The oncogenic roles of TRPM ion channels in cancer

Transient receptor potential (TRP) proteins are a diverse family of ion channels present in multiple types of tissues. They function as gatekeepers for responses to sensory stimuli including temperature, vision, taste, and pain through their activities in conducting ion fluxes. The TRPM (melastatin) subfamily consists of eight members (i.e., TRPM1–8), which collectively regulate fluxes of various types of cations such as K+, Na+, Ca2+, and Mg2+. Growing evidence in the past two decades indicates that TRPM ion channels, their isoforms, or long noncoding RNAs encoded within the locus may be oncogenes involved in the regulation of cancer cell growth, proliferation, autophagy, invasion, and epithelial–mesenchymal transition, and their significant association with poor clinical outcomes of cancer patients. In this review, we describe and discuss recent findings implicating TRPM channels in different malignancies, their functions, mechanisms, and signaling pathways involved in cancers, as well as summarizing their normal physiological functions and the availability of ion channel pharmacological inhibitors.


| TRPM PROTEINS IN MALIGNANCIES
Accumulating evidence has demonstrated the oncogenic roles of several TRPM proteins as shown through gene knockdown, knockout, or overexpression methodologies both in human cancer cell lines and experimental animal models, while others may function as tumor suppressors. In this review, we discuss and summarize the association of each TRPM ion channel with various cancers, and their potential as therapeutic targets in human malignancies. The normal physiological functions of each TRPM are first introduced before their relevance with cancers are described and discussed. In addition, the list of pharmacological inhibitors against each TRPM protein member is listed in Table 1. The inhibitors were selected based on evidence showing TRPM currents or ion flux activities were reduced by the treatment with each antagonist, and IC 50 value of ≤10 μM (except TRPM1, TRPM5, and TRPM6). The 10 μM cut-off was used to shortlist the inhibitors as it was considered to represent both marginally and highly active inhibitors (Koutsoukas et al., 2013;Mervin et al., 2015).

| TRPM1: Reduced expression in melanoma progression
The founding member of the TRPM family, TRPM1, was initially identified as a gene whose transcript expression was downregulated in a metastatic mouse melanoma cell line (Duncan et al., 1998). The ion channel is also known as melastatin because of its expression in melanin-producing melanocytes (Duncan et al., 1998;Zhiqi et al., 2004).
TRPM1 is a nonselective cation channel (permeable to Na + and Ca 2+ ) that is thought to be constitutively open and its closure is activated by heterotrimeric G-proteins . Physiologically, TRPM1 plays crucial roles in triggering the depolarization of ON-bipolar cells of the retina through the metabotropic glutamate receptor 6 (mGluR6) cascade, enabling synaptic transmission of light in the eye and maintaining normal vision (Koike et al., 2010). Indeed, F I G U R E 1 Normal physiological functions of activated TRPM ion channels. In TRPM1, glutamate released from rods binds to the metabotropic glutamate receptor 6 (mGluR6) of ON-bipolar cells of the retina, subsequently activates heterotrimeric G-protein subunits (G o α and Gβγ) and causes the closure of TRPM1 channel, which allows the visual signal to be transmitted in low-light condition. Closure of the channel prevents the influx of Na + and Ca 2+ ions. In TRPM2, the vascular endothelial growth factor (VEGF)-induced angiogenesis activates reactive oxygen species (ROS) to release adenosine diphosphate (ADP)-ribose. TRPM2 activated by ADP-ribose allows the influx of Ca 2+ ion. This channel has sensitivity toward heat (body temperature). TRPM3 is activated by steroid pregnenolone sulphate (PS), allowing influx of Ca 2+ ion. TRPM4 is activated by increased concentration of intracellular Ca 2+ [Ca 2+ ] I which allows the influx of monovalent ions only such as K + and Na + ion. TRPM5, another monovalent cation-selective channel activated by increased [Ca 2+ ] I , allows the influx of Na + , K + , and Cs + . TRPM6 is a channel kinase with a TRP channel permeable to Mg 2+ and Ca 2+ . The channel is activated by depletion of Mg 2+ or autophosphorylation of its Ser/Thr-rich kinase domain. TRPM7 is another channel kinase that allows the influx of Mg 2+ or Ca 2+ . TRPM8 is sensitive to increased levels of intracellular pH, and it is permeable to Ca 2+ , Na + , and K + . TRPM8 is activated by cold temperatures and chemical cooling compounds such as menthol and eucalyptol [Color figure can be viewed at wileyonlinelibrary.com] T A B L E 1 Summary of TRPM inhibitors, types of cells examined and IC 50 values   TRPM  member  Inhibitors  Types of cells examined  IC 50 Effects on other ion channels a TRPM1 Voriconazole ON-bipolar cells of mice injected with 0.24 mg/g body weight voriconazole  90 ± 4% inhibition of capsaicin-activated currents b Inactive against mGluR6; 92.3 ± 6.3% inhibition against TRPM3 TRPM2 2-APB TRPM2-overexpressing HEK293 cells (Togashi et al., 2008)~1 µM Not tested; known to inhibit other TRPC and TRPM (TRPM3, TRPM7, TRPM8) channels Curcumin TRPM2-overexpressing HEK293T cells (Kheradpezhouh et al., 2016)~5 0 nM Not tested; known to inhibit other CRAC channels with 10-100 times higher IC 50 Scalaradial TRPM2-overexpressing HEK293 cells (HEK293-TRPM2); Rat insulinoma cells (INS-1) (Starkus et al., 2017) 210 nM (HEK293-TRPM2); 330 nM (INS-1) Inactive against CRAC, TRPM4, and TRPV1. Inhibited TRPM7 (IC 50 : 760 nM) 2,3-dihydroquinazolin-4(1H)-one derivative (compound D9) TRPM2-overexpressing HEK293 cells  3.7 µM Inactive against TRPM8 Adenosine 5′-diphosphoribose analogues (compounds 7i and 8a) TRPM2-overexpressing HEK293 cells (Luo et al., 2018) 5.7 μM (compound 7i) 5.4 μM (compound 8a) Inactive against TRPM7, TRPM8, TRPV1, and TRPV3
In malignancies, studies have focused on the association of reduced TRPM1 expression in melanoma progression. TRPM1 transcripts were commonly expressed in benign melanocytic proliferations but lost in 51% of primary cutaneous melanomas, 53% of invasive melanomas, and 100% of melanoma metastases, indicating a gradual loss of TRPM1 expression during progression to invasive and metastatic disease (Deeds, Cronin, & Duncan, 2000). Subsequent studies utilizing in situ hybridization demonstrated that higher TRPM1 mRNA expression was significantly associated with prolonged disease-free survival (DFS) (p < 0.0001), and that TRPM1 status together with tumor thickness could identify patients with a high risk of metastatic disease (Duncan et al., 2001).
These findings were also reproduced in a recent study demonstrating loss of TRPM1 in the progression of melanoma to a metastatic phenotype that was significantly associated with worse DFS (p < 0.0001) and overall survival (OS) (p < 0.0001; Brozyna et al., 2017). Moreover, TRPM1 autoantibodies targeting exons 9 and 10 of human TRPM1 gene were present in melanoma-associated retinopathy, a paraneoplastic syndrome associated with melanomas (Duvoisin et al., 2017). Interestingly, intron 6 of TRPM1 encodes the microRNA miR-211, which has been shown to disrupt melanoma metastasis through transcriptional suppression of multiple oncogenes (Levy et al., 2010), suggesting a miR-regulated tumor suppressive mechanism linked to the TRPM1 locus. 2.2 | TRPM2: Oncogenic nuclear localization and the long noncoding RNA TRPM2-AS TRPM2 (permeable to Ca 2+ , Na + , and K + ) is gated by adenosine diphosphate (ADP) ribose, and it is potently activated by reactive oxygen species (ROS) (Kolisek, Beck, Fleig, & Penner, 2005). In neurons, TRPM2 is required to generate heat sensitivity (Tan & McNaughton, 2016), and the ion channel also limits fever and drives hypothermia (Song et al., 2016). TRPM2 is also expressed in various immune cells including monocytes, macrophages, neutrophils, dendritic cells, and lymphocytes, playing crucial roles in both innate and adaptive immunity but its excessive activity in immune cells contributes to inflammatory and autoimmune diseases (Syed Mortadza, Wang, Li, & Jiang, 2015). For instance, TRPM2 was required by the type II transmembrane protein CD38 to induce chronic