17.1. INTRODUCTION

Many proteins in cancer cells exhibit increased or decreased expression compared to their levels in normal cells. Some of these proteins, i.e., those encoded by oncogenes and tumor suppressor genes, play key roles in tumorigenesis and in the development of metastases, while others, most likely including those involved in intracellular Ca²⁺ homeostasis (reviewed in Refs. 1 and 2), are associated with cancer progression but are not causative in further development of the tumor and/or malignant cells (reviewed in Ref. 3). Most cancers are heterogeneous with respect to rates of growth and degrees of aggression. This most likely reflects the fact that, for a given cancer, there can be different combinations of oncogenes and tumor suppressor genes that are mutated, different sequences in which these mutations occur, and variations in the time over which the mutations accumulate.³

Some of the most important signaling pathways altered in tumorigenesis enhance cell proliferation and inhibit apoptosis. Ca²⁺ homeostasis controls these cellular processes, including proliferation, apoptosis, gene transcription, and angiogenesis.⁴ Ca²⁺ signaling is thus required for cell proliferation in all eukaryote cells, while some transformed cells and tumor cell lines show reduced dependence on Ca²⁺ to maintain proliferation.^{5, 6} Furthermore, the regulation of cell cycles, apoptosis, or proliferation depends on the amplitude and temporal-spatial aspects of the Ca²⁺ signal,^{7, 8} thus highlighting the importance of Ca²⁺ signaling components such as Ca²⁺ channels. Indeed, dysfunctions in Ca²⁺ channels are involved in tumorigenesis because increased expression of plasma membrane Ca²⁺ channels amplifies Ca²⁺ influx with consequent promotion of Ca²⁺-dependent proliferative pathways.^{4, 7, 9}

Transient receptor potential (TRP) channels contribute to changes in intracellular Ca²⁺ concentrations, either by acting as Ca²⁺ entry pathways in the plasma membrane or via changes in membrane polarization, modulating the driving force for Ca²⁺ entry mediated by alternative pathways,¹⁰ as well as the activity of voltage-gated Ca²⁺ channels. In addition, TRP channels are expressed on the membranes of internal Ca²⁺ stores^{11– 13} where they may act as triggers for enhanced proliferation, aberrant differentiation, and impaired ability to die, leading to the uncontrolled expansion and invasion characteristic of cancer.

All these normal as well as abnormal misregulations are within the scope of this chapter, which is aimed at the study of TRP channels at all known levels, such as mRNA transcription, splicing variants, mRNA translation into protein, further protein processing in Golgi, regulation of channel trafficking to the plasma membrane, and the final modulation of TRP channel activity at the plasma membrane.

17.1.1. TRP Channels and Cancer

The extent to which TRP channels are associated with cancer has been increasingly clarified in recent years. The approximately 30 TRPs identified to date are classified in six different families: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin), and TRPA (ankyrin transmembrane protein).¹⁴ The expression levels of members of the TRPC, TRPM, and TRPV families are correlated with the emergence and/or progression of certain epithelial cancers.^{15– 18} It has not yet been established whether these expression changes are drivers, required to sustain the transformed phenotype. Usually, the progression of cells from a normal, differentiated state to a tumorigenic, metastatic state involves the accumulation of mutations in multiple key signaling proteins, encoded by oncogenes and tumor suppressor genes, together with the evolution and clonal selection of more aggressive cell phenotypes.

Several recent works have shown that changes in the expression of TRP channels contribute to malignancy. The first evidence that TRP channel expression is correlated with different types of cancers came from the analysis of the expression of TRPM1. The expression of the TRPM1 gene is inversely correlated with the aggressiveness of melanoma malignant cells, which suggests that TRPM1 may behave as a tumor suppressor gene.^{18, 19} A second member of the TRPM subfamily, TRPM5, was shown to be responsible for the Beckwith-Wiedemann syndrome, a disease characterized by a childhood predisposition to tumors.²⁰ TRPC channels are often coupled to a membrane receptor with which they work in synergy. Thus, it was demonstrated in prostate cancer cells that a Ca²⁺ signal can promote either cell proliferation or apoptosis,^{12, 15} depending on the type of TRPC channel involved: Ca²⁺ entry via TRPC6 channels stimulates cell proliferation, whereas TRPC1 and TRPC4 are mostly involved in apoptosis induction.

