**5. KCNMA1 splicing in glioma**

**Figure 3.** High-grade glioma cells, U-87 MG were transfected to make stable cell line over-expressing KCNMA1. In addition, we transfected U-87 MG cells with shKCNMA1; this suppressed basal KCNMA1 expression. A microarray was performed on these cells. The heat map shows the differential expression of genes that were directly or indirectly affected by upregulation or down regulation of KCNMA1. We found 8102 and 7259 significant features at p < 0.05, respectively, for overexpression and suppression of *KCNMA1*. From these, features having at least a signal value of 255

of the protein being investigated as well as its associated molecules, crystal structure of the proteins with its PDB ID, and combined score [confidence score, neighborhood score, fusion score, homology score] on the basis of some parameters like experimental results, text-mining,

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

cance of up- and down-regulation of *KCNMA1* on associated genes, we performed microarray using the Affymetrix Human Genome U133 Plus 2.0. Array analyses of U-87 MG cell lines where *KCNMA1* was either overexpressed or suppressed showed significant changes in genes involved in cell proliferation, angiogenesis, cell cycle, and invasion (**Figure 3**). Class comparison tests indicated significant changes in global expression patterns. Twenty genes highly downregulated by suppression but upregulated by overexpression of *KCNMA1* or vice versa are shown in **Figure 3**. This data support our rationale that *KCNMA1* plays a critical role in

Array analyses of U-87 MG cell lines where *KCNMA1* was either overexpressed or suppressed showed significant changes in genes involved in cell proliferation, angiogenesis, cell cycle,

. In order to study the biological signifi-

**4. Possible KEGG pathway following activation and suppression of** 

co expression, databases, and co-occurrence (**Figure 2**).

amino acids. Cells were maintained at 37°C in 5% CO<sup>2</sup>

**KCNMA1 in glioma cells**

230 Brain Tumors - An Update

the above cellular processes.

were selected to reduce false positives (false discovery rate < 0.0079).

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 [14–16]. Gating properties and kinetics with regard to the voltage and Ca2+ dependence of gating are altered by alternative splicing in these regions [17–19]. Expressions of different BKCa isoforms have been implicated in auditory processing [20] and alter the sensitivity of BKCa to modulation by phosphorylation [21] and other processes [22]. However, the role of BKCa isoforms in cancer is now being investigated [23]. More specifically, KCNMA1 is altered in a wide variety of cancers, and their overexpression liked to increased malignancy in gliomas [4–7]. 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 [4–9] and play an important role in glioma invasion and migration [24, 25].

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 [23]. Several sites of alternative pre-mRNA splicing of *KCNMA1* have been described, and majority of them are located within the intracellular C-terminal domain of the channel [19]. In the past a novel splice variant of *KCNMA1* (gBK) with an additional 34-amino-acid exon at splice site 2 in the

Role of an Alternatively Spliced *KCNMA1* Variant in Glioma Growth

http://dx.doi.org/10.5772/intechopen.74509

233

In addition to the above studies, we present herein the cloning, functional characterization, and splicing of a novel *KCNMA1* splice variant. *KCNMA1* encodes the alpha-subunit of human BKCa channels and is known to form BKCa channel isoforms. Here, we report hitherto unknown *KCNMA1* splice variant, which has a 108-base-pair deletion at the splice site on one of its exons, which we termed as *KCNMA1v*. More importantly, *KCNMA1v* expression correlates positively with the relative degree of malignancy of the glioma cell lines (under publication). Moreover, we found that *KCNMA1v* was expressed only in high-grade glioma samples and not in normal brain tissues as evidenced by examination of human biopsy specimens (under publication). Expression of *KCNMA1v* in HEK (null type) revealed that the pharmacological and biophysical properties of the variant were consistent with the properties of wild-type *KCNMA1* gene in glioma cells suggesting that *KCNMA1v* is likely to encode the principal wild-type BKCa channels (under publication). Although we have not separated wildtype and splice variant isoform for sequence and structure analysis, the biological properties of both wild-type and isoform protein appear to be similar. However, when overexpressed in glioma cell line (under publication), the variant showed distinct biological properties such as

enhanced Ca2+ sensitivity at physiologically relevant [Ca2+]i levels (under publication).

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 [35]. For example, miR-182 was found to be a valuable *marker of glioma progression* and that high miR-182 expression is associated with poor prognosis [36]. Such a multistep process of tumorigenesis has been proposed to involve a series of mutational events which ultimately lead to development and progression of neoplasia [35]. Aberrant pre-mRNA splicing is an important factor in tumor progression and has been proposed to result in the loss of a normal pathway of differentiation, which could lead to tumor progression. Several studies have implicated BKCa channel expression to oncogenic cell transformation [37, 38]. Increased activity of BKCa channels appeared to be required for the mitogenic stimulation of non-transformed cells and may play a role in cell proliferation [39]. Consistent with the above studies, we show that *KCNMA1v*-induced effects promote proliferation in glioma cell lines when the variant was overexpressed. The upregulation of *KCNMA1v* in glioma cell lines provides an opportunity to determine variant-specific changes that enhance gliomagenesis in vivo. The overexpression of *KCNMA1v* resulted in increased proliferation in glioma cell lines. It has also been suggested that cell invasion into narrow brain spaces may

C-terminal has already been described in gliomas [6].

