Possible regulatory mechanisms responsible for the high expression of serpin protease inhibitor PI-9 in ER+ -derived breast cancer stem cells.

Breast cancer (BC) is the most common endocrine cancer and the second leading cause of cancer-related death in women. About 75% of BCs expresses high levels of estrogen receptors that sustain the tumor growth. Moreover, in BC estrogens prevent apoptosis induced by granzyme B released by cytotoxic T lymphocytes and NK cells through the production of the granzyme B inhibitor PI-9. As a consequence, cancer cells acquire the ability to escape immune surveillance’s signaling. Although some studies explored the role of PI-9 in BC cells, its presence has not been investigated in cancer stem cells so far. In this research, tertiary tumorspheres were obtained from estrogen receptor-alfa positive (ERalfa+) BC MCF7 cells and studies were performed to evaluate their stem identity. These tumorspheres showed high levels of stemness 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 as well as the level of the proliferating cell nuclear antigen (PCNA). The analysis of the three isoforms (66, 46 and 36 kDa) of ERalfa disclosed that tertiary tumorspheres exhibit a marked increase in ERalfa36, while the level of ERalfa66, which is highly expressed in MCF7 cells, dropped. Then, we analyzed the granzyme B inhibitor PI-9, which is transcriptionally regulated by ERalfa66. Surprisingly, we found that tertiary tumorspheres express a higher level of both PI-9 protein and mRNA than MCF7 cells, despite the reduced level of ERalfa66. The high content of PI-9 might be ascribed to the activation of proliferative CXCR4/phospho-p38 axis which was observed only in tertiary tumorspheres. Taken together, these events could supply a selective advantage to BC stem cells by interfering with immune surveillance systems and open the way to new possible targets for BC treatment.