The regulatory and modulatory roles of TRP family channels in malignant tumors and relevant therapeutic strategies

2.1. TRPV channels

Transient receptor potential vanilloid (TRPV) channels are a family of ligand-gated ion channels that mainly own sensory receptors that respond to thermal, mechanical, or chemical stimulations. This subfamily is constituted by six members, and the most investigated channel is TRPV1 as an ion channel stimulated by vanilloid capsaicin, shedding light on the nomination of this subfamily⁴. Those TRPV subfamilies own comparable structures with an ankyrin-repeat domain in the wake of six-transmembrane helix domains within the amino-terminal region. The carboxyl-terminal in the cytoplasmatic side comprises the TRP domain and other binding sites for those lipid regulators with identified architectures⁵. In several cell types, TRPV channels are ubiquitously expressed throughout the body and associated with neuronic disease such as stress, anxiety, depression, schizophrenia, etc.⁶. Apart from the roles of temperature and pain-sensing, TRPV channels could modify the advent of Rho GTPase^7,8.

2.2. TRPC channels

The mammalian TRPC members resemble the Drosophila TRP and TRPL since they could be stimulated from receptors that signal through phospholipase C (PLC). The involvement of TRPC channels in cancer cell proliferation, invasion, and chemoresistance has also been discovered in recent years. This channel is nominated as “canonical” since the shared similarities of the TRP channel from Drosophila⁹. This channel has intracellular N- and C-terminal domains and six trans-membrane-spanning domains. Hetero-multimerization of those channels with the TRPC or other sub-families is reported, resulting in the functional non-selective ion channels with the relative concentration of calcium and sodium. What's more, PIP(4,5)₂ and PIP₂-derivated materials like diacylglycerol (DAG), inositol triphosphate (IP₃), PI(4)P, and PI regulate the activity of TRPC channels. TRPC exists in various tissues like the kidney, hippocampus, salivary glands, pancreatic β cells, vascular smooth muscle, and heart with a wide range of physiological functions¹⁰.

2.3. TRPM channels

Eight members consist of the TRPM subfamily defined as TRPM1–8 due to the first member. Higher-grade human prostate cancer (PCa) samples were accompanied by TRPM2 overexpression, among which distinct autophagic–apoptotic gene expression levels were observed. In addition, both monovalent and divalent cationic channels were identified with the former as TRPM4 and TRPM5 and the latter of TRPM7 and TRPM8. Other TRPM channels are non-selective cationic current channels^11,12.

Within the amino-terminal region, TPRM channels have a common melastatin homology region (MHR) domain which is likewise in other TRP members. However, diversified carboxyl-terminal are found between the TRPM membranes¹³. For TRPM4 and TRPM8 channels, they own coiled-coil domains and binding motifs for varied regulatory molecules. Therefore, the TRPM subfamily is quite a diversified subgroup within the TRP channel family for the particular structures and physical functions¹⁴.

2.4. TRPA channels

TRPA is a subtype of TRP ion channels, which is widely studied in the field of pain and neurogenic inflammation, and A stands for ankyrin. This subfamily of channels comprises around 1000 amino acids with the ratio of P_Ca/P_Na of 0.8–1.4. It also contains a TRP-box-like motif, including an inositol phosphate-binding region with a coiled-coil region¹⁵. This channel type could be activated by diversified metabolites of oxidative stress, i.e., reactive oxygen species (ROS), methylglyoxal, and acrolein. In addition, these Ca²⁺-permeable cation channels are activated by a broad range spectrum of hazardous external/internal stimuli like sharp coldness, stimulating compounds, reactive chemical species, and endogenous signals associated with cell damage¹⁶.

As far as our knowledge could reach, TRPA1 is the only member within the TRPA family in mammals. However, in Caenorhabditis elegans and Drosophila, there are respectively two and four members¹⁷. The pain-detecting sensory nerves within the plasma membrane contain TRPA1, and it would activate the pain-sensing pathways to stimulate avoidance behaviors and promotion of long-lasting responses like inflammation. As a result, blockade TRPA1 is promising to reduce pain¹⁸.

2.5. TRPP channels

Three human genes nominated as polycystic kidney disease 2 (PKD2, TRPP2), PKD2-like 1 (PKD2L1, TRPP3), and PKD2-like2 (PKD2L2, TRPP5) encode the TRPP channel proteins. This protein channel is a 110 kDa protein, participating in autosomal dominant polycystic kidney disease (ADPKD) as a calcium-permeable non-selective channel. The transmembrane segments of S1–S6 share the identical sequential similarity of homology, whereas the amino and carboxyl terminals have few similarities. There are conserved orthologues within the mammalian TRPP channels with ∼90% similarity for TRPP2/3 and ∼80% for TRPP5. The polycystin-1 family proteins modify the TRPP ion channels into the receptor–channel complexes, establishing the core of the signaling pathway in which calcium is the second messenger¹⁹.

2.6. TRPN channels

This type of TRP channel doesn't encode any mammal proteins, only to be present in worms, flies, and zebrafish. This channel is appropriate for mechano-transduction, and it affects the behaviors of insects and nematodes as well as the transduction of vibratory stimuli in zebrafish hair cells²⁰.