In addition, TRP channels could also play a key role in cancer progression. Indeed, this seems to be the case for TRPM8 and TRPV6 channels in prostate cancer. TRPM8 has originally been cloned from cancerous prostate tissue¹⁶ and was thereafter identified as an ion channel responding to cold stimuli.²¹ TRPM8 is expressed in normal prostate; however, its expression is increased in ADCaP. TRPV6 is strongly expressed in advanced prostate cancer, with little or no expression in healthy and benign prostate tissues.^{17, 22}

Thus, to date, most changes involving TRP proteins do not involve mutations in the TRP gene but rather increased or decreased expression levels of the wild-type TRP protein, depending on the stage of the cancer. Table 17.1 summarizes these changes in cancer and metastatic cells.

TABLE 17.1

Expression Profile of TRPs in Cancer

17.2. ROLE OF TRP CHANNEL ISOFORM EXPRESSION AND FUNCTION IN THE STUDY OF THE CANCER INITIATION AND PROGRESSION

TRP proteins form tetrameric channel complexes, and at least the closely related members of one subfamily are capable of building heteromeric channels.³⁷ The diversity of native TRP-related channels might be considerably increased by combining different TRP channel subunits to build a common ion-conducting pore. There is growing evidence that transcriptional regulation and alternative mRNA processing also contribute to the diversity of TRP channels. Some TRPs are expressed in two or more short splice variants, which may also exhibit different expression profiles in cancer as compared to the full-length forms. Alternative splicing enables the same gene to generate multiple mature mRNA types for translation, resulting in multiple-channel proteins. This leads to functional diversity, which, in turn, may have consequences for cellular function. Alternative splicing generates protein isoforms with different biological properties, such as a change in functionality, protein/protein interaction, or subcellular localization.³⁸ Many of the splice variants are not functional and may not even be efficiently translated, so they may be considered negligible populations of incomplete or aberrantly spliced transcripts. Nevertheless, alternative splicing, as a regulatory process, contributes to biological complexity, not only by proteome expansion but also through its ability to control the expression of functional proteins. This may be achieved by producing nonfunctional isoforms of the gene by altering the domains necessary for TRP channel opening, membrane localization, or association.

This is the case for TRPV2, which expresses two transcripts in normal human urothelial cells and bladder tissue specimens: full-length TRPV2 and a short-splice variant, s-TRPV2. Analysis of TRPV2 gene and protein expressions in distinct superficial and invasive grades and stages indicates that the mRNA of TRPV2 increases gradually at increasing grades and stages, while that of s-TRPV2 gradually decreases.²⁰ The authors suggested that the differences observed in the short/full TRPV2 ratio during tumorigenesis implied that s-TRPV2 was lost as an early event in bladder carcinogenesis, whereas the enhanced expression of full-length TRPV2 in high-stage muscle-invasive urothelial cancer is a secondary event. In a similar study, a different s-TRPV2, lacking the pore-forming region and the fifth and sixth transmembrane domains, was characterized in human macrophages.³⁹ As for TRPV1, these naturally occurring alternative splice variants may act as dominant-negative mutants⁴⁰ by forming a heterodimer with TRPV2 and inhibiting its trafficking and translocation to the plasma membrane.

In the case of TRPV1, the short isoform (TRPV1β) is produced by alternative splicing of the TRPV1 gene, with 10 amino acids missing near the end of the cytoplasmic N-terminus.⁴⁰ TRPV1β does not form a functional channel when it is heterologously expressed alone, but exerts a dominant-negative effect on TRPV1 when they are co-expressed. Stability is affected when TRPV1β is assembled with the full-length channel, making less TRPV1 protein available at the plasma membrane. The residual amount of TRPV1β on the plasma membrane is not activated by factors known to stimulate TRPV1, but there are two other possibilities.⁴⁰ Either the residual proteins are not properly assembled into tetrameric channels or channels that contain TRPV1β subunits cannot be opened. It should be noted, however, that TRPV1 Western blot analysis of the urothelium revealed two bands of equal intensity at 100 and 95 kDa, which decreased as the cancer progressed.¹⁹ Further investigation is required to determine whether these are the two splice TRPV1 isoforms and to analyze their expression regulation as cancer progresses. A similar mechanism is present in normal and benign melanocytes, which express the full-length TRPM1 mRNA, along with some shorter products.¹⁸ Heterologous co-expression of the full-length and short TRPM1 isoforms results in retention of the full-length channel in the endoplasmic reticulum (ER).¹³ However, it is currently unclear whether TRPM1 expression in metastasizing lines inhibits their growth. Metastatic melanomas lack the full-length transcript, but express several short fragments of TRPM1,¹⁸ probably owing to proteolysis of the full-length protein.⁴¹

