Smoothened activates breast cancer stem-like cell and promotes tumorigenesis and metastasis of breast cancer

Abstract

Smoothened (Smo) is a G protein-coupled receptor protein encoded by the Smo gene of the hedgehog signalling pathway, which is thought to play an important role in maintaining organ patterning, cell differentiation and self-renewal. The possible role of Smo in the process of tumorigenesis and metastasis of breast cancer still remains unclear. The present experiments were to investigate the effect of Smo on activating breast cancer stem-like CD44⁺CD24⁻ cells and the tumorigenesis and metastasis of breast cancer. By injected CD44⁺CD24⁻ cells (1 × 10⁴) into the cleared fat pad of NOD/SCID mice, it was observed that CD44⁺CD24⁻ cells possess higher tumor-initiating capacity and metastasis properties than equal numbers of non-CD44⁺CD24⁻ cells. The mRNA and protein expressions of Smo in CD44⁺CD24⁻ cells were higher than those in non-CD44⁺CD24⁻ cells, indicating that Smo may play a role in maintaining breast cancer stem cell features. qRT–PCR results revealed that expressions of STAT3, Bcl-2 and cyclinD1 mRNA in MCF-7 cells were decreased after transfected by Smo siRNA. In addition, the expressions of MMP-2 and MMP-9 were downregulated in MCF-7 cells after Smo expression was inhibited. Smo inhibition may be a possible therapeutic target that potentially suppresses breast tumor formation and development.

Introduction

Breast carcinoma is the leading female malignancy and the leading cause of death in women all over the world [1]. Although the early detection and treatments has significantly reduced the breast cancer mortality over the past decades, the incidence rate of breast cancer was increasing over the period in China. The breast cancer stem-like cells as CD44⁺CD24^−/low cells can initiate tumor formation when a few hundreds cells were injected into the mammary fat pad of NOD/SCID mice [2]. These cells possess the characteristics of cancer stem cells (CSCs), such as the potential of self-renewal and multilineage differentiation. The mechanisms whereby the neoplastic cells invade and destroy tissues are poorly understood, but emerging evidence suggests that these cells are responsible for breast cancer initiation, proliferation, drug resistance and relapses [3], [4].

The Hedgehog (Hh) signalling pathway plays a crucial role in vertebrate embryogenesis by controlling cell fate, proliferation, and differentiation by sequentially activating downstream target genes. Hh signal transduction is initiated by the binding of the processed and lipid modified Hh ligand to its receptor Patched (Ptch), a 12-pass transmembrane protein. In the absence of the Hh protein, Ptch1 represses signal transduction by inhibiting the seven transmembrane domain containing protein, Smoothened (Smo) [5]. Upon Hh binding, the inhibitory function of Ptch1 on Smo is abolished, resulting in Smo activation and thereby sequential activating downstream target genes. The activation of Hh signalling has been identified in a variety of human tumors, including tumors of brain, lung, pancreatic, digestive tract, liver, ovarian and skin [6], [7], [8], [9], [10], [11], [12]. Moreover, many investigations have showed that self-renewal and proliferation of cancer stem cells are associated with the activity of the Hh pathway [13], [14], [15].

It has been reported that the expression of constitutively activated Smo is related to the incidence of many kinds of carcinomas [16]. In human breast cancer, the expression of Smo is altered in clinical samples of human breast cancers, as well as in breast cancer cell lines [17], [18], [19], activated Smo leads to increased proliferation, altered differentiation, and ductal dysplasias, while cyclopamine, an antagonists of Smo, can inhibit breast cancer growth in vitro [20]. In addition, the metastatic tumors in some advanced basal cell carcinoma patients who were treated with Smo antagonist were inhibited [21], implicating that Hh signalling may play a role in facilitating the maintenance and movement of tumor cells. These results suggest that stimulation of Hh signalling in the mammary gland may be sufficient to induce breast cancer, but that additional molecular mechanisms are required to be explored.

In this study, we were to investigate the role of Smo in maintaining breast cancer stem-like cell and explore the potential molecular mechanism of Smo in breast cancer cell proliferation and invasion.