin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003eTo further understand the role of SHMT2 isoforms in cisplatin resistance, SHMT2 knockdown cells were restored with SHMT2 isoform 1 and 3, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Restoration of SHMT2 isoform 1, but not isoform 3, significantly decreased the cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) and colony formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H) of SKOV3 and A2780 cells under cisplatin exposure. SHMT2 isoform 3 increased colony formation of SKOV3 cells, but not A2780 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). Neither isoform 1 nor isoform 3 demonstrated any effects on viability and colony formation of SKOV3/DDP and A2780/DDP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003cb\u003eOpposite roles of SHMT2 isoforms in maintenance of stem cell like features of ovarian cancer cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs mentioned above, SHMT2 isoform 3 only expressed under metabolic stress situation, in order to further understand the roles of SHMT2 isoforms, a low-glucose and hypoxic cell culture environment were constructed for the following experiments. SHMT2 isoform 3, but not isoform 1, significantly promoted survival of both cisplatin-sensitive and cisplatin-resistant cells under low-glucose and hypoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). SHMT2 isoform 1 decreased, while isoform 3 increased stem cell like features of ovarian cancer cells, as identified by spheroid formation ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D), expression of stemness markers CD44 and CD133 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), as well as tumor formation rate and stem cell frequency using the tumor-limiting dilution assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSelective utilization of alternative promoters of\u003c/b\u003e \u003cb\u003eSHMT2\u003c/b\u003e \u003cb\u003ein cisplatin-resistant ovarian cancer cells under metabolic stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSHMT2 gene contains three promoters, and different variants will be transcribed due to selective utilization of different promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). The reporters containing \u0026minus;\u0026thinsp;841/+72, -841/+448, -841/+724 fragments showed highest and similar activities, while no obvious activity of reporter containing\u0026thinsp;+\u0026thinsp;82/+448 or +\u0026thinsp;520/+724 fragments was observed in both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells under normal condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), indicating that ovarian cancer cells preferentially utilized promoter 1, while promoter 2 and promoter 3 were rarely activated under normal culture condition. The activity of reporter containing \u0026minus;\u0026thinsp;841/+72 fragment significantly decreased, while the activity of reporter containing\u0026thinsp;+\u0026thinsp;82/+448 fragment significantly increased in SKOV3/DDP and A2780/DDP under hypoglycemic and hypoxic condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The enrichment of RNA Pol II pSer2 and Pol II pSer5 of SKOV3/DDP and A2780/DDP to the promoter 1 region was significantly decreased under the low-glucose and hypoxic condition, while there was no significant difference in SKOV3 and A2780 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSelective utilization of\u003c/b\u003e \u003cb\u003eSHMT2\u003c/b\u003e \u003cb\u003epromoter 2 by HIF1α and TFE3 complex\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo understand the regulation of SHMT2α by transcription factors, the specific transcription factor binding sites around promoter 2 which were associated with metabolic stress were surveyed and XBP1, HIF1α and TFE3 were screened out. Knockdown of TFE3 and HIFα in low-glucose and hypoxic condition decreased the expression of SHMT2 isoform 3, while XBP1 knockdown demonstrated no obvious effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). ChIP confirmed significant enrichment of HIF1α and TFE3 to the promoter 2 region in cisplatin-resistant cells under low-glucose and hypoxic culture condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Knockdown of TFE3 and HIFα decreased the activity of promoter 2, and knockdown of XBP1 had no significant difference to the activity of promoter 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). To further