Evidence of calcium‑activated potassium channel subunit alpha‑1 as a key promoter of glioma growth and tumorigenicity

Introduction

Brain tumors are the most common type of solid tumor in both children and adults. It is estimated that 78,980 new cases of primary malignant and nonmalignant brain tumor and other central nervous system tumors will be diagnosed in the United States in 2019. This includes an estimated 23,830 primary malignant brain tumors and 55,150 nonmalignant brain tumors.[1] The most common form of malignant glioma is glioblastoma multiforme (GBM). The treatment of brain tumors is problematic, in terms of both cure rates and long-term quality of life.[2] Gliomas are insidious due to their highly invasive and destructive manifestation, and thus, GBM patients’ median survival is <15 months.[3] The low-grade gliomas often manifest as astrocytomas or anaplastic astrocytomas and sometimes progress dramatically to a highly malignant GBMs. There is a critical need to develop reliable biomarkers for the early detection and successful treatment to attenuate gliomas’ progression into GBM. Gene expression profiling studies have identified many GBM specific genes, involved in epigenetic inactivation, that drive glioma transformation.[4] GBM has distinct gene expression pattern among different histological types and grades of gliomas. [5] Increasing evidence shows that potassium channels are integral part of glioma cell growth and survival.[6]

The importance of voltage-gated potassium channels in tumor biology has aroused great interest in recognition of ion channels as potential targets for tumor therapy.[5‑8] Several potassium channels have been implicated in tumor progression and cell proliferation. Specifically, large-conductance, voltage-sensitive, Ca2+-activated potassium (BKCa) channels are overexpressed in human glioma cells.[7] Calcium-activated potassium channel subunit alpha‑1 (KCNMA1) encodes the α-subunit of the BKCa channels. They play a key role in cellular functions and have recently emerged as regulators of tumorigenesis. These channels respond to changes in intracellular calcium ([Ca2+]i ) and membrane potential, and their expression correlates with increased malignancy in gliomas.[8‑10] Studies have shown that pharmacological inhibition of BKCa channel by iberiotoxin abolished the activation of K+ ion channel currents and attenuated migration of glioma cells.[11‑13] Elucidation of the molecular mechanisms of KCNMA1 regulation is critical for the understanding of a variety of physiological and pathological conditions. The aim of this study was to study the effect of KCNMA1 modulation in glioma cells, thus offering a promising biomarker for early diagnosis and prognostication of high-grade gliomas.

The entire cloned fragment was sequenced using multiple primers to ensure whether each sequence has a good overlap with the adjacent sequence. This sequence was then compared to the KCNMA1 sequence NM 002247.2 (NCBI, Bethesda, MD, USA). Subsequently, the TOPO clones were digested with HindIII and XbaI to release the 3.7 kb fragment, which was gel purified and ligated into the HindIII-XbaI site of pcDNA6-V5/HIS expression vector (Invitrogen). The resulting plasmid pcDNA6/KCNMA1‑expressing KCNMA1 from a T7 promoter containing blasticidin resistance marker was used for selection of stable transfectants