TRPV Family Ion Channels in the Mammary Epithelium: Role in Normal Tissue Homeostasis and along Breast Cancer Progression

Abstract

Calcium homeostasis directs various intracellular cascades and therefore strict spatio-temporal control of calcium influx is also crucial for diverse physiological processes. In the mammary gland, calcium is important for the specialized tasks of this organ during lactation, but it also guides other structural and functional features of the mammary epithelium and in this way the maintenance of the whole tissue. Transient receptor potential, TRP, family ion channels are cationic channels, permeable to both monovalent and divalent cations and play a role in the influx of calcium mainly through the plasma membrane. These channels also represent vital calcium entry routes in the mammary epithelium and may thus act as central players in the preservation of calcium balance within this tissue. Moreover, TRP family channel proteins are abnormally expressed in breast cancers and may promote cancer progression through deregulation of intracellular signaling, consequently triggering several hallmarks of cancer. This chapter concentrates on the role of transient receptor potential vanilloid, TRPV, a subfamily of proteins in the calcium-dependent functions of normal mammary epithelium and the evident role of these channel-forming proteins along breast cancer progression.

1. Introduction

In adult individuals, the mammary gland is composed of bilayered epithelial structures, forming a branched ductal tree within an adipocyte-rich stroma [1]. These tree-like structures consist of distinct epithelial cell populations that form secretory alveoli, organized into lobules and a branched network of ductal structures. Development of the mammary epithelial cell populations within these structures, occurs hierarchically through specific intermediates and coordinated expression of several lineage-specific markers [2, 3, 4]. Functionally these distinct cell populations are organized into an inner luminal epithelial (LE) cell layer and outer basal cell layer, the basal layer containing both mature myoepithelial (ME) and stem/progenitor cell populations [5]. These specific cell populations within the bilayered mammary epithelium can be distinguished by the expression of various markers, including the cytokeratin expression pattern [6].

The basal cell layer is responsible for the regenerative potential of the mammary epithelium due to the colonization of the mammary stem cells with multilineage potential, within this compartment [5, 7, 8]. Contractile ME cells, localized to the same cell layer, provide a niche for these stem cells. Additionally, ME cells have an important role in synthesizing and maintaining normal basement membrane (BM), controlling polarization and proliferation of the LE cells as well as directing branching and differentiation of the developing structures [9, 10]. Upon gestation, epithelial cell populations further undergo directed differentiation and proliferation, consequently leading to side-branching and formation of alveolar, lactating units within lobular clusters [11, 12, 13]. In such functionally mature mammary epithelial structures, the inner luminal cell population produces and secretes milk into the lumen [14, 15], whereas the outer, smooth muscle actin (α-SMA)-expressing myoepithelial cells provide contractile forces for milk ejection in response to oxytocin [14, 16, 17]. When lactation is over, the alveolar cells undergo programmed cell death and the epithelium is returned to its pregestational state [18, 19, 20].

Calcium is crucial for various physiological processes through activation of specific intracellular cascades and by modulating the integrity of cellular junctions [21, 22]. Alterations in the activity or expression levels of different Ca²⁺ channels, or factors involved in their regulation can therefore significantly change cellular responses to various cues that direct tissue homeostasis [23, 24]. Consequently, deregulation of calcium signaling is therefore associated with several pathological conditions, including cancers. In cancers, abnormal calcium signaling has been linked to high proliferation, inhibition of apoptosis and invasive migration through the epithelial-to-mesenchymal transition, EMT [25, 26]. As for any other tissue, calcium signaling is likewise crucial for the regulation of mammary epithelium, its various calcium-dependent intracellular functions, the integrity of the epithelial sheets and mammary tissue-specific task, lactation [27]. The functional maintenance of the bilayered mammary epithelium is importantly also guided by various hormones and growth factors, which may also cooperate with calcium-triggered pathways [28, 29, 30]. In this review, the role of TRPV, vanilloid subgroup of transient receptor potential family ion channels are discussed in respect of their significance in the regulation of normal mammary epithelial homeostasis and along breast cancer progression.

2. TRPV family channels

TRPV channel proteins belong to the transient receptor potential, TRP, the family of proteins [23]. This superfamily of proteins is formed by over 30 different cationic channel proteins, which are further divided into seven subfamilies: TRPV (vanilloid), TRPA (ankyrin), TRPC (canonical), TRPM (melastatin), TRPML (mucolipin), TRPN (NOMPC), and TRPP (polycystin) families [23, 31]. TRP proteins possess crucial functions in various tissues, in both non-excitable cells as well as in the cells of the nervous system [23, 31]. The members of this family display both structural and functional similarities and many of them are voltage- and temperature-sensitive for functioning as sensors in the peripheral and central nervous systems. Besides these, they can sense various other extracellular cues, both biochemical and physical ones, leading to their spatio-temporal activation, ion influx and adjustment of the downstream signaling cascades [32, 33].

The subfamily of TRPVs has six members, TRPV1-6 that can form both homo-and heterodimeric channels. Of these subfamilies, TRPV1-4 do not display high Ca²⁺-selectivity [34, 35, 36, 37]. On the opposite, TRPV5 and TRPV6 are highly selective for Ca²⁺ [38, 39, 40]. Most of these TRPV family members can sense and respond to various stimuli, consequently activating multiple intracellular signaling cascades [31, 41, 42]. TRPV1 is found in the plasma membrane and prominently expressed in sensory neurons but it is also clearly expressed in other cell types [43]. TRPV1 is involved in nociception and triggered by heat, pH and some compounds, including capsaicin [44, 45, 46]. TRPV2 is found in all tissues, and is highly expressed in sensory neurons. Its main localization in cells is not at the plasma membrane but in the intracellular membranes [47, 48, 49]. TRPV2 displays various physiological functions through its actions as a thermo-, lipid- and mechanosensor. Additionally, it can also respond to growth factors, hormones and cytokines [50, 51, 52], leading to a wide range of functions that play a role in healthy tissues and in pathophysiological conditions.

