**3. Ion channels and transporters with clinical relevance in solid cancer**

An overview of the main ion channels and transporters expressed in different solid tumors is reported in **Figure 1**.

#### **3.1 Potassium channels**

*Translational Research in Cancer*

will respond to a define treatment (Predictive).

targeting specific cancer-related biomarkers.

used in cancer diagnostics are based on this technique.

**2.2** *In vivo* **cancer diagnosis: molecular imaging**

**2.1** *In vitro* **cancer diagnosis**

*2.1.2 Flow cytometry (FC)*

*2.1.3 Omics profiles*

*2.1.1 Immunohistochemistry (IHC)*

greatly improved diagnosis through their application for either *in vitro* diagnosis (on tumor specimens or in blood samples) or *in vivo* molecular imaging. According to the National Cancer Institute (NCI) definition (NCI Dictionary of Cancer Terms, http://www.cancer.gov/dictionary?cdrid=46636), a biomarker may be used either to help diagnosis, for example, to identify early stage cancers (Diagnostic) or to forecast how aggressive a condition is (Prognostic), or to predict how well a patient

For the purposes of this chapter, we will briefly summarize the main techniques, either *in vitro* or *in vivo*, which take advantage of the use of biomarkers to obtain diagnostic, prognostic and predictive data on the cancer under study. Notably, most, although not all, of these techniques are based on the use of antibodies,

IHC represents an indispensable diagnostic tool to assess the presence or absence, as well as the amount, of a specific molecular tumor marker in a tissue. After appropriate assessment of categorical scoring system and proper validation of the immunohistochemical assay, a given marker can be proposed as a potential diagnostic or prognostic factor. Indeed, many of the cancer biomarkers routinely

Using a multiparametric approach, FC immunophenotyping plays an indispensable role in the diagnosis and subclassification of leukemias, as well as for minimal residual disease detection. FC, in fact, provides a rapid and detailed determination of antigen expression profiles; these information along with morphologic assessment, allow to diagnose a particular type of leukemia and/or help in distinguishing from other subtypes. Also, the identification of specific antigens has prognostic and therapeutic relevance in acute leukemias. Moreover, FC immunophenotyping is useful to monitor response to therapy, recurrence and minimal residual disease.

While IHC and FC represent the standard of care in solid cancers and hematologic malignancies, respectively, some remarkable technological breakthroughs of the last 10 years have greatly contributed to improve cancer diagnostics through either the definition of "Omics profile" or the assessment of plasma-based cancer biomarkers:

The study of tumor genomes using high throughput profiling strategies including (but not limited to) DNA copy number, DNA methylation, and transcriptome and whole-genome sequencing—technologies that may collectively be defined as "omics"—has led to identifying genes and pathways deregulated in cancer, hence revealing those that may be useful for the detection and management of disease. In the near future, such discoveries will lead to the discovery of novel diagnostic, prognostic and predictive markers that will ultimately improve patient outcomes.

Besides *ex vivo* procedures (either on surgical/bioptic samples or blood), cancer

*diagnosis* is mainly based on imaging procedures, such as *computed tomography,*

**44**

K+ channels are the class of ion channels mostly de-regulated in cancers. Among them, *KCa 1.1* channels (also known as BK channels, encoded by the *KCNMA1* gene) have shown a clinical relevance in breast (BC) and prostate cancer (PCa). In both tumor types, BK overexpression can be traced back to the amplification of the *KCNMA1* gene located in 10q22: in BC, the amplification is restricted to invasive ductal tumors, and is associated with high stage, high grade and unfavorable prognosis [14]. In BC, KCa 1.1 positively correlates with the expression of estrogen

