The expression of REDD-1 mediated mTOR pathway proteins in the human breast cancer

Abstract

Breast cancer was mainly classified into 5 molecular subtypes, namely normal breast-like, luminal A, luminal B, HER2 overexpression, and basal-like phenotypes [triple negative breast cancer (TNBC)] according to gene profiling studies. Among these, HER-2 overexpression and TNBC show high tumor grade, increased proliferation activity and increase in the area of geographic tumor necrosis, representing hypoxic tumor microenvironment. In hypoxic condition, REDD1 (regulated in development and DNA damage response 1)-mediated regulation of the mammalian target of rapamycin (mTOR) pathway is involved in cell proliferation and survival. The purpose of present study was intended to investigate the expression of REDD1-mediated mTOR pathway proteins and hypoxia related proteins such as AMPK and HIF-1α in breast cancer through in vitro cell line and tissue microarray studies. For in vitro cell line studies, 4 types cell lines were selected showing breast cancer phenotype; MDA-MB-231(TNBC), SKBR3 (HER-2), MCF-7 (luminal A) and BT474 (luminal B). Through the western blot analysis and siRNA treatment for REDD1 and AMPK, the expressions of S6K1, phospho-S6KI, AMPK, REDD 1, and HIF-1α were evaluated in which hypoxic condition was made by cobalt chloride (CoCl2) treatment of 100, 200, 300, and 400 μM. Tissue microarray was done from the samples collected from 224 patients with breast cancer, and 30 cases of papilloma as the control group. The immunohistochemistry (IHC) for estrogen receptor (ER), progesterone receptor (PR), HER-2, epithelial growth factor receptor (EGFR), cytokeratin (CK) 5/6, Glut-1, HIF-1α, REDD 1, AMPK α 1, 14-3-3 σ, PTEN, phospho-Akt, phospho-mTOR, phospho-S6, and Ki-67 were performed. Phenotype classification of breast cancer was performed based on the results of IHC for ER, PR, and HER-2: luminal A (ER or/and PR +, HER-2 -), luminal B (ER or/and PR +, HER-2

+), HER-2 overexpression (ER or/and PR -, HER-2 +), and TNBC (ER or/and PR -, HER-2 -). In cell line study, the expression of HIF-1α and REDD 1were induced in all 4 cell lines under hypoxic condition. However, the expression of REDD1 was decreased or lost under severe hypoxic condition. The expression of HIF-1α was increased as hypoxic status was severe in MDA-MB-231. Under hypoxic condition the expression of phospho-S6K1 was higher with inhibition of REDD1 or AMPK by siRNA than without inhibition in MDA-MB-231 and MCF-7. The expression of HIF-1α was induced under hypoxic condition, irrespective of the inhibition of REDD1 or AMPK through siRNA. The human tissue microarray study showed that Glut-1 and HIF-1α were highly expressed in TNBC, HER-2 overexpression type breast cancer, and papilloma than luminal A and B type (p < 0.000). REDD1 expression was higher in papilloma than breast cancer (p < 0.000), but no difference was found among the 4 breast cancer phenotypes (p = 0.307). HIF-1α was significantly associated with poor disease free survival (DFS) and overall survival (OS) in multivariate Cox regression analysis (DFS; p = 0.034, odd ratio: 6.3), OS; p=0.033, odd ratio=5.2).In conclusion, loss of REDD1 expression in TNBC and HER-2 overexpression type play important role to maintain tumor cell proliferation and survival under severe hypoxic condition. HIF-1α would be an important hypoxia related protein especially in high grade breast cancer