study whether there was a regulation between TFE3 and HIFα, ChIP showed that TFE3 knockdown inhibited the recruitment of HIF1α in both SKOV3/DDP and A2780/DDP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). HIF1α knockdown decreased recruitment of TFE3 in A2780/DDP, while had no effect in SKOV3/DDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Molecular docking model predicted direct interaction of HIF1α and TFE3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). DuoLink confirmed interaction of TFE3 and HIF1α in cisplatin-resistant ovarian cancer cells under the low-glucose and hypoxic condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn 1955 Thomlinson first proposed the concept of tumor hypoxia, more than 60 years of clinical and experimental evidence have shown that hypoxia is a common feature of many types of solid tumors\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. As the tumor continues to proliferate, low-glucose that often occur at the same time as hypoxia is also a common change in the tumor microenvironment (TME). This is because cancer cells are characterized by a high proliferation rate and active metabolism, and they need to consume a lot of energy to support their increased proliferation and growth rate compared to normal cells\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. When the oxygen and glucose required by cell metabolism cannot be satisfied by organism, it will change TME into a hypoxia and low-glucose situation\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, and it might cause a series of metabolic disorders\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSHMT2 is a key mitochondrial enzyme in serine catabolism that converts serine to glycine\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that high expression of SHMT2 may promote tumor proliferation in bladder cancer, rhabdomyosarcoma, esophageal cancer and diffuse large B-cell lymphoma\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. SHMT2 induces stemness of head and neck cancer, Burkitt lymphoma\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. On the contrary, SHMT2 functions as a tumor suppressor and negatively regulates proliferation and metastasis in prostate cancer \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. These data indicate that SHMT2 might function as both tumor-promoting and tumor-suppressive functions in a cell-dependent context. The current study demonstrated that SHMT2 isoforms might exert paradoxical functions in ovarian cancer cells. SHMT2 isoform 1 might function as tumor suppressive function, as it suppressed cancer stem cell (CSC)-like features including increase of cisplatin responsiveness of parent ovarian cancer cells, suppression of spheroid formation, downregulation of CSC markers. On the contrary, SHMT2 isoforms 3 might function as tumor promoting function to promote CSC-like features. However, exact mechanisms underlying discrete roles of SHMT2 isoforms in ovarian cancer require further investigation. Anyway, the current study suggested that discrete isoform expression might represent another layer of paradoxical function of SHMT2 in different cancer.\u003c/p\u003e \u003cp\u003eIn order to survive under metabolic stress, cancer cells regulate metabolism, protein synthesis and cell cycle processes by the adjustment of transcription factors\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Hypoxia is a common characteristic of the tumor microenvironment found in most solid tumors, including ovarian cancer\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Cancer cells under hypoxic stress regulate protein translation through reprogramming gene expression, leading to microenvironmental changes\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. HIF1-α is an important regulatory transcription factor in the hypoxia and low-glucose situation. Accumulating data indicate complex implication of SHMT2 in hypoxia. Co-induction of SHMT2 by HIF1α and Myc maintains cell growth by balancing the NADPH/NADP\u003csup\u003e+\u003c/sup\u003e ratios in neuroblastoma tumors upon lack of oxygen\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Hypoxia stabilizes SHMT2 lactylation, thereby promoting glycolysis and stemness of esophageal cancer cells\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Furthermore, SHMT2 regulates HIF1α expression through both enzymatic and non-enzymatic functions in gastric cancer \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Identification of selective utilization of SHMT2 alternative promoter by the combination of HIFα and TFE3 in cisplatin-resistant ovarian cancer cells in the current study further strengthen the complicated interlinkage between hypoxia and SHMT2.