Abstract LB-189: A role of Notch2 in quiescence of mammary stem/progenitor cells

Notch signaling plays an important role in normal tissue development through its role in cell-fate determination, survival and proliferation. There are four mammalian homologues, Notch1-4. It has been suggested that in the normal human breast, Notch signaling has a role in self-renewal of mammary stem cells and/or expansion of luminal progenitor cells, however the roles of individual Notch receptors are not clear. Activation of the Notch pathway has been associated with several human cancers, including breast cancer. Transgenic animals overexpressing Notch1, 3, or 4 in the mammary gland develop tumors. Notch 2 was part of gene expression signature for cancer stem cells from breast cancer cell lines, but its role in breast development and breast cancer is poorly understood. In order to investigate the role of Notch2 in functional assays we performed shRNA knockdown in primary mammary epithelial cells and assessed the effect on mammosphere formation. We found an increase in primary, secondary and tertiary sphere formation. Similar results were seen after silencing Notch2 in breast cancer cell lines, in standard cultivation systems. We investigated the expression of Notch2 in the normal mammary stem/progenitor cell population by immunohistochemical staining of normal breast sections. We detected an overlap between high levels of Notch2 expression and the stem/progenitor cell marker ALDH1A1. Furthermore, we detected a strong correlation between ALDH1A1 positive cells and nuclear localization of Notch downstream target Hes1, indicating that Notch signaling is active in these cells. As Hes1 has been implicated in stem cell quiescence, we performed double stainings for Hes1, ALDH1A1 and markers associated with cell proliferation (MCM2, Ki67) and cell quiescence (p21, p27). We found that ALDH1A1 positive cells were predominantly resting or quiescent. We also detected co-expression of Hes1 and the cell cycle protein p27, which controls cell cycle arrest in G1. Similar results were seen in a breast tumor xenografted in immunodeficient mice where tumor cells with high Notch2 expression were negative for the proliferation marker MCM2 and positive for p27. Taken together, our data indicate that Notch2 has a role in keeping stem/progenitor cells in a quiescent state in the normal breast epithelium and possibly in a subset of breast cancers. This is in contrast to the other Notch receptors, which seem to enhance cell proliferation. These results imply that response of breast cancers to treatment with Notch inhibitors (currently in clinical trials) may be determined by the relative expression and activity of the four Notch receptors. Targeting individual Notch receptors may represent a more tailored and more efficacious intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-189. doi:1538-7445.AM2012-LB-189