inflammation and increase susceptibility to experimental lupus shown in Cd38 ─/─ and Trpm2 ─/─ murine models (Garcia-Rodriguez et al., 2018). TRPM2 also plays other crucial physiological roles including regulating neuronal growth and survival. The ion channel's expression was increased by neurotoxins (1-methyl-4phenylpyridinium) through ROS production that subsequently promoted death of neuronal cells , and Ca 2+ influx via TRPM2 promoted ROS production leading to death of rat cortical neuronal cells (Kaneko et al., 2006), suggesting a role of TRPM2 in neuronal degeneration and Parkinson's disease. SH-SY5Y cells in nude-FOXn1 nu mice; Bao et al., 2016). The mechanism was reported to be through TRPM2-mediated Ca 2+ entry that increased mitochondrial ROS and cellular bioenergetics. In gastric cancer cells (AGS and MKN-45), TRPM2 knockdown downregulated the c-Jun N-terminal kinase (JNK) signaling pathway that subsequently impaired autophagy and mitophagy, leading to mitochondria defects and cell death (Almasi et al., 2018). The authors also found that TRPM2 downregulation sensitized gastric cancer cells to paclitaxel and doxorubicin. In pancreatic ductal adenocarcinoma (PDAC), mutated (p = 0.010) or increased (p = 0.042) TRPM2 expression was significantly associated with inferior patient survival, and TRPM2 overexpression in PDAC cells (PANC-1) induced cell proliferation, invasion, and metastatic capabilities (Lin et al., 2018).
Inhibition of TRPM2 also induced cell cycle arrest resulting in the death of T-cell leukemia cells (Jurkat; Klumpp et al., 2016).
As both oncogenic and potentially tumor suppressive roles of TRPM2 have been demonstrated, depending on cancer types, care will need to be taken when considering TRPM2 as a bona fide target for cancer therapy. Moreover, as numerous normal cell populations express TRPM2 and the ion channel regulates a wide variety of important processes, targeting TRPM2 might result in organ toxicities or disruption of normal physiological processes.
We suggest the following future investigations to elucidate its potential as a cancer therapeutic target: (i) TRPM2 was found to be located in the nuclei of prostate cancer cells (PC-3 and DU-145) but not in benign prostate cells (BPH-1) where it showed membranous or cytoplasmic localization . In these cells, abundant TRPM2 was translocated into the nucleus that subsequently inhibited nuclear ADP-ribosylation, and the authors proposed that TRPM2 might have enzymatic function in the nucleus. In line with this observation, TRPM2 deletion inhibited migratory abilities and induced apoptosis of oral squamous carcinoma cells (SCC-9) where a significant amount of the protein was present in nuclei of SCC9 cells in contrast with its absence in the nuclei of nonmalignant human tongue samples (Zhao et al., 2016).
Therefore, the oncogenic roles of TRPM2 might be attributable to its activities dependent on its altered subcellular localization, and this warrants further investigation.
(ii) Instead of targeting TRPM2 per se, TRPM2-AS represents an alternative therapeutic avenue as the lncRNA is expressed at low levels in healthy tissues but it is upregulated in various human cancer cells (melanoma, breast, and lung cancers; Orfanelli et al., 2008), as well as prostate cancer and NSCLC (Huang et al., 2017a;Ma et al., 2017). Thus, investigations into TRPM2-AS-specific inhibitory agents may be desirable in the context of TRPM2-AS-associated cancer therapy.

| TRPM3: Promotes growth and autophagy in clear cell renal cell carcinoma (ccRCC)
TRPM3 (permeable to Ca 2+ , Zn 2+ , and Mn 2+ ) was the most recently identified ion channel of the TRPM family, and is activated by steroid or heat depending on the cell type. In pancreatic beta cells, TRPM3 is activated by the steroid pregnenolone sulfate that leads to Ca 2+ influx and increased insulin secretion (Wagner et al., 2008). Through induction by pregnenolone sulfate, TRPM3 regulates proliferation and contractility of vascular smooth muscle cells (Naylor et al., 2010), and TRPM3 mRNA has been shown to be upregulated by a set of nine circular RNAs in coronary artery disease patients (Pan et al., 2017).