**Figure 1.** *Schematic representation of the main ICTs expressed in solid tumors.*

receptors [15] and their levels are higher in BC metastatizing to brain [16]. In PCa, the *KCNMA1* gene is frequently amplified in late-stage tumors [17] and can be considered a potential biomarker [18]. Another Ca2+-dependent K+ channel often overexpressed in human cancers is *KCa3.1* (encoded by the *KCNN4* gene). KCa3.1 channels are upregulated in BC, especially in high grade tumors [19], in pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) [20], in colorectal cancer (CRC) [21] as well as in small cell lung cancer (SCLC) [22]. While the clinical relevance of KCa3.1 was hypothesized in CRC [23], although not validated [24], *KCNN4* hypomethylation turned out to be a negative prognostic factor in SCLC [22]. Kv channels are voltage-dependent K+ channels whose expression is often increased in cancer tissues [25]. For example, the expression of **Kv 1.3** (*KCNA3*), markedly increased in PCa in samples with Gleason score of 5–6 (GS5–6), but significantly decreased in the GS8–9 group. This malignancy grade-dependent K+ -channel expression pattern may provide a convenient marker to understand PCa progression level [26]. In PCa, Kv1.3 is mainly expressed in early stages of progression and down-regulated in high grade cancers [27]. Kv1.3 expression is lower in cancer compared with healthy pancreas. Kv1.3 downregulation could be traced back to promoter's methylation and was associated with the presence of metastases [28]. *K2P9.1* (KCNK9) belongs to the K2P family and genomic amplification of the gene was shown in a small fraction of BC [29]. **K2P5.1** (KCNK5) is a member of the same family and it was shown to be induced by estrogens in ER-positive BC cells; for this reason, it might represent a therapeutic target for ER-positive BCs [30]. The amplification of the *KCNK9* gene at the 8q23.4 locus justifies the over expression of K2P9.1 channels in BC. The overexpression of another K2p channel *K2p 2.1* has been demonstrated in PCa and it was shown that it regulates cell proliferation [31]. The expression of inward rectifiers K<sup>+</sup> channels, in particular **Kir3.1** (KCNJ3) channels positively correlated with lymph node metastases in BC [32]. The voltage-gated K<sup>+</sup> channels (VGKC) appear to exert a pleiotropic role in colorectal cancer. In primary human samples, the transcripts of *KCNA3*, *KCNA5*, *KCNC1*, *KCNH1* [33–35], *KCNH2* [36] and *KCNK9* [37] have been detected. A relevant family of VGKC, whose most important members are Kv 10.1 and Kv 11.1 was shown to be highly represented in human cancers. **Kv10.1** (KCNH1) was expressed in esophageal squamous cell carcinoma (ESCC) compared with the corresponding normal tissue, it was associated with depth of invasion and represented an independent negative prognostic factor [38].

**Kv11.1** (KCNH2) channels are expressed in gastric cancer (GC) cell lines and primary GCs. In GC cell lines, they regulate tumor proliferation [39]. Consistently, treatment with Kv11.1 blockers, like cisapride, and siRNA impairs tumor growth [40, 41]. It was also shown that the mean survival time was shorter in Kv11.1 positive patients thus Kv11.1 expression was proposed as an independent prognostic factor. We also showed that Kv11.1 regulates VEGF-A secretion, with a pathway similar to the one described in CRC [42]. *In vivo* analyses of xenografts obtained with GC cells demonstrated that the treatment with Bevacizumab and Kv11.1 blockers dramatically reduces greatly tumor growth. Kv11.1 is highly expressed in primary CRC and is associated with invasive phenotype [36]; moreover, along with Glut-1 absence, it represents a negative prognostic factor in TNM I and II CRC [43]. Kv11.1 expression is associated with chemosensitivity for several anti-tumor agents (such as vincristine, paclitaxel and hydroxy-camptothecin, doxorubicin). Such chemosensitivity is modulated by erythromycin that is also capable which, to inhibit Kv11.1 current [44]. Kv11.1 also regulates lung cancer (LC) cell proliferation [45]. Kv11.1 is expressed in precancerous and neoplastic lesions of the esophagus and it is associated with malignant progression [46]. Kv11.1 channel expression represents a negative prognostic factor in terms of ESCC patients' survival [47].

**47**

*Ion Channels and Transporters as Cancer Biomarkers and Targets for Diagnostics with Antibodies*

Kv11.1 are also expressed in PDAC cell lines and primary samples and it negatively

