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- Histone methyltransferase PRMT6 plays an oncogenic role of in prostate cancerPublication . Almeida-Rios, Diogo; Pinho dos Santos Graça, Maria Inês; Quintela Vieira, Ana Filipa; Ramalho-Carvalho, João; Pereira-Silva, Eva; Martins, Ana Teresa; Oliveira, Jorge; Gonçalves, Céline S.; Costa, Bruno M.; Henrique, Rui; Jerónimo, CarmenProstate cancer (PCa) is a major cause of morbidity and mortality. Until now the specific role of histone methyltransferases (HMTs) deregulated expression/activity in PCa is poorly understood. Herein we aimed to uncover the potential oncogenic role of PRMT6 in prostate carcinogenesis. PRMT6 overexpression was confirmed in PCa, at transcript and protein level. Stable PRMT6 knockdown in PC-3 cells attenuated malignant phenotype, increasing apoptosis and decreasing cell viability, migration and invasion. PRMT6 silencing was associated with decreased H3R2me2a levels and increased MLL and SMYD3 expression. PRMT6 silencing increased p21, p27 and CD44 and decreased MMP-9 expression and was associated with PI3K/AKT/mTOR downregulation and increased AR signaling pathway. In Sh-PRMT6 cells, AR restored expression might re-sensitized cells to androgen deprivation therapy, impacting in clinical management of castration-resistant PCa (CRPC). PRMT6 plays an oncogenic role in PCa and predicts for more clinically aggressive disease, constituting a potential target for patients with CRPC.
- Anti-tumoral effect of the non-nucleoside DNMT inhibitor RG108 in human prostate cancer cellsPublication . Pinho dos Santos Graça, Maria Inês; Sousa, Elsa Joana; Baptista, Tiago; Almeida, Mafalda; Ramalho-Carvalho, João; Palmeira, Carlos; Henrique, Rui; Jerónimo, CarmenCurrent therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-β2, by quantitative methylation-specific PCR and RT-PCR, respectively. Results: RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-β2 promoter hypermethylation levels, although mRNA re-expression was only attained GSTP1 and APC. RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy.
- Phenotypic impact of deregulated expression of class I histone deacetylases in urothelial cell carcinoma of the bladderPublication . Neto, Susana; Quintela Vieira, Ana Filipa; Montezuma, Diana; Costa, Natália R.; Antunes, Luís; Baptista, Tiago; Oliveira, Ana Isabel; Pinho dos Santos Graça, Maria Inês; Rodrigues, Ângelo; Magalhães, José S.; Oliveira, Jorge; Henrique, Rui; Jerónimo, CarmenDeregulated expression of histone deacetylases (HDACs) has been implicated in tumorigenesis. Herein, we investigated class I HDACs expression in bladder urothelial cell carcinoma (BUCC), its prognostic value and biological significance. Significantly increased transcript levels of all HDACs were found in BUCC compared to 20 normal mucosas, and these were higher in lower grade and stage tumors. Increased HDAC3 levels were associated with improved patient survival. SiRNA experiments showed decrease cell viability and motility, and increased apoptosis. We concluded that class I HDACs play an important role in bladder carcinogenesis through deregulation of proliferation, migration and apoptosis, constituting putative therapeutic targets.
- SMYD3 contributes to a more aggressive phenotype of prostate cancer and targets Cyclin D2 through H4K20me3Publication . Quintela Vieira, Ana Filipa; Costa-Pinheiro, Pedro; Almeida-Rios, Diogo; Pinho dos Santos Graça, Maria Inês; Reis, Sara; Simões-Sousa, Susana; Carneiro, Isa; Sousa, Elsa Joana; Godinho, Maria Inês; Baltazar, Fatima; Henrique, Rui; Jeronimo, CarmenProstate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.