In somatosensory neurons, TRPM3 functions as a nociceptor channel to detect noxious heat (Vriens et al., 2011) and to evoke pain through neuropeptide release from nerve terminals in the skin (Held et al., 2015). Owing to its involvement in triggering the activities of pain-sensing nerve cells, antagonizing TRPM3 has been proposed to be a potential analgesic strategy (Dembla et al., 2017). In osteoblast cells, downregulation of TRPM3 expression by puerarin (an isoflavonoid phytoestrogen) or siRNAs promoted their proliferation and differentiation, suggesting TRPM3 as a potential therapeutic target in osteoporosis (Zeng et al., 2018 miR-204 is expressed from intron 6 of the human TRPM3 gene, suggesting that they both share common regulatory mechanisms. miR-204 has been frequently documented to play tumor suppressive roles in various malignancies including gastric cancer (Sacconi et al., 2012), head and neck (Lee et al., 2010), and other tumor types (Li, Pan, & Li, 2016). miR-204 inhibited the translation of its host gene TRPM3 and the tumor suppressor miR-204 might share common expression regulators as miR-204 downregulation in gliomas was due to hypermethylation of TRPM3 promoter (Ying et al., 2013), and the expression of miR-204 was significantly associated with TRPM3 expression (Courboulin et al., 2011). Interestingly, miR-204 expression was positively associated with two shorter isoforms of TRPM3 but not with the full-length protein in ccRCC (Mikhaylova et al., 2012 (Launay et al., 2002;Launay et al., 2004). TRPM4 is expressed in numerous tissues including the central nervous system where it contributes to myogenic vasoconstriction of cerebral arteries (Earley, Waldron, & Brayden, 2004), and in various immune cells (e.g., dendritic, mast, and Th1 cells) where it mainly promotes their migration (Barbet et al., 2008;Weber, Hildner, Murphy, & Allen, 2010).
TRPM4 has been implicated in numerous cardiovascular disorders. Knockdown of TRPM4 via siRNA enhanced angiogenesis after ischemic stroke (Loh et al., 2014). TRPM4 regulates the development of cardiac hypertrophy (Kecskes et al., 2015) and is capable of limiting hypertension (Mathar et al., 2010). In addition, mutations of TRPM4 have been found in long QT syndrome (Hof et al., 2017) and progressive familial heart block type I (Daumy et al., 2016) where the latter disease having a gain-of-function TRPM4 mutation. One of the pathogenic mechanisms contributing to cardiac disorders is the presence of mutant TRPM4 proteins with altered TRPM4 half-life compared to the wild-type TRPM4 protein (Bianchi, Ozhathil, Medeiros-Domingo, Gollob, & Abriel, 2018). In rat models with stroke, TRPM4 inhibition demonstrated tissue salvage and improved blood-brain barrier integrity after ischemic stroke reperfusion, suggesting the use of TRPM4 inhibitors for early stroke reperfusion .
TRPM4 consists of at least two isoforms, TRPM4a and TRPM4b, the latter being the long form that has been referred to as TRPM4 since 2004 (Launay et al., 2004). Both TRPM4 and TRPM5 are activated by the rise of intracellular Ca 2+ and can be activated by heat (Talavera et al., 2005). However, both channels share some distinct features in which TRPM4 is expressed in wide variety of human tissues, whereas TRPM5 expression is mostly restricted in taste receptors; the temperature sensitivity of TRPM5 underlies the enhanced sweetness perception at high temperatures when this receptor is activated (Talavera et al., 2005). Nonetheless, it has recently been shown that TRPM5 is not solely responsible for TRPmediated taste signaling and that both TRPM4 and TRPM5 are required for taste transduction to detect bitter, sweet, or umami stimuli (Dutta Banik, Martin, Freichel, Torregrossa, & Medler, 2018).