Both TRPV3 and TRPV4 are highly ubiquitous and they are noticeably expressed in epithelial tissues [35, 37, 53]. TRPV3 is a non-selective cation channel and is especially abundant in the skin keratinocytes [54]. It can sense temperature and plays a role in various tasks, including maintenance of the skin barrier function, wound healing, pain sensation and itch [53, 55]. Therefore, TRPV3 seems to be particularly important for the skin health. Like TRPV3, TRPV4 is an abundant cationic channel in the epithelial tissues and can trigger ion-influx upon various cues, such as mechanical stretching, osmolarity and heat [56, 57, 58, 59, 60]. The activity of TRPV4 has been associated with various physiological functions, it has an important role in cell volume regulation, homeostasis of the vasculature, central nervous system and as a mechanosensor in a wide array of tissues [37, 61, 62].

Unlike the other TRPVs, TRPV5 and its close relative TRPV6 are the only highly calcium-selective channels among TRPVs and the whole TRP superfamily [38, 39, 40]. TRPV5 is highly expressed in the kidney, while TRPV6 has a broad expression pattern in some different tissues. TRPV5 and TRPV6 constitute the apical Ca²⁺ entry mechanism for active calcium transport in the kidney and intestine, respectively. Their roles in the active Ca²⁺-reabsorption and maintenance of cellular Ca²⁺-homeostasis are essential, loss of these proteins leading to reduced bone thickness, defects in the intestinal calcium absorption, reduced fertility, and hypocalcemia [63, 64, 65, 66, 67]. Interestingly, TRPV5 and TRPV6 are under the regulation of 1,25-dihydroxyvitamin D3, and hormones, such as parathyroid hormone, estrogen, and testosterone may participate in fine-tuning the calcium-uptake [68, 69, 70, 71, 72, 73].

The hormonal regulation of TRPV channels has mainly concentrated on the role of sex hormones, which can impact the expression of ion channels either directly or indirectly through intracellular signaling [74, 75]. Progesterone, a steroid hormone, is known to elevate TRPV6 levels in mammary carcinoma cells [76]. In human mammary epithelium, progesterone receptor, PR, is expressed in both luminal and basal epithelial cell populations, and it promotes the proliferation of the basal mammary epithelial cells. Luminal PR may also promote the proliferation of neighboring cells through paracrine signaling mechanisms [77]. In addition to TRPV6, TRPV4 is under the control of progesterone receptors in the mammary gland, airways and smooth muscle cells of the vasculature [78]. In the case of TRPV4, progesterone was found to decrease both mRNA and protein levels of TRPV4, while silencing of PR led to increased level and activity of TRPV4 in the T47D mammary epithelial cell model [78]. In adult individuals, the PR-positive cells are usually also ERα positive [79] and estrogen acts through ERα to induce the expression of PR [80, 81]. This interconnection between the hormone receptors and specific TRPV channel proteins should be further assessed in future studies, as they may also play a role in the disease progression.

3. TRPV channels in the structural maintenance of the mammary epithelium

Calcium signaling is known to direct developmental processes and is also crucial for both structural and functional maintenance of the mammary epithelium [82]. Different TRP family proteins serve as important calcium influx routes in the mammary epithelium and may thus act as central players in the maintenance of the mammary epithelium through calcium homeostasis.

Among the TRPV family channel members, the TRPV4 channel is probably the most well studied in respect of its role in epithelial integrity through the regulation of adherens- and tight junction proteins [83, 84, 85, 86, 87, 88, 89, 90]. In a mouse mammary epithelial cell line, HC11, TRPV4 localizes at the basolateral membrane to regulate calcium influx and permeability [86]. This TRPV4-mediated Ca²⁺-intake is known to trigger activation of some calcium-dependent voltage-gated potassium channels, BK channels, that have a major role in tight junction regulation through at least claudin family proteins. Mechanistically, TRPV4-mediated calcium influx leads to two separate cellular events: A fast elevation in the transcellular conductance via the activation of apically-located large BK potassium channels and a slower increase in paracellular permeability for small soluble molecules. Associated with these alterations, down-regulation of several claudin family tight junction proteins was detected together with large break formation in the tight junction strands [86]. In contrast, studies by Islam et al. showed that TRPV4 can also positively affect the expression of tight junction proteins through X-box-binding protein 1, XBP1, in the mammary epithelial cells upon heat induction [89]. Besides TRPV4, also TRPV6 may play a role in the homeostasis of the mammary epithelium, both during differentiation and maintenance of the intact epithelial structures: Zinc finger homeobox 3 (ZFHX3) is a transcription factor that directs numerous cellular processes, including differentiation. ZFHX3 was found to regulate calcium homeostasis in the mammary epithelium through positive regulation of TRPV6, leading to differentiation of MCF10A mammary epithelial cells in the 3D environment [91]. These observations support the role of TRPV6-mediated calcium influx in the differentiation and maintenance of the mammary epithelium, downstream of ZFHX3. As ZFHX3 is also linked to the function of hormones, including progesterone which can upregulate TRPV6, it would be interesting to investigate the possible connection between them. Furthermore, TRPV6 seems to be important for the maintenance of the junctional integrity of the mammary epithelium [92]. TRPV6 was found to localize at the cell-cell junctions together with adherens junction protein E-cadherin and its depletion led to the loss of epithelial integrity as detected with both MCF10A and 184A1 mammary epithelial cell cultures, treated with TRPV6 siRNA. This could be at least partially through the regulation of peripheral actomyosin bundles that maintain junctional tension as TRPV6 depletion affected pathways upstream of actomyosin assembly [92]. While there is evidence for the role of TRPV4 and TRPV6 in the structural maintenance of the mammary epithelium, the possible role of the other TRPV channel family members have not been properly assessed in this respect, at least in the mammary epithelial model. Additionally, it may be that these channel proteins respond differently to distinct cues to regulate the junctional integrity in the epithelial sheets. At least in the case of TRPV4, there seems to be dual modulation depending on the initial cues.