- Anti-neoplastic properties of hydralazine in prostate cancerPublication . Pinho dos Santos Graça, Maria Inês; Sousa, Elsa Joana; Costa-Pinheiro, Pedro; Quintela Vieira, Ana Filipa; Torres-Ferreira, Jorge; Martins, Maria Gabriela; Henrique, Rui; Jeronimo, CarmenProstate cancer (PCa) is a major cause of cancer-related morbidity and mortality worldwide. Although early disease is often efficiently managed therapeutically, available options for advanced disease are mostly ineffective. Aberrant DNA methylation associated with gene-silencing of cancer-related genes is a common feature of PCa. Therefore, DNA methylation inhibitors might constitute an attractive alternative therapy. Herein, we evaluated the anti-cancer properties of hydralazine, a non-nucleoside DNA methyltransferases (DNMT) inhibitor, in PCa cell lines. In vitro assays showed that hydralazine exposure led to a significant dose and time dependent growth inhibition, increased apoptotic rate and decreased invasiveness. Furthermore, it also induced cell cycle arrest and DNA damage. These phenotypic effects were particularly prominent in DU145 cells. Following hydralazine exposure, decreased levels of DNMT1, DNMT3a and DNMT3b mRNA and DNMT1 protein were depicted. Moreover, a significant decrease in GSTP1, BCL2 and CCND2 promoter methylation levels, with concomitant transcript re-expression, was also observed. Interestingly, hydralazine restored androgen receptor expression, with upregulation of its target p21 in DU145 cell line. Protein array analysis suggested that blockage of EGF receptor signaling pathway is likely to be the main mechanism of hydralazine action in DU145 cells. Our data demonstrate that hydralazine attenuated the malignant phenotype of PCa cells, and might constitute a useful therapeutic tool.
- Downregulation of miR-130b~301b cluster is mediated by aberrant promoter methylation and impairs cellular senescence in prostate cancerPublication . Ramalho-Carvalho, João; Pinho dos Santos Graça, Maria Inês; Gomez, Antonio; Oliveira, Jorge; Henrique, Rui; Esteller, Manel; Jerónimo, CarmenNumerous DNA-damaging cellular stresses, including oncogene activation and DNA-damage response (DDR), may lead to cellular senescence. Previous observations linked microRNA deregulation with altered senescent patterns, prompting us to investigate whether epigenetic repression of microRNAs expression might disrupt senescence in prostate cancer (PCa) cells. Differential methylation mapping in prostate tissues was carried using Infinium HumanMethylation450 BeadChip. After validation of methylation and expression analyses in a larger series of prostate tissues, the functional role of the cluster miR-130b~301b was explored using in vitro studies testing cell viability, apoptosis, invasion and DNA damage in prostate cancer cell lines. Western blot and RT-qPCR were performed to support those observations. We found that the miR-130b~301b cluster directs epigenetic activation of cell cycle inhibitors required for DDR activation, thus stimulating the senescence-associated secretory phenotype (SASP). Furthermore, overexpression of miR-130b~301b cluster markedly reduced the malignant phenotype of PCa cells. Altogether, these data demonstrate that miR-130b~301b cluster overexpression might effectively induce PCa cell growth arrest through epigenetic regulation of proliferation-blocking genes and activation of cellular senescence.
- Epigenetic disruption of miR-130a promotes prostate cancer by targeting SEC23B and DEPDC1Publication . Ramalho-Carvalho, João; Martins, João Barbosa; Cekaite, Lina; Sveen, Anita; Torres-Ferreira, Jorge; Pinho dos Santos Graça, Maria Inês; Costa-Pinheiro, Pedro; Eilertsen, Ina Andrassy; Antunes, Luís; Oliveira, Jorge; Lothe, Ragnhild A.; Henrique, Rui; Jerónimo, CarmenMicroRNAs (miRNAs) are small, non-coding RNAs that mediate post-transcriptional gene silencing, fine tuning gene expression. In an initial screen, miRNAs were found to be globally down-regulated in prostate cancer (PCa) cell lines and primary tumors. Exposure of PCa cell lines to a demethylating agent, 5-Aza-CdR resulted in an increase in the expression levels of miRNAs in general. Using stringent filtering criteria miR-130a was identified as the most promising candidate and selected for validation analyses in our patient series. Down-regulation of miR-130a was associated with promoter hypermethylation. MiR-130a methylation levels discriminated PCa from non-malignant tissues (AUC=0.956), and urine samples revealed high specificity for non-invasive detection of patients with PCa (AUC=0.89). Additionally, repressive histone marks were also found in the promoter of miR-130a. Over-expression of miR-130a in PCa cells reduced cell viability and invasion capability, and increased apoptosis. Putative targets of miR-130a were assessed by microarray expression profiling and DEPD1C and SEC23B were selected for validation. Silencing of both genes resembled the effect of over-expressing miR-130a in PCa cells. Our data indicate that miR-130a is an epigenetically regulated miRNA involved in regulation of key molecular and phenotypic features of prostate carcinogenesis, acting as a tumor suppressor miRNA.