TRPM8 also encodes some splice variants, comprising an altered N-terminus cloned from lung epithelia⁴² and cancerous prostate.⁴³ The lung epithelia splice variant localizes preferentially to the ER, and its activation controls cell responses to cold air-induced inflammation.⁴² It has not yet been clarified whether this newly identified variant is implicated in cancer, whereas it may constitute a regulatory mechanism for the full-length TRPM8 in tissues where they both localize, such as liver, colon, and testis.⁴² Little information is available concerning the cancerous prostate TRPM8 isoform. It has a truncated N-terminus⁴³ and may serve as a dominant negative regulator of full-length TRPM8, as suggested for TRPM1 truncated variants.¹³ Furthermore, a recent study by Bidaux and coworkers identified two TRPM8 isoforms with different androgen sensitivity and distinct localization on the plasma and ER membranes.¹¹ Figure 17.1 shows schematically the TRPM8 gene, TRPM8 mRNA, and the position of Q-PCR primers used to distinguish between the two isoforms. The relative ratio between the two PCR products is used to calculate the predominance of one isoform over the other. This strategy may be also used to selectively knock down either of the isoforms. The differential regulation of TRPM8 activity may be due to complex regulation of the two isoforms by androgen receptors: An alternative TRPM8 gene promoter may make the ER TRPM8 isoform less sensitive to androgens. However, this ER localization may also result from a variation in the primary sequence leading to the appearance of an ER retention signal or the involvement of other associated proteins affecting its trafficking. It should be noted that there is a controversy in the literature concerning the localization of ER TRPM8. Two studies proposed a TRPM8-independent ER Ca²⁺ release mechanism in LNCaP ⁴⁴ and PC3⁴⁵ cells when using high doses of menthol (3 mM)⁴⁴ versus the ER TRPM8 activation with 100–250 μM menthol.^{12, 46}

FIGURE 17.1

The TRPM8 gene encodes for classical TRPM8 channel and a putative truncated TRPM8 splice variant. (a) The human TRPM8 gene localizes on chromosome 2 in position 37.1. (b) Genomic map of TRPM8 (not to scale; numbered boxes denote exons) with its associated (more...)

Furthermore, immunocytochemistry experiments in LNCaP revealed contradictory results concerning the presence of TRPM8 on the ER.^{12, 47} Two scenarios may explain this incongruity: firstly, as TRPM8 is under androgenic control, culture conditions of LNCaP cells, especially with respect of the serum used, may be critical for channel expression and localization, and, secondly, the putative ER TRPM8 isoform is not necessarily detected by the different antibodies used in these studies. In any case, the presence of ER TRPM8 was demonstrated in freshly isolated primary epithelial prostate cancer cells.¹¹ Consequently, to clarify whether TRPM8 localizes into the ER, it is necessary to clone this putative ER-specific TRPM8 isoform and identify its distinguishing features, as compared to the two previously cloned variants.

Therefore, the abundant short or long mRNA forms in some cancers arise from a regulatory mechanism that produces either spliced or partially degraded nonproductive RNAs. These spliced transcripts form multimers and regulate targeting to the plasma or ER membrane, and consequently protein activity. Changes in TRP localization may have a causal or promoting role in cancer. The increases in constitutively active channels, such as TRPV6, in the plasma membrane of prostate cancer cells^{17, 30} may augment Ca²⁺ in the cytosol, thus promoting Ca²⁺-dependent proliferative pathways. The same may hold true for TRPM1 in melanocytes^{13, 18, 23} because Ca²⁺ imaging experiments on transfected HEK293 cells revealed an increase in intracellular Ca²⁺ concentrations in comparison to the nontransfected cells.¹³ However, in the absence of electrophysiological data, it would be premature to conclude that TRPM1 is a constitutively active channel. Alternatively, altered expression of the channels localizing on the internal stores such as the membranes of the ER may be an adaptive response or may offer a survival advantage, such as resistance to apoptosis. In that respect, the decrease in urothelial TRPV1¹⁹ and prostatic TRPM8^{12, 46} in intracellular stores in aggressive tumors probably reduces the Ca²⁺ release content and confers resistance to apoptosis.

