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

Evidence of BK_Ca Channelopathy-Driven Breast Cancer Metastasis to Brain

KCNMA1 encodes the a-subunit of the large conductance, voltage and Ca2+-activated and Voltage-dependent potassium channel (BKCa) and was shown by others and us to be a potential drug target gene in several cancers, including breast cancer. In addition, we studied the role of alternative pre-mRNA splicing events of KCNMA1 in migration, invasion, proliferation and dispersal of breast cancer cells. It is conceivable that by targeting gene variants we can attenuate processes such as distant metastasis and angiogenesis. Here we reviewed literature on the alternative splicing events specific to breast cancer metastasis to brain, its microenvironment, the biological activity of most alternatively spliced isoforms. We conclude that based on our and others’ work KCNMA1 and other such gene variants contribute to breast cancer dispersion, invasion, growth, and progression in the tumor microenvironment. Thus KCNMA1/BKCa channels and their variants are opportunistic diagnostic, prognostic and treatment targets in breast cancer

Role of KCNMA1 gene in breast cancer invasion and metastasis to brain

International audienceBACKGROUND: The prognosis for patients with breast tumor metastases to brain is extremely poor. Identification of prognostic molecular markers of the metastatic process is critical for designing therapeutic modalities for reducing the occurrence of metastasis. Although ubiquitously present in most human organs, large-conductance calcium- and voltage-activated potassium channel (BKCa) channels are significantly upregulated in breast cancer cells. In this study we investigated the role of KCNMA1 gene that encodes for the pore-forming alpha-subunit of BKCa channels in breast cancer metastasis and invasion. METHODS: We performed Global exon array to study the expression of KCNMA1 in metastatic breast cancer to brain, compared its expression in primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immunohistochemistry was performed to study the expression and localization of BKCa channel protein in primary and metastatic breast cancer tissues and breast cancer cell lines. We performed matrigel invasion, transendothelial migration and membrane potential assays in established lines of normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether BKCa channel inhibition attenuates breast tumor invasion and metastasis using KCNMA1 knockdown with siRNA and biochemical inhibition with Iberiotoxin (IBTX). RESULTS: The Global exon array and RT-PCR showed higher KCNMA1 expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased BKCa channel activity, leading to greater invasiveness and transendothelial migration, both of which could be attenuated by blocking KCNMA1. CONCLUSION: Determining the relative abundance of BKCa channel expression in breast cancer metastatic to brain and the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy

Epigenetic Regulation of MicroRNA Genes and the Role of miR-34b in Cell Invasion and Motility in Human Melanoma

Invasive melanoma is the most lethal form of skin cancer. The treatment of melanoma-derived cell lines with 5-aza-2\u27-deoxycytidine (5-Aza-dC) markedly increases the expression of several miRNAs, suggesting that the miRNA-encoding genes might be epigenetically regulated, either directly or indirectly, by DNA methylation. We have identified a group of epigenetically regulated miRNA genes in melanoma cells, and have confirmed that the upstream CpG island sequences of several such miRNA genes are hypermethylated in cell lines derived from different stages of melanoma, but not in melanocytes and keratinocytes. We used direct DNA bisulfite and immunoprecipitated DNA (Methyl-DIP) to identify changes in CpG island methylation in distinct melanoma patient samples classified as primary in situ, regional metastatic, and distant metastatic. Two melanoma cell lines (WM1552C and A375 derived from stage 3 and stage 4 human melanoma, respectively) were engineered to ectopically express one of the epigenetically modified miRNA: miR-34b. Expression of miR-34b reduced cell invasion and motility rates of both WM1552C and A375, suggesting that the enhanced cell invasiveness and motility observed in metastatic melanoma cells may be related to their reduced expression of miR-34b. Total RNA isolated from control or miR-34b-expressing WM1552C cells was subjected to deep sequencing to identify gene networks around miR-34b. We identified network modules that are potentially regulated by miR-34b, and which suggest a mechanism for the role of miR-34b in regulating normal cell motility and cytokinesis

The Regulation of miRNA-211 Expression and Its Role in Melanoma Cell Invasiveness

The immediate molecular mechanisms behind invasive melanoma are poorly understood. Recent studies implicate microRNAs (miRNAs) as important agents in melanoma and other cancers. To investigate the role of miRNAs in melanoma, we subjected human melanoma cell lines to miRNA expression profiling, and report a range of variations in several miRNAs. Specifically, compared with expression levels in melanocytes, levels of miR-211 were consistently reduced in all eight non-pigmented melanoma cell lines we examined; they were also reduced in 21 out of 30 distinct melanoma samples from patients, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic. The levels of several predicted target mRNAs of miR-211 were reduced in melanoma cell lines that ectopically expressed miR-211. In vivo target cleavage assays confirmed one such target mRNA encoded by KCNMA1. Mutating the miR-211 binding site seed sequences at the KCNMA1 3′-UTR abolished target cleavage. KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels. Two different melanoma cell lines ectopically expressing miR-211 exhibited significant growth inhibition and reduced invasiveness compared with the respective parental melanoma cell lines. An shRNA against KCNMA1 mRNA also demonstrated similar effects on melanoma cells. miR-211 is encoded within the sixth intron of TRPM1, a candidate suppressor of melanoma metastasis. The transcription factor MITF, important for melanocyte development and function, is needed for high TRPM1 expression. MITF is also needed for miR-211 expression, suggesting that the tumor-suppressor activities of MITF and/or TRPM1 may at least partially be due to miR-211's negative post transcriptional effects on the KCNMA1 transcript. Given previous reports of high KCNMA1 levels in metastasizing melanoma, prostate cancer and glioma, our findings that miR-211 is a direct posttranscriptional regulator of KCNMA1 expression as well as the dependence of this miRNA's expression on MITF activity, establishes miR-211 as an important regulatory agent in human melanoma

