A Novel Small Molecule WJ460 Inhibits Breast Cancer Metastasis By Targeting Myoferlin

Breast cancer represents the most common form of cancer in women. With improvements in early detection and effective treatment over the past decades, the death rates for breast cancer appear to be declining. However, tens of thousands of women still die from this disease each year. Breast cancer that has metastasized to other sites bears the worst prognosis, as breast cancer patients die directly or indirectly from tumor metastasis. Traditional breast cancer treatment includes surgery, radiation therapy, and systemic therapy. Surgery, in combination with radiation therapy, eliminates the majority of primary tumors. Systemic therapy (chemotherapy, hormone therapy and targeted therapy) administrated before or after surgery represses primary tumor growth and shrinks metastatic tumors. All these therapies, however, are aimed at tumor growth. Development of new therapies targeting metastasis cascade itself thus might provide a new strategy to improve the clinical management of metastatic disease and an opportunity to reduce the incidence of tumor metastasis.In 1999, Dawn Belt Davis and his colleagues identified and characterized a new ferlin family member that mediates muscular dystrophy and they named the new protein Myoferlin (MYOF). In recent years, the important role of MYOF in breast cancer is being increasingly recognized. Several groups of researchers have described the selective overexpression of MYOF in invasive breast carcinoma specimens. In parallel, studies have revealed the function of MYOF on breast cancer invasion and epithelial to mesenchymal transition (EMT), suggesting MYOF may act as a modifier of breast cancer metastasis. These findings led to the hypothesis that MYOF is a breast cancer related protein and highlighted myoferlin as a potential target for anti-metastasis therapy.The clinical setting for breast cancer treatment prompted us to screen our internal small molecule library. Using in vitro migration and invasion assay, we discovered 11 small molecules that possess anti-metastasis property. Next, we conducted classical Matrigel invasion assay to further assess the inhibitory effect of our candidate compounds on breast cancer metastasis, and we identified a small molecular compound, WJ460, with potent anti-breast cancer metastasis propensity. We employed spontaneous breast cancer metastasis mouse modelto evaluate whether WJ460 could inhibit breast cancer in vivo. Our data indicated that WJ460 markedly impaired breast cancer spontaneous metastasis to distant organs.Mechanistically, we used biotin as a probe to capture the target protein(s), and we successfully identified the target of WJ460 (MYOF). In addition, the results of our endogeneous and exogeneous affinity pull-down assay also reinforced this conclusion. In accordance with these results, immunofluorescence analysis revealed the colocalization between MYOF and biotin-WJ460.Different deletion vectors were constructed to identify the region(s) which is (are) responsible for the binding to WJ460. Pull-down assay deminstrated that construct containg C2D domain can bind directly to WJ460. Subsequently, we evaluated whether WJ460 could participate in the biological processes that mediated by MYOF.Addition of WJ460 markedly decreased the expression of MMP1 at the transcriptional and transcription level.Previous studies indicated MYOF is closely related to epithelial-mesenchymal transition. The results of 3D culture, immunofluorescence, real-time PCR and western blot assay revealed WJ460 could reverse epithelial-mesenchymal transition, leading to the decreased metastasis ability. In parallel, MYOF has been proved to envolve in receptor tyrosine kinase (RTKs) stability and RTKs are deemed essential mediators in breast cancer metastasis. Therefore, we examined the RTKs activity (phosphorylation form) in breast cancer cells after WJ460 treatment. The phosphorylation formof many RTKswas suppressed by WJ460. Finally, similar results were obtained when knocking down of MYOFand WJ460administration in breast cancer experimental pulmonary metastasis mouse model. Futhermore, MYOF silence and WJ460 treatment inhibited breast cancer cell extravasation and colonization.Taken together, our findings suggest that WJ460 can serve as the first lead compound for developing MYOF -targeted agents and provide the rationale for evaluation of WJ460 as anti-breast cancer therapy.