MCM5 as a target of BET inhibitors in thyroid cancer cells

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive thyroid cancer subtype, refractory to the current medical treatment. Among various epigenetic anticancer drugs, bromodomain and extra-terminal inhibitors (BETis) are considered to be an appealing novel class of compounds. BETi target the bromodomain and extra-terminal of BET proteins that act as regulators of gene transcription, interacting with histone acetyl groups. The goal of this study is to delineate which pathway underlies the biological effects derived from BET inhibition, in order to find new potential therapeutic targets in ATC. We investigated the effects of BET inhibition on two human anaplastic thyroid cancer-derived cell lines (FRO and SW1736). The treatment with two BETis, 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. Chromatin immunoprecipitation (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 BETis. Furthermore, MCM5 immunohistochemical evaluation in human thyroid tumor tissues demonstrated its overexpression in several papillary thyroid carcinomas and in all ATCs. MCM5 was also overexpressed 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

Exploiting the pathophysiological role of the Polycomb mutational landscape by CRISPR/Cas9 genome editing

Polycomb group of proteins (PcGs) are essential multiprotein complexes that regulate, through chromatin repression and compaction, cell identity and cell-fate transitions ensuring the correct establishment of lineage-specific transcriptional programs. Due to their key roles in cellular homeostasis and proliferation, it is not surprising at all that their deregulation, in terms of expression levels or activity, has been linked to the development and sustainment of several types of human cancers. Aberrations affecting subunits of both Polycomb Repressive Complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2), the two major PcGs complexes, have been reported. EZH2, the catalytic subunit of PRC2 responsible for its methylation activity on lysine 27 of histone H3 (H3K27), is often over-expressed in human cancers. This correlates with global increased H3K27 trimethylation (H3K27me3) levels and with tumor prognosis. Recently, mutations affecting critical residues within EZH2 catalytic SET domain and thus impairing its activity have been described. Interestingly, both hyper-activating and inactivating mutations have been shown to affect EZH2 histone methylation activity. A complex scenario in which EZH2 can act as oncogene or tumor-suppressor depending on the cell-context, is emerging. Up to now very little is known about the biological role of the mutated forms of EZH2 and subsequent alteration of methylation patterns and, therefore, the aim of this thesis is to try to unravel the molecular mechanisms underlying these tumorigenic mutations. I took advantage of mouse embryonic stem cells (mESCs), representing a simple model system where PcGs activity is well characterized, and of the new powerful CRISPR/Cas9 genome-editing tool. At the beginning of this project CRISPR/Cas9 technology had just emerged as a versatile and powerful tool to perform highly efficient genome-editing in a variety of cell-types. I applied this approach to mESC to obtain relevant genetic cellular models to study the role of mutations affecting EZH2 activity. I generated isogenic mESC lines harboring physiological EZH2 Y726D and R685C-inactivating aminoacidic substitution. Moreover, a cellular model for K27M mutation, that affects EZH2 substrate histone H3.3 thus inhibiting its enzymatic activity, was obtained. Ezh2 and Ezh1 knock-out cells combined with homozygous EZH2 Y641N expressing cells allowed me to clarify several aspects regarding EZH1 and EZH2 interplay and cooperation within PRC2 activity, suggesting a context-dependent EZH1-compensative role. My preliminary results demonstrate that the differentiation capabilities of mESCs relies on H3K27me3 deposition whereas PRC2 recruitment to target loci occurs in an H3K27me3-independent manner. I coupled differentiation assays with location analyses (ChIP-qPCR and ChIP-seq), aimed to map chromatin association of Polycomb components and specific deposition of histone modifications, to elucidate the molecular mechanisms by which distinct mutations affect the activity of PRC2

The Dual Role of EPOP and Elongin BC in Controlling Transcriptional Activity

Two studies published in this issue of Molecular Cell (Beringer et\uc2\ua0al., 2016; Liefke et\uc2\ua0al., 2016) characterize the novel interaction of EPOP with Elongin BC in regulating gene transcription at both H3K4me3-broad active and H3K27me3 Polycomb-repressed chromatin domains

