Gliomas develop genetic traits to rapidly form aggressive phenotypes. Hence, management of gliomas is complicated and difficult. Besides genetic aberrations such as oncogenic copy number variation and mutations, alternative mRNA splicing triggers prooncogenic episodes in many cancers. In gliomas, we found alternative splicing at the KCNMA transcription process. KCNMA1 encodes the pore forming α-subunit of large-conductance calcium-activated voltage-sensitive potassium (BKCa) channels. These channels play critical role in glioma invasion and proliferation. We identified a novel KCNMA1 mRNA splice variant with a deletion of 108 base pairs (KCNMA1v) mostly overexpressed in high grade gliomas. We found that KCNMA1 alternative pre-mRNA splicing enhanced glioma growth, progression and diffusion. The role of KCNMA1 and its splicing as a critical posttranscriptional regulator of BKCa channel expression is presented in this chapter. Our research implies that high grade gliomas express KCNMA1v and BKCa channel isoform to accelerate growth and transformation to glioblastoma multiforme (GBM). We demonstrated that tumors hardly develop in mice injected with KCNMA1v transfected cell line expressing short-hairpin RNA (shRNA) compared to mice injected with KCNMA1v transected glioma cells. We conclude that targeting the KCNMA1 variants may be a clinically beneficial strategy to prevent or at least slow down glioma transformation to GBM.
- BKCa channels
- potassium channels
KCNMA1-encoded BKCa channels in glioma
Brain tumors are the most common type of solid tumors. In the United States, an estimated 20,000 new primary brain tumor cases are reported . The most common form of malignant glioma is glioblastoma multiforme (GBM). The treatment of brain tumors is highly complicated due to their highly aggressive phenotypic and genotypic changes . The median survival among GBM patients is only 15 months or less . GBM contains heterogeneous subpopulations of glioma and other mixed supporting cells that are cancerous cells. They have the intrinsic ability that adapt in the brain tumor microenvironment and invade the normal brain. Gene expression profiling studies have identified many genes that have distinct expression patterns among different histological types and grades of gliomas . The response of “normal cells” to malignant transformation involves changes in gene expression and is thought to be regulated by transcription . The potassium ion channels are implicated in the malignant transformation to a higher grade in several cancers [5, 6, 7]. For example, we reported that low-grade gliomas might undergo certain epigenetic changes to develop into a GBM .
The physiological features of BKCa channels also known as maxi K or BK channels are well described [6, 7, 8, 9]. These channels are unique since its activity is triggered by depolarization and enhanced by an increase in μM range of cytosolic calcium (Figure 1). The BKCa channels provide a crucial link between the metabolic and electrical states of cells. The BKCa channel overexpression was observed in biopsies of patients with malignant gliomas compared with nonmalignant human cortical tissues and the level of expression correlated positively with increased malignancy . Studies have shown the importance of BKCa channels in brain tumor biology . Lastly, BKCa currents in glioma cells are more sensitive to intracellular [Ca2+] compared to BKCa channels in healthy glial cells [9, 10].
2. Diverse role of
3. KCNMA1: STRING analysis
In order to understand the possible interactions of
4. Possible KEGG pathway following activation and suppression of KCNMA1 in glioma cells
Glioma cell line U-87 MG was obtained from the American Type Culture Collection (Manassas, VA) and cultured in MEM supplemented with 10% FBS and 0.1 mM nonessential amino acids. Cells were maintained at 37°C in 5% CO2. In order to study the biological significance of up- and down-regulation of
Array analyses of U-87 MG cell lines where
5. KCNMA1 splicing in glioma
The KCNMA1 encodes the pore-forming α-subunits of large-conductance Ca2+-activated K+ (BKCa) channels. More than 20 variants of this gene are associated with alternative splicing at ten or more different sites [12, 13], while majority of the splice sites are located in the large cytoplasmic domain. This domain is called the C-terminal half of the channel that contains multiple Ca2+ binding sites [14, 15, 16]. Gating properties and kinetics with regard to the voltage and Ca2+ dependence of gating are altered by alternative splicing in these regions [17, 18, 19]. Expressions of different BKCa isoforms have been implicated in auditory processing  and alter the sensitivity of BKCa to modulation by phosphorylation  and other processes . However, the role of BKCa isoforms in cancer is now being investigated . More specifically, KCNMA1 is altered in a wide variety of cancers, and their overexpression liked to increased malignancy in gliomas [4, 5, 6, 7]. The BKCa protein isoform transcribed by its alternatively spliced mRNA in cancer cells is known as likely to respond differently to changes in intracellular calcium ([Ca2+]i) and membrane potential. We and others have demonstrated that BKCa channels are overexpressed in gliomas [4, 5, 6, 7, 8, 9] and play an important role in glioma invasion and migration [24, 25].
