Identification of TRPM2 as a prognostic factor correlated with immune infiltration in ovarian cancer

Ovarian cancer (OC) is one of the most common gynecologic malignant cancers in the world, with most of the patients diagnosed at an advanced stage and having poor outcomes [1]. It is the seventh most diagnosed cancer among women and the five-year survival rate is 46% [2]. Currently, the treatment for OC containing surgery, chemotherapy, immunotherapy, and targeted therapy such as Poly (ADP-ribose) polymerase (PARP) inhibitors, have improved the prognosis of OC patients. However, the survival rate remains low due to the complex molecular mechanisms and tumor microenvironment [3].

The transient receptor potential (TRP) is superfamily of ion channels involved in the modulation of physiological function and tumor progression [4]. TRP family contains TRPC, TRPV, TRPM, TRPP, TRPML, TRPA, and TRPN [5]. Among these, TRPM2 is a non-selective cationic channel, consisting of an approximate 800 amino acid N-terminal area, a C-terminal coiled-coil loop, three extracellular loops root in six transmembrane domains (S1-6), and ADP-ribose (ADPR). The N- and C-terminals of TRPM2 are located in intracellular, and the pore-forming loop is situated on S5 and S6 [6]. TRPM2 has been proved to increase the concentration of intracellular Ca^+ 2, which provides positive feedback for TRPM2 activation [7]. Additionally, TRPM2 also plays an important role in the reactive oxygen species (ROS) pathway in physiological and pathological activities [8, 9]. Early research illustrated that TRPM2 promoted cell apoptosis in the endothelial or neuronal cells and responded to oxidative stress in male-specific ischemic injury by modulating Ca^+ 2 [10]. Nowadays, some reports demonstrate that TRPM2 is correlated to innate immunity regulation and inflammation, resulting from the presence of TRPM2 on the cells of monocytic lineage, lymphocytes, and neutrophils [11‐13].

TRPM2 is also found to be involved in tumor development. Latest studies reported that TRPM2 was highly expressed on various cancer including bladder, breast, head and neck, lung, pancreatic, prostate, neuroblastoma, which might hint that TRPM2 could promote tumor progression [5, 14]. TRPM2 could sustain tumor cell viability by activating transcription factors such as hypoxia-inducible factor-alpha (HIF-1/2α), cAMP-responsive element-binding protein (CREB), and nuclear factor (erythroid-derived 2)-related factor-2 (Nrf2), subsequently modulating the downstream pathways including mitochondrial function maintenance, ATP production, cell autophagy, DNA repair, cellular bioenergetics, and ROS production [15‐20]. Inhibition of TRPM2 could cause tumor cell death, and promote the tumor drug sensitivity in T cell leukemia, gastric cancer, breast cancer cells, and neuroblastoma, to chemotherapeutic agents [16‐19, 21‐23]. TRPM2 inhibition also leads to increased DNA damage and cytotoxicity in triple-negative or estrogen-receptor-positive breast cancer, reducing survival and restrain migration of tongue carcinoma, and enhancing the radiation sensitivity in T-cell leukemia [14]. Thus, TRPM2 is a potential target for anti-cancer therapy in various tumors. However, the role and underlying mechanisms of TRPM2 in OC are still unclear.

In the present study, we found that TRPM2 was significantly highly expressed in most tumors including OC based on The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx). Survival analysis indicated that high expression of TRPM2 predicted poor survival in OC. Subsequent bioinformatics analyses found that TRPM2 expression was positively correlated with immune-associated pathway, immune score, majority immune checkpoints, and pyroptosis. These results suggested that TRPM2 could be a poor prognostic predictor in OC, which was possibly modulated by immune response suggested by the association between TRPM2 expression and the tumor immune microenvironment.

Ovarian cancer (OC) is the most lethal gynecological malignancy in the world with most patients diagnosed at an advanced stage [1], of which the survival rates remain unsatisfactory for drug resistance and immunosuppressive environment [3]. New therapeutic targets are crucial to improving the survival rate for ovarian cancer patients. Through a series of bioinformatics analyses based on the publicly accessible database, we explored the mRNA expression of TRPM2 in pan-cancer and corresponding normal tissues, and the correlation between TRPM2 expression and survival prognosis and immune microenvironment. Our results showed that TRPM2 was upregulated in most tumors containing ovarian cancer, and increased TRPM2 expression was correlated with poor OS and PFI in patients with OC. Additionally, upregulated TRPM2 expression was positively associated with various immune cell infiltration and immune checkpoints expression. Besides, a close correlation was observed in TRPM2 expression and M2 macrophage, pyroptosis-related genes. Our study demonstrated that TRPM2 was a hazard factor for OC patients which might result from immune regulation, and could be a potential prognosis-predict biomarker for ovarian cancer patients.

TRPM2 is a member of the TRPM ion channel subfamily. Previous reports have proved that TRPM2 promoted tumor progression via multiple mechanisms, such as drug resistance, autophagy, and immunosuppression [14]. Similar results were observed in previous reports, suggesting TRPM2 might act as a risk factor in various tumors through some kind of universal effect [5, 14]. It was reported that TRPM2 sustained cell viability, restore mitochondrial function, reduce reactive oxygen species (ROS) in neuroblastoma, and lead to NLRP3 inflammasome activation [18, 30]. TRPM2 was also proved to preserve gastric cancer cell survival via the JNK-signaling pathway [23]. In this study, we observed abnormal expression of TRPM2 expression in most tumors and survival correlation in some tumors containing ovarian cancer.