\u003c/p\u003e \u003cp\u003eMany genes have multiple promoters, which may even lead to different functions of the same gene. The utilization of alternative promoter produces different pre-mRNAs being transcribed from variable transcription start sites (TSSs) within a gene locus. The patterns of alternative promoter selection result in the regulation of diverse cell types, tissue types and complex developmental genes. The abnormal usage of alternative promoter plays an important role in the occurrence and development of various diseases. As a general mechanism, alternative promoter is diverse and flexible in gene expression regulation. In the current study, we reported that under hypoxia and hypoglycemia, the combination of HIFα and TFE3 leads to a reduction in the utilization of promoter 1, while the increase in the selection of promoter 2, resulting in transcriptional dysregulation and an increase in the transcription of variant 4, followed by increase of SHMT2 isoform 3 under metabolic stress conditions. In general, the high expression of SHMT2α and the low expression of SHMT2 in the hypoxia and low-glucose situation may be one of the causes of cisplatin resistance in ovarian cancer patients. However, further studies are needed to dissect, how SHMT2 isoforms exerted paradoxical functions in ovarian cancer.\u003c/p\u003e \u003cp\u003eCollectively, the current study demonstrated that ovarian cancer cells expressed multiple SHMT2 isoforms, which exerted complicated functions. Selective utilization of SHMT2 alternative promoter by HIF1α and TFE3 resulted in SHMT2 isoform expression shift, which might be implicated in adaptation of ovarian cancer cells under metabolic stress.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSHMT: Serine hydroxy methyltransferase\u003c/p\u003e\n\u003cp\u003eSAM: S-adenosylmethionine\u003c/p\u003e\n\u003cp\u003eHR: hazard ratio\u003c/p\u003e\n\u003cp\u003eCI: confidence intervals\u003c/p\u003e\n\u003cp\u003eCAF: cancer-associated fibroblast\u003c/p\u003e\n\u003cp\u003eOC: ovarian cancer\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eHuaqin Wang and Chuan Liu designed this study. Siqi Wang and Ning Liu performed all the experiments and analyzed the data. Baiqiang Li provided clinical samples and conducted pathological evaluation. Fuying Zhao and Chao Li provided the technical support. Siqi Wang and Ning Liu conceptualized and initiated the project and wrote the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJuarez-Mendez, S., et al., \u003cem\u003eSplice variants of zinc finger protein 695 mRNA associated to ovarian cancer.\u003c/em\u003e J Ovarian Res, 2013. \u003cstrong\u003e6\u003c/strong\u003e(1): p. 61.\u003c/li\u003e\n\u003cli\u003ePignata, S., et al., \u003cem\u003eTreatment of recurrent epithelial ovarian cancer.\u003c/em\u003e Cancer, 2019. \u003cstrong\u003e125 Suppl 24\u003c/strong\u003e: p. 4609-4615.\u003c/li\u003e\n\u003cli\u003ePignata, S., et al., \u003cem\u003eTreatment of recurrent ovarian cancer.\u003c/em\u003e Ann Oncol, 2017. \u003cstrong\u003e28\u003c/strong\u003e(suppl_8): p. viii51-viii56.\u003c/li\u003e\n\u003cli\u003eBookman, M.A., et al., \u003cem\u003eHarmonising clinical trials within the Gynecologic Cancer InterGroup: consensus and unmet needs from the Fifth Ovarian Cancer Consensus Conference.\u003c/em\u003e Ann Oncol, 2017. \u003cstrong\u003e28\u003c/strong\u003e(suppl_8): p. viii30-viii35.\u003c/li\u003e\n\u003cli\u003ePujade-Lauraine, E., et al., \u003cem\u003eBevacizumab combined with chemotherapy for platinum-resistant recurrent ovarian cancer: The AURELIA open-label randomized phase III trial.\u003c/em\u003e J Clin Oncol, 2014. \u003cstrong\u003e32\u003c/strong\u003e(13): p. 1302-8.\u003c/li\u003e\n\u003cli\u003eWilson, M.K., et al., \u003cem\u003eFifth Ovarian Cancer Consensus Conference of the Gynecologic Cancer InterGroup: recurrent disease.\u003c/em\u003e Ann Oncol, 2017. \u003cstrong\u003e28\u003c/strong\u003e(4): p. 727-732.\u003c/li\u003e\n\u003cli\u003eDeBerardinis, R.J., \u003cem\u003eSerine metabolism: some tumors take the road less traveled.\u003c/em\u003e Cell Metab, 2011. \u003cstrong\u003e14\u003c/strong\u003e(3): p. 285-6.