Voltage-gated sodium channels (VGSC) were among the first channels to be demonstrated mis-expressed in BC and PCa. In particular, the predominant VGSC in BC is the "neonatal" splice variant of *SCN5A* (**nNaV1.5**), whose activity promotes metastatization [49–51]; consistently, the nNAv1.5 was up-regulated in metastatic BC samples [49, 50, 52]. On the whole, VGSC and in particular nNav1.5 could represent a good specific target for BC treatment. In CRC [53–55], the clinical relevance of Nav 1.5 expression was established by IHC in CRC samples with respect to healthy colon. VGSC regulates invasiveness and it was shown that *SCNA5* gene modulates genes mediating, among others, cell migration and cell cycle control. Both nNav 1.5 and its "adult" counterpart are expressed in CRC and the local anesthetic Ropivacaine, blocks Nav 1.5 variants [56]. PCa show an aberrant expression of **Nav1.7** (SCN9A), associated with a strong metastatic potential and its activity potentiates cell migration, crucial for the metastatic cascade [57]. Hence, Nav1.7 could represent a useful diagnostic marker [58]. A recent paper [59] showed that EGFR and Nav1.7 are expressed in NSCLC cells and that EGFR-mediated upregulation of *SCN9A* is necessary for the invasiveness of such cells. Nav1.7 has clinical relevance and might represent a novel target for therapy and/or a prognostic biomarker in NSCLC [59]. A recent multicenter study identified two single nucleotide polymorphisms of VGSC genes (*SCN4A*-rs2302237 and *SCN10A*-rs12632942) that were associated with oxaliplatin-induced peripheral neuropathy development [60].

Calcium signal remodeling is one of the common features of proliferating cells, including cancer. Indeed many functional studies have provided different calcium signaling that can modulate cell proliferation and resistance to apoptosis [61–63]. Voltage-gated calcium channels (VGCC) that are involved in the regulation of BC cell proliferation. *CACNA2D3* gene (encoding the α2δ3 subunit of the voltage gated Ca2+ channel) is frequently up-regulated in BC, but in some metastatic cases, its expression is reduced [64]. The mechanisms of *CACNA2D3* contribution to the metastatic process has not being clarified yet. One possible mechanism for the overexpression of some calcium permeable ion channels is through the involvement of hormone receptors, such as ERα. Examples are **ORAI3** [65]. *CACNA2D3*, is frequently downregulated in primary BCs, as a result of methylation in CpG islands [64]. The influence of calcium channels in PCa has been known for over 30 years. Later research identified additional classes of channel proteins having an important regulatory role and affecting malignant transformation (reviewed in [66]). The expression of VGCC (mainly L-type) has been detected in the androgen-responsive LNCaP cells. In these cells Ca2+ currents are activated by androgens and mediate

the androgen-induced effects [67]. Part of the Ca2+ effects depend on K+

stimulation, for example, KCa3.1 blocking inhibits the proliferation of PCa cells [67]. An aberrant methylation of *CACNA2D1/3* gene (encoding the voltage-dependent calcium channel 2 subunit) was demonstrated in GC samples. *CACNA2D3* methylation is associated with diffuse type GC and shorter survival [68]. **ORAI1** and **STIM1**, belonging to the store operated calcium channels (SOC) family, are up-regulated in BC of the basal-like molecular subtype [69]. Moreover, another member of the same family, **STIM2**, is expressed at low levels in BC. Patients with

channels

*DOI: http://dx.doi.org/10.5772/intechopen.90401*

affects patients' prognosis [48].

**3.2 Sodium channels**

**3.3 Calcium channels**

*Ion Channels and Transporters as Cancer Biomarkers and Targets for Diagnostics with Antibodies DOI: http://dx.doi.org/10.5772/intechopen.90401*

Kv11.1 are also expressed in PDAC cell lines and primary samples and it negatively affects patients' prognosis [48].

## **3.2 Sodium channels**

*Translational Research in Cancer*

receptors [15] and their levels are higher in BC metastatizing to brain [16]. In PCa, the *KCNMA1* gene is frequently amplified in late-stage tumors [17] and can be

overexpressed in human cancers is *KCa3.1* (encoded by the *KCNN4* gene). KCa3.1 channels are upregulated in BC, especially in high grade tumors [19], in pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) [20], in colorectal cancer (CRC) [21] as well as in small cell lung cancer (SCLC) [22]. While the clinical relevance of KCa3.1 was hypothesized in CRC [23], although not validated [24], *KCNN4* hypomethylation turned out to be a negative prognostic factor in SCLC [22]. Kv channels are voltage-dependent K+ channels whose expression is often increased in cancer tissues [25]. For example, the expression of **Kv 1.3** (*KCNA3*), markedly increased in PCa in samples with Gleason score of 5–6 (GS5–6), but significantly decreased in