4. Functions of TRPV channels along gestation and lactation

In the course of gestation, the mammary gland and its epithelial structures undergo major architectural changes, leading to the formation of milk-producing alveolar structures. These morphological events are jointly guided by hormones and growth factors, alterations in the physical microenvironment as well as the paracrine signaling in between the mammary stroma and the bilayered epithelium [93, 94, 95]. Coinciding with the formation of alveoli, TRPV4 mRNA levels are known to be increased at the day 15 of gestation and to be downregulated immediately after lactation [89]. These findings suggest that at least TRPV4 could have a role in the pregnancy-linked developmental processes within the mammary gland. While other, TRPV channels are also responsive to hormones and changes in the mechanical microenvironment, their role along gestation-linked epithelial changes have not been assessed.

During lactation, the maternal calcium and magnesium homeostasis encounter significant alterations due to excessive need of the divalent cation Ca²⁺ in breast milk. Consequently, demineralization of the skeleton is observed together with changes in both renal and intestinal Ca²⁺ transport [96]. For this, several proteins, playing a role in the transcellular Ca²⁺ and Mg²⁺ transportation are upregulated along lactation. Vitamin D also contributes to this process by inducing intestinal hyperabsorption [97]. TRPV5 is highly expressed in the kidney epithelium, in the distal convoluted tubules and connecting tubules [98]. Structurally similar TRPV6 is more widely expressed but exhibits prominent expression in the intestine epithelium [99, 100]. Moreover, both TRPV5 and 6 are Ca²⁺-selective and also vitamin D-responsive [101], and in line with this connection to lactation-induced alterations in Ca²⁺-homeostasis, they are also upregulated in renal and intestinal epithelium upon lactation [97]. Furthermore, prolactin is known to regulate both vitamin D metabolism and induce TRPV6 levels to regulate calcium intake during lactation [102]. TRPV5 and TRPV6 thus participate to lactation by enabling the excessive need of calcium during this physiological phase.

Production of milk is triggered by heat as mammary epithelial cells can activate their milk generation at 39 degrees [103, 104]. Mammary epithelial cells also undergo heat-evoked proliferation and differentiation [105]. Interestingly, many TRP channels act in sensing heat and from the vanilloid subfamily of proteins, TRPV1-4 acts as major thermosensors [45, 106, 107, 108]. Upon heat-treatment, TRPV4 is also able to activate the expression of milk protein beta-casein and tight junction (TJ)-associated proteins, Zonula occludens-1 (ZO-1), Claudin 3 (Cldn3) and Occludin (Ocln) [89]. Permeability of TJs is known to be modulated upon milk production and immediately after parturition [109], and this feature may thus be dependent on TRPV4. Heat stress is also known to induce unfolded protein response, UPR [110] and UPR-associated transcription factor XBP1 plays a role in the differentiation of mammary epithelium together with the expression of milk protein beta-casein [104, 111]. Intriguingly, recent work by Islam et al. proposes that TRPV4 acts through XBP1 [89]. Besides heat, TRPV4 is activated by mechanical changes and stretching in the cell environment that are also known to take place along lactation. In addition to TRPV4, the TRPV2 channel can play a role in lactation as it localizes to oxytocinergic neurons [112].

After lactation is over, the milk-producing structures regress to the pre-pregnancy state in a complicated reverse action, involution [113]. Ca²⁺-dependent signaling may also impact this transfer from lactation to involution [14]. Whether any of the TRPV family members play a role in this process, remains to be studied.

5. Abnormal expression of TRPV channels along breast cancer progression

The characterization of breast cancers is based on different criteria, including the histopathological evaluation, grading and staging as well as defining the expression of estrogen (ER), progesterone (PR), and epidermal growth factor (HER2) receptors [114]. Additionally, gene expression profiles can be used to determine the molecular subtypes, which can be Basal-like, HER2-enriched, Claudin-low, Luminal A, Luminal B, or Normal-like. The heterogeneity of breast cancer as a disease is well seen also on the differences in Ca²⁺-channel expression that vary greatly in between specific breast cancer subtypes. Often the levels or activity of plasma membrane-embedded calcium channels can also reflect the metastatic potential and prognosis of distinct mammary carcinomas [26]. Abnormal activity of the Ca²⁺-channels in breast cancers could potentially take place due to mutations, deregulation of the channel gating or changes in the expression levels, triggering Ca²⁺-influx in unfavorable patterns, both spatially and temporally. As several calcium channels can respond to a wide variety of biochemical and mechanical cues in their microenvironment, any alterations in such could lead to deregulated calcium channel activity to sustain an elevated or abnormally low calcium entry. Additionally, a variety of the plasma membrane-associated calcium channels could be deregulated at the same time, within similar cancer types, further cooperating in adverse processes along the course of neoplastic progression.

