Abstract P6-05-25: Ratio of notch receptors is critical for response to notch inhibition by a gamma-secretase inhibitor in triple negative breast cancer cells

Abstract

Abstract Background: Women with triple-negative breast cancer have the worst prognosis, and have few targeted therapy options. Notch receptor genes are potent breast oncogenes, overexpressed in triple-negative breast cancer, and critical for survival of breast cancer stem cells. However, the contribution of each Notch receptor and the significance of the ratio of receptors to growth and sensitivity to Notch inhibitors have not yet been fully explored. We hypothesized that each Notch receptor has distinct roles in the etiology of triple negative breast cancer and designed a study to explore these roles in a panel of three triple negative cell lines and primary, human triple negative breast cancer tissue. Methods: Human cell lines BT-549, MDA-MB-231, and MDA-MB-468 were used to measure endogenous mRNA transcript levels of Notch ligands, receptors, and gene targets important for canonical Notch signaling (i.e. Hes-1, Hes-5, Hey-1, and Deltex-1), apoptosis (Noxa), inflammation (MMP-9 and IL-8), and cancer stem cells (ALDH1) by means of real time PCR under conditions where all four receptors were inhibited by a pan-inhibitor, gamma-secretase inhibitor or each receptor was individually knocked down using RNA interference. Furthermore, both anchorage-dependent and –independent growth were measured by counting cells and methylcellulose colony forming assay, respectively. In addition, the RNA from formalin-fixed, paraffin-embedded specimen from women-diagnosed with TNBC who had undergone breast surgery was also analyzed for comparable gene expression for similar targets following Laser capture micro-dissection. Results: The results showed that only MDA-MB-468 cells were sensitive to growth inhibition by a GSI while MDA-MB-231 and BT549 were resistant. Transcripts of Notch-4, Deltex-1, Hes-1 and Hes-5, as well as IL-8 and MMP-9 were significantly increased in MDA-MB-468 as compared to BT549 or MDA-MB-231 cells that were resistant. Interestingly, the cancer stem marker, ALDH1 was significantly increased only in MDA-MB-468 while the apoptotic marker, Noxa was decreased compared to resistant cells: BT549 and MDA-MB-231. The PCR and growth results from the Notch-1 knocked down were similar to GSI inhibition. However, knocked down of Notch-2 resulted in resistance to GSI. Notch-3 knocked down showed an increase in Notch-1, Notch-2, and Notch-4 transcripts implying that Notch-3 might suppress the other Notch receptors. Notch-4 knocked down alone had little effect on growth of any of the three cell lines. The results from human specimens showed an inverse relationship between Notch-1 and Notch-2, and Notch-1 and Notch-4. For example, Notch-1, Jagged-1, and Hes-5 genes were up-regulated in triple negative tumor tissue (n = 20), while the Notch-4 gene was down-regulated as compared to normal control specimens from reduction mammoplasty (n = 4). Conclusions: The results from this study indicate that Notch-1 is probably the growth driver in certain triple negative cancer types.. Furthermore, the ratios of Notch receptors and their activity states could predict sensitivity to Notch inhibition by a GSI or other inhibitors. Lastly, these results suggest that the best predictor genes for Notch activation are: Notch-1, Hes-1, and ALDH1. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-25.