Overactivation of AKT1 stimulates steroid hormone receptors promoting hormone-independent growth of IBH-6 and IBH-7 human breast cancer cell lines.

RIGGIO MARINA; POLO MARIA LAURA; BLAUSTEIN MATIAS; COLMAN-LERNER ALEJANDRO; LÜTHY ISABEL; LANARI CLAUDIA; NOVARO VIRGINIA

San Francisco

Congreso; AACR, Annual Meeting; 2011

In previous studies using a mouse mammary tumor model induced by medroxyprogesterone acetate (MPA) that transits through different stages of hormone dependence, we have reported that the activation of the PI3K/AKT pathway is critical for the growth of hormone-independent (HI) mammary tumors, but not for the growth of hormone-dependent (HD) tumors. Furthermore, primary cell cultures from HD tumors infected to express a constitutively active form of AKT1, myristoylated AKT1 (myrAKT1), were able to grow in vivo in the absence of MPA, acquiring a HI phenotype. These tumors were highly differentiated and displayed a ductal phenotype with a cytokeratin 8 and laminin-1 pattern expression typical of the HI counterpart. Ligand-independent phosphorylation of progesterone receptor (PR) in Ser 190 was increased in myrAKT1 tumors, supporting our hypothesis that overactivation of AKT1 cross talks with PR inducing hormone-independent tumor growth.             In the present study we analyzed the effect of AKT1 overactivation in two human breast cancer cell lines, IBH-6 and IBH-7. These cell lines, developed from two different primary invasive ductal carcinomas, express steroid hormone receptors and grow in immuno-compromised mice giving rise to invasive tumors. Thus, they are excellent models to study the regulatory mechanisms underlying breast cancer growth. In vivo IBH-6 cells grow independently of the administration of 17-b-estradiol (E2), and IBH-7 cells grow only in E2-treated mice with a 30% of incidence. Both cell lines were stably transfected with myrAKT1 to up-regulate PI3K/AKT pathway, or with the empty vector Acl4 (control) and inoculated subcutaneously (sc) into nu/nu female mice. In vivo, the growth rate of IBH-6-myrAKT1 cells was higher than that of IBH-6-Acl4 cells (p<0.001).  No morphological differences were found between IBH-6-myrAKT1 and control IBH-6-Acl4 cells, with a predominant spindle-shaped morphology. When control IBH-7-Acl4 cells were inoculated into untreated nude mice no tumor growth was observed. Surprisingly, IBH-7-myrAKT1 cells were able to induce E2-independent tumor growth in 70% of the animals. When we evaluated the effect of AKT1 overactivation on IBH-7 cells, we found that IBH-7-myrAKT1 cells in relation to IBH-7-Acl4 cells displayed higher levels of ERa and an increase in phosphorylation of PR in Ser 294. Furthermore, IBH-7-myrAKT1 cells exhibited a rounded shape morphology whereas control IBH-7-Acl4 cells or naïve IBH-7 cells show a spindle cell morphology. They also showed an increased expression of focal adhesion kinase (FAK) and E-cadherin. Moreover, in some IBH-7-myrAKT1 cell clusters, p-FAK showed a rudimentary polarization pattern. In conclusion, these results indicate that overactivation of AKT1 favors tumor growth by increasing steroid hormone receptors and modifying cellular morphology. Depending on the cell context, or other activated signaling pathways, the effects of activated AKT1 may differ between models, but finally they all collaborate inducing hormone independent tumor growth.