2.7. TRPML channels

Three subfamily members as TRPML1, TRPML2, and TRPML3, which consist TRPML group, share ∼75% amino acid similarity. For TRPM1, it is a 580 amino acid molecule with the weight of 65 kDa. Its mutation could be responsible for lysosomal storage disorder mucolipidosis IV, accounting for the iron ions going through the endosome/lysosome membrane into the cell²¹. On the other hand, 533 amino acids compose TRPML3 with a molecular weight of about 64 kDa. This channel is mainly expressed in the inner ear, being able to inwardly rectify the monovalent cation channel permeable to calcium proton secretion would alleviate its activity²².

2.8. Summary

To conclude, those major TRP channels in tumors would be the targets worth further investigation to shed light on possible therapeutic approaches to alleviate or even cure the diseases. However, due to the limited space in this review, we will not further discuss other types of TRP channels, and their functions and expressions in tumors will be discussed in the following sections. In Fig. 1, the general classifications and relevant subfamilies of TRP channels are chronologically presented for their discovery time with the phylogenetic tree. In Fig. 2, the detailed structural variances of each TRP channel subfamily type are stated respectively^14,17.

3.1. Dysregulated TRP channels expression in malignant tumor cells

In malignant tumors, dysregulated TRP channel expression is ubiquitous among the cells. In the following content, we will discuss several distinctive characteristics of tumor cells within which TRP channels play essential roles in such conditions.

3.2. Dysregulated TRP channels expression in vascular endothelial cells

Angiogenesis marks the generation progression and development of tumors, and recent investigations found that TRP channels have a tight linkage with angiogenesis among various types of tumors. The detailed content is illustrated in the following paragraphs.

In breast cancer tumor endothelial cells (BTECs), both TRPM8 and TRPV4 are down-regulated⁸⁰. However, a considerable up-regulation of TRPA1, TRPV2, and TRPC3 is observed in prostate-derived TECs (PTECs). The deregulation of those TRP channels associated with the prostate has prominent effects on endothelial cell biology, i.e., TRPV2 responsible for the proliferation, TRPC3 for TEC-mediated interplay with tumor cells, and TRPA1 for angiogenesis. Thus, they would be therapeutic targets to combat breast cancer⁸¹.

TRPC1 is also upregulated in the renal cancer-derived endothelial colony-forming cells (ECFCs), and an ensuing store operated calcium entry (SOCE) amplitude and stimulating ECFC proliferation are discovered⁸². When compared with normal human microvascular endothelial cells, the deregulation of TRP is quite weird and results in the sequencing intracellular calcium deregulation⁸³. TRPC1 has pro-metastatic characters, and it could be applied in many cancers. The ciRS-7/miR-135a-5p/TRPC1 axis activated by TRPC1 aggravated epithelial–mesenchymal transition (EMT) in gastric cancer (GC), and this TRPC1 would be a potential target for end-stage GC patients. The authors also found that in GC cells, miR-135a-5p regulated TRPC1 by targeting its 3′-UTR. Thus, ciRS-7 acted as a sponge of miR-135a-5p in GC⁸⁴. In Trpv4-KO mice, the tumor vasculature showed a more significant percentage of the hyper-permeability without the generation of pericytes or the dilation of microvessels, which is an effective approach to alleviate the therapeutic outcome of anti-cancer treatments⁸⁵. In tumor-derived ECFCs, TRPC1 is strongly remodeled for vascular endothelial growth factor (VEGF) is not competent enough to cause the oscillations of pro-angiogenetic of calcium oscillations. This characteristic is essential to consolidate the viewpoint that anti-VEGF drugs incur primary and secondary resistance due to TRPC1 (and SOCE) inhibition could offer a bright future to interfere with tumor vascularization⁸⁶.

Tumor proliferation, migration and survival demand angiogenesis under both physiological conditions such as the growth and renewal of blood vessels and pathological conditions like cardiovascular diseases and the initiation of tumor progression. There would be a close association between the TRP channels and the angiogenesis effect. The ensuing focus would be whether those TRP-generated calcium signals induce various cellular functions and other aspects. The Trpv4-knockout mice show the novel tumor blood vessels with enlarged diameters and densities. Meanwhile, the shrinkage of tumor capillary endothelial cells within the surrounding areas took place. Thus, the abnormalized formation of blood vessels could enhance the progression of lung cancer. In some sense, the activation of TRPV4 with the agonist of GSK1016790A normalized the vascular endothelium and the permeability of the chemotherapeutic drugs, ultimately reducing tumor cell secretion and suppressing the tumor growth⁷⁷. Up-regulation of TRPV4 channels in the endothelium may essentially impact the early phases of angiogenesis in breast cancer. A significant enhancement of TRPV4 expression is seen in BTECs for malignant angiogenesis. Meanwhile, arachidonic acid (AA) or 4α-phorbol-12,13-didecanoate (4α-PDD)-induced migration accompanied with TRPV4 stimulation in BTECs instead of normal human cardiac microvascular endothelial cells⁸⁰. During the initial stage of the tubulogenic process rather than the capillary-like network, AA promotes the access of extracellular Ca²⁺ entry into BTECs⁸⁷. Substantial reduction of TRPV4 resulted in the VE-cadherin expression for the intercellular contacts, further contribute to the increased vascular leakage⁸⁸. TRPC1 in tumor-derived ECFC might be considerably undergone vascular remodeling process. A significant upregulation of TRPC and SOCE was seen in renal carcinoma cells (RCC)⁸².