may provide a convenient marker to understand PCa progression level [26]. In PCa, Kv1.3 is mainly expressed in early stages of progression and down-regulated in high grade cancers [27]. Kv1.3 expression is lower in cancer compared with healthy pancreas. Kv1.3 downregulation could be traced back to promoter's methylation and was associated with the presence of metastases [28]. *K2P9.1* (KCNK9) belongs to the K2P family and genomic amplification of the gene was shown in a small fraction of BC [29]. **K2P5.1** (KCNK5) is a member of the same family and it was shown to be induced by estrogens in ER-positive BC cells; for this reason, it might represent a therapeutic target for ER-positive BCs [30]. The amplification of the *KCNK9* gene at the 8q23.4 locus justifies the over expression of K2P9.1 channels in BC. The overexpression of another K2p channel *K2p 2.1* has been demonstrated in PCa and it was shown that it regulates cell proliferation [31]. The expression of inward rectifiers K<sup>+</sup> channels, in particular **Kir3.1** (KCNJ3) channels positively correlated with lymph

a pleiotropic role in colorectal cancer. In primary human samples, the transcripts of *KCNA3*, *KCNA5*, *KCNC1*, *KCNH1* [33–35], *KCNH2* [36] and *KCNK9* [37] have been detected. A relevant family of VGKC, whose most important members are Kv 10.1 and Kv 11.1 was shown to be highly represented in human cancers. **Kv10.1** (KCNH1) was expressed in esophageal squamous cell carcinoma (ESCC) compared with the corresponding normal tissue, it was associated with depth of invasion and

**Kv11.1** (KCNH2) channels are expressed in gastric cancer (GC) cell lines and primary GCs. In GC cell lines, they regulate tumor proliferation [39]. Consistently, treatment with Kv11.1 blockers, like cisapride, and siRNA impairs tumor growth [40, 41]. It was also shown that the mean survival time was shorter in Kv11.1 positive patients thus Kv11.1 expression was proposed as an independent prognostic factor. We also showed that Kv11.1 regulates VEGF-A secretion, with a pathway similar to the one described in CRC [42]. *In vivo* analyses of xenografts obtained with GC cells demonstrated that the treatment with Bevacizumab and Kv11.1 blockers dramatically reduces greatly tumor growth. Kv11.1 is highly expressed in primary CRC and is associated with invasive phenotype [36]; moreover, along with Glut-1 absence, it represents a negative prognostic factor in TNM I and II CRC [43]. Kv11.1 expression is associated with chemosensitivity for several anti-tumor agents (such as vincristine, paclitaxel and hydroxy-camptothecin, doxorubicin). Such chemosensitivity is modulated by erythromycin that is also capable which, to inhibit Kv11.1 current [44]. Kv11.1 also regulates lung cancer (LC) cell proliferation [45]. Kv11.1 is expressed in precancerous and neoplastic lesions of the esophagus and it is associated with malignant progression [46]. Kv11.1 channel expression represents a negative prognostic factor in terms of ESCC patients' survival [47].

channel often


channels (VGKC) appear to exert

considered a potential biomarker [18]. Another Ca2+-dependent K+

the GS8–9 group. This malignancy grade-dependent K+

node metastases in BC [32]. The voltage-gated K<sup>+</sup>

represented an independent negative prognostic factor [38].

**46**

Voltage-gated sodium channels (VGSC) were among the first channels to be demonstrated mis-expressed in BC and PCa. In particular, the predominant VGSC in BC is the "neonatal" splice variant of *SCN5A* (**nNaV1.5**), whose activity promotes metastatization [49–51]; consistently, the nNAv1.5 was up-regulated in metastatic BC samples [49, 50, 52]. On the whole, VGSC and in particular nNav1.5 could represent a good specific target for BC treatment. In CRC [53–55], the clinical relevance of Nav 1.5 expression was established by IHC in CRC samples with respect to healthy colon. VGSC regulates invasiveness and it was shown that *SCNA5* gene modulates genes mediating, among others, cell migration and cell cycle control. Both nNav 1.5 and its "adult" counterpart are expressed in CRC and the local anesthetic Ropivacaine, blocks Nav 1.5 variants [56]. PCa show an aberrant expression of **Nav1.7** (SCN9A), associated with a strong metastatic potential and its activity potentiates cell migration, crucial for the metastatic cascade [57]. Hence, Nav1.7 could represent a useful diagnostic marker [58]. A recent paper [59] showed that EGFR and Nav1.7 are expressed in NSCLC cells and that EGFR-mediated upregulation of *SCN9A* is necessary for the invasiveness of such cells. Nav1.7 has clinical relevance and might represent a novel target for therapy and/or a prognostic biomarker in NSCLC [59]. A recent multicenter study identified two single nucleotide polymorphisms of VGSC genes (*SCN4A*-rs2302237 and *SCN10A*-rs12632942) that were associated with oxaliplatin-induced peripheral neuropathy development [60].