The PARP inhibitor PJ34 modifies proliferation, NIS expression and epigenetic marks in thyroid cancer cell lines

Since PARP-1 is supposed to be part of a multimeric repressor of sodium iodide symporter (NIS) expression, in this study the effect of the PARP inhibitor PJ34 on several properties of thyroid cancer cell lines was investigated. In TPC1, BCPAP, FRO, WRO cell lines PJ34 induced a strong increase in NIS mRNA levels. In BCPAP and TPC1 cells also significant increase of radio-iodine uptake was induced. Accordingly, in transfection experiments performed in TPC1 cells, treatment with PJ34 increased NIS promoter activity without affecting PARP-1 binding to the promoter sequence. We also investigated the epigenetic status of NIS promoter after PJ34 treatment in TPC1 cell line: in addition to an increase of histone modification activation marks (H3K9K14ac, H3K4me3), surprisingly we observed also an increase of H3K27me3, a classical repressive mark. Our data demonstrate that in various thyroid cancer cell lines PARP inhibition increases NIS gene expression through a particular modulation of transcriptional regulatory mechanisms. Therefore, we suggest that PARP inhibitors may deserve future investigations as tools for medical treatment of thyroid cancer. (C) 2012 Elsevier Ireland Ltd. All rights reserved

Epigenetic bivalent marking is permissive to the synergy of HDAC and PARP inhibitors on TXNIP expression in breast cancer cells

Studies on stem cell differentiation led to the identification of paused genes, characterized by the contemporary presence of both activator and repressor epigenetic markers (bivalent marking). TXNIP is an oncosuppressor gene the expression of which was reduced in breast cancer. In the present study, we evaluated whether the concept of epigenetic bivalent marking can be applied to TXNIP gene in breast cancer cells. Using chromatin immunoprecipitation (ChIP), three histone modifications were investigated: two associated with transcriptional activation, lysines 9-14 acetylation of H3 histone (H3K9K14ac) and lysine 4 trimethylation of H3 histone (H3K4me3), and one associated with transcriptional silencing, lysine 27 trimethylation of H3 histone (H3K27me3). According to the bivalent marking model, TXNIP gene appears to be paused in MDA157 cells (markers of active and repressed transcription are present), but are definitively silenced in MDA468 cells (presence of only markers of transcription repression). This was proven by evaluating TXNIP mRNA and protein levels after the treatment of cell lines with a histone deacetylase inhibitor (SAHA) and a poly-ADPribose polymerases inhibitor (PJ34). In MDA157 cells, SAHA and PJ34 showed a synergistic effect: a large increment was observed in TXNIP mRNA and protein levels. By contrast, in MDA468 cells, synergy between the two compounds was not observed. Therefore, the pausing epigenetic signature was permissive for synergy between SAHA and PJ34 on TXNIP gene expression. The synergy between SAHA and PJ34 on TXNIP expression was associated with variation in cell viability and apoptosis. In MDA157 cells, but not in MDA468 cells, combined treatment of SAHA and PJ34 induced a decrease in cell viability and an increase of apoptosis. Thus, our data support the hypothesis that TXNIP is an effective target for the treatment of breast cancer

Cyclic AMP-Response Element Modulator Inhibits the Promoter Activity of the Sodium Iodide Symporter Gene in Thyroid Cancer Cells

