Expression profiling of ion channel genes predicts clinical outcome in breast cancer

Ion channels are membrane proteins expressed in various tissues that allow the passage of ions across biological membranes. Ion transport is a key component in a wide variety of biological processes including electrical impulse generation and conduction along nerves, fluid balancing within cells and across cell membranes, and signal transduction within and among cells. In addition, ion channels are known to play critical roles in gene expression, hormone secretion, muscle contraction, immune response, cell volume regulation, and cell proliferation [1‐7]. Because of the involvement of ion channels in diverse biological functions, defects in the expression and functional activity of ion channels can cause disease in many tissues [8]. The number of human diseases related with ion channel malfunction has grown rapidly over the past few years [4, 9, 10]. In particular, there is increasing evidence that ion channels, including both voltage-gated and ligand-gated channels, are involved in the progression and pathology of diversified human cancers [6, 7, 11‐17]. For example, voltage-gated potassium (K⁺) (Kv) channels and calcium (Ca²⁺)-activated K⁺ (K_Ca) channels are known to control tumor cell proliferation through the modulation of membrane potential in breast, colon, and prostate cancers [12, 14, 15]. Transient receptor potential (TRP) channels are involved in vascular permeability and angiogenesis and have been implicated in tumor growth and metastasis [18, 19]. Several ligand-gated channels, such as nicotinic acetylcholine receptors, affect neoplastic progression by regulating tumor cell proliferation, apoptosis, and angiogenesis [20‐23]. More importantly, the expression level of ion channels is potentially able to serve as a prognostic index in human cancers for clinical purposes. For instance, TRPM1, a TRP cation channel, is an indicator of melanoma aggressiveness [24] and expression of the Ca²⁺-selective cation channel TRPV6 is a prognostic marker for tumor progression in human prostate cancer [25]. In addition, the long TRP channel TRPM8 might serve as a prognostic marker for androgen-unresponsive and metastatic prostate cancer [26] and the expression of SCN9A, a voltage-gated sodium (Na⁺) channel, is also useful for prognostic purposes in prostate cancer [27].

Breast cancer is the most common invasive cancer in women worldwide. It is also the principal cause of death from cancer among women globally [28]. A large number of breast cancer studies sought to understand the molecular mechanisms of cancer origin, progression, and invasion that lead to metastasis, and many of these studies have underlined the involvement of ion channels in breast cancer. For example, K_Ca channels contribute to breast tumor migration and progression [29, 30] and TRP channels are strongly correlated with breast tumor cell proliferation [31]. In addition, the upregulation of several voltage-gated Na⁺ (NaV) channels is associated with metastatic process in breast cancer [32]; however, most of these studies focus on only one ion channel or one type of ion channel. So far, there is no clear picture on the overall expression profiling of different ion channel genes in breast cancer. High-throughput “omic” technologies make it possible to scan all ion channel genes rather than focusing on a single gene or gene family [33]. In this study, we looked to identify molecular signatures consisting of multiple genes from different ion channel families that are implicated in the pathology of human breast cancer. Firstly, we investigated the association of ion channel genes with p53 mutation status in breast tumors. The tumor suppressor p53 is known to play a critical role in regulating the cell cycle and is thus involved in preventing cancer. Mutations in p53 are strongly associated with poor clinical outcome in breast cancer patients [15, 23]. Comparison between the p53 mutant and wild-type groups showed that ion channel genes are associated with more aggressive and therapeutically refractory tumors [15]. Secondly, we identified the ion channel genes that were differentially expressed between estrogen receptor (ER) positive and negative breast cancer patients. About 75% of all breast cancers are ER positive, which grow in response to the hormone estrogen. ER is a powerful prognostic marker and an efficient target for the treatment of hormone-dependent breast cancer [26]. Identification of the ER-related ion channels helps us understand the role of ER in the development and progression of breast cancer. Thirdly, we investigated the relationship between ion channel gene expression and histological tumor grade in breast cancer. We identified a molecular signature consisting of 30 ion channel genes (IC30), which significantly correlated with tumor grade. We demonstrate that IC30 is a robust prognostic biomarker to predict clinical outcome in breast cancer, and is independent of standard clinical and pathological prognostic factors including patient age, lymph node status, tumor size, tumor grade, ER status, and progesterone receptor (PR) status. The performance of IC30 was also validated in several independent cohorts from different parts of the world (Table 1).