### **3.3 Calcium channels**

Calcium signal remodeling is one of the common features of proliferating cells, including cancer. Indeed many functional studies have provided different calcium signaling that can modulate cell proliferation and resistance to apoptosis [61–63]. Voltage-gated calcium channels (VGCC) that are involved in the regulation of BC cell proliferation. *CACNA2D3* gene (encoding the α2δ3 subunit of the voltage gated Ca2+ channel) is frequently up-regulated in BC, but in some metastatic cases, its expression is reduced [64]. The mechanisms of *CACNA2D3* contribution to the metastatic process has not being clarified yet. One possible mechanism for the overexpression of some calcium permeable ion channels is through the involvement of hormone receptors, such as ERα. Examples are **ORAI3** [65]. *CACNA2D3*, is frequently downregulated in primary BCs, as a result of methylation in CpG islands [64]. The influence of calcium channels in PCa has been known for over 30 years. Later research identified additional classes of channel proteins having an important regulatory role and affecting malignant transformation (reviewed in [66]). The expression of VGCC (mainly L-type) has been detected in the androgen-responsive LNCaP cells. In these cells Ca2+ currents are activated by androgens and mediate the androgen-induced effects [67]. Part of the Ca2+ effects depend on K+ channels stimulation, for example, KCa3.1 blocking inhibits the proliferation of PCa cells [67]. An aberrant methylation of *CACNA2D1/3* gene (encoding the voltage-dependent calcium channel 2 subunit) was demonstrated in GC samples. *CACNA2D3* methylation is associated with diffuse type GC and shorter survival [68]. **ORAI1** and **STIM1**, belonging to the store operated calcium channels (SOC) family, are up-regulated in BC of the basal-like molecular subtype [69]. Moreover, another member of the same family, **STIM2**, is expressed at low levels in BC. Patients with

high STIM1 and low STIM2 have unfavorable prognosis, suggesting that the SOC family has a role in aggressiveness and in the metastatic process [69]. **ORAI3** has recently been associated with ER-positive BC [65] and could represent a novel target for ER-positive BCs [70].

### **3.4 Transient receptor potential (TRP) channels**

TRP channels are non-selective cation channels that can be activated by different stimuli such as pH variations, temperature and pressure among others [71, 72]. Since TRP channels are involved in migration and invasiveness, they contribute to the metastatic process in different tumors [73]. Ca2+ influx through TRPCs also occurs and promotes either cell proliferation or apoptosis, depending on TRPC subtype. **TRPC1** whose levels are high in BCs with low proliferation capacity, may not be the optimal target for therapies against aggressive BCs [74]. Significantly elevated (up to 200-fold) mRNA levels of *TRPC6* were shown in BC samples compared with paired control samples [74, 75], but no correlations with clinicopathological features emerged [74]. A similar behavior characterizes TRPC1, whose expression levels decrease during the progression of PCa from androgen-dependent to androgen-independent phase [75]. TRPC6 is overexpressed in ESCC with respect to normal esophageal tissue at both protein and mRNA levels [76]. A recent report evidenced correlations of TRPC6 with T and staging and an association between *TRPC6* mRNA and poor prognosis [77]. **TRPV6** is up-regulated in PgR and ER-negative BCs [78]. Basal-like BCs with high TRPV6 mRNA levels are associated with poor survival [79]. *In vitro* data suggest that TRPV6 may be a potential therapeutic target [79]. TRPV6 is highly expressed in PCa and are associated with the Gleason score and metastatisation [80]. The expression of **TRPV4** is decreased by progesterone [81]. **TRPM7** is highly expressed in BC, and such over expression is associated with poor prognosis in terms of distant metastasis- and recurrence-free survival [82]. In accordance with these observation, *TRPM7* mRNA levels are higher in BC metastases with respect to primary tumors. Also, TRPM7 are overexpressed in pancreatic ductal adenocarcinomas and are associated with lymph node metastases [83]. TRPM7 mRNA and protein are also overexpressed in bladder cancer with respect to normal tissue and are associated with poor prognosis [84]. **TRPA1** is overexpressed also in SCLC patients compared with NSCLC and since it is associated with SCLC patients' survival representing a potential therapeutic target [85].