Thus, the TRP channel plays an indispensable role in tumor vasculature and the spread of angiogenesis. Therefore, proper treatment of the TRP channel would be a useful tool to alleviate tumor growth.

3.3. Dysregulated TRP channels expression in tumor-associated immunocytes within the microenvironment

The solid tumor has a complicated mixture of cells, both cancerous and non-cancerous. In general, these non-cancerous cells, plus factors including the ECM, cytokines, growth factors, and hormones, consisting of the microenvironment of tumors. Cytokines are the general name of bioactive small molecular peptides and glycoproteins. The function of cytokines is to regulate the immunological functions, mediate inflammatory response, stimulate the proliferation and differentiation of hematopoietic stem cells (HSCs), and participate in tissue repair and wound healing. Macrophages are essential in initiating immunological reactions such as immune surveillance stimulation, bactericide, remodeling, and repairment of tissues with both beneficial and hazardous effects in various diseases relying on the cell activation state and the microenvironment where they are present⁸⁹.

The expression of those TRP members within the monocytes and macrophages⁹⁰. Those channels enable the identification of exogenous signals such as exogenous damage-associated molecular pattern molecules as well as the endogenous danger signaling transductions during the injury period⁹¹. Microarray-based studies demonstrated that an up-regulation of TRPM4 transcripts was observed in human large B cell lymphoma. Also, within lymphoid tissues, human normal B cells could express a relatively lower level of TRPM4, while in diffuse large B cell lymphoma, higher TRPM4 protein levels were observed. An overexpression of TRPM4 level was kept in a quarter of all the cases in large B cell lymphoma when compared with human normal B cells within lymphoid-rich tissues (reactive tonsil, lymph node, and appendix)⁹².

TRP channels might regulate the varied macrophages differentiation. In Helicobacter pylori-infected Trpm2 knockout mice, alleviated bacterial colonization and aggravated gastric inflammation were diagnosed when compared with their counterparts. The absence of TRPM2 in the H. pylori-infected macrophages stimulates elevated inflammatory cytokines as well as M1 polarization. Triggering TRPM2-deficient macrophages with H. pylori resulted in calcium overload, extracellular signal-regulated kinase 1/2 (ERK1/2), and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activation⁹³. Within lymphoid tissues (reactive tonsil, lymph node, and appendix), an insufficient level of TRPM4 was observed. However, a higher TRPM4 protein level was confirmed in diffuse large B cell lymphoma accounting for 26% of all cases, akin to prostate cancer with aggressive clinical parameters, resulting in a worse prognosis⁹². Differentially regulated TRPM7 expression and activity in bone-marrow-derived murine M1 and M2 macrophages⁹⁴. The IL-4 triggered M2 macrophages have a more prominent TRPM7 current (∼4.7 fold), but the TRPM7 mRNA levels keep static upon cell polarization⁹⁴.

The TRPM7 inhibitors of NS8593 and FTY720 would suppress the IL-4 and M-CSF-induced macrophage proliferation to suppress M2 polarization. Furthermore, suppression of TRPM7 diminished the levels of phosphorylation. Phosphoinositide 3-kinase (PI3K) and the downstream ERK1/2 phosphorylation levels were suppressed when TRPM7 was inhibited, causing apoptosis in the rat hepatic stellate cells^95,96. An adoptive transfer of macrophages within the TRPM8-deficient mice exacerbates colitis. Meanwhile, the overexpression of IL-10 would rescue the sub-population of M2 macrophage. The M1 macrophage phenotype and pro-inflammatory activity were facilitated via tumor necrosis factor-α (TNF-α) generation in TRPM8-positive macrophages. As a result, triggering the TRPM8 channel in the murine peritoneal macrophages stimulates the calcium transient currents in wild-type rather than TRPM8-deficient mice, demonstrating defective phagocytosis and enhanced motility⁹⁷. The TRPC6 channel translocated into the phagosomal membrane to elevate its function since in cystic fibrosis patients could restore the alveolar macrophage function via the enhancement of TRPC6⁹⁸.

For the TRPC1 channels, TLR4-dependent TRPC1 activation stimulates the ER storage. In the wake of activation of PLCγ, the protein kinase C-alpha (PKCα) is mediated by the calcium entry of TRPC1, leading to NF-κB/Jun kinase nuclear translocation as well as cytokine secretion-induced tissue destruction⁹⁹.

Therefore, it is an efficient approach to investigate the roles of TRP channels in the immunocytes and tumor microenvironments, and it is believed that certain modifications within such microenvironments could facilitate potent therapeutic outcomes.

3.4. Summary of the characteristics and functions of TRP channels in tumors

TRP channels are often the effectors of dysregulated physiological functions for the altered expression, making a severe modification in the cells’ major signaling pathways. Likewise, altered expression of TRP channels in certain pathological functions such as tumor and inflammation could be fully utilized to regulate the entry of suitable reagents to unveil the non-permeable potential drugs and improve the therapeutic index of existing drugs via favorably aiding the uptake in affected cells and tissues. We just reviewed the apparent features of TRP channels in tumors, and thereafter, further investigations of the underlying intracellular and intranuclear mechanisms are prone to be elucidated.

4.1. The effects of TRP channels in ER stress in tumor cells

Interference of calcium homeostasis in the ER is an essential stimulation of ER stress, and the majority of the TRP family has been involved and mediate the intra- and extra-cellular homeostasis to initiate ER stress via diversified mechanisms.