TRPV channel family, among the other TRP family members, has been linked to the progression of a variety of human cancers [115, 116]. These cationic channels can also mediate Ca²⁺-influx and have been shown to contribute to several hallmarks of cancers, including the potential to proliferate, resistance to apoptosis, angiogenesis, and invasion [117, 118]. Additionally, these channel proteins may have different roles, as either cancer promoters or suppressors, depending on the cancer type and its genetic background as well as the expression levels of distinct channel proteins. The primary Ca²⁺-triggered pathways that play a role in promoting these cancer-associated features through specific TRP channels, include CaMKII, NF-κB, calpains and calcineurin pathways [119, 120], but other less studied signaling cascades may as well be involved.

While the members of TRPV family channels are frequently deregulated in many cancers and associated with certain cancer-specific cellular features, their regulation along the breast cancer progression is still poorly understood. TRPV1 channel is often upregulated in breast cancers and its high expression correlates with the tumor grade [121]. Some studies have shown no differences in between distinct breast cancer sub-types and expression levels of TRPV1 [122, 123, 124]. Aggregated TRPV1 in the intracellular compartment has, however, been linked to poor prognosis in breast cancer patients [125]. TRPV2 expression also seems to display oncogenic activity in various cancers [126, 127]. In triple-negative breast cancers (TNBCs), TRPV2 levels are especially prominent but correlate interestingly with high relapse-free survival in this case [122, 128]. Additionally, the study by Elbaz et al. [128], proposed the therapeutic potential of high TRPV2 to elevate the uptake and efficacy of chemotherapeutic agents in patients with TNBC. The role of TRPV4 in cancer progression has been investigated by several labs and its expression in breast cancers is highest in the basal-like cancer subtype [122, 129]. High TRPV4 expression has also been detected in IHC stainings from the metastatic lesions of invasive ductal carcinomas and its levels correlate with the tumor grade and size [130].

TRPV6 channel is likewise overexpressed in various cancers, including cancers of the mammary tissue [76, 131, 132, 133, 134]. The levels of overexpressed TRPV6 vary a lot depending on the breast cancer subtype, and as with the TRPV4 channel also TRPV6 levels are highest in the basal-like breast cancers and HER2-enriched molecular subtypes [76, 135, 136]. In line with this, ER receptor-negative breast cancers and cancer cell lines with several overlapping features with the basal and HER2-enriched subtypes display significant amounts of TRPV6 [136]. High TRPV6 in the patients is also associated with lower survival in comparison to patients that express lower TRPV6 levels [136].

Of the TRPV family, TRPV3 and TRPV5 have been studied to less extent. TRPV3 is known to be expressed at low levels in different types of breast cancer subtypes and its possible association with cancer progression has not been well assessed [122]. Likewise, there are no reports on TRPV5 and its link to the progression of distinct breast cancer types [122]. While these two subtypes may not be important in respect of breast cancer progression, more studies are needed on the field to understand how the deregulation of the other TRPV forms takes place along cancer progression and whether for instance hormonal regulation or stromal changes could impact their expression and activity.

6. TRPV family channels: implications for cancer cell-associated features along breast cancer progression

6.1 Excessive proliferation

Various studies have shown the significance of calcium signaling in the uncontrolled proliferation of cancer cells [25, 137, 138]. Various plasma membrane-embedded calcium channels are acting as major sources of calcium for the regulation of such pathways that lead to elevated cell amounts [139, 140]. Among the TRP family, also TRPV channels contribute to these processes, related to malignant growth [120].

TRPV1 channel can mediate both Ca²⁺ and Na⁺-influx and trigger cell proliferation by two separate mechanisms: It can contribute to the activation of serine-threonine kinase Akt as well as to the activation of ERK1/2 downstream of the epidermal growth factor (EGFR) [141]. However, studies in the MCF-7 breast cancer cell line show that both agonists and antagonists of the TRPV1 channel can inhibit cell growth through yet unidentified mechanisms [142]. Thus, it may be that the balance in the expression of this protein is important for controlled cell proliferation through distinct intracellular pathways in a cell type-specific manner. MCF-7 cell line has also been utilized as a model to study TRPV2 in respect of cell proliferation. TRPV2 was shown to be responsive to insulin-like growth factor-I (IGF-I) [143] and tranilast, an anti-inflammatory agent, was reported to inhibit IGF-1-induced cell growth by blocking the calcium influx through TRPV2 [144]. Significant overexpression of TRPV4 is also linked to breast cancers and this seems to correlate with the tumor grade and size, leading to poor survival [122, 130, 145, 146]. However, evidence from studies performed with 4 T07, MDA-MB-231 and MDA-MB-468 breast cancer cell lines show that TRPV4 is dispensable for the proliferative potential of these specific breast cancer cell lines, since its silencing or pharmacological inhibition was not anti-proliferative [145, 147]. In contrast, high expression of TRPV6 is linked to the proliferation through Ca²⁺-triggered intracellular pathways and the high levels also act as prognostic factors together with potential resistance to chemotherapy [76, 134, 135]. Depletion of TRPV6 from the T47D breast cancer cell line, displaying high endogenous TRPV6, also decreases the proliferation of these cells [76, 136]. The precise mechanisms behind this are not understood but may involve PI3K/pAKT pathway that regulates cell proliferation, survival and therapeutic resistance in some breast cancer subtypes, including the HER2-enriched subtype. In line with this, depletion of TRPV6 was associated with lower levels of active, phosphorylated AKT in HCC-1569 breast cancer cells [148]. Studies in breast cancer cell lines, MCF-7 and MDA-MB-231, additionally showed the link in between TRPV6 and PI3K/Akt pathway as a functionally auto-inhibitory intramolecular interaction between S5 and S6 helices of TRPV6 was shown to contribute to TRPV6/PI3K association and the activation of PI3K/Akt/GSK-3β pathway [149].

