CURRENT SCIENTIFIC INTERESTS
Since many years, my scientific interest is focused on the role of UHRF1 (alias Np95 or ICBP90) and its close structural homologue UHRF2, in DNA methylation and in the epigenetic regulation of onco-suppressors during tumour progression. The SRA (Set and RING-finger-associated domain) domain containing protein UHRF1 is master epigenetic regulator and transcriptional repressor essential to maintain global and local DNA methylation and required for the epithelial to mesenchimal transition (EMT) in tumours,
During the last 5 years, my research group has shown that UHRF1 is deeply implicated in the control of epigenetic modifications during DNA replication and in EMT in prostate and colon cancers. Our current scientific hypothesis is that UHRF1 and UHRF2 are implicated in (at least) prostate and colon cancer pathogenesis and are critical players within the complexes that act at the interplay between DNA replication, chromatin remodelling and transcription to regulate epigenetic modifications and transcriptional silencing of tumour suppressors and other genes during prostate and colon tumour progression.
Three main lines of research are carried out in my laboratory of General Pathology at the University of Insubria in collaboration with three laboratories.
1) DNA hypo/hypermethylation in cancer: decrypt the epigenetic basis of the paradox
2) The involvement of UHRF1 and UHRF2 in prostate and colon cancer pathogenesis
3) The role of UHRF1 and UHRF2 in the epigenetic control of tumour suppressor genes during tumour progression and epithelial to mesenchymal transition
1) DNA hypo/hypermethylation in cancer: decrypt the epigenetic basis of the paradox
An early global DNA hypo- and a later site-specific hyper-methylation are hallmarks of the tumorigenic process as they often co-exist during progression, but answers to this apparent paradox have not been found yet. As the vast majority of the genome is composed of non-coding regions and up to 38% of the human genome consists of Alu and LINE-1 methylated repetitive sequences, DNA hypomethylation affects mainly the genomic regions that contain these non-coding and transposable elements, although demethylation of oncogenes is also observed. Demethylation of these sequences is a source of strong chromosomal instability and an important cause of insertional mutagenesis of genes critical for preventing (tumour-suppressors, both coding and non-coding) or driving malignant transformation (oncogenes, both coding and non-coding). DNA hypermethylation, instead, mainly affects the promoter regions of tumour suppressor genes, which leads to unchecked proliferation, reduced response to apoptotic stimuli and metastasis. The co-existence of DNA hypo- and hyper-methylation in tumours implies that factors required for DNA methylation are still active in transformed cells and that DNA demethylation cannot be simply caused by loss of DNMTs activity. This is exemplified by the finding that UHRF1, a required component of the methylating complexes, and DNMTs are overexpressed in many tumours in which genome-wide hypo- and hyper-methylation of tumour-suppressors genes coexist.
DNA methylation patterns are better correlated with histone modification patterns rather than with the underlying genome sequence context in all genomic regions, including repetitive sequences, suggesting that these two types of epigenetic modifications are tightly co-regulated. Whether DNA methylation drives histone methylation or vice-versa has not been definitely established, although binding of UHRF1 to H3K9me3 is required for DNA methylation. UHRF1 (ubiquitin-like with PHD and ring finger domains 1) is a member of a subfamily of RING-finger type E3 ubiquitin ligases required for DNA methylation, as it controls methylation of virtually all CpG sequences in the genome by recruiting all three DNMTs to sites of methylation. Altogether, genome-wide methylation sites almost totally coincide with UHRF1/DNMTs binding sites. Interestingly, UHRF1/DNMTs are found overexpressed in many tumours where hypo- and hyper-methylation co-exist.
We assume that genes that can be methylated might have a common epigenetic signature that must be red by UHRF1 in order to recruit the methylating complexes. We hypothesise that, following DNA demethylation occurred for whatever reason, UHRF1 and the complexes required for DNA methylation are no longer capable of binding demethylated DNA because of an epigenetic switch. This switch would generate a chromatin configuration that impedes UHRF1 to recruit the methylating complexes to these sites, i.e. ‘non-competent’ chromatin. The methylating enzymes driven by UHRF1 should now be available to relocate to other chromosomal regions that have a ‘competent’ epigenetic configuration and unmethylated DNA, thereby promoting UHRF1-mediated DNA methylation at these sites.
Our plan is to gain a ‘photograph’ of the stable end point epigenetic configuration of the binding properties of UHRF1 and DNMTs by performing ChIP-seq, BS-ChIP-seq and high-resolution mass spectrometry analysis of histones, in combination with stable isotope labeling with amino acids in cell culture (SILAC), to quantitatively track with an unbiased approach the differences of histone post translational modifications, possibly identifying new epigenetic markers involved in the binding. We will study the epigenetic events that dynamically occur to allow UHRF1 binding to the chromatin during the epithelial to mesenchymal transition (EMT). We expect to unveil potential epigenetic signatures at genome level that define ‘competent’ and ‘non-competent’ chromatin configurations for the binding of UHRF1/DNMTs methylating complexes, that will de facto identify positive and negative epigenetic configurations for DNA methylation.
To verify whether a potential ‘competent’ and ‘non-competent’ chromatin for DNA methylation does indeed exist in vivo, we will investigate if the epigenetic configurations identified in the in vitro experiments are also present in human tissues. ChIP from paraffin-embedded tissues (Pat-ChIP) from a well-characterized tumour series previously published will be used to compare samples of normal colorectal mucosa with specimens of adenomas and of carcinomas of colon-rectum.
