Abstract 557: Identification of mitogen-activated protein kinases which are required for the growth of triple-negative breast cancers.

Background: Breast cancer is the most frequently diagnosed malignancy (besides non-melanoma skin cancer) and is the second leading cause for cancer-related death among US women. Most breast cancers overexpress the estrogen receptor (ER) or, to a lesser extent, the HER2 receptor and thus are responsive to hormonal therapies. However nearly a quarter of patients do not express ER, HER2, or progesterone receptor (“triple-negative breast cancer”) and are clinically managed as a single disease with nonspecific chemotherapies. Novel targeted therapies are urgently needed to treat these aggressive cancers. Recent large-scale studies integrating multiple dimensions of genomic data have revealed that alterations to the MAP kinase pathways are critical for the development and progression of triple-negative cancers (TNBCs). In particular, mutations in MAP3K genes have been associated with receptor-negative tumors. In this study, we hypothesized that TNBCs depend on unique MAP3K proteins distinct from those required by non-TNBCs to transduce growth signals. Methods: We performed an unbiased screen of the MAP3K family to identify genes critical for growth and tumorigenicity in six TNBC and six ER-positive cell lines. Pools of siRNA were deposited into optical microplates and cell lines were liposomally reverse-transfected. Depending on the growth rate, cells were stained with DAPI at 4 or 6 days post-transfection and nuclei were detected by high-throughput microscopy. To test for inhibition of anchorage-independent growth, cells were transfected with siRNA and the next day seeded in soft agar in 6-well dishes. Colonies were counted by machine at 1-3 weeks and compared to control siRNA-treated cells. For death assays, siRNA-treated cells were grown for 2 days and collected for western blot detection of annexin V and cleaved caspase 3. Results: We determined that MAP3K3 (MEKK3) and TAO2 (MAP3K17) are essential for anchorage-dependent growth of TNBC cells, but are not required for the proliferation of ER-positive cell lines. Among the TNBC cells, there were variable responses to knockdown of these kinases. While the growth of MDA-MB-231, HCC70, and HCC1937 cells were greatly inhibited after MAP3K3 knockdown, the growth of HCC1937 cells was unaffected by MAP3K3 knockdown but was instead suppressed by TAO2 inhibition. Knockdown of MAP3K3 and TAO2 also resulted in a dramatic reduction of anchorage-independent colony formation in these TNBC cells but not in ER-positive lines. We demonstrated that this reduction in growth is due to suppression of proliferation, and not by induction of apoptosis via annexin V apoptotic assay and detection of cleaved caspases. Conclusion: These data suggest that TNBCs evolve a dependence on specific MAP kinase pathways, and that targeting MAP3K3 and TAO2 may be an effective strategy to treat the subset of TNBCs that rely on these molecules to potentiate growth. Citation Format: Graham M. Poage, Abhijit Mazumdar, Ivan P. Uray, Powel H. Brown. Identification of mitogen-activated protein kinases which are required for the growth of triple-negative breast cancers. [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 557. doi:10.1158/1538-7445.AM2013-557