TRPV6 activity and expression are known to be controlled by estrogen, progesterone, and 1,25-vitamin D that all play a role in the proliferation of breast cancer cells [76, 120]. Treatment of breast cancer cell line T47D with estrogen receptor antagonist, tamoxifen, also led to lower activity and expression of TRPV6 calcium channel protein. Further, the effect of tamoxifen on the functionality of TRPV6 was shown in EYFP-C1-TRPV6-transfected MCF7 breast cancer cells by Fura-2 calcium imaging [150]. Calcium levels in the transfected cells were found to be higher than in non-transfected cells and the calcium levels were lowered by 50% with tamoxifen-treatment. Interestingly, tamoxifen also played a role in TRPV6 inhibition in MDA-MB-231 cells that are estrogen receptor-negative [150], suggesting a direct impact on TRPV6-mediated Ca²⁺-influx. Besides tamoxifen, TRPV6 activity can also be negatively regulated by a protein called Numb1 [151]. Numb1 is maybe more known for its role in the stabilization of tumor suppressor protein p53 [152], affecting both cell cycle progression and apoptosis. Studies on the Numb1-TRPV6 link in MCF-7 breast cancer cells showed that Numb1-depleted cells displayed elevated TRPV6 expression and calcium influx as well as enhanced proliferation. TPV6 thus has interesting connections to the pathways of the major tumor suppressor protein as well as to hormones that play a role in breast cancer progression through the proliferative potential of the cells.

6.2 Resistance to apoptosis

Apoptosis can be characterized as a programmed cell death process, which leads to the fragmentation of DNA. This strictly controlled process can take place through cell death-receptors or through mitochondria-mediated apoptotic pathways [153]. Apoptosis is also controlled by calcium-dependent pathways [154, 155, 156]. Changes in intracellular Ca²⁺-levels are known to influence the two major apoptotic pathways through gene expression [157, 158, 159, 160, 161]. For instance, the calcium/calmodulin-dependent signaling cascades can affect the balance in between cell cycle progression and apoptosis [160].

TRPV1-triggered calcium influx has been shown to act as a determinator of the balance in between cell proliferation and apoptosis. TRPV1-mediated apoptosis can take place through the mitochondrial mechanism, while its proliferation-supportive actions usually involve other cell membrane receptors or specific intracellular signaling cascades [141]. Studies with MCF-7 breast cancer cell line have also shown that high TRPV1 sensitizes cells to programmed cell death, induced by TRPV1 activator capsaicin [162, 163]. Likewise, capsaicin is involved in the induction of cell death in breast cancer cell line, SUM149PT through TRPV1 activation [121].

The role of TRPV4 in apoptosis has as well been investigated during the past few years and these studies support the role of high TRPV4 expression in inducing cell death. In breast cancer cell lines, MDA-MB-468 and HCC1569, activation of TRPV4 by pharmacological compounds reduced the viability of the cells [147]. Both cell lines display high endogenous TRPV4 levels and its activation was able to promote cell death by apoptosis or oncosis, while the same phenomenon was not detected in breast cancer cell lines with low TRPV4 levels. Moreover, the studies by Peters et al. found that TRPV4 activation has therapeutical relevance in vivo and can inhibit the growth of tumors [147]. Similarly, to TRPV4, overexpression of TRPV2 and its pharmacological activation with cannabidiol have been linked to inducing cytotoxic impact in SUM159 and MDA-MB-231 breast cancer cells via doxorubicin-treatment [128]. In contrast, the TRPV6 calcium channel seems to act oppositely and its high levels are rather protecting from apoptosis: TRPV6 calcium channel is known to get transported to the plasma membrane in an Orai1-mediated mechanism to control the survival of the cancer cells [164]. On the other hand, TRPV6 depletion from breast cancer cells with high expression of this protein can be used for decreasing the viability of the cells, as shown by studies in T47D breast cancer cells [76].

6.3 Tumor microenvironment and angiogenesis: connection to TRPV channels

The tissue microenvironment undergoes drastic alterations along breast cancer progression [165, 166, 167]. Besides stiffness and composition of the matrix, there are also changes for instance in the amount of growth factors and acidicity of the environment that may trigger specific calcium channels [168, 169]. How TRPV channels, among other ion channels on the plasma membrane, respond to such cancer-linked cues from the extracellular space, is poorly understood. Additionally, stromal cells, such as fibroblasts, immune cells, or adipocytes that also express channel proteins, may be functionally altered and contribute to abnormal signaling from the stroma.