The TRP channels and ER stress have a mutual function, reciprocally facilitating each other to amplify those actions. TPPC channels have been initially identified to play a role in ER stress-induced apoptosis; thus, TRP channels play crucial roles in maintaining ER calcium homeostasis and disrupting channel function, leading to ER stress¹⁰². TRPC1 is essential for maintaining ER calcium homeostasis and the reduction leading to prolonged unfolded protein response (UPR) pathway activation with a deterioration of AKT, resulting in neuro-degradation¹⁰³. Be that as it may, mammalian target of rapamycin (mTOR) is also involved in such processes, and functional TRPC1 overexpression prevented neurotoxin-induced ER stress as well as UPR via AKT/mTOR signaling restoration and increasing dopaminergic (DA) neuron survival. Thus, prolonged ER stress might induce cellular death, launching a protective response mediated partially by autophagy. Meanwhile, TRPC3 also impacts ER stress in the epithelial cells extracted from mouse pancreatic and parotid glands; the absence of TRPC3 function ameliorates ER stressed induced-UPR via pERK signaling¹⁰⁴. In tumor cells, ER-induced activation of calcium/calmodulin dependent protein kinase II (CAMKII) in human coronary artery endothelial cells (HCAECs) demonstrated that calcium influx via TRPC3 is indispensable UPR and the ultimate ER-stress-driven apoptosis^105,106. Excessive calcium entry into cells under the condition of albumin overload results in the calcium entry and the ensuing ER stress protein glucose-regulated protein 78 (GFP78) accumulation as well as the ultimate apoptosis¹⁰⁷.

Human lung cells have witnessed that TRPV1 agonists inside the ER deteriorated calcium homeostasis so as to trigger eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2αK3)-dependent ER stress responses¹⁰⁸. Overexpression of stress response-induced genes such as growth arrest and DNA damage-inducible gene 153 (GADD153), GADD45α, ATF3, CCNG2, and BiP/GRP78 mRNA and the simultaneous decrease of cyclin D (CCND) indicates the stress induced-agents like dithiothreitol (DTT) and thapsigargin¹⁰⁹. Furthermore, phenomena such as ER stress, GADD153 expression, and lung cell injury are generated by inflammation-induced TRPV1 agonists¹¹⁰. However, ER-bound TRPV1 channels have a relatively lower sensitivity for its agonist capsaicin, indicating the critical safety mechanism as protecting neurons from calcium exhaustion from ER, which results in ER stress, abnormal folding of protein response, and cell death¹¹¹.

ER stress and the crosstalk with autophagy are vital for the maintenance of cellular energetics and cell survival. The spatially restricted subcellular domain of calcium entry into the cells is achieved by many proteins and ion channels. Further investigation is needed to detect these intricate relationships, and a complicated signaling pathway for autophagic activation would be further analyzed.

4.2. The effects of TRP channels in tumor cell proliferation and progression

Tumor cells are characterized as unrestrained proliferation and duplication. There are intertwined underlying mechanisms worth investigating. It is believed that calcium could be one promoter to regulate the extent and rate of tumor cell proliferation. Thus, we will carefully review the roles of TRP channels in cellular proliferation.

The resultant suppression of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mouse xenograft models was subsequently observed¹¹². Hepatocellular carcinoma (HCC) cells, Huh-7, and hepatocytes from HCC patients are harvested, and a research on TRPC6-mediated Ca²⁺ entry in HCC cell proliferation was carried out. Cell proliferation and store-operated calcium entry were detected in the wake of TRPC6 overexpression, which could be attenuated by its knockdown. Similar effects happened by the knockdown of STIM and Orai. This effect is more prominent in HCC when compared with normal liver tissue, suggesting the TRPC6-mediated calcium accession into HCC cells is essential for the HCC cells proliferation and oncogenesis¹¹³. The elevated expression of TRPV4 in healthy or inflamed skin when compared with lower or absent expression in skin cancer like precancerous lesions and non-melanoma. The discharge of IL-8 was activated via the triggering of TRPV4, which in turn downregulates the TRPV4 expression. The hypothesis made that lower expression in skin cancer would affect the ATP secretion along with autocrine communication between those keratinocytes via the regulation of calcium homeostasis. Thereafter, the diminished extracellular calcium concentration was also suppressed, resulting in the cells’ intact and eventual tumor formation^114,115.

The basic biological events of cells contain proliferation and differentiation, and cancer cells have the capability of unlimited replicative potential, unlike most of the normal cell lineages with a limited number of successive growth-and-division cycles. The imitation point of tumorigenesis is the moment when those cancerous cells become immortal. TRPC6 was crucial in cell growth with a positive relationship of its expression when compared with the tumor grade. Inhibition of this TRP channel reduced cell growth, leading to glioblastoma arrest at the G₂ phase, with promoted anti-tumor levels. As a result, the antiproliferative therapeutic efficacy of ionizing radiation has been further advanced to clonogenic ability with the state transition of the G2/M phase². For TRPC6, its association with the pathophysiology in breast cancer when investigating the role of calcium-sensing receptor (CaR) within the cell line of MCF7 in the luminal A ER ⁺ breast cancer. The dual distinct cationic current induced by CaR activation through a highly Ca²⁺-sensitive and another secondary Na⁺ influx with the latter quite be dependent on the former, which is uniquely active upon the stimulation of calcium current. Thus, those two CaR-activated cationic influx TRPC1 and/or TRPC6-dependent mediated by hetero-multimers, making contributions to the biophysical characteristic of the channels and the intracellular calcium depletion^116,117.

