Essential role of the cancer stem/progenitor cell marker nucleostemin for indole-3-carbinol anti-proliferative responsiveness in human breast cancer cells

Background: Nucleostemin is a GTPase residing in the nucleolus that is considered to be an important cancer stem/progenitor cell marker protein due to its high expression levels in breast cancer stem cells and its role in tumor initiation of human mammary tumor cells. It has been proposed that nucleostemin may represent a valuable therapeutic target for breast cancer; however, to date evidence supporting the cellular mechanism has not been elucidated. Results: Expression of exogenous HER2, a member of the EGF receptor gene family, in the human MCF-10AT preneoplastic mammary epithelial cell line, formed a new breast cancer cell line, 10AT-Her2, which is highly enriched in cells with stem/progenitor cell-like character. 10AT-Her2 cells display a CD44(+)/CD24(-/low) phenotype with high levels of the cancer stem/progenitor cell marker proteins nucleostemin, and active aldehyde dehydrogenase-1 (ALDH-1). The overall expression pattern of HER2 protein and the stem/progenitor cell marker proteins in the 10AT-Her2 cell population is similar to that of the luminal HER2+ SKBR3 human breast cancer cell line, whereas both MCF-7 and MDA-MB-231 cells display reduced levels of nucleostemin and no detectable expression of ALDH-1. Importantly, in contrast to the other well-established human breast cancer cell lines, 10AT-Her2 cells efficiently form tumorspheres in suspension cultures and initiate tumor xenograft formation in athymic mice at low cell numbers. Furthermore, 10AT-Her2 cells are highly sensitive to the anti-proliferative apoptotic effects of indole-3-carbinol (I3C), a natural anti-cancer indole carbinol from cruciferous vegetables of the Brassica genus such as broccoli and cabbage. I3C promotes the interaction of nucleostemin with MDM2 (murine double mutant 2), an inhibitor of the p53 tumor suppressor, and disrupts the MDM2 interaction with p53. I3C also induced nucleostemin to sequester MDM2 in a nucleolus compartment, thereby freeing p53 to mediate its apoptotic activity. Small interfering RNA knockdown of nucleostemin functionally documented that nucleostemin is required for I3C to trigger its cellular anti-proliferative responses, inhibit tumorsphere formation, and disrupt MDM2-p53 protein-protein interactions. Furthermore, expression of an I3C-resistant form of elastase, the only known target protein for I3C, prevented I3C anti-proliferative responses in cells and in tumor xenografts in vivo, as well as disrupting the I3C-stimulated nucleostemin-MDM2 interactions. Conclusions: Our results provide the first evidence that a natural anti-cancer compound mediates its cellular and in vivo tumor anti-proliferative responses by selectively stimulating cellular interactions of the stem/progenitor cell marker nucleostemin with MDM2, which frees p53 to trigger its apoptotic response. Furthermore, our study provides a new mechanistic template that can potentially be exploited for the development of therapeutic strategies targeted at cancer stem/progenitor cells.