At least TRPV4 and TRPV6 are known to be responsive to stromal stiffening [92, 170, 171, 172, 173] and could be triggered by cancer-associated mechanical changes in the extracellular space. Furthermore, TRPV4 has been shown to control the expression of some extracellular matrix proteins, in this way contributing itself to the stiffness of the environment [130]. Stiffening may impact various processes along cancer progression and one of these features is the growth of new vasculature, angiogenesis. The first evidence that TRPV4 could also be involved in angiogenesis along breast cancer progression was presented in the work by Fiorio Pla et al. [174]. The authors discovered the role of TRPV4 in mediating arachidonic acid (AA)-promoted migration of endothelial cells (ECs), derived from breast tumors. These endothelial cells displayed high endogenous TRPV4 and were enhancing the migration of ECs, a key step in the growth of new vessels. This step could be inhibited by antagonist or siRNAs against TRPV4 and the opposite was detected with TRPV4 stimulation [174].

Support for the role of TRPV4 in angiogenesis has also been shown in studies by Adapala et al. [170]. TRPV4 seems to control the mechanosensitivity of tumor endothelial cells (TECs), and the angiogenetic process all the way to the maturation of the vessels. Interestingly, the authors found that these TECs display lower TRPV4 levels than normal endothelial cells, leading to angiogenesis through the altered ability of the cells to sense the mechanical environment. Besides, they discovered that normalizing TRPV4 levels could be acting as an anti-angiogenetic therapy to normalize the vasculature and enhance drug efficiency. Moreover, studies by Thoppil et al. have shed light on the mechanisms that TRPV4 could utilize in the regulation of the angiogenetic process [175]. These studies also linked low TRPV4 levels of endothelial cells to enhanced migration and disturbed angiogenesis. This could be reversed by the treatment of cells with Rho kinase inhibitor, Y-27632, suggesting that TRPV4 action in angiogenesis involves modulation of mechanosensitivity of ECs via Rho pathway [175]. Based on these data, TRPV4 may therefore be a significant regulator of angiogenesis and this information could potentially be utilized in therapeutical approaches. TRPV4 thus has an important role in the modulation of tumor stroma by affecting both its mechanical features as well as the growth of new blood vessels in the stroma. Interestingly, TRPV4 is this far the only channel protein among the TRP superfamily that has been implicated in the growth of new vessels along cancer progression.

6.4 Invasion and metastasis

Abnormal expression of distinct TRPV channels has also been linked to invasive migration and metastasis. Several TRP channel family members are connected to Rho-pathway and display the potential to promote invasive migration through Rho-dependent cytoskeletal reorganization [174, 176]. Of the TRPV family members, at least TRPV2 appears to be under the control of Rho-kinase as the treatment of breast cancer cells with Rho-inhibitors lowers the levels of TRPV2 [177]. Another factor, known to impact cell migration through activation of TRPV2, is the antimicrobial peptide, LL-37. LL-37 can influence cancer progression through various ways, including its positive impact on cancer cell migration [178]. The expression of LL-37 correlates with the expression levels of TRPV2 in breast cancer cell lines and has been shown to promote invasive migration of MDA-MB-435, MCF-7 and MDA-MB-231 cells dependently on TRPV2 [179]. Mechanistically, LL-37 increases calcium influx through TRPV2 and enhances cell migration via PI3K/AKT signaling [180]. Activation of PI3/Akt pathway as such leads to recruitment of TRPV2 into pseudopodia, impacting the migration of specific breast cancer cell types [179].

TRPV4 has also been associated with invasive migration and has been linked to EMT and lower relapse-free survival in basal breast cancers with lymph node involvement [181]. In MDA-MB-468 breast cancer cells, TRPV4-mediated calcium-influx plays an important role in the EGF-triggered EMT: activation of TRPV4 by chemical compounds was able to drive the upregulation of various EMT markers in these cells [181]. In line with these results, TRPV4 depletion from a murine mammary cancer cell line, 4T07, lowered the migration capability and 3D invasion of these normally high TRPV4-expressing cells [145]. Furthermore, determining TRPV4 levels from database information of human clinical samples as well as phosphoproteomic analyses of xenograft-derived in vitro models, indicated the role of TRPV4 in breast cancer metastasis, high expression of TPV4 in basal breast cancers and its association with poor prognosis [145]. Additionally, TRPV4 KD decreased the levels of metastatic nodules in mouse xenografts [145].

Interestingly, TRPV4 also implies to determine the stiffness of cancer cells through actin dynamics, in this way affecting deformability and metastasizing potential of breast cancer cells [130, 145]. TRPV4 was regulating the compliance of cancer cells through Ca²⁺-mediated AKT-E-cadherin signaling [130]. Additionally, TRPV4 was involved in the expression of extracellular matrix proteins and the modeling of the matrix [130]. Knowing the mechanosensitive nature of TRPV4, there seems to be a functional feedback loop in between TRPV4 and its mechanical environment that plays a role along cancer progression. TRPV4 may therefore impact invasion and metastasis of breast cancer cells through various means.

Besides TRPV2 and -4, also TRPV6 has been linked to invasion and metastasis in breast cancers. Overexpression of TRPV6 is very common in breast carcinomas and TRPV6 levels have been shown to be very high in the invasive regions of the mammary carcinoma samples [76, 135]. The mechanisms of how TRPV6 could impact invasive progression, are not well understood. However, it seems to be linked to both actomyosin dynamics and the expression of EMT markers that could be critical along the development of invasive disease [92]. Further, inhibition of TRPV6-mediated calcium-influx by lidocaine, led to lower migration and invasion ability of the MDA-MB-231 breast cancer cells [182]. The exact molecular pathways, affecting TRPV6-mediated invasion in breast carcinomas, needs still to be further clarified.

