KCNMA1 encodes the a-subunit of the large conductance, voltage and Ca2+-activated and Voltage-dependent potassium channel (BKCa) and was shown by others and us to be a potential drug target gene in several cancers, including breast cancer. In addition, we studied the role of alternative pre-mRNA splicing events of KCNMA1 in migration, invasion, proliferation and dispersal of breast cancer cells. It is conceivable that by targeting gene variants we can attenuate processes such as distant metastasis and angiogenesis. Here we reviewed literature on the alternative splicing events specific to breast cancer metastasis to brain, its microenvironment, the biological activity of most alternatively spliced isoforms. We conclude that based on our and others’ work KCNMA1 and other such gene variants contribute to breast cancer dispersion, invasion, growth, and progression in the tumor microenvironment. Thus KCNMA1/BKCa channels and their variants are opportunistic diagnostic, prognostic and treatment targets in breast cancer.
- KCNMA1 pre-mRNA splicing
- BKCa channelopathy
- breast cancer-dispersion
- treatment target
1.1 Metastatic breast cancer etiology
Breast cancer is the most common type of cancer affecting women. Despite great advances in primary breast cancer treatment a significant number of women develop metastases in different organs of the body, especially brain , possibly as a result of the emergence of targeted and aggressive systemic cancer therapy. The actual incidence of brain metastases is not precisely known; however, studies suggest that 6–16% of patients with metastatic breast cancer develop brain metastases during their lifetime. Furthermore, autopsy studies have reported brain metastases in 18–30% of patients dying of breast cancer . The majority of women who develop brain metastases have undergone aggressive treatment for stage IV disease [3, 4, 5]. Although brain metastasis is the leading cause of breast cancer death, its pathogenesis is poorly understood and the predictors of breast metastasis to brain are yet to be characterized. Albeit recent studies found genes that mediate breast cancer metastasis to brain [6, 7]. Targeting metastatic breast cancer cells in brain is extremely difficult as brain provides a “safe haven” for cancer cells. Gene expression profiling has been used to predict metastatic gene-expression signature that is present in a subset of primary breast tumors . However, a reliable profile has not yet been identified that specifically predicts brain metastases. Therefore, it is extremely important to study the genetic changes in breast cancer cells that metastasize to brain and develop specific targeted therapeutic molecular agents.
2. Channelopathy promote breast cancer metastasis
Cancer research is not only focusing on understanding the possible role of transmembrane-BKCa channels in cancer development and progression but also on development of BKCa channel modulator drugs to attenuate cancer growth. Several researchers, including us have shown that brain tumor cells express BKCa and ATP-sensitive potassium (KATP) channels that are highly responsive to minute changes in intracellular Ca2+ and ATP levels. This allows the brain tumor cells to develop pseudopodia for migration through constricted spaces in the brain parenchyma, as depicted in Figure 1. Several articles have described the efficacy of BKCa channel-inhibiting drugs or molecules in reducing tumors in preclinical mouse tumor models. A recent study has shown the role of intracellular BKCa channels (mitoBKCa) in cancer cell biology [9, 10].
2.1 Ion channels in breast cancer metastasis
Even now the metastatic breast cancers are incurable. Extensive research has shown that breast cancer metastasis to other organs, including brain is a complicated process. It is widely believed that breast cancer cells escape the primary site and migrate by lymphatic route to lymph nodes and vascular route to colonize in other organs including brain [11, 12]. Gene-expression profiling studies of breast cancer cells indicate that specific molecular pathways are associated with dissemination of primary tumor cells through a vascular route and not by lymphatic dissemination . There is much interest in studying how and when the cancer cells initiate the metastatic cascade so that a therapeutic intervention can be developed to stop or delay the metastasis. Some cancer researchers  believe that targeted treatment of breast cancer with ER/PR modulators (Aromatase inhibitors) and targeted biologics such as Herceptin (Her-2 neu inhibitor)  and bevacizumab  (anti-vascular). Others argue that the metastasis of cancer cells is triggered by a dysregulated cellular Ca2+ homeostasis and altered Ca2+ signaling caused by imbalanced fluxes through ion channels and transporters [10, 16]. The BKCa channels are more sensitive to Ca2+ ions in cancer cells. In this regard, we studied whether the increased sensitivity of potentially new BKCa channel variant protein encoded by splice variants (Figure 2)
Furthermore, a recent computational analysis of human genomic sequence identified mutations that cause pathogenic splicing abnormalities in breast cancer susceptibility genes, BRCA1, BRCA2 and other genes . Several investigations have reported that voltage gated ion channels are expressed in several cancers and contribute significantly to cell signaling, cell cycle progression and cell volume regulation, cancer cell proliferation, as well as metastasis. Hence, there is a great deal of interest in possible therapeutic potential of voltage gated ion channels as pharmacological targets [20, 21].
2.2 Metastatic breast cancer in brain microenvironment
Cancer cells have the innate ability to “exploit” the “chaotic” environmental challenges surrounding them and grow uninterrupted by manipulating transportome that regulate proliferation, apoptosis, metabolism, growth factor signaling, migration and invasion. Ion channels and transporters are some of the key modulators of cancer progression in hostile tumor microenvironment that includes hypoxia. It has been suggested that modulation of ion channels by the hypoxic environment may contribute to the aggressive phenotype observed in GBM cells residing in a hypoxic environment . In hostile microenvironment such as hypoxia, BKCa channels are modulated to aid cancer cell invasion and neovascularization. Affymetrix Array analyses of brain tumor cell lines where KCNMA1 was either overexpressed or suppressed showed significant changes in genes involved in cell proliferation, angiogenesis, cell cycle, and invasion .
