Role of an Alternatively Spliced KCNMA1 Variant in Glioma Growth

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.

Introduction

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 [1]. 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 [2]. The median survival among GBM patients is only 15 months or less [3]. 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 [4]. The response of “normal cells” to malignant transformation involves changes in gene expression and is thought to be regulated by transcription [5]. The potassium ion channels are implicated in the malignant transformation to a higher grade in several cancers [5–7]. For example, we reported that lowgrade gliomas might undergo certain epigenetic changes to develop into a GBM [8].

rain tumors are the most common type of solid tumors. In the United States, an estimated 20,000 new primary brain tumor cases are reported [1]. 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 [2]. The median survival among GBM patients is only 15 months or less [3]. 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 [4]. The response of “normal cells” to malignant transformation involves changes in gene expression and is thought to be regulated by transcription [5]. The potassium ion channels are implicated in the malignant transformation to a higher grade in several cancers [5–7]. For example, we reported that lowgrade gliomas might undergo certain epigenetic changes to develop into a GBM [8].

The physiological features of BKCa channels also known as maxi K or BK channels are well described [6–9]. 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 [7]. Studies have shown the importance of BKCa channels in brain tumor biology [5]. Lastly, BKCa currents in glioma cells are more sensitive to intracellular [Ca2+] compared to BKCa channels in healthy glial cells [9, 10].

Diverse role of KCNMA1 in glioma

KCNMA1-encoded BKCa channel plays a pivotal role in cancer cell proliferation. Amplification of KCNMA1 was observed in breast, ovarian, and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3–7%) and gliomas. KCNMA1 amplification was significantly associated with high tumor stage, highgrade, high tumor cell proliferation, and poor prognosis. Due to the large number of protein interactions and activating factors influencing BKCa channel function, intracellular Ca2+, membrane voltage, pH, shear stress, carbon monoxide, phosphorylation states, and steroid hormones, it is generally difficult to predict its direct role in a given tissue. However, in many diseases including cancers, defective regulation and/or expression of BKCa channels have repeatedly been associated with altered cell cycle progression [11], cell proliferation [11], and cell migration [11]. These altered cell functions are implicated in development of malignancy [11].

KCNMA1: STRING analysis

In order to understand the possible interactions of KCNMA1 with other genes and molecules, we used the tool STRING 9.1. It is a database consisting of known and possible protein– protein interactions with a gene of interest. The gene may have a direct (physical) or indirect (functional) association with other molecules. With this tool we can easily identify possible interaction of KCNMA1 with other associated molecules. We can derive detailed information 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, co expression, databases, and co-occurrence.