17.3. TRP PROTEIN TRANSLATION FROM ALTERNATIVE START CODONS: AN UNKNOWN MECHANISM OF CANCER CELL-ENHANCED RESISTANCE AND SURVIVAL

There is not so much known so far about TRP protein translation from alternative start codons (ATGs) situated downstream of the first methionine codon of the predicted full-length sequence. These ATG codons are preceded by the Kozak consensus sequence (gcc)gccRccAUGG, where R is a purine (adenine or guanine), three bases upstream of the start codon (AUG), which is followed by another “G.”⁴⁸ The Kozak consensus sequence occurs on eukaryotic mRNAs and plays a major role in the initiation of the translation process.⁴⁹ In vivo, this site is often not matched exactly on different mRNAs, and the amount of protein synthesized from a given mRNA is dependent on the strength of the Kozak sequence. There are examples in vivo of each type of Kozak consensus, and they probably evolved as yet another mechanism of gene regulation. Lmx1b is an example of a gene with a weak Kozak consensus sequence.⁵⁰ For initiation of translation from such a site, other features are required in the mRNA sequence in order for the ribosome to recognize the initiation codon. It seems likely that the above mechanisms may be engaged by the cancer cell to enhance its survival and resistance to apoptosis. It may also be an alternative mechanism to increase the cancer cell plasticity in the accelerated process of evolution and adaptation to the environment.

The cloning and expression pattern of bCCE 1Δ514, a 5′ truncated splice variant of the bovine TRPC4 (formally bCCE1, as supported by the evidence that it functioned as a capacitative calcium entry channel), provided the first example of native TRP protein expression using a downstream ATG codon,⁵¹ although it was shown to represent an alternatively spliced product of the TRPC4 gene that gives rise to an ~1.9-kb transcript rather than protein translation from an alternative ATG. It is interesting that in contrast to the six transmembrane segments predicted to be present in the full-length bTRPC4, bCCE 1Δ514 contains only three hydrophobic segments corresponding to transmembrane segments 5 and 6 and the putative pore-forming region in between. This membrane topology is reminiscent to that of the inward rectifier-type K⁺ channel (K_ir) gene family, which also encodes proteins of less than 500 amino acids with two putative transmembrane spanning domains M1 and M2 and a hydrophobic segment in between contributing to ion conducting pore formation.⁵² Accordingly, the ion conducting properties of the bTRPC4 protein might still be preserved in bCCE 1Δ514, although with different regulation mechanisms arising from the lack of the greater part of the N-terminus of bTRPC4.

By utilizing recently developed full-length cDNA technologies, large-scale cDNA sequencing is carried out by several cDNA projects. Now full-length cDNA resources cover the major part of the protein-coding human genes. Comprehensive analyses of the collected full-length cDNA data reveal not only the complete sequences of thousands of novel gene transcripts but also novel alternatively spliced isoforms of hitherto identified genes. However, it is not as easy as expected to deduce their encoded amino acid sequences based solely on the full-length cDNA sequences. It is neither always the case that the longest open reading frame corresponds to the real protein coding region nor that the first ATG should be the translation initiation codon. Also, proteome-wide mass spectrometry analysis has shown that there is an unexpectedly large population of small proteins, encoded by so-called upstream open reading frames, within the cell (for review see Ref. 53). Figure 17.2 shows the methodological approach to study the expression of the TRPV6 channel from the cloned cDNA. The TRPV6 molecule contains several ATG codons preceded by the functional consensus Kozak sequences theoretically capable of producing different size proteins. As can be seen from the blots, at least five alternatively translated protein molecules may be produced. Site-directed mutagenesis should be used thereafter to prove that putative protein molecules result from proper alternative ATG codons. This method may also be used to verify the specificity of the antibody. Some other examples follow hereafter.

FIGURE 17.2

TRPV6-YFP protein expression from different ATGs. HEK293 cells were transfected with the pTRPV6-YFP plasmid, the total protein lysates were subject to SDS-PAGE, blotted, and revealed with either anti-TRPV6 or anti-GFP antibodies.