TRPM4 channel expression is observed in several cancer types such as prostate, urinary bladder, liver, cervical, colorectal, as well as large B cell lymphoma. For prostate cancer, benign and malignant prostate tissues with TRPM4 protein are observed. The ensuing studies confirmed TRPM4 upregulation in prostate cancerous tissues. Essentially, a higher level of TRPM4 protein has a positive correlation with a higher risk of recurrence following radical prostatectomy¹¹⁸. For the hepatic stellate cells (HSC), its proliferation and upregulation would be regulated by TRPV4. The mRNA and protein expression levels of TRPV4 in rat HSC-T6 cell line far outweighed the control group in the wake of the addition of transforming growth factor β1. Nevertheless, Ruthenium Red (non-specific antagonist of TRPV4) or synthetic siRNA targeting TRPV4 suppressed TGF-β1-induced HSC-T6 cell proliferation¹¹⁹. Upon the activation of TRPV4, HSCs could be transformed into the myofibroblasts and subsequently secrete a considerable amount of collagen, resulting in fibrosis and causes damages to liver tissue, cirrhosis, and liver cancer. Inhibition of TRPV4 with its antagonist of HC-067047 in hepatocellular carcinoma diminished the cellular proliferation, resulting in apoptosis with decreased migration capability via the alleviation of the EMT process both in vitro and in vivo¹²⁰. Intraperitoneal injection of HC-067047 in oligodendrocytes, the proliferation is also promoted upon TRPV4 activation with increased mRNA levels in both primary cultures and in vivo models. The triggering of TRPV4 by GSK1016790A elevated OPC proliferation which could also be abolished by the co-treatment with HC-067047¹²¹. When it comes to the TRPM7 channel, this tension-triggered enhances the calcium entry via IP₃R2 stimulation with the resultant local calcium concentration fluctuating for cellular migration¹²². Its overexpression is associated with the loss of cellular adhesion via calpain II¹²³. The bladder cancer cells have a significantly elevated level of TRPM8, and the knockdown/inhibition of those cells could result in attenuated cell proliferation and migration. What's more, synthetic siRNA targeting TRPM8 treatment enhanced the ROS levels together with enhanced levels of catalase, heme oxygenase-1 (HO-1), and superoxide dismutase 2 (SOD2). Moreover, transplantation of the mouse model with stable TRPM8-deficient T24 cell line suggests such knockdown of TRPM8 has holdback the in vivo tumor growth¹²⁴.

Strangely, TRPA1 is linked to tumor suppression with the inhibition of cancer progression as the down-regulation takes place in tumor cells¹²⁵. Subsequently, TRPA1 showed tumor-promoting effects in small cell lung cancer (SCLC) cell lines, with well-stated roles in neuroendocrine functions¹²⁶.

Thus, proper regulation of TRP channels would be an effective approach to combat cancer to restrain tumor growth via suppressing cellular proliferation.

4.3. The effects of TRP channels in tumor cell migration and metastasis

EMT refers to the tumor cells’ properties tightly linked with the invasiveness as well as the metastatic dissemination. It also associates with the degeneration of components of the extracellular matrix. During this process, the cells become spindle-shaped fibroblast-like ones with enhanced motility¹²⁷.

The occurrence of EMT is mediated via TRPV4, and E-cadherin is one of the vital inhibitors to suppress the neonatal epithelial tumor cells by their movement and invasion. Diminished expression of E-cadherin is regarded as the prominent marker of EMT¹²⁸.

For migration and metastasis, TRPV4 enhances the abilities of the relevant proteins associated with the tumor metastasis. Constant stimulation of TRPV4 enables the gene expression for the ability of metastasis while reducing the metastasis inhibition genes. The administration of TRPV4 agonist 4α-PDD causes the adhesion-associated tumor suppressor genes (i.e., Fn1, Clu, Tubb2c, and Spp1) to diminish in the mouse breast cancer cell line 4T07⁷⁸. Meanwhile, the metastasis-promoting gene of talin secreted via exosomes demonstrates an increment¹²⁹. In mice cerebral edema, the expression of hippocampus matrix metalloproteinases (MMP) such as MMP-2/MMP-9 (associated with the metastasis of many types of cancers) levels were considerably suppressed by the TRPV4 antagonist of HC-067046¹²⁹. For vasoactive intestinal peptide receptor subtype 1 (VPAC1) as a G-protein coupled receptor (GPCR), it is predominantly activated by vasoactive intestinal peptide (VIP). Its activation facilitates the migration and invasion of the gastric cancerous cells via TRPV4-dependent calcium entry, amplifying the VIP expression. Thus, the VPAC1/TRPV4/calcium axis signaling axis caused substantial lung metastasis of gastric cancer cells in vivo via VIP elevation⁶⁰.

