Structure-Function Studies of the bHLH Phosphorylation Domain of TWIST1 in Prostate Cancer Cells

Rajendra P. Gajula, Sivarajan T. Chettiar, Russell D. Williams, Katriana Nugent, Yoshinori Kato, Hailun Wang, Reem Malek, Kekoa Taparra, Jessica Cades, Anvesh Annadanam, A. Rum Yoon, Elana Fertig, Beth A. Firulli, Lucia Mazzacurati, Timothy F. Burns, Anthony B. Firulli, Steven S. An, Phuoc T. Tran

Research output: Contribution to journalArticle

Abstract

The TWIST1 gene has diverse roles in development and pathologic diseases such as cancer. TWIST1 is a dimeric basic helix-loop-helix (bHLH) transcription factor existing as TWIST1-TWIST1 or TWIST1-E12/47. TWIST1 partner choice and DNA binding can be influenced during development by phosphorylation of Thr125 and Ser127 of the Thr-Gln-Ser (TQS) motif within the bHLH of TWIST1. The significance of these TWIST1 phosphorylation sites for metastasis is unknown. We created stable isogenic prostate cancer cell lines overexpressing TWIST1 wild-type, phospho-mutants, and tethered versions. We assessed these isogenic lines using assays that mimic stages of cancer metastasis. In vitro assays suggested the phospho-mimetic Twist1-DQD mutation could confer cellular properties associated with pro-metastatic behavior. The hypo-phosphorylation mimic Twist1-AQA mutation displayed reduced pro-metastatic activity compared to wild-type TWIST1 in vitro, suggesting that phosphorylation of the TWIST1 TQS motif was necessary for pro-metastatic functions. In vivo analysis demonstrates that the Twist1-AQA mutation exhibits reduced capacity to contribute to metastasis, whereas the expression of the Twist1-DQD mutation exhibits proficient metastatic potential. Tethered TWIST1-E12 heterodimers phenocopied the Twist1-DQD mutation for many in vitro assays, suggesting that TWIST1 phosphorylation may result in heterodimerization in prostate cancer cells. Lastly, the dual phosphatidylinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor BEZ235 strongly attenuated TWIST1-induced migration that was dependent on the TQS motif. TWIST1 TQS phosphorylation state determines the intensity of TWIST1-induced pro-metastatic ability in prostate cancer cells, which may be partly explained mechanistically by TWIST1 dimeric partner choice.

Original languageEnglish (US)
Pages (from-to)16-31
Number of pages16
JournalNeoplasia
Volume17
Issue number1
DOIs
StatePublished - Jan 1 2015

ASJC Scopus subject areas

  • Cancer Research
  • Medicine(all)

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    Gajula, R. P., Chettiar, S. T., Williams, R. D., Nugent, K., Kato, Y., Wang, H., Malek, R., Taparra, K., Cades, J., Annadanam, A., Yoon, A. R., Fertig, E., Firulli, B. A., Mazzacurati, L., Burns, T. F., Firulli, A. B., An, S. S., & Tran, P. T. (2015). Structure-Function Studies of the bHLH Phosphorylation Domain of TWIST1 in Prostate Cancer Cells. Neoplasia, 17(1), 16-31. https://doi.org/10.1016/j.neo.2014.10.009