Sequence analysis of various human papillomavirus types associated with particular clinical outcomes has revealed that L1 protein sequences of the major cervical cancer-associated viruses generally possess the ability to encode a longer translation product whilst the non-cancer-causing viruses do not.⁵⁴ Equally intriguing, the upstream initiation codon is always separated by 78 nucleotides from the initiation codon that produces L1 protein, which efficiently assembles into viral particles. The authors conclude that the longer L1 protein could play a role in the development of cervical carcinoma and that human papillomaviruses with the potential to cause cervical cancer may be identified by the presence of an in-frame ATG situated 78 nucleotides upstream.

A novel form of the E2F-3 protein, termed E2F-3B, has been identified.⁵⁵ In contrast to the full-length E2F-3, which is expressed only at the G1/S boundary, E2F-3B is detected throughout the cell cycle with peak levels in G0 where it is associated with Rb. Transfection and in vitro translation experiments demonstrated that a protein identical to E2F-3B in size and isoelectric point is produced from the E2F-3 mRNA via the use of an alternative translational start site. Owing to this alternative initiation codon, the E2F-3B is missing 101 N-terminal amino acids relative to the full-length E2F-3. This region includes a moderately conserved sequence of unknown function that is present only in the growth-promoting E2F family members, including E2F-1, E2F-2, and full-length E2F-3. These observations make E2F-3B the first example of an E2F gene giving rise to two different protein species and also suggest that E2F-3 and E2F-3B may have opposing roles in the cell cycle control.⁵⁵

Therefore, the alternative initiation of translation may represent an important evolutionary mechanism for a cancer cell to survive, to escape apoptosis, and to invade the body. TRP channels represent a potential “cancer target” because they are involved in all of them and therefore are subject to additional variability and regulation.

17.4. TRP PROTEIN POSTTRANSLATIONAL MODIFICATIONS: DIRECT OR INDIRECT MODULATION OF THE CHANNEL ACTIVITY

Protein posttranslational modifications are the chemical modifications of a protein after its translation. These modifications are largely used in the cell to regulate not only the protein activity but also its trafficking and stability at the plasma membrane in the case of the channel. Several types of posttranslational modifications exist, including the addition of functional groups such as acetylation, glycosylation, and phosphorylation; the addition of other proteins and peptides such as SUMOylation and ubiquitination; modifications involving changing the chemical nature of amino acids such as deamidation and deimination, as well as modifications involving structural changes such as disulfide bridges, proteolytic cleavage, etc. Modification and/or misregulation of posttranslational modifications may play a significant role in carcinogenesis and therefore should be considered in the case of TRP channels and cancer.

Nothing is known so far as to whether particular posttranslational modifications are explicitly used by the cancer cell for its needs. However, some data exist as to the posttranslational changes the channels are subjected to in cancer. In the case of Morris hepatoma H5123 cells, the cell-to-cell channel protein, connexin-43 (Cx43), is little expressed, and these cells lack gap junctional communication.⁵⁶ The authors found that the inhibition of glycosylation by tunicamycin induced open channels in these cells. Although tunicamycin caused the formation of open channels, channels were not found aggregated into gap junctional plaques, as they are when they have been induced by elevation of intracellular cAMP. The results suggest that although Cx43 itself is not glycosylated, other glycosylated proteins influence Cx43 posttranslational modification and the formation of Cx43 cell-to-cell channels.⁵⁶ Below, we will consider the two most frequent posttranslational modifications, namely, glycosylation and phosphorylation.

17.5. CONCLUSIONS

The progression of cells from a normal, differentiated state to a tumorigenic, metastatic state involves the accumulation of mutations in multiple key signaling proteins, encoded by oncogenes and tumor suppressor genes, together with the evolution and clonal selection of more aggressive cell phenotypes. These events are associated with changes in the expression of numerous other proteins. To date, most changes involving TRP proteins do not involve mutations in the TRP genes but rather increased or decreased expression levels of the wild-type TRP proteins, depending on the stage of the cancer. On the other hand, several common tuning pathways lead to this divergence in expression. In this respect, TRP channels may be regulated at different levels: (1) transcriptional, (2) translational, (3) trafficking to the plasma membrane, or (4) direct channel modulation on the plasma membrane. Modulation of TRP expression/activity on one of these levels affects intracellular Ca²⁺ signaling and, consequently, the processes involved in carcinogenesis, such as proliferation, apoptosis, and migration.

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