BKCa channels show a variety of electrophysiological properties due to alternative splicing of their α-subunits. In glioma cells, Liu et al. [6] 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 [6]. 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) [10] 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 [28]. 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 [30]. 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 [33]. 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 [6]. 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 [29]. Venables et al. [34] 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 [23]. Several sites of alternative pre-mRNA splicing of *KCNMA1* have been described, and majority of them are located within the intracellular C-terminal domain of the channel [19]. In the past a novel splice variant of *KCNMA1* (gBK) with an additional 34-amino-acid exon at splice site 2 in the C-terminal has already been described in gliomas [6].

calcium ([Ca2+]i) and membrane potential. We and others have demonstrated that BKCa channels are overexpressed in gliomas [4–9] and play an important role in glioma invasion and

BKCa channels show a variety of electrophysiological properties due to alternative splicing of their α-subunits. In glioma cells, Liu et al. [6] 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 [6]. 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) [10] 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 [28]. 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 [30]. 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

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 [33]. 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 [6]. 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 [29]. Venables et al. [34] 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.

migration [24, 25].

232 Brain Tumors - An Update

brain tumors is a matter of great interest.

In addition to the above studies, we present herein the cloning, functional characterization, and splicing of a novel *KCNMA1* splice variant. *KCNMA1* encodes the alpha-subunit of human BKCa channels and is known to form BKCa channel isoforms. Here, we report hitherto unknown *KCNMA1* splice variant, which has a 108-base-pair deletion at the splice site on one of its exons, which we termed as *KCNMA1v*. More importantly, *KCNMA1v* expression correlates positively with the relative degree of malignancy of the glioma cell lines (under publication). Moreover, we found that *KCNMA1v* was expressed only in high-grade glioma samples and not in normal brain tissues as evidenced by examination of human biopsy specimens (under publication). Expression of *KCNMA1v* in HEK (null type) revealed that the pharmacological and biophysical properties of the variant were consistent with the properties of wild-type *KCNMA1* gene in glioma cells suggesting that *KCNMA1v* is likely to encode the principal wild-type BKCa channels (under publication). Although we have not separated wildtype and splice variant isoform for sequence and structure analysis, the biological properties of both wild-type and isoform protein appear to be similar. However, when overexpressed in glioma cell line (under publication), the variant showed distinct biological properties such as enhanced Ca2+ sensitivity at physiologically relevant [Ca2+]i levels (under publication).

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 [35]. For example, miR-182 was found to be a valuable *marker of glioma progression* and that high miR-182 expression is associated with poor prognosis [36]. Such a multistep process of tumorigenesis has been proposed to involve a series of mutational events which ultimately lead to development and progression of neoplasia [35]. Aberrant pre-mRNA splicing is an important factor in tumor progression and has been proposed to result in the loss of a normal pathway of differentiation, which could lead to tumor progression. Several studies have implicated BKCa channel expression to oncogenic cell transformation [37, 38]. Increased activity of BKCa channels appeared to be required for the mitogenic stimulation of non-transformed cells and may play a role in cell proliferation [39]. Consistent with the above studies, we show that *KCNMA1v*-induced effects promote proliferation in glioma cell lines when the variant was overexpressed. The upregulation of *KCNMA1v* in glioma cell lines provides an opportunity to determine variant-specific changes that enhance gliomagenesis in vivo. The overexpression of *KCNMA1v* resulted in increased proliferation in glioma cell lines. It has also been suggested that cell invasion into narrow brain spaces may require tumor cells to shrink and squeeze through tight interstitial space [40]. Cell shrinkage requires the efflux of K<sup>+</sup> and Cl<sup>−</sup> ions [41], and BKCa channels may serve as pathway for regulated K<sup>+</sup> efflux [42]. Consistent with these findings, the overexpression of *KCNMA1v* increased the invasion potential of glioma cells (under publication). The role of BKCa channels in cell migration was already described [43]. The changes in proliferation and migration of cells over-expressing *KCNMA1v* were mostly attributed to increased levels of *KCNMA1* and BKCa channel protein expression in transfected cells. Additionally, overexpression of *KCNMA1v* in glioma cells may assist them to diffusely invade the normal brain. Due to this phenomenon, GBM patients typically show high propensity to recur as the cancer cells expressing *KCNMA1v* might survive surgical and therapeutic treatment. The xenograft tumors in mice likewise demonstrated increased growth, which correlated well with Ki-67 expression (under publication). The overexpression of *KCNMA1v* resulted in increased angiogenesis in the tumor xenografts, supporting the angiogenic role of *KCNMA1v*. The observation that the overexpression of *KCNMA1v* in human gliomas correlates with increased angiogenesis in high-grade gliomas further supports that *KCNMA1* splicing event is an important biological process for glioma progression. Consistent with this observation, we found that glioma cells over-expressing *KCNMA1v* secreted significantly the high level of angiogenic factor VEGF (under publication).

**Author details**

Bangalore, India

**References**

and Research, Bangalore, India

Divya Khaitan1,2, Nagendra Ningaraj2,3,4\* and Lincy B. Joshua5

1 Department of Molecular Oncology, Scintilla Academy for Applied Sciences' Education

Role of an Alternatively Spliced *KCNMA1* Variant in Glioma Growth

http://dx.doi.org/10.5772/intechopen.74509

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4 BGS-Global Institute of Medical Sciences, Bangalore, India 5 BGS-Global Institute of Medical Sciences, Bangalore, India

## **6. Conclusion**

Further investigation into the mechanisms and cellular events caused by *KCNMA1* splicing may lead to the development of future therapies for this highly deadly disease. Splice variants that are found in high-grade gliomas have clear diagnostic and prognostic values besides providing potential targets for anticancer drug development. Clinical outcome of *KCNMA1v* expression in high-grade glioma is expected to reveal the variants' clinical importance. This analysis is being performed in our laboratory. In conclusion, the results presented here might suggest that quantifying the levels of *KCNMA1v* could be useful to identify biological process that increases the malignancy and affect prognosis of high-grade glioma patients.
