Inflection point in glioma growth and angiogenesis driven by potassium channels

Background and Aim

The overexpression and alternative splicing of calcium-activated potassium channel subunit alpha-1 (KCNMA1) that encodes large-conductance calcium-activated voltage-sensitive potassium (BKCa) channels are implicated in the development of human cancers. Dysfunctional angiogenesis in hypoxic tumors is a challenge to intravenous anticancer drug treatments. Hypoxic factors also lead to abnormal vascular functions posing hurdle for anticancer drug delivery to tumors. The aim of this study was to explore the role of BKCa channels in tumor angiogenesis, specifi cally with regard to release of vascular endothelial growth factor (VEGF). Materials and Methods: We subjected the glioma cells under hypoxia and normoxia and studied the expression and activity of BKCa channels in in vitro and in vivo tumor models. Then, we studied the proangiogenic factor, VEGF, in tumors and monitored the neoangiogenic process. Results: We presented in vivo and cell based in vitro experimental evidence on the direct and indirect interactions of BKCa channels with VEGF signaling. There was evidence that under hypoxia, glioma cells overexpressed KCNMA1 and increased VEGF secretion. By inhibiting KCNMA1, we showed that VEGF secretion was signifi cantly reduced, thus potentially controlling angiogenesis, which has implications for vascular permeability and anticancer drug delivery. Moreover, there were differences in alternate splicing of KCNMA1 between normal and malignant cells under hypoxia and normoxia. Conclusion: We conclude that BKCa channels regulate hypoxia-induced angiogenesis. Therefore, serious effort is needed to better understand the molecular mechanisms of BKCa channelopathies triggering angiogenesis and progression of glioma. The modulators of BKCa channels could be viable in new anticancer therapeutics. The study protocol was approved by the Institutional Animal Care and Use Committee, Mercer University, Atlanta, GA, USA (approved No. A0706007_01) on July 20, 2007.

INTRODUCTION

Cancer is a chronic disease characterized by uncontrolled cell growth.[1] Cancer cells typically go through four stages – initiation, proliferation, invasion, and metastasis. There are over 100 different types of cancer, and each is classifi ed by the type of cells that are initially affected.[2] Cancer cells divide uncontrollably to form lumps or masses of tissue called tumors that can grow and interfere with several bodily functions. Cell signaling involving vascular endothelial growth factor (VEGF) and its VEGF receptor (VEGFR) plays a major role in cancer progression by promoting new blood vessels formation called neoangiogenesis.[3] Disruption of the genes encoding either VEGF or any of the three receptors of the VEGF family results in embryonic lethality because of failure of blood vessel development.[4] VEGFR2 is the main signal transducing VEGFR for angiogenesis and mitogenesis of endothelial cells, which is directly related to cell cancer. VEGFs are combined with VEGFRs to activate the VEGF signaling cascade leading to angiogenesis. As shown in Table 1, specifi c isoforms of VEGFs are combined with specifi c VEGFRs to regulate several critical cell functions and also impact on human health and diseases.[5-8]

VEGF can be detected in both plasma and serum samples of patients, with much higher level in serum. Platelets release VEGF upon aggregation and may be a major source of VEGF delivery to tumors.[9] Many tumors release cytokines that can stimulate the production of megakaryocytes in the marrow and elevate the platelet count. This can result in an indirect increase of VEGF delivery to tumors.[10,11] The autocrine VEGF signaling is crucial for tumor initiation and transformation into highly aggressive cancers.[3] The blocking of autocrine VEGF secretion provides a promising strategy to develop new therapeutic approaches.[12] VEGF is implicated in several other pathological conditions associated with enhanced angiogenesis, such as cancer, psoriasis, and rheumatoid arthritis. Direct role of VEGF in tumor growth has been shown using dominant negative VEGFRs to block in vivo proliferation, as well as blocking antibodies to VEGF or to VEGFR2.[13] Interference with VEGF function by targeting the VEGF signaling pathway is a major interest in drug development for blocking angiogenesis in primary and metastatic brain tumors [Figure 1].

The calcium-activated voltage-sensitive potassium (BKCa) channels interact with a variety of proteins both at the plasma membrane and with intracellular organelles including the endoplasmic reticulum, nucleus, and mitochondria. However, the role of BKCa channels in tumor microenvironment including hypoxia is yet to be explored. Hypoxia promotes vessel growth by upregulating multiple proangiogenic pathways that mediate key aspects of endothelial, stromal, and vascular support cell biology.[14] In general, uncontrolled cancer growth and subsequent neoangiogenesis lead to hypoxic tumor microenvironment.[15] VEGF expression increases dramatically in hypoxic conditions due to a number of activated oncogenes that are overexpressed in hypoxia. VEGF induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis.[16] Deregulated VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis and to the etiology of several additional diseases that are characterized by abnormal angiogenesis. Consequently, inhibition of VEGF signaling abrogates the development of a wide variety of tumors.[17] The second-generation multitargeted tyrosine kinase inhibitor targets VEGFR, platelet-derived growth factor receptor, and c-kit as key proteins responsible for tumor growth and survival.[18] Pazopanib exhibits good potency against all of the human VEGFRs and closely relate to tyrosine receptor kinases in vitro and demonstrates antitumor activity in several human tumor xenografts. Therefore, VEGFRs are attractive therapeutic targets.[19]

Recent work has shown the central role of K+ channels affect multiple conditions of the tumor microenvironment including hypoxia and adenosine.[20] It has long been known that the interaction of tumor cells with their host microenvironment, including endothelial cells and the extracellular matrix, plays an important role in tumor growth and invasion.[21,22] Hypoxia induces the transcriptional activation of signaling pathways and regulates tumor growth through differential alternative splicing.[23] Nonetheless, very little is known about the effect of hypoxia on the alternative splicing of calcium-activated potassium channel subunit alpha-1 (KCNMA1) either in tumor cells or in endothelial cells. Understanding the role of hypoxia in KCNMA1 splicing is extremely crucial to study blood–brain tumor barrier (BTB) function and improve drug delivery. Our previous studies[24-30] have revealed that human brain microvascular endothelial cells adjacent to glioma cells overexpress BKCa channels, as opposed to human brain microvascular endothelial cells in normal brain. Our aim is to seek whether the tumor cells (with or without physical contact) overexpress KCNMA1 or its splice variants to increase secretion of VEGF to induce angiogenesis.