PI3K/Akt and Stem Cells in two breast cancer cell lines.

CERLIANI JP; GARGINI R; CALVO JC; LANARI C; IZQUIERDO M

San Diego, California, USA

Congreso; 98th Annual Meeting of the American Association of Cancer Research; 2008

The mammary gland is a dynamic organ that undergoes major developmental changes during pregnancy, lactation and involution. Mammary gland stem cells play a pivotal role in this organ remodeling. It has been speculated that these stem cells may represent the cellular origin of cancer, the cancer stem cells (CSC), because they exist quiescently for long periods of time being able to accumulate multiple mutations over the lifespan of the organism, ultimately giving rise to tumors when stimulated to proliferate. Recently, it has been reported that highly tumorigenic cells with properties consistent with those of CSC can be isolated from human breast cancers. These breast cancer stem cells have a CD 44+/CD24-/low phenotype. Conceivably, CSC may be more resistant to conventional breast cancer therapies, which may ultimately result in recurrence or metastasis even when remarkable initial responses are clinically observed. The aim of this study was to evaluate the CSC characteristics of the two human breast cancer cell lines:  MCF-7, which is estrogen receptor alpha positive, and MDA-MB 231 which is estrogen receptor alpha negative and to evaluate the PI3K/Akt pathway in the self-renewal and stem cells phenotype of both cell lines. We cultured the cells in medium without serum to avoid attachment and to favor the growth of CSC in mammospheres. After a few passages we confirmed the selection of CD 44+/CD24-/low cells by flow cytometry. Then we evaluated the effects of the PI3K inhibitor LY 294002 (LY; 10 µM) and/or the inhibitor of mTOR, rapamicin (Rap; 100 nM) on the CSC phenotype after 1 week of treatment. MCF-7 and MDA-MB 231 cells incubated with the PI3K/Akt inhibitors changed their growth. Cells growing in mammospheres attached now to the plastic and showed an inhibition in cell proliferation (p<0.01) and a differentiated phenotype. In MCF-7 cells this was accompanied by an inhibition in Bmi, Cyclin D1 and b catenin expression. However, only Ly treatment induced an increase in apoptosis evaluated by FACS: control 24.76 ±3.54%, Ly 61.09 ±3.04%, Rap 25.12±8.05%  (p<0.005). In MDA-MB 231 cells total levels of these proteins did not change, however a re-localization of b-catenin to the cell membrane was observed. Next we evaluated the percentage of CD 44+/CD24-/low cells which remained after 7 days of treatment. A significant decrease in the SC phenotype was observed with both inhibitors in both cell lines (p<0.05). However, MDA-MB-231 cells proved to be more sensitive to Rap whereas MCF-7 to LY (p<0.05). These results demonstrate for the first time that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of breast stem cells