In terms of its role in malignancy, growing evidence has shown TRPM4's association with prostate cancer. TRPM4 was identified as one of five candidate driver genes involved in androgen-independent prostate cancer (characterized by the resistance to the standard androgen-deprivation therapy; Schinke et al., 2014). TRPM4 levels were overexpressed in prostate cancer tissues compared to nonmalignant or benign prostate tissues at both the mRNA (Schinke et al., 2014) and protein levels (Berg et al., 2016;Holzmann et al., 2015). TRPM4 was weakly expressed in nonmalignant or benign prostatic hyperplasia cells, but moderately or strongly expressed in prostatic intraepithelial neoplasia and prostate cancer (Holzmann et al., 2015). Increased TRPM4 protein expression was also significantly associated with higher risk of biochemical recurrence in prostate cancer patients (p = 0.043; Berg et al., 2016).
Functionally, in prostate cancer cell lines (DU145 and PC3), TRPM4 knockdown by siRNA conferred decreased migration without effects on proliferation (Holzmann et al., 2015). In DU145 cells, this might be due to TRPM4 allowing large Na + influx, leading to depolarization of the membrane potential that subsequently decreased the driving force for Ca 2+ entry and reduced storeoperated Ca 2+ entry (SOCE) (Holzmann et al., 2015). In a recent independent study by Sagredo et al. (2018) Gene expression profiling also identified upregulation of TRPM4 transcripts in cervical cancer (Narayan et al., 2007) and diffuse large B-cell lymphoma (DLBCL; Suguro et al., 2006

| TRPM5: A potential oncochannel in melanoma
Similar to its structurally closest TRPM family member (i.e., TRPM4), TRPM5 is a monovalent cation-selective channel (permeable to Na + and K + ) activated by increased [Ca 2+ ] I but itself is impermeable to Ca 2+ . The ion channel is a taste signaling receptor for response to sweet, bitter, and umami taste (Damak et al., 2006).
TRPM5 is also expressed in olfactory epithelium where it is required to trigger responses to odorants and pheromones (Lemons et al., 2017). Additionally, TRPM5 is present in pancreatic cells where it promotes insulin secretion and TRPM5 SNPs are associated with increased prediabetic phenotypes including plasma glucose, defects in insulin secretion, and decreased insulin sensitivity (Vennekens, Mesuere, & Philippaert, 2018). Induction of TRPM5 through the sweetening food additive steviol glycosides (SGs) has recently been proposed to treat type 2 diabetes (Philippaert et al., 2017). SGs potentiate TRPM5 channel activities that subsequently activate glucose-induced insulin secretion from pancreatic islets, and long-term SGs consumption prevents the development of highfat-diet-induced diabetes in mice in a TRPM5-dependent manner (Philippaert et al., 2017).
TRPM5 mRNA was found to be highly expressed in Wilms' tumor and rhabdomyosarcoma (Prawitt et al., 2000). In contrast, TRPM5 transcript was significantly lower in bladder cancer tissues compared to normal bladder (p = 0.003) but TRPM5 protein was not detected in the bladder tissues of either cancer patients or control subjects (Ceylan et al., 2016). Additionally, SNP present in TRPM5 (CG or GG genotype of rs2301696; intron) was reported to be significantly associated with decreased risk of childhood leukemia compared with the CC genotype (p = 0.004; Han et al., 2012).
In publicly-available databases, high TRPM5 mRNA expression was significantly associated with shorter survival in melanoma   (Chubanov et al., 2016). TRPM6 mutations cause familial hypomagnesemia with secondary hypocalcemia, and abnormal renal magnesium excretion (Katayama et al., 2015;Walder et al., 2002) as well as insulin resistance (Nair et al., 2012). heteromer (Zhang et al., 2014a). Such heteromerization leads to further functional diversification and assessment of both TRPM6 and TRPM7 oncogenic properties might also need to consider the presence and relevance of TRPM6/7 heteromers especially when both channels are expressed in the same cancer cells.