In the bladder cancer cells, the inhibition of the overexpressed TRPV4 could significantly downregulate the expression of E-cadherin. TRPV4 suppression-induced AKT and FAK activation would further alter the E-cadherin expression level¹³⁰. The cytoskeletal remodeling is triggered by TRPV4 activation in breast cancer cells. TRPV4 not only regulates the microtubule–microfilament polymerization but also causes the dynamic alterations of the microvilli and pseudopods, affecting the cellular motility. Contributing to the occurrence of tumor EMT modified via intracellular calcium signaling¹³¹. It is prominent that the straight binding of TRPA1 N-terminal ankyrin repeats into the C-terminal proline-rich motif of fibroblast growth factor receptor 2 (FGFR2), which subsequently activates the receptor, leading to the lung adenocarcinoma progression and metastasis. Conversely, the depletion of TRPA1 happens by the transfer of TRPA1-targeting exosomal microRNA (miRNA1723 142-3p) from brain astrocytes to cancer cells¹³².

Ryanodine receptor (RyR) stimulation results in the TRPM7 to migration of human nasopharyngeal cancer cells, and a subsequent increment of intracellular calcium levels and migration occur. This proves the hypothesis that TRPM7 has a direct impact on various cancer cells through the mechano-transduction mediated by intracellular calcium. Thus, TRPM7 overexpression is linked to the poor prognosis and metastasis as well as the enhancement of both phenomena in nonmetastatic cancer cells¹³³. Overexpression of VPAC1 is considerably reported, and the VPAC/TRPV4/Ca²⁺ signaling axis has been confirmed involved in gastric cancer. The secretion of intrinsic VIP would enforce a positive feedback regulatory system via a Ca²⁺-dependent manner. VPAC1 triggered the TRPV4 channel through the PLC/DAG/PKC signaling pathway. Proper interference of VPAC1/TRPV4/Ca²⁺ signaling or VIP suppression would be helpful to be a potential prognostic marker to cure gastric cancer to suppress the metastasis degree⁶⁰.

The progressive and invasive manner of pancreatic ductal adenocarcinoma (PDAC) is also partially attributed to TRPM7. TRPM7 silencing in the PDAC cells alleviates the cancer cell invasion. The MMP-2 released via Hsp90a/uPA/MMP-2 proteolytic axis triggers TRPM7, breaking the ECM to promote the invasive capacity of the tumor cells¹³⁴. TRPM2 modifies the migration and invasion of the gastric cancerous cells in vitro, as well as that representative EMT markers such as N-cadherin, snail, slug, integrins, and MMPs to modify their expressions in vivo¹³⁵.

Ovarian cancer patients with shorter survival periods have higher EMT processes, and TRPM7 channel overexpression is observed in such a process. Intracellular Ca²⁺ reduction via TRPM7 silencing or inhibition attenuated the EMT process, migration, invasion, and wound healing of ovarian cancer cells by inhibiting the PI3K/AKT activation, suggesting the preventive approach of metastasis in ovarian cancer¹³⁶.

For TRPC1, it mediates the calcium entry and the MAPK and PI3K/AKT signaling pathways; it also modifies HIF1α, calpains as well as those MMPs. The directionality of the migrating cells such as the renal transformed epithelial cells¹³⁷. The epidermal growth factor (EGF)-evoked migration is also aroused by TRPC1 in aggressive glioma cells, and the suppression of the channel ameliorates the state of cells¹³⁸. Besides, in the leading edge of migration cells, TRPC1 facilitates the localization of the lipid rafts. Therefore, TRPC1 has been regarded as an essential player in both normal and cancerous cellular function¹³⁹. TRPC2-regulated migration is dependent on Rac, calpain, and MMP-2 during embryogenesis in the thyroid cells¹⁴⁰. Furthermore, activation of Ca²⁺ activated K⁺ channel 3.1 (KCa3.1) elicited TRPC3-evoked calcium entry with elevated calpain activity and concomitant cell migration¹⁴¹. For melanoma cell migration, it is enhanced by TRPC3 stimulation both in vitro and in vivo¹⁴². Both TRPC3 and TRPC6 account for the invasive manner of cancerous bladder cells, and within those cells, the regulation of histone variant macroH2A expressional levels arouse¹⁴³. In follicular thyroid cancer ML-1 cells, TRPC1 is a strong regulator in both migration and invasion. The underlying mechanisms seem to be dependent on the MAPK/ERK1/2, MMP-2 and-9, and HIF1α¹⁴⁴. As for the TRPC2 channel, it mainly regulates normal cells. But TRPC3 channel is a very interesting subject worth further investigation in various tumor cells, such as human ovarian cancer cells, in which an enhancement of TRPC3 expression is observed. Furthermore, the micro-injection of TRPC3-knockdown human ovarian adenocarcinoma SKOV3 cells diminished the tumor formations in nude mice²⁸. Both cell and animal studies showed TRPC3 promoted tumor formation and cell migration in melanoma, causing deleterious results to the patients¹⁴². In stellar pancreatic cells, TRPC3-triggered calcium entry and calpain activity along with concomitant cell migration¹⁴¹. For bladder cancer cells, the migration and invasion behavior is also found dependent on both TRPC3 and TRPC6, and to our interest, the expressional levels in those cells are tightly regulated by the histone variant macroH2A¹⁴³.

Thus, the intertwined relationship between metastasis and TRP channels are crucial targets for further investigations to seek effective and efficient approaches to cure or at least alleviate cancer.

