Chromosome Instability Associates with Radiation and Doxorubicin Resistance in Triple Negative Breast Cancer Cell Lines.

Background Triple negative (TN) breast cancers often have a worse prognosis, despite receiving appropriate chemotherapy and radiation treatment. Thus, one of the most pressing issues in breast cancer research is investigating the molecular basis for the inherent resistance of TN cells to standard of care treatment. Chromosome instability strongly associates with chemo-resistance and poor prognosis in breast cancer. We hypothesized that genetic heterogeneity imparted by chromosome instability provides the basis for chemotherapy and radiotherapy resistance in TN cells. Methods Four TN cell lines (HS578, HCC1806, MB231, MB468) and a benign breast tissue line (MCF10) were treated with radiation or doxorubicin. The effects of the treatments were evaluated by the colony forming unit (CFU), MTT survival, Ki-67 immuno-histochemistry (IHC), caspase-3 IHC, micronuclei, and γ-H2AX assays. Results Radiation and Chemotherapy Dose Responses: Table I reveals the data from the CFU and MTT assays after treatment with radiation at 0, 1, 2, 4, 6, 8 Gy or doxorubicin at 0.5, 1, 2 μg/ml, respectively. The analyses revealed a range of sensitivities to radiation and doxorubicin, where HS578 was more sensitive to both treatments.Apoptosis and Proliferation: Because the basis for chemo-and radio-resistance of TN cells may be uncontrolled proliferation frequencies and/or decreased apoptosis, proliferation assays (assessed by Ki-67 IHC), as well as apoptotic indexes (i.e. cleaved caspase 3) were performed. The Ki-67 assay showed that 2 Gy reduced proliferation by 13% in the HS578 line, but by only 2% with the MB231cells.After receiving 2 Gy, there was a wide range of apoptotic activity among the TN cell lines. The largest percentage of apoptosis occurred with the MB468 cells at 19% vs the lowest apoptotic activity seen with the HCC1806 line, 7%. Importantly, apoptotic activity was ≤ 7% when these cell lines were treated with 1 μg/ml of doxorubicin.Chromosome instability: The micronuclei and the γ-H2AX assays showed that 3 of the 4 TN cell lines harbored active genomic instability in the absence of doxorubicin or irradiation. The unstable TN cell lines had 15-30% γ-H2AX activity and 20-40% micronuclei formation, whereas the MCF10 and HCC1806 cells had approximately 11% γ-H2AX activity and 10% micronuclei present. To assess the molecular markers contributing to chromosome instability, we performed a real time PCR screen, as well as Western blot analyses of the cyclins, cyclin-dependent inhibitors and other signaling molecules that may contribute to chromosome instability. All cell lines that had high frequencies of chromosome instability had increased levels of cyclin D1, whereas a subset also de-regulated cyclin A. Conclusion Our results show that radio- and chemo- resistance in TN breast cancer cells closely associates with a de-regulated cell cycle and active chromosome instability. We conclude that the molecular basis for chromosome instability, chemotherapy and radiation resistance may be secondary to the unregulated expression of cyclins D1 and A. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1143.