7. Pain sensation

TRPV channels have been indicated to function in nociception, the sensation of pain [41, 44]. Although, not directly linked to the function of the mammary gland, it plays a role in breast cancer progression in the form of bone pain as a consequence of bone metastasis formation.

One of the main TRPV channels, playing a role in nociception, is TRPV1 [44, 46]. Interestingly, the formation of a tumor within a bone is known to increase the expression of TRPV1 in a specific population of dorsal root ganglion neurons [183]. In addition, TRPV1 is important for both the development and maintenance of cancer pain [184]. Likewise, it has been observed that extracellular cues within the bone microenvironment, developed during the formation of breast cancer-derived metastasis, are contributing to the pain sensation via TRPV1 activation [185]. In line with these data, experiments with rat models have revealed that when mammary carcinoma cells are injected to the rat bones, TRPV1 expression is upregulated within the dorsal root ganglion cells [184, 186]. Further, MDA-MB-231 breast cancer cells have been shown to promote sensory neuronal growth and elevate sensitivity to active TRPV1 [187]. TRPV1 may therefore be important in the sensation of pain upon metastatic breast cancer and its pharmacological targeting has also been pursued for instance by blocking the capsaicin receptor [188].

The mechanisms through which TRPV1 is induced upon bone cancer and -metastasis have been studied as well: in a rat bone cancer-pain model, utilizing mammary carcinoma cells injected to the bone cavity, TRPV1 was upregulated and activated through induction of Insulin-like Growth Factor 1, IGF-1 [184]. Additionally, TGF-β1 is known to contribute to pain upon bone cancer via upregulation and sensitization of TRPV1 in sensory neurons [189, 190]. In conjunction with these observations, TGFβRI and TGFβRII are known to be upregulated in this rat bone cancer-pain model upon inoculation of rat mammary carcinoma cells [190]. Furthermore, lysophosphatidic acid, LPA, triggers TRPV1 through a PKCε-dependent signaling cascade in dorsal root ganglion neurons upon bone cancer formation in rats [191]. TRPV1 may thus be a central player along the pathways that are behind bone cancer pain in advanced breast cancers.

8. Potential for therapeutical targeting

The emerging role of TRP channels in cancer progression has been widely admitted. Abnormal expression of several TRPV family members, along with the altered expression of other TRP family channels, direct various cancer-linked features, including proliferation, apoptotic control, angiogenesis, and invasive migration leading to distant metastasis [76, 122, 129, 133, 191] (see also Figure 1). For that, these calcium channel proteins can also serve as biomarkers and as attractive objectives for therapeutical targeting. The fact that these ion channels can be activated by small pharmacological compounds, also supports their potential for therapeutical approaches and several studies have been performed with potential modulators against the activity of these proteins to target cancer cells [191, 192, 193, 194, 195].

Figure 1.

The role of TRPV family members in the maintenance of normal mammary epithelium and in the induction of hallmarks of cancer. The connection of distinct TRPV family members to the structural and functional maintenance of the normal mammary epithelium as well as their connections to specific steps along breast cancer progression are summarized in this figure. The corresponding references are found within the brackets.

TRPV1 channel is activated by a natural compound, capsaicin, the primary pungent component of the chili pepper, and there is evidence for its potential anticancer activity and ability to induce apoptosis [196]. In breast cancer models, ectopic expression of TRPV1 combined with capsaicin-treatment, leads to mitochondrial Ca²⁺ accumulation and necrosis [197]. TRPV1 expression alone was able to stop cell proliferation and induce apoptosis via activation of caspase-3 activity in breast cancer cell lines [197]. As capsaicin, through its impact on TRPV1 activity, also causes pain sensation, it cannot be used as a therapeutical compound in high doses to induce apoptosis. However, a chemical compound, dihydropyridine derivative MRS1477, operates as a modulator of TRPV1 activity, and can be used together with capsaicin to promote apoptosis in breast cancer cells [163]. A study by Wu et al. investigated the mechanisms behind capsaicin-mediated apoptosis by utilizing a TRPV1-inducible MCF-7 breast cancer cell line [162]. They found that the cell death upon capsaicin-treatment was necrotic and linked to elevated levels of c-Fos and receptor-interacting serine/threonine kinase 3, RIP3 that plays a role in the inflammatory mode of cell death, necroptosis [162]. Additionally, an alkyl sulfonamide analogue of capsaicin, RPF151, shows potential for targeting breast cancer cells as shown by studies with MDA-MB-231 cells [198]. In this study, capsaicin analogue was found to downregulate p21, cyclins A, D1, and D3, subsequently leading to arrest in the S-phase and induction of apoptosis [198]. Furthermore, modulation of TRPV1 activity in sensory neurons by pharmacological compounds may also lead to an anti-tumoral immune response [199]. Systemic treatment with low-dose of capsaicin was shown to trigger an anti-inflammatory response against metastatic breast carcinomas and have potential as a therapy choice [199]. On the other hand, a synthetic antagonist against TRPV1, capsazepine, CPZ, has also been shown to possess anti-cancer effects in vivo through its impact on cell proliferation in several cancer cell types, including breast cancer cells [200]. Capsazepine and its analogues may thus act as potential therapeutic compounds in the future [200].