Ion channels are implicated in many physiological processes and also play a pivotal role in the development of cancers; however, it is currently difficult to assign a specific mechanism for each ion channel in the proliferation, invasion, and metastasis of tumor cells [17, 24]. Here, we investigated the pathological role of ion channel genes in breast cancer with respect to gene expression level. We tested the association of ion channel genes with p53 mutation status, ER status, and tumor histological grade: 22 ion channel genes were found to be dysregulated between p53 mutant and wild-type tumors, 24 ion channel genes were differentially expressed between ER positive and negative patients, and the expression level of 30 ion channel genes was significantly correlated with histological grade. There is a large overlap between the three differentially expressed gene lists, which suggests a common ion channel-related biological mechanism underlying the different pathological phenotypes. The prognostic value of p53 mutation status has been well characterized in previous studies [15, 29‐31, 35]. The frequency of p53 mutations is higher in ER negative breast cancer [25, 35]. Mutant p53 and/or negative ER status are often associated with a high rate of proliferation, a high histological grade, and a poor prognosis [32, 33, 36, 37]. Therefore, it is reasonable that several common ion channel genes are associated with p53 mutation status, ER status, and tumor histological grade, although the causal relationship between these three factors is still controversial.

Gene expression-based molecular signatures have been proven as prognostically valuable in several human cancers [38‐42]. Gene signatures that work cooperatively with known clinicopathological factors may enhance prediction accuracy when identifying patients at higher risk for relapse and death. Our proposed molecular signature that is composed of 30 ion channel genes (IC30) associated with tumor grade is a promising prognostic marker. IC30 was solely developed based on the discovery cohort and its prognostic power of IC30 was validated in seven independent validation cohorts. We demonstrated that there were no significant multivariate interactions between IC30 status and other clinicopathological covariates. When grouped by age, tumor size, tumor grade, or PR status, the expression of IC30 further stratified breast cancer patients with significant differences in survival. A significantly increased risk of death was also observed in IC30 positive patients with positive lymph node status or positive ER status. However, a significant difference between IC30 positive and negative groups among the patients with negative ER status was not detected, which may be due to the relatively smaller sample size in this category within the USA1 cohort. In addition, we only detected a marginal significant association for patients with negative lymph node status in the USA1 cohort, and a similar result was reproduced in the GER cohort. In fact, all patients from the GER cohort had negative lymph node status [34] and a marginally significant difference in the risk of death was observed between IC30 positive and negative patients in this cohort. Taken together with previous data, these results confirm that IC30 is not dependent on specific values of the respective covariates status, which enhances the identification of cancer patients at greater risk for death.

Although IC30 gene signature was identified using the tumor grade information, both multivariate and univariate Cox regressions of survival reveal that IC30 is superior to tumor grade, which suggests that the prognostic information contained in IC30 is not limited to tumor grade. Tumor grade only explains a small proportion of variation (less than 25%) in expression of each IC30 gene. The significant association between tumor grade and IC30 gene expression specifically implies that IC30 genes are actively involved in tumor pathology in breast cancer. The combination of 30 genes contains more quantity of information than tumor grade itself. Therefore, IC30 is based on tumor grade but not limited to tumor grade.

The involvement of ion channels in human cancer has been intensively studied in the past years. However, there is no broad consensus on the role and interplay between ion channels and cancer. Generally, ion channels are thought to “assist” cancer by tumor-related cellular behaviors such as proliferation, apoptosis, migration, or angiogenesis [14, 17, 43, 44]. However, it is difficult to assign a detailed role for each ion channel in cancer pathology. In breast cancer, accumulating evidence indicates that K⁺ channels play important roles in regulating tumor cell proliferation, cell cycle progression, and apoptosis [12, 45, 46]. Although a significant over-expression of K⁺ channels has been correlated with human breast cancer cells [45, 47‐51], here we report a heterogeneous expression profiling in K⁺ channel genes. Dysregulation in both directions was observed in K⁺ channel genes within the IC30. Four K⁺ channel genes in the IC30 were upregulated in high-grade tumors. For example, KCNN4, encoding an intermediate conductance K_Ca channel (K_Ca3.1), is among the upregulated K⁺ channel genes in the IC30 in high-grade tumors. The expression pattern of K_Ca3.1 was confirmed by a recently published study where K_Ca3.1 mRNA and protein were more highly expressed in grade 3 tumors than in both grades 1 and 2 [52]. On the contrary, three K⁺ channel genes within the IC30 were downregulated in tumors with higher grade, which includes KCNMA1 encoding the BK channel alpha subunit. The negative correlation for KCNMA1 between expression and tumor grade was in accord with four independent cohorts. However, increased expression of KCNMA1 was found in metastatic breast cancer in the brain compared to metastatic breast cancers in other organs [53], which suggests a more complicated pathological role for the BK channel in tumor metastasis.