4.4. TRP channels on novel patterns of programmed cellular death in cancer cells

Three novel modules of cell death patterns as pyroptosis, necroptosis and ferroptosis were found in recent decades. Discovered and defined by Yuan's laboratory in 2005, necroptosis is a type of death modality linked with inflammation and immunal responses, demonstrating many morphological similairities to necrosis, i.e., organelle swelling, cytoplasmic membrane breakage, and cellular collapse¹⁴⁵. Pyroptosis, associated with inflammation, has the features of plasma membrane pores, is featured by the disruption of plasma membrane wholeness, and in the wake of those above characterizations, there is an ouflow of cytologic inflammatory contents¹⁴⁶. In such processes, caspase plays an indispensable role, with the classic caspase-1/NLR family pyrin domain containing 3 (NLRP3)/gasdermin D(GSDMD) pathway srecting IL-1β and IL-18¹⁴⁷ and atypical caspase-4/5/11 pathways¹⁴⁸. As for ferroptosis, it is a Fe-dependent affregation of lipid peroxides, leading to the degradation of intracellular antioxidant pool with an elevation of intracellular iron¹⁴⁹. When ferroptosis takes place, the integrity of cellular membrane is jeopardized with the mitochondrial membrane shrinkage, rupturing the out membrane of mitochondria as well the chromatin condensation of nulei¹⁵⁰.

There are limited amount of literature reports indicating the association of those three novel pattern of cellular death with TRP channels. For pyroptosis, the TRPM2/NLRP3 inflammasone pathway induced by mitochondrial ROS generation is reported to own a tight linkage with gout^151,152. Also TRPM2 demonstrated inhibitory effects in lipopolysaccharide (LPS)–ATP-induced pyroptosis via downregulation of ROS generation in macrophages stemmed from bone marrow, Trpm2^−/− mice group showed higher caspase-1-P10 with elevated ASC oligomerization. Thus, ablation of TRPM2 woule be beneficial to trigger caspase-1 and pyroptosis through regulation of ROS generation as an approach of immunal adjustment¹⁵³.

RIP-induced mixed lineage kinase domain-like protein (MLKL, a trimer) phosphorylation constitutes a homotrimer sited at the plasma membrane in the TNF-induced necroptosis. Thereafter, TRPM7 worked as its downstream substrate and mediates the calcium influx, resulting in the disruption of plasma membrane and shedding light on the role that TRPM7 plays in the association of MLKL with the receptor-interacting protein kinase 3 (RIP3) necrosome¹⁵⁴. Moreover, TRPM4 is involved in LPS-induced vascular endothelial necrosis by mediating Na⁺ influx, cell depolarization, and increased cell volume¹⁵⁵.

As to ferroptosis, it would be linked with oxidative glutamate toxicity or oxytosis, TRPC channels might be involved in such process via the 12/15-lipoxygenase (12/15-LOX) and the 12/15 LOX as the substrates¹⁵⁶.

Despite substantial efforts have been invested to TRP channels in novel mechanisms of cell death, the above-mentioned patterns of cancer cell death is poorly elucidated. To achieve deeper apprehension within this field, TRP channels on the novel patterns of cellular death in tumors would be potential areas of further investigation.

4.5. Summary of the intracellular mechanisms of TRP channels in tumors

The essential interactions between tumor cells and tumor microenvironments are tightly regulated and modified by those transports and channels. We have briefly summarized the potential underlying mechanisms to tell which signaling pathways might be involved within such processes. By far, many factors are to be uncovered, and thorough investigation towards the underlying mechanisms would facilitate the therapeutic approaches of cancer.

5.1. TRPC channels

The luminal A ER⁺ breast cancer cell lines such as MCF7 and triple-negative breast cancer (TNBC) MDA-MB-231 have overexpression of TRPC6 when compared with the epithelial breast cell line of MCF10A. For cellular proliferation and invasion that TRPC6 takes part in, the physiological and pathological functions were uniquely and considerably diminished in the tumoral cell lines. The proper intervention of TRPC6 would adjust the calcium cation hemostasis state in breast cancer¹⁷⁷. Non-selective TRPC channel blockers SKF96365, MRS1845 and 2-aminoethoxydiphenyl borate (2-APB) suppressed cell proliferation and cytokinesis in D54MG glioblastoma cells²⁷. TRPC1 channels promote the proliferation of human malignant gliomas intrinsically. Suppression of TRPC1 via shRNA or SKF96365 in D54MG glioma cells inhibits cell proliferation and incomplete cell division. In vivo results also indicated that shRNA suppression of TRPC1 expression reduced flank tumor size. In the D54MG glioma cells, shRNA knockdown comprised the EGF gradient-dependent chemo-tactical migration. Caveolar lipid rafts mark the TRPC1 channels, associating with cholesterol exhaustion, mediating glioma chemotactic metastasis, which could be the target by pharmacological inhibitors for TRPC channels¹⁷⁸. SKF-96365 is an imidazole compound belonging to antimycotics that could inhibit both CRACs and some TRP channels. Initially, in human blood cells such as platelets, neutrophils, and endothelial cells, this chemical substance suppresses receptor-mediated Ca²⁺ entry, while before long, TRPCs blockade makes this compound suppress ovarian cancer cell growth and tumorigenesis to a larger extent²⁷.