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

Background and Aim: Mechanisms of glioma progression are poorly understood. Upregulation of calcium-activated potassium channel subunit alpha-1 (KCNMA1), which encodes the α-subunit of maxi-calcium-activated potassium (BKCa) channels, is shown to be a novel mechanism for the malignant phenotype of brain tumor cells. The aim of this study was to establish the functional role of KCNMA1 in glioma biology. Materials and Methods: U-87-MG (U-87) cells were transfected to increase BKCa channel expression and activity. Glioma cell proliferation, invasiveness, and transendothelial migration were then measured. BKCa channels were blocked with iberiotoxin or short hairpin RNA (shRNA), which significantly inhibited K+ currents and growth of U-87 cells. It was tested whether KCNMA1 overexpression enhanced tumorogenecity in glioma xenograft mouse models by injecting wild-type and KCNMA1- overexpressing U87-MG cells. In parallel experiment, it was studied whether shRNA KCNMA1-expressing U-87 cells show attenuated glioma growth in mice. The study protocol was approved by the Institutional Animal Care and Use Committee, Mercer University (A0706007_01), Atlanta, GA, USA on July 20, 2007. Results: The effect of KCNMA1 overexpression in glioma growth as well as on associated cell biology functions such as proliferation, invasion, and migration was presented in this study. Messenger RNA and protein analyses revealed that KCNMA1 was amplified in 90% of high-grade gliomas and in high-grade glioma cell line U-87. In contrast, KCNMA1 amplification was not found in normal brain tissues. These data indicate that KCNMA1 plays critical role in glioma biology by interacting with several cellular processes. The data demonstrate that KCNMA1 amplification drives glioma cell proliferation and growth, which can be attenuated by its downregulation. Conclusion: KCNMA1 is a regulator of glioma cell proliferation and growth and thus qualifies as a promising diagnostic and therapeutic target in the treatment of glioma

Endogenous and Induced Oxidative Stress in Multi-cellular Tumour Spheroids: Implications for Improving Tumour Therapy

16-24The endogenous oxidative stress in tumours is determined by the status of mitochondrial, metabolic, oxygen (hypoxia) and inherent enzymatic as well as non-enzymatic antioxidant defense systems, which influence tumour growth and respond to anticancer therapeutics. Induced oxidative stress is one of the important determinants of the outcome of treatment with certain chemotherapeutic drugs and ionizing radiation. The mild to moderate levels of reactive oxygen species (ROS) have often been found to trigger prosurvival responses, thereby contributing to the resistance against therapy. The higher levels of ROS stimulate multiple death pathways viz. typical and atypical apoptosis, necrosis etc, thereby enhancing the therapeutic efficiency. Therefore, approaches employing therapeutic agents that generate ROS efficiently in the tumour cells and enhance the antioxidant defense system in the normal cells could significantly enhance the therapeutic gain. Multi-cellular tumour spheroids (MCTS) offer an excellent in vitro system that mimics endogenous oxidative stress often observed in tumours, arising due to a number of factors (gradients of oxygen and nutrients, altered intercellular interaction and tumour necrosis factor), besides antioxidant defense systems similar to tumours in vivo. More importantly, MCTS resemble tumours in vivo with reference to the induced oxidative stress related responses, particularly following combinations of certain chemotherapeutic drugs and metabolic inhibitors and differs significantly from the responses in monolayer cultures. Therefore, MCTS appear to be excellent in vitro models, ideally suited for developing novel therapies that are based on the generation of oxidative stress in tumours. The present review provides a modest account on the utility of MCTS in understanding the role of oxidative stress in treatment-induced responses of tumours for designing therapies and therapeutics

Inhibitors of topoisomerases as anticancer drugs: Problems and prospects

649-659DNA topoisomerases, which solve topological problems associated with various DNA transactions, are the targets of many therapeutic agents. Various topoisomerase inhibitors especially, topo-poisons, camptothecin (topo-I) and etoposide (topo-II) are some of the drugs that are used in the current treatment protocols, particularly for the treatment of leukemia (AML, ALL etc). However, tumor resistance, normal and non-specific tissue cytotoxicity are the limitations for successful development of these drugs as one of the primary therapeutic agents for the treatment of tumors in vitro. This brief review presents the current understanding about cytotoxicity development and outlines various approaches to overcome the limitations for enhancing the efficacy of topo-poison based anticancer drugs