Besides capsaicin, another natural compound, cannabidiol, has an impact on the induction of apoptosis in MDA-MB-231 breast cancer cells through the TRPV1 channel [201]. These studies showed that besides inducing apoptosis through vanilloid transient receptor potential vanilloid type-1 receptors, cannabidiol can act in the induction of apoptosis via cannabinoid receptor type 2, CB2 and through cannabinoid/vanilloid receptor-independent mechanisms [201]. The interconnection between cannabinoid receptors and TRPV1 has also been investigated in another study that utilized MDA-MB-231 cells as a model. In this study, the role of these receptors in cancer cell invasion was assessed and the results linked activation of both CB1 and TRPV1 by agonist to reduced invasion capability of the MDA-MB-231 cells [202].

Intriguingly, it has also been noticed that some common chemotherapeutic agents interact with TRPV-dependent pathways: The combination of selenium and cisplatin operate through overlapping intracellular pathways and can also modulate TRPV1 activity to induce apoptosis in MCF-7 breast cancer cell line [203]. In addition, combination therapy with alpha-lipoic acid, ALA and cisplatin benefits from the activation of the TRPV1 channel to induce apoptosis in MCF-7 breast cancer cells [204]. Furthermore, in the same breast cancer cell line, chemotherapeutic agent 5-Fluorouracil induces mitochondrial cytotoxicity and apoptosis upon TRPV1 activation [205]. The effectiveness of chemotherapy, combined with the activation of transient receptor potential channel activity, has also been demonstrated with TRPV2: activation of TRPV2 with cannabidiol, CBD, sensitized aggressive triple-negative breast cancer (TNBC) cells to the chemotherapeutic drug, doxorubicin, consequently inhibiting tumor growth in in vitro and in vivo models [128]. TRPV2 may thus act as a positive prognostic marker for TNBC patients who are undergoing chemotherapy.

Besides induction of apoptosis and inhibition of cell proliferation, TRPV channels have been investigated as potential targets to block invasive migration. TRPV2 has been associated with the function of antimicrobial peptide hCAP18/LL-37, which stimulates both proliferation and migration of various cancer cell types, including breast cancer cells [206]. In line with these previous findings, TRPV2 silencing was found to diminish the LL-37-dependent migration of MCF-7 and MDA-MB-231 breast cancer cells [179]. As TRPV4 is involved in invasive migration as well, and modulation of its activity is possible through several compounds, the potential of targeting this protein should be assessed for reducing metastasis in breast cancer models. Several animal studies have already shown the effectiveness of TRPV4 antagonists as therapeutic agents for treating several other diseases [207, 208]. In addition, TRPV6 is overexpressed in breast cancers and could be targeted in estrogen receptor-negative subtype of mammary carcinomas [136]. Specific TRPV6-targeting compounds have been developed that could be used for manipulating TRPV6 activity and such compounds have also shown promising results in various cancer types, including breast cancer cells [209, 210, 211, 212]. While the utilization of TRPV modulators to induce apoptosis or inhibition of either cell proliferation or migration has shown very promising results, one should also consider the risk of other unwanted side-effects through toning of some critical signaling cascades. Such problems can be caused due to the unspecificity of certain antagonists and agonists against the ion channel proteins, leading to the deregulation of several channel protein types. In addition, the wide expression of many of the ion channel proteins throughout various tissues will create challenges in the modulation of ion channel activities at specific sites. For instance, TRPV1 is very widely expressed and most often linked to toxicity in the trials [213, 214]. The balance in the expression and activity of these proteins is though decisive for such a variety of cellular processes.

9. Conclusions

The mammary epithelium is strictly regulated by hormonal signaling, growth factors and cytokines that direct its development, growth and functional organization. In addition, the mammary epithelium is exposed to various physical alterations in the microenvironment that may be sensed by the plasma-membrane embedded structures, such as the ion channels in the mammary epithelial cell populations. Calcium ion pumps and influx through them play a central role in decoding many of the extracellular cues into intracellular signaling. Therefore, these channel proteins greatly impact all essential processes in the maintenance of normal mammary tissue and participate to the development of pathophysiological conditions.

TRPV channels, among the TRP superfamily, are abundantly expressed in discrete tissues, also in the mammary tissue. Of these channel proteins, at least TRPV4-6 have identified functions in the structural and functional maintenance of the normal mammary epithelium, both directly in the mammary epithelium or indirectly through the control of ion influx in other tissues that impact physiological functions of the mammary gland. Whether the other TRPV channels have importance in the structural maintenance of the mammary epithelium or along with lactation, remains to be studied.

Abnormal expression of TRPV channels is also abundantly found in human breast carcinomas and these channel proteins are involved in triggering many of the typical hallmarks of cancers. How TRPV channels are deregulated or aberrantly expressed along breast cancer progression, is still poorly understood. However, as these proteins are sensitive to any physical or biochemical changes in the microenvironment, it is more than likely that they would be affected by the cancer-associated changes in the stroma. This topic certainly requires more investigations in the future. As inducers of the cancer-linked features, such as proliferation, inhibition of apoptosis, invasive migration and angiogenesis, TRPV family members also act as attractive targets for therapeutical choices. A number of known natural and synthetic modulators of TRPV activity already exist and some of them have given promising results in the trials that aim for pharmacological intervention of breast cancers. However, as these proteins are upstream of numerous intracellular pathways that guide cellular functions, there are challenges in such attempts. Furthermore, one should consider the possible interplay in between distinct plasma-membrane embedded calcium channels, several of which may be deregulated along cancer development and impact overlapping intracellular pathways. Such a phenomenon creates a more complex picture on the role of specific ion channels in cancer progression and requires extensive studies in the future.