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

Background: 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 α-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

Background

A significant number of patients in the U.S with metastatic brain tumors face a dismal prognosis and high mortality. Increasing numbers of breast cancer patients are being diagnosed with brain metastases, possibly as a result of the emergence of targeted and aggressive systemic cancer therapy. In overall frequency, breast cancers and lung cancers are by far the most common cancers that metastasize to brain [1,2]. Brain metastasis generally arises in women diagnosed with aggressive breast cancer or in men with advanced lung cancer. The actual incidence of brain metastases is not precisely known, however studies suggest that 6–16% of patients with metastatic breast cancer develop brain metastases during their lifetime. Furthermore, autopsy studies have reported brain metastases in 18–30% of patients dying ofbreast cancer [3]. The majority of women who develop brain metastases have presented with debilitating neurological symptoms, and have undergone aggressive treatment for stage IV disease [4-6]. Although brain metastasis is the leading cause of breast cancer death, its pathogenesis is poorly understood and the predictors of breast metastasis to brain are yet to be characterized.

Accumulating evidence suggests that human epidermal growth factor receptor 2 (HER2) overexpression and consequent trastuzumab (Herceptin)-based therapy might be associated with a higher rate of brain metastases [7]. Retrospective studies in the U.S. in women with HER2 overexpressing breast cancer receiving trastuzumab-based treatment have indicated that approximately one-third of patients had developed brain metastases [7-9]. Palmeri et al. suggested that HER2 overexpression increases metastatic outgrowth of breast cancer cells in the brain [10], and that HER2 overexpression might be a predictor of asymptomatic, occult brain cancer. Therefore, it is extremely important to study the genetic changes in breast cancer cells that lead to brain metastasis and to develop specific targeted molecular agents. Many genetic aberrations are reported in human breast cancers, including altered splice variants and loss of genomic imprinting. Specifically, altered BKCa channels, which respond to changes in intracellular calcium and membrane potential, are described in a wide variety of tumor cell types. For example, Huang [11] reported that p21ras plays a pivotal role in controlling oncogenic transformation [12], and with its immediate downstream target, Raf kinase, is required for the induction of BKCa channels. Although ubiquitously present in most human organs, the pore forming α-subunit of the BKCa channel is encoded by the KCNMA1 gene, and is significantly unregulated and altered in cancer cells. In human glioma cells, up-regulation and constitutive activation of the KCNMA1 gene and its alternate splice variants are correlated with increased malignancy [13]. Up-regulation of BKCa channels was shown to be a novel mechanism for the malignant phenotype of human tumor cells [14]. However, in osteosarcoma, KCNMA1 was shown to have antitumor property, suggesting that KCNMA1 may have diverse roles in different tumor types [15]

In the present study, our results from global exon array analysis showed higher expression of KCNMA1 in metastatic breast cancers located in brain than in metastatic breast cancers in other organs. This observation prompted us to investigate the potential for invasiveness and brain metastasis of KCNMA1 expression in normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), nonbrain metastatic breast cancer cells (MDA-MB-231), and brain specific metastatic breast cancer cells (MDA-MB361). Our results clearly show that metastatic breast cancer cells exhibit increased BKCa channel activity, leading to greater invasion and transendothelial migration. We showed that the invasion and transendothelial migration of breast cancer cells can be attenuated by blocking BKCa channel activity. Therefore, determining the relative abundance of KCNMA1 in breast cancer metastases to brain and understanding its role in brain metastasis may allow us to target KCNMA1 for its prognostic and possibly therapeutic potential.