Characterization of Twist1 transcriptional targets in thyroid cancer cells

Abstract

Follicular cell derived thyroid carcinoma comprise a heterogeneous group of cancers with different clinical and morphological characteristics. Anaplastic thyroid carcinomas (ATC) represent less than 5% of all thyroid cancers but are responsible for more than 50% of thyroid cancer mortality, with a mean survival time from diagnosis of ∼3–6 months. ATC is characterized by local invasion, distant metastasis, chemo and radio-resistance. We showed that ~ 50% of ATCs up-regulated Twist1 with respect to normal thyroids as well as to poorly and well-differentiated thyroid carcinomas. Twist1 is a highly conserved basic helix-loop-helix transcription factor that plays an important role in the development and progression of human cancer. Knockdown of Twist1 by RNA interference in ATC cells reduced cell migration and invasion and increased sensitivity to apoptosis. The ectopic expression of Twist1 in papillary thyroid carcinoma cells (TPC) induced resistance to apoptosis and increased cell migration and invasion. In this dissertation, we have investigated the molecular mechanisms by which Twist1 exerts its biological effect in thyroid cancer cells. To this aim, we analyzed gene expression profiles of thyroid cancer cells ectopically expressing Twist1 in comparison to vector control cells. According to gene expression profile, the top functions enriched in TPC-Twist1 cells were: cellular movement, cellular growth and proliferation, cell death and survival. Silencing of the top ten up-regulated genes reduced viability of TPC-Twist1 and CAL62 cells. Silencing of COL1A1, KRT7, PDZK1 induced also cell death. Silencing of HS6ST2, THRB, ID4, RHOB, and PDZK1IP, also impaired migration and invasion of TPC-Twist1 cells. Chromatin immunoprecipitation showed that Twist1 directly binds the promoter of the ten up-regulated genes. q-RT-PCR on thyroid cancer samples showed that HS6ST2, COL1A1, F2RL1, LEPREL1, PDZK1 and PDZK1IP1 are over-expressed in thyroid carcinoma samples compared to normal thyroids. More recently we have conducted a miRNome expression profile of TPC-Twist1 cells in comparison to control. We identified a set of miRNA potentially regulated by Twist1. We then focused on miR-584 up-regulated by Twist1 in TPC cells. We showed that miR-584 is up-regulated in ATCs respect to normal thyroids and papillary thyroid carcinomas. The ectopic expression of miR-584 protected TPC cells from apoptosis induced by Doxorubicin and Staurosporine. Finally, we showed that miR-584 targets TUSC2 (tumor suppressor candidate 2). Thus, we have identified a set of mRNA and miRNA that mediates Twist1 biological effects in thyroid cancer cells. Transcription factors as Twist1 are generally believed to be difficult to target due to nuclear localization and lack of a specific groove for tight binding of a small molecule inhibitor. Thus, downstream targets of Twist1 could be more realistic for therapeutic intervention.