A Potential Role of CACNA1A, a Voltage-Gated Calcium Channel, in Chemotherapy Resistance in Triple Negative Breast Cancer
Triple negative breast cancer (TNBC), characterized by the lack of hormone receptors and HER2, is the most aggressive form of breast cancer. Chemotherapy is used to treat TNBC, as no targeted therapies currently exist, but resistance to chemotherapeutic agents is a major obstacle against effective treatment. Our lab obtained and sequenced twelve TNBC tumor samples from patients treated with neoadjuvant chemotherapy. Five patient samples possessed mutations in the calcium voltage-gated channel subunit alpha1 A (CACNA1A) gene, suggesting the frequent mutation of CACNA1A may confer chemotherapy resistance. In addition, a previous study showed that CACNA1A is under expressed in breast tumors. Collectively, these suggested that CACNA1A functions as a tumor suppressor. Based on these previous observations, the hypothesis of this study was that CACNA1A deficiencies resulted in chemotherapy resistance in TNBC. In order to test the hypothesis, two TNBC cell lines, MDA-mb-231 and MDA-mb-468 were used. First, the cell lines were established to delete CACNA1A by using the lentiviral delivery system of two independent CRISPR guide RNA constructs, named CAC-1 and CAC-3. Western blots were performed to verify CACNA1A protein expression in each cell line. The cell lines were tested for chemotherapy response by using drug dose inhibition assays and determining the half maximal inhibitory concentration (IC50) of the chemotherapeutic agents doxorubicin (DOXO) or paclitaxel (PTX) of each cell line. Western blot analysis showed that CACNA1A expression was reduced in CAC-1 and CAC-3 cells by 0.5-fold and 0.2-fold, respectively, compared to the vector control V2, indicating that CACNA1A was partially deleted in each cell line. The results of the drug assays showed that the IC50 value of DOXO in MDA-mb-231 vector control was 84nM, whereas CAC-1 and CAC-3 had IC50 values of 120nM and 135nM, respectively. These results demonstrated CACNA1A deleted cells were 1.4 to 1.6-fold higher in the IC50 values, indicating CACNA1A deleted cells were more resistant to DOXO. The IC50 value of PTX in MDA-mb-231 vector control was 62pM, while CAC-1 and CAC-3 had IC50 values of 65pM and 53pM, respectively, indicating that PTX sensitivity was not affected by CACNA1A deletion. Taken together, CACNA1A deficiency resulted in increased resistance to doxorubicin, while paclitaxel sensitivity was not affected by CACNA1A deficiency in TNBC cells. These results suggest that CACNA1A may play a role in response to doxorubicin chemotherapeutic drug, proposing the CACNA1A pathway as a potential target for therapy resistant TNBC. Drug response assays need to be repeated in MBD-mb-468 cell lines. Additionally, we observed that CACNA1A deleted cells grew faster than the vector control, suggesting that CACNA1A deficiency may increase cell proliferation, a phenotype related to aggressive cancer.