Gene expression of Cl^- channels also demonstrated a heterogeneous pattern in breast cancer. We reported two up- and two downregulated Cl^- intracellular channel genes in high-grade tumors in the IC30. Among them, CLIC4 was found to be involved in skin cancer [54]; however, the exact role of CLIC4 is unclear. Besides Cl^- intracellular channels, the Ca⁺ activated Cl^- channel CLCA2 was downregulated in breast cancer and is a candidate tumor suppressor gene [55]. We show here that the CLCA2 gene was upregulated in p53 mutant and/or ER negative breast tumors. In fact, the tumorigenicity of breast cancer was related with a loss of CLCA2[56, 57]. CLCA2 is a p53-inducible inhibitor of breast tumor proliferation [55]. However, the reason why CLCA2 expression is associated with p53 mutation status is beyond the scope of this study.

Ca²⁺ is an essential regulator of the cell cycle and is indispensable for cell proliferation [14]. Increased expression of voltage-gated Ca²⁺ channels has been observed in colon cancer cells [58] and small cell lung cancers [59]. However, all 3 voltage-gated Ca²⁺ ion channel genes in the IC30 were downregulated in p53 mutant tumors and/or high-grade tumors. Apart from these voltage-gated Ca²⁺ channels, the TRP family is also important to provide a Ca²⁺ influx pathway such that Ca²⁺ influx may occur through voltage-gated Ca²⁺ channels [14]. Several studies have demonstrated that the expression of TRP channels is significantly upregulated in breast tumor tissue and breast cancer cell lines [60‐63], which is related to malignant growth and cancer progression [64]. However, a paradoxical result was observed in our study. PKD1, PKD2, and TRPC1, which are within the IC30 and belong to the TRP family, were downregulated in patients with p53 mutant tumors and/or of higher histological grade. This expression pattern is consistent in several of the validation cohorts.

Conflicting results were also seen in Na⁺ channel genes. Decreased expression in high-grade tumors was found for the three Na⁺ channel genes in the IC30, which was confirmed by the validation cohorts. However, increased expression of voltage-gated Na⁺ channels has been reported in several cancer types, including breast, prostate, and lung cancer [65‐68]. Na⁺ channels were thought to enhance the invasiveness of cancer cells by increasing H⁺ efflux [66] and by stimulating cysteine cathepsin activity [69]. The precise mechanism of Na⁺ channels in tumor development remains unclear [70]. The discrepancy between our results and previous observation may be due to the discrepancy between mRNA expression, protein expression, and channel activity. Our study focused on mRNA abundance. However, protein expression and activity is not directly correlated to mRNA expression. Post-transcriptional regulatory mechanisms predominantly control cellular mRNA to protein abundance ratios [71].

Voltage-dependent anion channels (VDACs) are a class of ion channel located in the outer mitochondrial membrane [72]. We observed a consistent and significant positive correlation between gene expression and tumor grade for the three VDAC genes in the IC30. A recently published study indicated that higher VDAC1 expression level predicts poor outcome in non-small cell lung cancers [73]. Here, we expanded this finding to breast cancer and the other 2 genes in VDAC family.

In summary, we investigated the gene expression profile of ion channels in breast cancer with respect to p53 mutation status, ER status, and histological grade. We show that there are numerous common ion channel genes, including ANO1, CACNA1D, CACNA2D1, CACNA2D2, CLIC6, GLRB, KCND3, KCNE4, KCNMA1, KCNN4, MCOLN2, P2RX4, SCN7A, SCNN1A, and TPCN1, that are differentially expressed with a change in p53 mutation status, ER status, and histological grade. The expression pattern of some ion channels, including several potassium, calcium, and sodium channels, is contradictory to previously published results derived from breast cancer cell lines, animal models, and/or human patients. We also identified a molecular gene signature IC30, which represents a promising diagnostic and prognostic biomarker in breast cancer. Further investigation into the role of ion channels in tumor pathology could provide new targets for therapy in multiple human cancers.