\u003c/li\u003e\n\u003cli\u003ePan, S., et al., \u003cem\u003eSerine, glycine and one\u003c/em\u003e\u003cem\u003e‑\u003c/em\u003e\u003cem\u003ecarbon metabolism in cancer (Review).\u003c/em\u003e Int J Oncol, 2021. \u003cstrong\u003e58\u003c/strong\u003e(2): p. 158-170.\u003c/li\u003e\n\u003cli\u003eKim, D., et al., \u003cem\u003eSHMT2 drives glioma cell survival in ischaemia but imposes a dependence on glycine clearance.\u003c/em\u003e Nature, 2015. \u003cstrong\u003e520\u003c/strong\u003e(7547): p. 363-7.\u003c/li\u003e\n\u003cli\u003eNing, S., et al., \u003cem\u003eSHMT2 Overexpression Predicts Poor Prognosis in Intrahepatic Cholangiocarcinoma.\u003c/em\u003e Gastroenterol Res Pract, 2018. \u003cstrong\u003e2018\u003c/strong\u003e: p. 4369253.\u003c/li\u003e\n\u003cli\u003eWoo, C.C., et al., \u003cem\u003eDownregulating serine hydroxymethyltransferase 2 (SHMT2) suppresses tumorigenesis in human hepatocellular carcinoma.\u003c/em\u003e Oncotarget, 2016. \u003cstrong\u003e7\u003c/strong\u003e(33): p. 53005-53017.\u003c/li\u003e\n\u003cli\u003ePranzini, E., et al., \u003cem\u003eSHMT2-mediated mitochondrial serine metabolism drives 5-FU resistance by fueling nucleotide biosynthesis.\u003c/em\u003e Cell Rep, 2022. \u003cstrong\u003e40\u003c/strong\u003e(7): p. 111233.\u003c/li\u003e\n\u003cli\u003eCui, X., et al., \u003cem\u003eSHMT2 Drives the Progression of Colorectal Cancer by Regulating UHRF1 Expression.\u003c/em\u003e Can J Gastroenterol Hepatol, 2022. \u003cstrong\u003e2022\u003c/strong\u003e: p. 3758697.\u003c/li\u003e\n\u003cli\u003eParsa, S., et al., \u003cem\u003eThe serine hydroxymethyltransferase-2 (SHMT2) initiates lymphoma development through epigenetic tumor suppressor silencing.\u003c/em\u003e Nat Cancer, 2020. \u003cstrong\u003e1\u003c/strong\u003e: p. 653-664.\u003c/li\u003e\n\u003cli\u003eWeatheritt, R.J. and T.J. 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Platinum-based chemotherapy constitutes the first-line therapeutic regime. However, primary or acquired resistance seriously affects the survival rate of patients with ovarian cancer. Serine hydroxy methyltransferase (SHMT) catalyzes conversion of serine to glycine and is responsible for production of S-adenosylmethionine (SAM) for methylation. There are cytosolic SHMT1 and mitochondrial SHMT2 in human. Alternative promoter usage is a proteome-expanding mechanism that allows multiple pre-mRNAs to be transcribed from a single gene. The current study demonstrated that cisplatin-sensitive and cisplatin-resistant ovarian cancer cells expressed discrete SHMT2 isoforms, which was ascribed to the selective utilization of \u003cem\u003eSHMT2\u003c/em\u003e alternative promoters. SHMT2 isoforms exerted somewhat paradoxical roles in ovarian cancer cells, with tumor-suppressive role of isoform 1, and tumor-promotive role of isoform 3. In addition, the current study demonstrated that SHMT2 alternative promoter usage mediated by HIF1α and TFE3 might represent adaptive response of ovarian cancer cells to metabolic stress. Collectively, regulation of SHMT2 isoform expression via alternative promoter usage by transcription factors HIF1α and TFE3 provides a novel basis and potential drug targets for the clinical treatment of platin-resistant ovarian cancer.\u003c/p\u003e","manuscriptTitle":"TFE3 and HIF1a regulates the expression of SHMT2 isoforms via alternative promoter utilization in ovarian cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-18 06:58:01","doi":"10.21203/rs.3.rs-4981006/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"25e0f302-b5fe-441f-8232-ab079fccb9f5","owner":[],"postedDate":"October 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36916983,"name":"Biological sciences/Cancer/Gynaecological cancer/Ovarian cancer"},{"id":36916984,"name":"Biological sciences/Drug discovery/Drug regulation"}],"tags":[],"updatedAt":"2025-03-18T07:10:30+00:00","versionOfRecord":{"articleIdentity":"rs-4981006","link":"https://doi.org/10.1038/s41419-025-07445-y","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-03-17 04:00:00","publishedOnDateReadable":"March 17th, 2025"},"versionCreatedAt":"2024-10-18 06:58:01","video":"","vorDoi":"10.1038/s41419-025-07445-y","vorDoiUrl":"https://doi.org/10.1038/s41419-025-07445-y","workflowStages":[]},"version":"v1","identity":"rs-4981006","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4981006","identity":"rs-4981006","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}