Hedgehog Signaling is a Possible Therapeutic Target in Cd24-Negative Breast Cancer Cells

Abstract

ABSTRACT Aim: Breast cancer still continues to be a leading cause of cancer-related death among women worldwide. Consequently, the development of a new effective therapeutic strategies for breast cancer is urgently required. Previously, we have shown that Hedgehog (Hh) signaling is activated in CD24-CD44+ breast cancer cells which are candidates of cancer stem-like cells and it may play an important role in these cells. However, the precise mechanim of Hh signaling activation in CD24-CD44+ breast cancer cells is still unclear. In this study, we focused on the CD24 molecule and investigated the mechanism of Hh signaling activation in CD24-negative breast cancer cells. Methods: Breast cancer cell lines, MCF-7 and HCC-1937 were used in this study. CD24 and Hh ligand; sonic Hh (Shh) were inhibited using small interfering RNA (siRNA). Expressions of CD24 and Hh signaling-related molecules were assessed by real time RT-PCR, flow cytometry and fluorescent immunochemistry. Invasiveness and colony formation were estimated by matrigel invasion assay and soft agar colony formation assay, respectively. Five-week-old NOD/SCID mice were used in xenograft experiment. Gli1 expression was used as an index of Hh signaling activation. Results: 1) CD24 siRNA transfected cells significantly increased invasiveness and colony formation compared with controls. 2) CD24 siRNA transfected cells significantly increased Hh signaling componens; Shh and Gli1 expresions compared with controls. 3) Invaded cells in matrigel invasion assay showed lower CD24 expression and higher Shh expression than non-invaded cells. 4) Shh siRNA and CD24 siRNA concurrent transfected cells significantly decreased Gli1 expression, invasiveness and colony formation compared to CD24 siRNA transfected cells in vitro. 5) Tumor volume and tumorigenesis in Shh siRNA and CD24 siRNA concurrent transfected cells was significantly lower than those in CD24 siRNA transfected cells in the xenograft mice model. 6) In fluorescent immunohistochemical staining using surgically resected specimens from cancer patients, Shh expression was negatively correlated with CD24 expression. Conclusions: Our results suggest that CD24 negative breast cancer cells may induce proliferation and invasiveness through upregulation of Shh. Therefore, Hh signaling may be a suitable therapeutic target in CD24-negative breast cancer. Disclosure: All authors have declared no conflicts of interest.