The tumor microenvironment is responsible for tumorigenesis and progression, and affects the efficiency of immunotherapy to a certain extent [31]. Adaptive immune cells, including T cells and B cells, are thought to be tumor-killing effector cells, while multiple innate immune cells are thought to play an immunomodulatory function in the tumor microenvironment [32]. Previous studies have proved that TRPM2 existed on monocytes, macrophages, lymphocytes, and neutrophils [11, 12]. In our study, we utilized the GSEA method to analyze the biological process and KEGG pathways. The outcomes demonstrated that high TRPM2 expression was closely related to immune-related pathways such as adaptive immune response, neutrophil mediated immunity, Th1 and Th2 cell differentiation, Th17 cell differentiation, and Natural killer cell mediated cytotoxicity. Additionally, we found that TRPM2 had a positive correlation with multiple immune cells including B cell, T cell, macrophage, neutrophil, and myeloid dendritic cell in OC. We observed that TRPM2 had a stronger correlation with innate immune cells than with adaptive immune cells. In the subsequent correlation analysis of immune cell markers, we also observed a stronger correlation between TRPM2 and innate cell markers and immune regulatory cell markers such as CD86, CSF1R for monocyte, CD11b for neutrophil, CD11c for dendritic cell, and FOXP3 for Treg. The results indicated TRPM2 might mainly participate in immune regulation instead of the direct immune killing effect. Besides, we also observed that TRPM2 was positively correlated with the biomarker genes of M2 macrophages, not M1 macrophages. M1 macrophages are usually considered as tumor suppressor cells, while M2 macrophages are thought to be tumor promoter cells with immunosuppression function [26]. Thus, we speculated that TRPM2 might be involved in the process of promoting tumor progression mediated by M2 macrophages. Together, these results indicated that TRPM2 played an essential role in regulating ovarian cancer immune infiltrating cells.

Immunotherapy, especially immune checkpoints inhibitors, has drawn public attention in the field of cancer treatment [31]. For example, anti-programmed cell death-1 (anti-PD-1), anti-programmed death-ligand 1 (anti-PD-L1), or anti-cytotoxic T-lymphocyte associated protein 4 (anti-CTLA4) have been generally used for non-small cell lung cancer (NSCLC), bladder cancer, heck and neck squamous cell cancer, renal cell carcinoma, melanoma, classical Hodgkin lymphoma, and ovarian cancer [1, 33, 34]. Our result illustrated TRPM2 was significantly positively correlated with ICOSLG, TNFRSF18, CTLA4, CD86, TNFRSF4, CD40, TIGIT, LAIR1, CD48, TNFRSF14, CD27, LGALS9, HAVCR2, KIR3DL1, TMIGD2, CD40LG, ICOS, IDO2, IDO1, PDCD1LG2, and CD80 in OC. This result might explain the high level of immune cell infiltration but the low survival rate of OC patients with high TRPM2 expression. Among the immune checkpoints above, TIGIT is a promising new immunotherapy target, upregulated in activated T cells, natural killer cells, and regulatory T cells [35]. The TIGIT inhibitor tiragolumab has shown effects in multiple tumors [36]. Our study showed that increased TRPM2 was positively correlated to TIGIT, suggesting the potential function of TRPM2 in TIGIT related pathways. Based on the results above, we hope our study on TRPM2 could be helpful for immunotherapy in OC in the future.

Besides, previous reports suggest that TRPM2 can mediate inflammasome-dependent pyroptosis by activating ROS-dependent NLRP3 [27, 28]. Our results also showed that the correlation analysis showed a strong positive correlation between TRPM2 and pyrolysis-related genes including NLRP3, NLRC4, NOD2, NOD1, CASP1, CASP5, IL1B, and GSDMD. Real-time quantitative PCR results validated that patients with high TRPM2 expression had both higher NLRC, NLRP3, NOD2, NOD1, GSDMD, CASP5 and CASP1 (Fig. 6J), which was consistent with our results by bioinformatics analysis. Pyroptosis, often triggered by perturbations of extracellular or intracellular homeostasis related to innate immunity, has been proved to play a dual role in promoting and inhibiting tumor progression in multiple tumors [37, 38]. Combined with the correlation between TRPM2 and innate immunity, we speculated that TRPM2 might be involved in the innate immune-related pyroptosis pathway in OC, resulting in the poor survival of OC patients.

It should be emphasized that there are some limitations to our study. Our study is based on data analysis to mine potential data and functional biomolecules, providing a potential study direction for future research. Experimental studies are needed to verify our results based on designing PCR, Western blotting, and immunohistochemistry tests. More clinical data is needed to prove the relationship between TRPM2 and survival.

In conclusion, our result found that TRPM2 was upregulated in OC, and high expression of TRMR2 was related to poor prognosis in OC patients. In addition, TRPM2 was mainly related to immune pathways, with a positive correlation with immune cells especially innate immune cells, immune checkpoints, and pyroptosis. It may regard as a new prognostic predictor for OC patients and could be a potential therapeutic target based on further research.