KCNMA1/BKCa channel splice variants in breast cancer
During the past decade, a number of genes associated with breast cancer have been cloned and identified. Gene expression levels alone cannot fully explain gene function as alternative splicing produce multiple mRNAs and protein isoforms. New molecular insights indicate that the metastatic capacity of breast tumors is an inherent feature, and not necessarily a late, acquired phenotype [23, 24]. Breast cancer cells show alternative mRNA splicing and have prognostic and therapeutic value . Although there are many reports of alternative splicing events specific to breast cancer [25, 26], the biological activity of majority of alternatively spliced isoforms, and specifically their contribution to metastatic breast cancer biology, remains to be investigated. As many researchers are focusing on “Understanding and Preventing Brain Tumor Dispersal”, we recently reported on a novel
Identifying the most optimal and novel biomarker(s) for breast cancer metastasis to brain is ideal [27, 28] yet challenging because of the multi-factorial nature of the disease. The roles of roles of different ion channels in the development of cancer have been reported . The identification of a potential new biomarker has relied heavily on an increase or decrease in gene expression, but these changes may not always result in altered protein expression. Growing evidence indicates that alternative or aberrant pre-mRNA splicing resulting in protein isoforms with diverse functions occurs during the development, progression, and metastasis of breast cancer . Earlier, we have reported that the BKCa channels play a role in human breast tumor progression, cell proliferation, invasion, and micro-metastases [11, 17]. Nevertheless, the precise role of
The PCR results validated the findings of Exon array study. Two distinct splice variants expressed in breast cell line (MDA-MB-361) metastatic to brain were identified (i) deletion of exon 2 (
|Cell line||Proliferation (at 72 h)||Invasion||Trans-endothelial migration||Functional activity||Tumor volume at the 5th week|
|Untransfected||1 ± 0.09||1 ± 0.10||1 ± 0.17||1 ± 0.11||1 ± 0.21|
|Vector-transfected||0.98 ± 0.04||1.01 ± 0.12||0.95 ± 0.13||1.2 ± 0.15||1 ± 0.27|
|1.4 ± 0.12||1.9 ± 0.15||1.5 ± 0.19||1.6 ± 0.21||3.8 ± 0.42|
3. BKCa channels and neovascularization
Altered ion channels could play a pivotal role in physiological angiogenesis in including cancer [30, 31]. BKCa channel inhibitor modulated the tumorigenic ability of hormone-independent breast cancer cells via the Wnt pathway . Our work shows an association between the BKCa channel isoform expression and VEGF secretion by breast tumor cells, which might be exacerbated under hypoxia that has implications for vascular permeability and anticancer drug delivery (to be published). Understanding the underlying mechanism and splicing patterns of
We rationalize that
In addition, alternate splicing of
4.1 Splicing in health and disease
Many human diseases are implicated to errors in mRNA splicing. These aberrant splicing also provides an opportunity to develop targeted treatment to correct the faulty gene in some genetic disorders, or target aberrant protein encoded by these gene variants in human cancers. Breast cancer-specific biomarkers might generate specific epitopes that offer targets for developing diagnostic, prognostic and immunotherapy . Articles on alternative pre-mRNA splicing regulation in cancer  and misregulation of mRNA splicing in cancer  highlights the important roles in promoting aberrant splicing, which in turn contributes to all aspects of tumor biology.
4.2 BKCa channels as target to attenuate breast cancer metastasis
4.3 Alternate splicing of BKCa channels in diagnosis and prognosis
Several articles have highlighted the use of alternative splicing as a promising source for new diagnostic, prognostic, predictive, and therapeutic tools [38, 39, 40]. The diversity of RNA species detected through RNA-seq holds the potential of extracellular RNAs as non-invasive diagnostic indicators of disease [41, 42, 43, 44]. We recently reported that targeting the KCNMA1 variants may be a clinically beneficial strategy to prevent or at least slow down glioma transformation to GBM . In both human and mouse lymphoma models, researchers have shown that MYC directly induced the transcription of genes encoding core splicing machinery components. They also showed that PRMT5 is involved in MYC-driven tumorigenesis in mice with lymphoma and discovered that tumor development was delayed . Now due to high-throughput New Generation Sequencing (NGS) technologies the splicing diagnostic methodologies have improved. Hence NGS can be utilized in clinical diagnostics of splice variants in diagnosis, prognosis and treatment of breast cancers.
4.4 Perspective of
We believe that future therapies for metastatic breast cancer depend on further investigation into the mechanisms and cellular events caused by oncogene splicing such as
Perhaps the discovery and validation of brain specific metastasis-associated
The authors thank the Scintilla Group, Bangalore, India; Anderson Cancer Institute and Mercer University Medical Center, Savannah, GA, USA; Vanderbilt-Ingram Cancer Center, Nashville, TN, USA; Cedars-Sinai Medical Center, Los Angeles, CA, USA; American Cancer Society, USA; Georgia Cancer Coalition, Atlanta, GA, USA; and NIH for providing opportunity and research grant support. We also thank Dr. Nagendra of MVIT, Bangalore, for assisting us with the STRING software for the analysis and Michigan State University Research Center, Grand Rapids, MI, USA, for generating KKEG pathway using Affymetrix analysis data.