Selection of metastatic breast cancer cells based on adaptability of their metabolic state.

A small subpopulation of highly adaptable breast cancer cells within a vastly heterogeneous population drives cancer metastasis. Here we describe a function-based strategy for selecting rare cancer cells that are highly adaptable and drive malignancy. Although cancer cells are dependent on certain nutrients, e.g., glucose and glutamine, we hypothesized that the adaptable cancer cells that drive malignancy must possess an adaptable metabolic state and that such cells could be identified using a robust selection strategy. As expected, more than 99.99% of cells died upon glutamine withdrawal from the aggressive breast cancer cell line SUM149. The rare cells that survived and proliferated without glutamine were highly adaptable, as judged by additional robust adaptability assays involving prolonged cell culture without glucose or serum. We were successful in isolating rare metabolically plastic glutamine-independent (Gln-ind) variants from several aggressive breast cancer cell lines that we tested. The Gln-ind cells overexpressed cyclooxygenase-2, an indicator of tumor aggressiveness, and they were able to adjust their glutaminase level to suit glutamine availability. The Gln-ind cells were anchorage-independent, resistant to chemotherapeutic drugs doxorubicin and paclitaxel, and resistant to a high concentration of a COX-2 inhibitor celecoxib. The number of cells being able to adapt to non-availability of glutamine increased upon prior selection of cells for resistance to chemotherapy drugs or resistance to celecoxib, further supporting a linkage between cellular adaptability and therapeutic resistance. Gln-ind cells showed indications of oxidative stress, and they produced cadherin11 and vimentin, indicators of mesenchymal phenotype. Gln-ind cells were more tumorigenic and more metastatic in nude mice than the parental cell line as judged by incidence and time of occurrence. As we decreased the number of cancer cells in xenografts, lung metastasis and then primary tumor growth was impaired in mice injected with parental cell line, but not in mice injected with Gln-ind cells.

Overexpression of COX-2 in celecoxib-resistant breast cancer cell lines.

BACKGROUND: Cyclooxygenase-2 (COX-2) plays a key role in breast cancer progression and metastasis. Effective therapeutic targeting of COX-2 would require the knowledge of whether a tumor is addicted to COX-2, and if we can counter the potential resistance to anti-COX-2 therapy. Herein we tested the hypothesis that celecoxib-resistance involves selection of cancer cells that overexpress COX-2. MATERIALS AND METHODS: We selected celecoxib-resistant (CER) variants from two metastatic cell lines, SUM149 inflammatory breast cancer (IBC) cell line and MDA-MB-231-BSC60 cell line, by culturing them in the presence of celecoxib. We measured the relative levels of COX-2 protein and its network components Bcl-2, Bcl-xL, and Bax in the parental cell lines and their CER variants by Western blotting. To determine whether celecoxib resistance would increase tumorigenicity, we performed an in vitro clonogenicity assay. We determined the statistical significance of differences between the groups using the two-sample t-test. RESULTS: Both the celecoxib-resistant cell lines SUM149-CER and BSC60-CER produced significantly higher levels of COX-2 protein than their parental counterparts (P < 0.05). The CER variants produced a reduced level of pro-apoptosis protein Bax (both cell lines) and increased levels of anti-apoptosis proteins Bcl-2 (BSC60) or Bcl-xL (SUM149). Importantly, the CER variants had significantly higher clonogenicity than their parental cell lines (P < 0.05). The siRNA-mediated COX-2 knockdown in SUM149-CER cell line resulted in a significant decrease in clonogenicity and in Bcl-xL and Bcl-2 protein levels, thus supporting our hypothesis. CONCLUSION: Celecoxib-resistant variant cells present in breast cancer cell lines overexpress COX-2, which is robustly linked with survival pathways and clonogenicity. Since COX-2 is important in the variant cancer cells of aggressive nature, it represents a good therapeutic target.