2) The involvement of UHRF1 and UHRF2 in prostate and colon cancer pathogenesis
In collaboration with with Dr. Carlo Catapano and Dr. Giuseppina Carbone of the Laboratory of Experimental Oncology of the Institute of Oncological Research in Bellinzona (CH), we have studied the pathological role of UHRF1 in prostate cancer. By integrating genomic data and functional assays in normal and prostate cancer cells, we have examined the expression profile of UHRF1 and found that it is over-expressed in prostate tumours and its expression is strictly correlated with the epigenetic effector EZH2. Analysis of a large cohort of tissue microarrays indicated that UHRF1 staining was absent in benign and increased in clinically localized prostate cancer where it was significantly associated with elevated Gleason score (≥8) and poor prognosis.
By immunohistochemistry (IHC) analysis, we discovered that UHRF1 expression was elevated in prostate cancer cell lines more aggressive and androgen independent. UHRF1 knockdown in prostate cancer cells (PC3) reduced proliferation, clonogenic capability, and anchorage independent growth and resulted in re-expression of several tumour suppressor genes frequently repressed and significantly inversely correlated to UHRF1 in prostate tumours. UHRF1 deregulation was associated with clinical outcome in prostate cancer patients. Follow-up data for 203 patients treated with radical prostatectomy and for which UHRF1 protein level had been assessed by IHC in the TMAs, where tested for clinical outcome. We found that patients with highest positivity for UHRF1 had significantly reduced overall survival (Log Rank (Mantel-Cox) p-value=0.07) compared to the patients with low or negative staining. Thus, high levels of UHRF1 might signal tumours more prone to progression and with a negative impact on patient survival. These data may have important clinical implications since UHRF1 may help to identify a subgroup of prostate cancer patients with poor prognosis.
Similar results have been obtained in colon cancer models. By IHC, we have found that UHRF1 expression was high in 60% of the samples of a cohort of 110 primary colorectal tumors, that generally displayed a less differentiated phenotype and a subverted histologic structure. High UHRF1 expression is associated with poorly differentiated and/or undifferentiated CRC (high grade, G3/G4 tumors).
3) The role of UHRF1 and UHRF2 in the epigenetic control of tumour suppressor genes during tumour progression
Analysis of a gene expression data set from human normal prostate and organ-confined prostate cancer samples and in prostate and colon cancer cells, showed that UHRF1 is a key epigenetic regulator and promotes epigenetic cross-talks at the promoters of tumour suppressor genes and is implicated in prostate and colon cancer progression:
a) UHRF1 is required for the methylation of DNA and H3K9me3 on the promoters of at least three oncosuppressors (CDH1, RARb and Nkx3.1) in prostate cancer cells
b) UHRF1 is required for the silencing of the PPARg in colon cancer cells RKO and recruits Suv39H1 to determine methylation of H3K9 at the promoter that tumour suppressor.
c) UHRF1 is required for the recruitment of Dnmt3a to the promoter of CDH1 in prostate cancer cells
d) UHRF1 and EZH2 are co-overexpressed in prostate and in colon cancer cell lines and in tumours of patients
e) UHRF1 is among the transcripts with statistically significant increase in tumours compared to normal prostate, with about 50% of tumour samples having elevated UHRF1 mRNA compared to normal prostate and with very high expression in about 20% of the cases.
f) UHRF1 also inversely correlates with a large number of many known tumour suppressor genes. An inverse correlation was also observed for prostate-specific genes, like microseminoprotein (PSP94), prostatic acid phosphatase (PAcP/ACPP), secretoglobin proteins (SCGB-A1, -1D1 and -1D2), acireductone dioxygenase 1 (ADI1) and human prostate-specific transglutaminase (TGM4).
PRINCIPAL COLLABORATIONS
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Kirsten EDEPLI, New York University Abu Dhabi Saadiyat Campus P.O. Box 129188 Abu Dhabi, United Arab Emirates
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Prof. Vittorio COLANTUONI, D.S.B.A. - Università degli Studi del Sannio, Benevento
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Dr. Carlo CATAPANO e Dr.ssa Giuseppina CARBONE, Laboratory of Experimental Oncology – Istituto Oncologico della Svizzera Italiana, Bellinzona, Svizzera.
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Dr. David MOLLEVI , Translational Research Laboratory, Institut Català d'Oncologia-ICO, IDIBELL. Barcelona, Spain
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Dr. Tiziana BONALDI, IFOM-IEO Campus – Milan - Italy
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Prof. Enrico AVVEDIMENTO, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli
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Dr. Magdalena GÖTZ, Helmholtz Zentrum Munich Institute of Stem Cell Research - Ludwig-Maximilians-Universität München (LMU); Munich, Germany.
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Dr. Denti Michela, ‘Centre for Integrative Biology’, Università di Trento
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Dr. Fabio SPADA and Prof. Heinrich LEONHARDT, Ludwig-Maximilians-Universität München (LMU); Munich, Germany.
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Dr. Françoise DANTZER, Departement Intégrité du génome UMR 7175 – ESBS. Illkirch Strasbourg. France
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Dr. Laurent BRINO and Prof. Oudet Transfected Cell Array platform, CEGBS-IGBMC, Strsbourg France