BACKGROUND: Comprehension of the regulatory mechanism involved in the sodium iodide symporter (NIS) expression is of great relevance for thyroid cancer. In fact, restoration of NIS expression would be a strategy to treat undifferentiated thyroid cancer. Previous in vitro findings suggest that the cyclic AMP-response element (CRE) modulator (CREM) is involved in control of NIS expression. In this work, we examined the expression of CREM in a series of thyroid cancer tissues and its action on NIS promoter in human thyroid cancer cells. METHODS: Expression of mRNA levels for CREM, PAX8 and NIS was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 6 normal thyroid tissues, 22 papillary, 12 follicular and 4 anaplastic thyroid cancers. The effect of CREM on transcriptional activity of the NIS promoter was investigated by transient transfection of human thyroid cell lines. RESULTS: Compared to normal tissues, NIS and PAX8 mRNA levels were significantly reduced in all types of thyroid cancer. As expected, the maximal decrease was detected in anaplastic thyroid cancer. Conversely, CREM mRNA levels were increased in all types of thyroid cancer, reaching statistical significance for follicular and anaplastic thyroid carcinoma (p=0.0157 and 0.0045, respectively). Transfection experiments showed an inhibitory effect of CREM on NIS promoter activity in various thyroid cancer cell lines. CONCLUSIONS: These data demonstrate that CREM expression is increased in thyroid cancer tissue and may play a role in the downregulation of NIS expression in thyroid cancer acting at the transcriptional level

Molecular Differences Between Human Thyroid Follicular Adenoma and Carcinoma Revealed by Analysis of a Murine Model of Thyroid Cancer

Mouse models can provide useful information to understand molecular mechanisms of human tumorigenesis. In this study, the conditional thyroid mutagenesis of Pten and Ras genes in the mouse, which induces very aggressive follicular carcinomas (FTCs), has been used to identify genes differentially expressed among human normal thyroid tissue (NT), follicular adenoma (FA), and FTC. Global gene expression of mouse FTC was compared with that of mouse normal thyroids: 911 genes were found deregulated +/- 2-fold in FTC samples. Then the expression of 45 deregulated genes in mouse tumors was investigated by quantitative RT-PCR in a first cohort of human NT, FA, and FTC (discovery group). Five genes were found significantly down-regulated in FA and FTC compared with NT. However, 17 genes were found differentially expressed between FA and FTC: 5 and 12 genes were overexpressed and underexpressed in FTC vs FA, respectively. Finally, 7 gene products, selected from results obtained in the discovery group, were investigated in a second cohort of human tumors (validation group) by immunohistochemistry. Four proteins showed significant differences between FA and FTC (peroxisomal proliferator-activated receptor-gamma, serum deprivation response protein, osteoglycin, and dipeptidase 1). Altogether our data indicate that the establishment of an enriched panel of molecular biomarkers using data coming from mouse thyroid tumors and validated in human specimens may help to set up a more valid platform to further improve diagnosis and prognosis of thyroid malignancies

Overexpression of genes involved in miRNA biogenesis in medullary thyroid carcinomas with RET mutation

Abnormal expression of non-coding micro RNA (miRNA) has been described in medullary thyroid carcinoma (MTC). Expression of genes encoding factors involved in miRNA biogenesis results often deregulated in human cancer and correlates with aggressive clinical behavior. In this study, expression of four genes involved in miRNA biogenesis (DICER, DROSHA, DCGR8, and XPO5) was investigated in 54 specimens of MTC. Among them, 33 and 13 harbored RET and RAS mutations, respectively. DICER, DGCR8, and XPO5 mRNA levels were significantly overexpressed in MTC harboring RET mutations, in particular, in the presence of RET634 mutation. When MTCs with RET and RAS mutations were compared, only DGCR8 displayed a significant difference, while MTCs with RAS mutations did not show significant differences with respect to non-mutated tumors. We then attempted to correlate expression of miRNA biogenesis genes with tumor aggressiveness. According to the TNM status, MTCs were divided in two groups and compared (N0 M0 vs. N1 and/or M1): for all four genes no significant difference was detected. Cell line experiments, in which expression of a RET mutation is silenced by siRNA, suggest the existence of a causal relationship between RET mutation and overexpression of DICER, DGCR8, and XPO5 genes. These findings demonstrate that RET- but not RAS-driven tumorigenic alterations include abnormalities in the expression of some important genes involved in miRNA biogenesis that could represent new potential markers for targeted therapies in the treatment of RET-mutated MTCs aimed to restore the normal miRNA expression profile