MCM5 as a target of BET inhibitors in thyroid cancer cells

Abstract

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive thyroid cancer sub-type, refractory to current medical treatment. Among various epigenetic anticancer drugs, BET inhibitors (BETi) are considered an appealing novel class of compounds. BETi target the Bromodomain and Extra-Terminal (BET) proteins that act as regulators of gene transcription, interacting with histone acetyl groups. The goal of this study is to delineate which pathway underlie the biological effects derived from BET inhibition, in order to find new potential therapeutic targets in ATC. We investigated effects of BET inhibition on two human anaplastic thyroid cancer-derived cell lines (FRO and SW1736). The treatment with two BETi, JQ1 and I-BET762, decreased cell viability, reduced cell cycle S-phase and determined cell death. In order to find BETi effectors, FRO and SW1736 were subjected to a global transcriptome analysis after JQ1 treatment. A significant portion of deregulated genes belongs to cell cycle regulators. Among them, MCM5 was decreased at both mRNA and protein levels in both tested cell lines. ChIP experiments indicate that MCM5 is directly bound by the BET protein BRD4. MCM5 silencing reduced cell proliferation, thus underlining its involvement in the block of proliferation induced by BETi. Furthermore, MCM5 immunohistochemical evaluation in human thyroid tumor tissues demonstrated its over-expression in several papillary thyroid carcinomas and in all ATCs. MCM5 was also over-expressed in a murine model of ATC, and JQ1 treatment reduced Mcm5 mRNA expression in two murine ATC cell lines. Thus, MCM5 could represent a new target in the therapeutic approach against ATC.

BET proteins regulate homologous recombination‐mediated DNA repair: BRCAness and implications for cancer therapy

“…Total RNA was extracted, reverse transcribed and qPCRs were performed as previously described . Each sample was run in triplicate and actin was used as control for input RNA; data analysis was based on the ΔΔCt method and experiments were repeated at least three times.…”

“…Nuclear proteins were extracted as in Mio et al . For Western Blot analysis, proteins were electrophoresed and then transferred to nitrocellulose membranes.…”

“…Interestingly, BRD4 is described to be more actively associated with enhancers and promoter regions, regulating RNA polymerase II dependent transcription. BET inhibition is currently being explored as a promising anticancer strategy focusing mainly on three small molecular compounds, JQ1, GSK1210151 and GSK525762 . Recently, it has been shown that BRD4 is able to control RAD51 promoter and that JQ1 treatment contributed to downregulate numerous DNA repair genes .…”

MCMs in Cancer: Prognostic Potential and Mechanisms

“…Also, Widdrol, an odorant compound, can activate DNA damage checkpoint through the signaling Chk2-p53-Cdc25A-p21-MCM4 pathway in HT29 cells [63]. Besides, Genistein, BETi, and Breviscapine were reported to, respectively, downregulate MCM2, MCM5, and MCM7 [61,64]. The second strategy is to inhibit the helicase activity of MCMs.…”

Biological and molecular effects of bromodomain and extra‐terminal (BET) inhibitors JQ1, IBET‐151, and IBET‐762 in OSCC cells

“…Bromodomain-containing protein 4 (BRD4) is a member of bromodomain and extra-terminal (BET) protein family, and its abnormal expression has been described in several human malignant tumors. [8][9][10] In the study of He et al, 8 it has been demonstrated that BRD4 silencing by siRNA suppresses OSCC cells proliferation, migration, and invasiveness. These data point to the possibility of using BRD4 inhibition as a therapeutic strategy in OSCC.…”