Genomics The Twist Box Domain Is Required for Twist 1-induced Prostate Cancer Metastasis

Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial–mesenchymal transition (EMT) that promotes cancer metastasis. Structure–function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer. Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in prostate cancer cells using in vitro assays, which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extrathoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for prostate cancer cells to colonize metastatic lung lesions and extrathoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in prostate cancer cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and prostate cancer metastasis. Implications:Targeting the Twist box domain of Twist1may effectively limit prostate cancer metastatic potential. Mol Cancer Res; 11(11); 1387–400. 2013 AACR. Introduction Prostate cancer is the most common cancer diagnosed in men in the United States and is responsible for the second most cancer deaths in men (1). Patterns of disease failure in prostate cancer suggest understanding the determinants that confer progression of localized presentations to metastatic disease will result in the largest therapeutic gains (2). One mechanism by which cancer cells may acquire the characteristics necessary for metastasis is the epithelial–mesenchymal transition (EMT). EMT is a transcriptional program, crucial in early embryonic development that is coopted by some cancer cells to facilitate aggressive and metastatic behavior (3). Twist1 is a basic helix-loop-helix (bHLH) multidomain transcription factor which directly mediates EMT by transcriptional activation and repression of E-box-regulated target genes (4, 5). A role for TWIST in prostate cancer pathogenesis has been suggested (6, 7), but the role of EMT and Twist1 in prostate cancer disease Authors' Affiliations: Departments of Radiation Oncology andMolecular Radiation Sciences, and Oncology; Division of Biostatistics and Bioinformatics, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center; Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science; Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health; Departments of Urology and In Vivo Cellular and Molecular Imaging Center, Johns Hopkins University School of Medicine; Physical Sciences in Oncology Center, Johns Hopkins University, Baltimore, Maryland; and; Division of Hematology-Oncology, Department of Medicine, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). R.P. Gajula and S.T. Chettiar contributed equally to this work. CorrespondingAuthor:PhuocT. Tran, Department of RadiationOncology & Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins Hospital, 1550 Orleans Street, CRB2 Rm 406, Baltimore, MD 21231. Phone: 410-614-3880; Fax: 410-502-1419; E-mail: tranp@jhmi.edu doi: 10.1158/1541-7786.MCR-13-0218-T 2013 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals. org 1387 on June 19, 2017. © 2013 American Association for Cancer Research. mcr.aacrjournals. org Downloaded from Published OnlineFirst August 27, 2013; DOI: 10.1158/1541-7786.MCR-13-0218-T