In CNE2 nasopharyngeal tumor cells, siRNA-mediated silencing downregulates the TRPC1 and significantly attenuated the adhesive and invasive abilities, indicating the efficacy of suppressing TRPC1 would be a potential approach of tumor therapy¹⁷⁹. Overexpression of TRPC3 increment was observed in breast cancer cells. A specific TRPC3 blocker Pyr3 and dominant negative TRPC3 reduced proliferation, induced apoptosis and sensitized cell death to chemotherapeutic agents²⁹.

5.2. TRPA channels

For the oxidative stress-dependent TRPA1, anti-cancer treatments with chemicals like bortezomib, oxaliplatin, cisplatin, and paclitaxel induces severe cold and mechanical allodynia. Further, blockade of TRPA1 alleviated cold and mechanical allodynia induced by anti-cancer chemicals like paclitaxel or oxaliplatin¹⁸⁰. For the pro-inflammatory/proalgesic effects, TRPA1 also serves as the keynote mediator when the three aromatase inhibitors as exemestane, letrozole, and anastrozole, are used in breast cancer therapy¹⁸¹. In addition, TRPA1 has been reported to promote resistance to ROS-producing chemotherapies, TRPA1 inhibitor AM-0902 showed a significant inhibition of xenograft tumor growth and enhanced chemosensitivity²³.

5.3. TRPV channels

TH-1177 was then modified to own higher specificity for TRPV6 as the primary category in cancer compared with TRPV5, and this novel chemical compound has been further demonstrated to be more efficient in combating prostate and breast cancer¹⁸². TRPV4 channel regulates the vessel maturation and angiogenesis in tumors, and GSK1016790A is its agonist of best activity with over hundreds folds potency than the routine clinical application of 4α-PDD. Combined usage of GSK1016790A with other anti-tumor reagents such as cisplatin infiltrated the tumor tissues with more depth. Melanoma A2058 and A375 cells showed decreased viability by TRPV2 and TRPV4 agonists, which would lead to cellular apoptosis or necrosis¹⁸³. Soricimed-C13 (SOR-C13) and SOR-C27 are two TRPV6 inhibitors that resembling the C-terminus of soricidin analog, and they demonstrate competitive affinity in ovarian cancer cells. TRPV6 channel overexpression has been applied in phase I clinical trial for advanced cancer¹⁸⁴. Resiniferatoxin (RTX), as a potent TRPV1 activator, is applied to own a permanent analgesic effect via the impairment of the signaling potential of susceptible sensory neurons¹⁸⁵. RTX administration witnessed the fantastic decline in the pain behaviors in the wake of RTX intrathecal administration within a variety of species, including canine with osteosarcoma-induced pain¹⁸⁶. The TRPM8 antagonists would induce probable hypothermia in the patients. The identification and optimization of those antagonists could make the PF-05105679 undergo clinical trials with ensured efficacy in one test akin to the opioid medication derivate oxycodone for severe pain alleviation^187,188.

When it comes to malignant tumors, TRPV channels differentially contribute to diversified breast cancer, the specific activators as well as inhibitors of TRPV4 and TRPV6 channels would contribute to exert therapeutic effects to combat breast cancer. TRPV channels have mutating genetic modifications such as TRPV4 of S823D, S824D, and T813D, and TRPV6 of S691D, S692D, and T702. The mutant has more potent invasiveness in colon adenocarcinoma SW480 cells. S692D and T702D mutants show elevated mutant channel activity with TRPV6 rather than their TRPV4 counterparts. Thus, varied mechanisms would converge into the identical goal to the global effect in the deleterious phenotype. This highlights the potential importance of searching for TRP mutations involved in cancer¹⁸⁹. On the contrary, shRNA targeting TRPV4 caused the knockdown of TRPV4 and inhibited AA-generated BTECs migration; within this process, AA-induced intracellular calcium entry has a larger extent of migration instead of non-migrating BTECs since the TRPV4 expression in the cellular surface overexpression by a mere exposure of AA itself⁸⁰. Overexpression of TRPV1 ion channel can promote cisplatin to take part in the esophageal squamous cell carcinoma (ESCC) chemotherapy via inhibition tumor cells proliferation¹⁹⁰.

5.4. TRPM channels

D-3263 is a TRPM8 activator and induces cancer cell apoptosis, which is a proper candidate for patients with solid tumors. For gastric cells, TRPM2 knockdown suppressed cellular proliferation, resulting in apoptosis and mitochondrial dysfunction. Within this process, ATP generation along with autophagy was attenuated, which would be an ensuing target for tumor therapy. As for human prostate cancer cells of DU145 and PC3, resveratrol diminished TRPM7 expression and function as well as alleviated TGF β-induced cell migration². Another feature that might underpin the primary and secondary resistance to anti-VEGF drugs, TRPC1 (and SOCE) inhibition, could represent a promising strategy to interfere with tumor vascularization¹⁹¹.

Apart from the conventional drugs, proper interference of the TRP channel would be an appropriate approach to perform as the means to combat cancer. Prostate patients presented higher gene expression of TRPM4. The knockdown of the TRPM4 channel would deactivate the migration rather than proliferation in the prostate cells such as DU145 and PC3 cells¹⁹². The silencing of TRPM7 via proper knockdown of MDA-MB-231 or MDA-MB-435 in breast cancer cells would enhance the contractility and the amount of focal adhesions, which greatly diminishes the migratory and invasive potentials⁷⁴.

To summarize, both small molecular drugs and gene delivery-associated therapy are also efficient and novel approaches to target the therapy of tumors.