Abstract 5116: The calcium channel subunit CACNG4 plays a role in breast cancer metastasis.
Authors: Kanwar Nisha , Nair Ranju , Wang Dong-Yu , Done Susan J. 
Published: August 2014

Introduction: Previously, we used aCGH to analyze genomic alterations in primary breast tumors with and without lymph node (LN) metastasis. A significant correlation was found between gain of chromosome 17q24 and LN metastasis. Selected genes including L-type calcium channel voltage dependent gamma subunit 4 (CACNG4) were further validated for increased protein expression in invasive breast tumors. CACNG4 is involved in calcium channel regulation in excitable cells of muscles and the brain. It is the only gamma subunit that also shows expression in non-excitable cells, such as glial and intestinal epithelial cells, suggestive of additional functions. It is over-expressed in glioblastoma multiforme cell lines resistant to EGFR inhibitors. Breast cancer gene expression datasets (NKI and IPC) identified an association of CACNG4 expression with LN metastasis and ER positivity. In the present study, we characterized CACNG4 in breast cancer with particular attention to its metastasis-related ability.

Methodology: RNA interference was used for knockdown studies in cell lines followed by cell migration, invasion, aggregation and adhesion assays. Intracellular calcium influx in response to calcium channel agonists and antagonists was measured using the Fluo-4 calcium indicator dye.

Results: CACNG4 showed variable expression across a panel of breast cancer cell lines. MCF10A (a non malignant cell line) does not express CACNG4, while it is highly expressed in ER positive cell lines (MCF7, MDA-MB-361, CAMA1) and lower in EGFR high cell lines (MDA-MB-468 and MDA-MB-231). CACNG4 knockdown inhibited cell migration and invasion in MCF7 and MDA-MB-231 cells. Conversely, cell adhesion and cell-cell aggregation was increased. RT-PCR revealed an inverse relation between CACNG4 expression and PKC-zeta specifically, which is involved in tight junction formation and shows low expression in breast, prostate and renal cancers. Thus, CACNG4 may play a role in de-adhesion and cell dissemination during metastasis. Blocking calcium channels with amlopidine had an anti-proliferative affect. Serum stimulation of EGFR high cell lines resulted in calcium influx, that was blocked in a dosage dependent manner with amlodipine, and synergistically blocked further with the EGFR specific tyrosine kinase inhibitor tryphostin AG1478. Knockdown of CACNG4 resulted in increased calcium influx suggesting a role in keeping the channel closed. Cells also showed resistance to amlodipine, requiring higher doses to block calcium influx.

Conclusions: L-type channels are active in non-excitable breast cancer cells, and are affected by EGFR activity. CACNG4 maintains these channels in a closed state, modulating calcium influx, and inhibiting PKC-zeta expression which may result in metastatic function. The ability of calcium channel blockers to target channels in a CACNG4 dependent manner suggests novel treatment possibilities for invasive breast cancer.

Citation Format: Nisha Kanwar, Ranju Nair, Dong-Yu Wang, Susan J. Done. The calcium channel subunit CACNG4 plays a role in breast cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5116. doi:10.1158/1538-7445.AM2013-5116