Inhibition of SALL4 suppresses epithelial-to-mesenchymal transition and tumorigenicity of breast cancer cells by targeting β-catenin and vimentin

Abstract

Breast cancer is the second most common malignancy behind lung cancer with more than 1.3 million cases and 450,000 deaths each year worldwide. Although the survival rates for breast cancer have been improved strikingly over the decades, treatments of 'triple-negative' breast cancer (TNBC) which has a higher metastatic potential and poorer prognosis than other types of breast cancer remain challenging. SALL4 plays an important role in early mammalian development. Homozygous loss-of-function mutants are embryonic lethal. It has been reported recently that SALL4 is de-regulated and aberrantly expressed in various cancers, affecting multiple cellular processes including tumorigenesis, tumor growth and metastasis. Several studies have also discovered that SALL4 is overexpressed in breast cancer and contributes to breast cancer progression, but the molecular mechanism remains unknown. Therefore, we performed multiple experiments and demonstrated for the first time that SALL4 facilitated tumorigenicity and metastasis of 'triple-negative' breast cancer cell MDA-MB-231 by direct upregulation of vimentin and indirect activation of Wnt/β-catenin signaling pathway. Here, tissue microarray was conducted to explore the correlation between SALL4 and common clinical features of breast cancer. It was found that SALL4 was associated with higher grade and higher lymph node status of breast cancer. Knockout of SALL4, using CRISPR-Cas9 system, in 'triple-negative' breast cancer cell MDA-MB-231 was performed. It was demonstrated that decrease of SALL4 led to impaired ability in cell proliferation, migration, sphere formation as well as reduced tumor burden and lung metastasis in nude mice. On the other hand, SALL4 gain-of-function study demonstrated that upregulation of SALL4 enhanced cell migration and sphere formation in vitro and increased tumorigenicity in vivo. The mechanism by which SALL4 affected epithelial-to-mesenchymal transition (EMT) and tumorigenicity was investigated. It was found that there was a positive correlation between SALL4 and EMT markers including ZEB1, vimentin, Slug, and Snail. The Chromatin immunoprecipitation (CHIP) experiment indicated that SALL4 enhanced vimentin expression by directly binding to the promoter region (-778 to -550 bp). SALL4 also activated Wnt/β-catenin signaling pathway to enhance cell migration and tumorigenesis through inhibition of GSK-3β kinase activity. Knockout of SALL4 led to reduced GSK-3β (ser9), indicating more active GSK-3β to phosphorylate β-catenin, Snail and Slug for proteasomal degradation. In summary, SALL4 is overexpressed in breast cancer and positively correlated with histological tumor grade and lymph node status of breast cancer patients. Functional studies have demonstrated that inhibition of SALL4 suppressed breast cancer cell proliferation, migration, sphere formation in vitro, tumorigenicity and metastasis in vivo through direct inhibition of vimentin and indirect inactivation of Wnt/β-catenin pathway, probably explaining the clinical relevance of SALL4 and breast cancer.