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The distal-less homeobox gene 4 (DLX4) is a member of the DLX family of homeobox genes. Although absent from most normal adult tissues, DLX4 is widely expressed in leukemia, lung, breast, ovarian and prostate cancers. However the molecular targets, mechanisms and pathways that mediate the role of DLX4 in tumor metastasis are poorly understood. In this study, we found that DLX4 induces cancer cells to undergo epithelial to mesenchymal transition (EMT) through TWIST. Overexpression of DLX4 increased expression of TWIST expression in cancer cell lines, resulting in increased migratory and invasive capacity. Likewise, knocking down expression of DLX4 decreased TWIST expression and the migration ability of cancer cell lines. DLX4 bound to regulatory regions of the TWIST gene. Both western blotting and immunohistochemistry staining showed that the expression of DLX4 and TWIST are correlated in most of breast tumors. Taken together, these data from both cell models and tumor tissues demonstrate that DLX4 not only upregulates TWIST expression but also induces EMT and tumor metastasis. Altogether, we propose a new pathway in which DLX4 drives expression of TWIST to promote EMT, cancer migration, invasion and metastasis.

Loss of estrogen receptor α (ERα) expression and gain of TWIST (TWIST1) expression in breast tumors correlate with increased disease recurrence and metastasis and poor disease-free survival. However, the molecular and functional regulatory relationship between TWIST and ERα are unclear. In this study, we found TWIST was associated with a chromatin region in intron 7 of the human ESR1 gene coding for ERα. This association of TWIST efficiently recruited the nucleosome remodeling and deacetylase (NuRD) repressor complex to this region, which subsequently decreased histone H3K9 acetylation, increased histone H3K9 methylation and repressed ESR1 expression in breast cancer cells. In agreement with these molecular events, TWIST expression was inversely correlated with ERα expression in both breast cancer cell lines and human breast ductal carcinomas. Forced expression of TWIST in TWIST-negative and ERα-positive breast cancer cells such as T47D and MCF-7 cells reduced ERα expression, while knockdown of TWIST in TWIST-positive and ERα-negative breast cancer cells such as MDA-MB-435 and 4T1 cells increased ERα expression. Furthermore, inhibition of histone deacetylase (HDAC) activity including the one in NuRD complex significantly increased ERα expression in MDA-MB-435 and 4T1 cells. HDAC inhibition together with TWIST knockdown did not further increase ERα expression in 4T1 and MDA-MB-435 cells. These results demonstrate that TWIST/NuRD represses ERα expression in breast cancer cells. Therefore, TWIST may serve as a potential molecular target for converting ERα-negative breast cancers to ERα-positive breast cancers, allowing these cancers to restore their sensitivity to endocrine therapy with selective ERα antagonists such as tamoxifen and raloxifene.