2.7 | TRPM7: An oncochannel in numerous cancer types TRPM7, the closest homolog to TRPM6, is also another channel kinase that enables the entry of Mg 2+ and Ca 2+ , and it is crucial for magnesium absorption (Ryazanova et al., 2010). Apart from maintaining whole body Mg 2+ homeostasis, TRPM7 plays diverse roles including axonal growth and maturation of hippocampal neurons (Turlova et al., 2016), mediates the mineralization of craniofacial hard tissues (Nakano et al., 2016), as well as regulating mast cell degranulation and release of histamine through its kinase activity (Zierler et al., 2016).  (Leng et al., 2015). Conversely, treatment of glioblastoma cells (U87) with naltriben, a pharmacological activator of TRPM7, induced TRPM7-like current via Ca 2+ influx, and enhanced migration and invasion but not viability and proliferation of U87 cells.
These were in conjunction with increased activation of proteins involved in MAPK/ERK but not of PI3K/AKT signaling pathway (Wong, Turlova, Feng, Rutka, & Sun, 2017).
In aggressive PDAC cells (PANC-1 and MIA PaCa-2), TRPM7 mediated the entry of Mg 2+ and was required for cell invasion without effects on cell viability as shown by TRPM7 knockdown by siRNA or shRNA (Rybarczyk et al., 2017). Mg 2+ entry was reported to be associated with the invasiveness of these cells and MMP secretions, and higher expression levels of TRPM7 in primary human PDAC tumors were associated with metastasis of PDAC cells that disseminated from the pancreas to lymph node. The authors proposed that Mg 2+ entry induced heat-shock protein 90α (Hsp90α) secretion that in turn stabilized both pro-MMP2 and urokinase plasminogen activator pathway that subsequently promoted degradation of extracellular matrix and PDAC cell invasiveness.
In breast cancers, higher TRPM7 mRNA levels were associated with significantly inferior recurrence-free survival (p = 0.042) and For other cancer types, readers are directed to a recent review on TRPM7 in malignancies (Yee, 2017

| TRPM8: Oncogenic cytoplasmic localization and short isoforms
TRPM8 is a thermally-regulated ion channel (permeable to Ca 2+ , Na + , and K + ) that it is activated by cold temperatures and chemical cooling compounds such as menthol and eucalyptol (Liu et al., 2013). Its roles in somatosensory neurons have been well-established where it primarily functions to detect cold temperatures (Bautista et al., 2007) and is responsible for normal eye blinking in mice (Quallo et al., 2015). In addition, TRPM8 is highly expressed in prostate tissues where the ion channel functions as testosterone receptor, suggesting a role in regulating androgen responses (Asuthkar et al., 2015a).
Activities of TRPM8 were required for the chemotaxis and migration of glioblastoma cells (T98G and U-87MG) as shown by siRNA-mediated downregulation of TRPM8, pharmacological inhibition by the TRP channel blocker BCTC or TRPM8 activation by icilin (Klumpp et al., 2017). The authors reported that TRPM8 promoted migration of these glioblastoma cells possibly through activation of Ca 2+ -regulated K + channel, that is, big conductance (BK) K + channel. The same study also demonstrated that TRPM8 TRPM8 was also significantly overexpressed in osteosarcoma at both mRNA and protein levels associated with higher clinical stage (p = 0.007), distant metastasis (p = 0.030), worse OS (p = 0.008), and DFS (p = 0.008; Zhao & Xu, 2016). In addition, TRPM8 appears to play important roles in the following other cancer types: (i) In pancreatic cancer, where TRPM8 silencing induced replicative senescence of pancreatic adenocarcinoma cell lines BxPC-3 and PANC-1 (Yee et al., 2012); (ii) TRPM8 was highly expressed in breast cancer cell lines compared to normal breast epithelial cells and the protein promoted breast cancer metastasis (Liu et al., 2014). Notably, TRPM8 channel activity in breast cancer might be hormone-dependent. TRPM8 expression was regulated by estrogen receptor alpha (ERα) and estrogens in MCF7 cell line, and its overexpression in breast adenocarcinoma correlated with estrogen receptor status of the tumors (Chodon et al., 2010). However, a recent study (Yapa et al., 2018) demonstrated that TRPM8 was not expressed in MDA-MB-231 cells, and the authors highlighted that applicability of TRPM8 as a therapeutic target in breast cancers might be limited due to absence of TRPM8 transcript in four of seven (57.1%) breast cancer cell lines investigated; (iii) Menthol-induced TRPM8 augmented the migration and invasion capabilities of oral squamous carcinoma cell lines, HSC3 and HSC4 (Okamoto, Ohkubo, Ikebe, & Yamazaki, 2012). Conversely, TRPM8 was expressed in the human bladder cancer cells (T24) and induction of TRPM8 activity by menthol promoted death of T24 cells (Li, Wang, Yang, Wang, & Li, 2009). These apparent opposing roles of TRPM8 might be due to the differential expression of TRPM8 fulllength or isoforms, and it warrants future investigations.