BKCa channels show a variety of electrophysiological properties due to alternative splicing of their α-subunits. In glioma cells, Liu et al.  reported that BKCa channels exhibit distinct electrophysiological properties due to alternate splicing of its α-subunits. These BKCa variants showed higher Ca2+ sensitivity in glioma cells compared to BKCa channels present in normal glial cells. The amplified sensitivity to intracellular [Ca2+i] was shown in a novel splice isoform (gBK) of hSlo, the gene that encodes the α-subunits, specifically expressed in glioma . We have recently shown (submitted for review) that KCNMA1 that encodes α-subunit (pore forming) of BKCa channel undergoes specific splicing at mRNA to form a variant (KCNMA1v) that encodes for a novel BKCa channel isoform only in glioblastoma multiforme (GBM). Other types of Ca2+-activated K+ channels such as intermediate (IKCa) and small (SKCa)  have been characterized in human glioma cells, but their roles in brain tumor biology are yet to be explored.
The alternative RNA splicing might increase protein expression levels and functions. In cancer, it was shown that abnormal mRNA splicing often leads to tumor-promoting splice variants that are translated into activated oncogenes or inactivated tumor suppressors [26, 27]. Interestingly, the brain appears to have maximum alternative splicing of exons . The present knowledge suggests that alternative or aberrant pre-mRNA splicing results in oncoproteins with diverse functions in the development, progression, and dispersal of glioma cells [29, 30]. Further, genomic studies have shown that gliomas often have splice isoforms than in normal brain . For instance, KCNMA1 was shown to undergo alternative pre-mRNA splicing at several sites in humans and mice [31, 32] to generate physiologically diverse BKCa channels. These altered BKCa channels respond differently to calcium/voltage changes. Often, these channels show abnormal regulation of cellular signaling pathways in glioma cells [13, 19]. Hence, the cause–effect of KCNMA1 splicing in functional modification of BKCa channels in brain tumors is a matter of great interest.
We have described an unknown KCNMA1 mRNA splice variant with a deletion of 108 base pairs of exon 22 (KCNMA1v) between the S9 and S10 protein subunits (C-terminus) overexpressed in high-grade gliomas. This serendipitous finding prompted to study the role of KCNMA1v as a critical posttranscriptional regulator of BKCa channel isoform expression and altered channel function in gliomas (submitted for review). The complex interaction between various ions and their respective ion channels at the invadopodia of the malignant gliomas is speculated to explain some of the invasive properties of gliomas [24, 25]. The role of various ions and their respective ion channels in glioma is recently well documented . Among many ion channels, BKCa channels have many known spliced variants. Liu et al. have initially described a spliced variant, glioma BK (gBK), channel in human glioma cells . Inherited and acquired changes in pre-mRNA splicing have been shown to play a significant role in human disease development (pre-mRNA splicing and human disease . Venables et al.  showed that alternative splicing of pre-mRNA increases the diversity of protein functions in ovarian and breast cancer samples. Specifically, they found that expression of FOX2 was downregulated in ovarian cancer and its splicing is altered in breast cancer samples affecting cell proliferation.
However, studies on the association of changes in gene splicing pattern and malignancy are rare. However, few studies have shown the presence of BKCa channels at the invadopodia of the malignant gliomas that lead to speculation that these channels may help the invasive properties of gliomas. A recent study found a clinical relevance where the investigators found T cells derived from GBM patients who were sensitized to the gBK peptide could also kill target cells expressing gBK. This study shows that peptides derived from cancer-associated ion channels maybe useful targets for T-cell-mediated immunotherapy . Several sites of alternative pre-mRNA splicing of
In addition to the above studies, we present herein the cloning, functional characterization, and splicing of a novel
Progression of brain tumor from localized, slow-growing tumors to more aggressive brain tumors capable of invading the surrounding brain most likely involves a series of stepwise biological events . For example, miR-182 was found to be a valuable
Further investigation into the mechanisms and cellular events caused by
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.