Establishment and characterization of multicellular spheroids from a human glioma cell line; Implications for tumor therapy

Abstract Background Multicellular spheroids, an appropriate in vitro system for simulating 3-D tumor micro-milieu can be used for evaluating and predicting tumor response to therapeutic agents including metabolic inhibitors. However, detailed understanding of the nature, distribution and sensitivity/responses of cellular sub-populations to potential therapeutic agents/strategies is required for using this unique model with optimal precision. Spheroid characteristics may also vary considerably with the origin and type of cell line used, and thorough characterization of viable and dissociated glioma cell spheroids is not yet completely known. In order to evaluate in vivo responses of gliomas to various therapeutic strategies, especially the metabolic inhibitors capable of penetrating the blood brain barrier, we have characterized continuously growing spheroids of a human glioma cell line (BMG-1) with respect to organization, growth, viability, cell survival, cell death, metabolic and mitochondrial status, oxidative stress and radiation response using microscopy, flow cytometry and enzymatic assays. Spheroids were fed daily with fresh medium in order to maintain nutrient supply to outer cellular layers while hypoxia/necrosis developed in the innermost cells of enlarging spheroids. Results Volume of spheroids, fed daily with fresh medium, increased exponentially during 7–28 days of growth through three population doublings. Proportion of G1-phase cells was higher (~60%) than exponentially growing monolayer cells (~48%). A significant fraction of S-phase cells turned metabolically inactive (disengaged in DNA synthesis) with increasing age of the spheroids, unlike in quiescent monolayer cultures, where the fraction of S-phase cells was less than 5%. With increasing spheroid size, increasing sub-populations of cells became non-viable and entered apoptosis or necrosis revealed by Annexin-V-FITC/PI staining. PI positive (necrotic) cells were not confined to the centre of the spheroid, but distributed at certain discrete foci. Average glucose consumption and lactate production were 2–3 folds higher in viable spheroid cells compared to monolayer cells, implying a compensatory increase in glycolysis possibly due to hypoxic environment. HIF-1α was expressed only in spheroids and increased in an age-dependent manner, whereas c-Myc (known to induce apoptosis in glucose-deprived cells) levels were three times higher than monolayer cells. Mitochondrial mass and activity decreased significantly during first 14 days of growth but increased with age, and were not associated with increase in ROS levels. Bcl-2 and Bax levels were higher (~2 folds) than monolayers, while the ratio (Bcl/Bax) remained unaltered. Radiation-induced oxidative stress was considerably less in spheroids as compared to monolayers, and corresponded well with increase in radioresistance demonstrated by the clonogenic assay, similar to hypoxia induced radioresistance observed in tumors. Conclusion Development of S-negative cells and reduced endogenous and radiation-induced ROS coupled with higher levels of anti (Bcl2) as well as pro (Bax) apoptotic regulators observed in spheroids suggest the intricate/complex nature of endogenous as well as induced stress resistance that could exist in tumors, which contribute to the treatment resistance.</p

Heterogeneity in the radiosensitizing effects of the DNA ligand hoechst-33342 in human tumor cell lines.

The AT specific minor grove DNA binding ligands bisbenzimidazole derivatives like hoechst-33342 and hoechst-33258 which scavenge free radicals and stabilize macromolecular structure have been shown to afford radioprotection by reducing the induction of DNA damage. However, their ability to inhibit topoisomerases I &amp; II, which play important roles in damage response pathways including DNA repair can enhance radiation damage under certain conditions. Since pool sizes of the topoisomerases differ not only between normal and tumor cells, but also among different tumors, it is anticipated that radiosensitization by hoechst-33342 can vary among tumors. The present studies were, therefore, undertaken to verify this proposition in human glioma (BMG-1 &amp; U-87) and squamous carcinoma (4197 &amp; 4451) cell lines which differ in their biological behavior (ploidy, p53, cyclins, bcl, bax etc).Isotoxic concentrations of hoechst-33342 (IC50 i.e producing 50% cell kill) administered immediately following irradiation resulted in the radiosensitization of all cell lines, with a 4&amp;7 fold increase in the cell death (loss of clonogenic cell survival) in U-87&amp; BMG-1 and a 3 fold increase in 4197 &amp;4451 cells. Growth inhibition and increase in cytogenetic damage (micronuclei formation) as well as delayed apoptosis observed under these conditions corroborated well with the enhanced cell death. The ligand induced a significant cell cycle delay, particularly in the late S and G2 phases of BMG-1, U-87 and 4197 cells, while no significant changes could be observed in 4451 cells. Higher endogenous levels of cyclin B1 found in both the glioma cell lines, was enhanced further by the ligand as compared to the squamous carcinoma cells. These results clearly demonstrate that the radiosensitizing effects of the ligand are indeed heterogeneous among different human tumor cell lines. The radiaosensitization is p53 independent and accompanied by enhanced mitotic death (linked to cytogenetic damage) as well as induction of cyclin B1 mediated apoptosis