The Analysis Of Estrogen Receptor-Alpha Positive Breast Cancer Stem-Like Cells Unveils A High Expression Of The Serpin Proteinase Inhibitor Pi-9: Possible Regulatory Mechanisms

Breast cancer stem cells seem to play important roles in breast tumor recurrence and endocrine therapy resistance, although the underlying mechanisms have not been well established. Moreover, in some tumor systems the immunosurveillance failure against cancer cells has been related to the presence of the granzyme B inhibitor PI-9. This study explored the status of PI-9 in tumorspheres isolated from estrogen receptor-alpha positive (ER alpha(+)) breast cancer MCF7 cells. Studies were performed in tertiary tumorspheres which possess high levels of sternness markers (Nanog, Oct3/4 and Sox2) and self renewal ability. The exposure to estrogens (17-beta estradiol and genistein) increased the number and sizes of tumorspheres, promoting cell proliferation as demonstrated by the increase in the proliferating cell nuclear antigen (PCNA). The study of the three isoforms (66, 46 and 36 kDa) of ER alpha disclosed that tertiary tumorspheres exhibit a marked increase in ER alpha 36, while the level of ER alpha 66, which is highly expressed in MCF7 cells, declines. Although it is known that PI-9 is a transcriptional target of ER alpha 66, surprisingly in tertiary tumorspheres, despite the reduced level of ER alpha 66, the protein and mRNA content of PI-9 is higher than in MCF7 cells. Treatment with estrogens further increased PI-9 level while decreased that of ER alpha 66 isoform thus excluding the involvement of this receptor isoform in the event. Moreover, our studies also provided evidence that tertiary tumorspheres express elevated levels of CXCR4 and phospho-p38, suggesting that the high PI-9 content might be ascribed to the activation of the proliferative CXCR4/phospho-p38 axis. Taken together, these events could supply a selective advantage to breast cancer stem cells by interfering with immunosurveillance systems and open up the avenue to new possible targets for breast cancer treatment.