Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Development of a Bioassay for Continuous Monitoring of TWIST1 Activity

Version 1 : Received: 5 March 2021 / Approved: 9 March 2021 / Online: 9 March 2021 (11:41:34 CET)

How to cite: Czapiński, J.; Kałafut, J.; Przybyszewska-Podstawka, A.; Pawlicka, M.; Roszkowska, A.; Kiełbus, M.; Borkiewicz, L.; Dudziak, K.; Czerwonka, A.; Stepulak, A.; Rivero-Muller, A. Development of a Bioassay for Continuous Monitoring of TWIST1 Activity. Preprints 2021, 2021030265 (doi: 10.20944/preprints202103.0265.v1). Czapiński, J.; Kałafut, J.; Przybyszewska-Podstawka, A.; Pawlicka, M.; Roszkowska, A.; Kiełbus, M.; Borkiewicz, L.; Dudziak, K.; Czerwonka, A.; Stepulak, A.; Rivero-Muller, A. Development of a Bioassay for Continuous Monitoring of TWIST1 Activity. Preprints 2021, 2021030265 (doi: 10.20944/preprints202103.0265.v1).

Abstract

TWIST1 is a transcription factor that affects cell behavior during development and cell differentiation. Yet, it is better known for its roles in neoplasia through regulation of cell plasticity. The pathological contributions of TWIST1 in tumor initiation, angiogenesis, invasion, metastasis, and chemo-resistance have been the focus of much research. To-date, the only way to quantitatively measure the abundance of TWIST is by immunoblots. Yet, no bioassay exists that can detect TWIST1 activity. Thus, we present here a TWIST1 cell-based assay that allows measuring the amount of active TWIST1 non-invasively in living cells. The bioassay was characterized against previously described TWIST1 “inhibitors”, as well as by epigenetic modulators of TWIST1 gene expression. Moreover, we tested multiple cell lines, showing that the level of TWIST1 mRNA resembles that of the bioassay. We show that prostate cancer cells (PC3) undergoing EMT, migrate out of 3D-spheroids and have increased TWIST1 activity. This fast and reliable system to detect active TWIST1 in different biological conditions allows a detailed analysis of this factor, as well as it can be used for drug discovery, since TWIST1 is a potential target for cancer chemotherapeutics.

Keywords

TWIST1, reporter system, Gaussia Luciferase, EMT, cell-based assay

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