The cryo-EM structure of the full-length TRPM8 derived from collared flycatcher has recently been revealed whereby the binding site for its activator menthol is located within a voltage-sensor-like domain, providing molecular cues of cold and menthol sensation (Yin et al., 2018).  .

| INHIBITORS OF TRPM CHANNELS
In terms of TRPM2 inhibitors, the plant-derived polyphenol in turmeric spice, curcumin, inhibited TRPM2-mediated elevation of cytosolic [Ca 2+ ] in rat hepatocytes suggesting that the hepatoprotective effects of curcumin from oxidative stress might be partially attributable to inhibition of TRPM2 channel activities (Kheradpezhouh, Barritt, & Rychkov, 2016). The authors also reported that curcumin inhibited activation of TRPM2 in HEK293T cells overexpressing human TRPM2 (IC 50 :~50 nM). The antitumor properties of curcumin have been welldocumented and it inhibits numerous cancer cell signaling pathways including NF-κB, STAT3, PI3K/AKT/mTOR, and Wnt/β-catenin pathways, furthermore it potentiates the effects of chemotherapeutic agents and radiation against multiple tumor types Liu, Wang, & Li, 2018a;Tian et al., 2017). This nutraceutical is capable of interacting with the targets modulated by a wide variety of FDA-approved drugs against cancer (Kunnumakkara et al., 2017), and TRPM2 is thus unlikely to be its sole target in cancer cells that express high levels of TRPM2.
The phenanthrene derivative compound 9-phenanthrol has been the most commonly used TRPM4 inhibitor to investigate the ion channel's roles in physiology and autoimmune or cardiovascular diseases; however, it lacks potency with a reported IC 50 of 20 μM (Grand et al., 2008). Flufenamic acid (FFA) and glibenclamide have also been shown to inhibit TRPM4 but both compounds have yielded even lower potency or specificity than 9-phenanthrol (Guinamard, Hof, & Del Negro, 2014). Recently, fluorescence cell-based screening assay to monitor TRPM4-induced Na + influx has been utilized to screen for TRPM4-specific inhibitors through a ligand-based virtual screening approach of a catalog of 900,000 compounds available commercially to uncover analogs of 9-phenanthrol, FFA, and glibenclamide . The authors found aryloxyacyl-anthranilic 5 (compound 5) to be the most potent and specific TRPM4 inhibitor (IC 50 : 1.5 ± 0.1 μM) with approximately 20 times stronger inhibition than 9-phenanthrol (IC 50 = 29.1 ± 5.8 μM). In an aequorin bioluminescence-based assay, the compound NS8593 was identified to be a potent TRPM7 inhibitor (IC 50 : 1.6 μM), in TRPM7-overexpressing HEK293 cells, that disrupted Mg 2+ fluxes and interrupted TRPM7-like currents in a variety of cell types including smooth muscle cells, primary podocytes and ventricular myocytes (Chubanov et al., 2012). In the context of cancer cells, a TRPM7 kinase assay was performed to identify potent TRPM7 inhibitors in breast cancer cells known to require F I G U R E 2 Oncogenic functions and signaling pathways mediated by TRPM2, TRPM3, and TRPM4. TRPM2: LC3A/B II, ATGs and BNIP3 are autophagy-and mitophagy-associated proteins (Almasi et al., 2018). The lncRNA TRPM2-AS exerting its effects in prostate cancer (Orfanelli et al., 2015) and NSCLC (Huang et al., 2017a;Ma et al., 2017) are depicted here independent of TRPM2 channel activity. TRPM3: miR-204 directly or indirectly (through CAV1) inhibits TRPM3. Zn 2+ fluxes via TRPM3 inhibit miR-214 which directly suppress transcription of LC3A/B (required for autophagy) by binding their 3′ UTRs. Autophagy involves degradation of protein aggregates, mitochondria, and cytoplasmic components to generate nutrients for ccRCC growth (Hall et al., 2014). TRPM4: TRPM4 was a negative feedback regulator of SOCE in the prostate cancer cells DU145 through large influx of Na + that reduced the driving force for Ca 2+ influx. The reduced migration of TRPM4depleted DU145 cells by small interfering RNA (siRNA) might be due to negative regulation by Ca 2+ -dependent mechanism such as SOCE (Holzmann et al., 2015). This remains to be validated by future independent assessments as another study (Sagredo et al., 2018) showed a decrease in SOCE in TRPM4-depleted PC3 cells by shRNA, hence the symbol "?" was presented in between the inhibition and activation arrows involving Na + and TRPM4, respectively, directed towards SOCE As one of the sentinels of human sensations, TRPM family of proteins are receptive to a relatively wide range of natural or synthetic chemical compounds, a similar phenomenon recognized in other TRP ion channels (Vriens, Nilius, & Vennekens, 2008). While this broadens the avenues available to target TRPM channels, identifying inhibitors that specifically target an individual TRPM channel without off-target effects on its closest family member (e.g., TRPM4 and TRPM5) or on other non-TRP ion channels with similar pharmacological properties (e.g., ability to interact with compounds of similar chemical structures) poses a challenge because of a multitude of ion channels being present. Development of focused assays to test the specificity of inhibitors against a large set of ion channels, or uncovering specific inhibitors with IC 50 at low nanomolar concentrations is thus recommended. Furthermore, assessing inhibitors against endogenous TRPM channels in addition to the heterologous overexpression system, which has dominated most of the assessed inhibitors (Table 1), is evidently desirable.
F I G U R E 3 Oncogenic functions and signaling pathways mediated by TRPM5, TRPM7, and TRPM8. TRPM5: Activities of TRPM5 increased acidic extracellular pH (pH e ) signal. TPPO (triphenylphosphine oxide, inhibitor of TRPM5) reduced NF-kB activity and expression of EMT-associated genes (Mmp9, Vim, and Cdh2; Palmer et al., 2010). TRPM7: Induction of Hsp90α secretion promoted stabilization of pro-MMP2 and urokinase plasminogen activator (uPA) that induced production of MMP2, triggering increased degradation of the extracellular matrix (ECM) and invasion of PDAC cells (Rybarczyk et al., 2017); transforming growth factor β (TGFβ)-induced cell migration likely required Mg 2+ influx by TRPM7 that induced expression of proteins involved in reduced cell-cell adhesion (E-cadherin) but increased mesenchymal phenotype such as vimentin (and N-cadherin) that promoted EMT and invasion of prostate cancer cells (Sun et al., 2018a); In breast cancer cells, activities of TRPM7 decreased cytoskeletal tension that subsequently increased expression of the EMT transcription factor SOX4 that promoted EMT through reduction of cell adhesion molecules such as E-cadherin (Kuipers et al., 2018). TRPM8: Ca 2+ influx through TRPM8 upregulated activation of CaMKII, cdc25c, and cdc2 that inhibited G2/M arrest. Increased BK K + channel activity contributed to increased migration and chemotaxis (Klumpp et al., 2017); knockdown of full-length TRPM8 and sM8 isoforms induced ER stress and mitochondrial oxidative stress that increased PERK (protein kinase RNA [PKR]-like ER kinase) pathway that contributed to induction of p21 (restricts cell cycle progression) and apoptosis of prostate cancer cells (Bidaux et al., 2016 TRPM2-AS, TRPM3, TRPM4, TRPM5, TRPM7, or TRPM8

ACKNOWLEDGMENTS
The authors would like to thank the staff members at Department of Immunology

CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.