Abstract Purpose: The Twist1 gene has diverse roles during development and pathologic states such as cancer. Twist1 is a bHLH transcription factor that has both repressor and transactivation functions. Twist1 is known to repress transcription by several mechanisms and is therefore considered to mediate its function mainly through transcriptional repression. The Twist1 transactivation domain has been reported but the functional significance of this domain is still unclear. In cancer, Twist1 is best known for its roles in facilitating tumor progression by inducing an epithelial-mesenchymal transition (EMT) transcriptional program implicated in facilitating tumorigenesis, tumor progression and treatment resistance. We hypothesized Twist1 may mediate these various functions using distinct structural domains and/or motifs. Here we have investigated the role of the Twist box domain of Twist1 in prostate cancer. Methods: We disrupted the putative transactivation domain (TD) of Twist1 by mutating a critical phenylalanine residue (F191) to glycine. We then created stable isogenic prostate cancer cell lines overexpressing wildtype and F191G versions of Twist1. We assessed the role of the Twist box using in vitro and in vivo assays, which mimic the various stages of cancer progression to metastasis. These include loss of homotypic cell-cell contacts, cell migration and invasion, anoikis resistance and soft agar colony formation. We also observed single-cell biophysical traction forces on a fabricated substratum and finally performed experimental lung metastasis assays. Results: The overexpression of Twist1 in prostate cancer cells lead to an EMT biomarker phenotype and the F191G mutant lacked expression of some of these markers. The F191G mutant was deficient for transcriptional activity using promoter reporter based assays. Using single cell measurements we found that Twist1 overexpressing prostate cancer cells exert more force on the substratum than vector control cells. Additional in vitro assays suggest Twist1 can confer cellular properties associated with increased tumor cell aggressiveness including increased migration/invasion, cell death/anoikis resistance and in vitro tumorigenic potential by soft agar colony formation. The Twist box mutant, F191G, displayed compromised activity compared to wildtype Twist1 in many of the in vitro assays described above revealing that the Twist box is necessary for many of the pro-metastatic functions of Twist1. We compared the gene expression profile of Twist1 and F191G overexpressing prostate cancer cells by microarray and observed that the F191G mutant had an expression profile that was similar to wildtype Twist1 but attenuated in key gene sets. Lastly, Twist1 overexpression compared to vector control prostate cancer cells showed an increased frequency of metastatic lung tumors using the experimental lung metastasis assay. Interestingly, Twist1 overexpression also resulted in the appearance of extra-thoracic metastases. The F191G mutant was less able to confer prostate cancer cells the ability to colonize metastatic lesions in the lung and resulted in no extra-thoracic metastases. Conclusions: Our results show that the Twist1-F191G mutant behaves as a loss of function and consequently that the Twist1 box is necessary for Twist1-induced metastasis of prostate cancer cells. Citation Format: Rajendra Gajula, Sivarajan Chettiar, Russell Williams, Saravanan Thiyagarajan, Khaled Aziz, Nishant Gandhi, Aaron Wild, Tarek Salih, Yoshinori Kato, Jessica Cades, Elana Fertig, Christine Chung, Joseph Herman, Russell Hales, Charles Rudin, Steven An, Phuoc T. Tran. The Twist box domain is required for Twist1-induced metastasis of prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B45.