- Epigenetic modulators as therapeutic targets in prostate cancerPublication . Pinho dos Santos Graça, Maria Inês; Pereira-Silva, Eva; Henrique, Rui; Packham, Graham; Crabb, Simon J.; Jerónimo, CarmenProstate cancer is one of the most common non-cutaneous malignancies among men worldwide. Epigenetic aberrations, including changes in DNA methylation patterns and/or histone modifications, are key drivers of prostate carcinogenesis. These epigenetic defects might be due to deregulated function and/or expression of the epigenetic machinery, affecting the expression of several important genes. Remarkably, epigenetic modifications are reversible and numerous compounds that target the epigenetic enzymes and regulatory proteins were reported to be effective in cancer growth control. In fact, some of these drugs are already being tested in clinical trials. This review discusses the most important epigenetic alterations in prostate cancer, highlighting the role of epigenetic modulating compounds in pre-clinical and clinical trials as potential therapeutic agents for prostate cancer management.
- Regulation of histone H2A.Z expression is mediated by sirtuin 1 in prostate cancerPublication . Baptista, Tiago; Pinho dos Santos Graça, Maria Inês; Sousa, Elsa Joana; Oliveira, Ana Isabel; Costa, Natália R.; Costa-Pinheiro, Pedro; Amado, Francisco; Henrique, Rui; Jerónimo, CarmenHistone variants seem to play a major role in gene expression regulation. In prostate cancer, H2A.Z and its acetylated form are implicated in oncogenes’ upregulation. SIRT1, which may act either as tumor suppressor or oncogene, reduces H2A.Z levels in cardiomyocytes, via proteasome-mediated degradation, and this mechanism might be impaired in prostate cancer cells due to sirtuin 1 downregulation. Thus, we aimed to characterize the mechanisms underlying H2A.Z and SIRT1 deregulation in prostate carcinogenesis and how they interact. We found that H2AFZ and SIRT1 were up- and downregulated, respectively, at transcript level in primary prostate cancer and high-grade prostatic intraepithelial neoplasia compared to normal prostatic tissues. Induced SIRT1 overexpression in prostate cancer cell lines resulted in almost complete absence of H2A.Z. Inhibition of mTOR had a modest effect on H2A.Z levels, but proteasome inhibition prevented the marked reduction of H2A.Z due to sirtuin 1 overexpression. Prostate cancer cells exposed to epigenetic modifying drugs trichostatin A, alone or combined with 5-aza-2’-deoxycytidine, increased H2AFZ transcript, although with a concomitant decrease in protein levels. Conversely, SIRT1 transcript and protein levels increased after exposure. ChIP revealed an increase of activation marks within the TSS region for both genes. Remarkably, inhibition of sirtuin 1 with nicotinamide, increased H2A.Z levels, whereas activation of sirtuin 1 by resveratrol led to an abrupt decrease in H2A.Z. Finally, protein-ligation assay showed that exposure to epigenetic modifying drugs fostered the interaction between sirtuin 1 and H2A.Z. We concluded that sirtuin 1 and H2A.Z deregulation in prostate cancer are reciprocally related. Epigenetic mechanisms, mostly histone post-translational modifications, are likely involved and impair sirtuin 1-mediated downregulation of H2A.Z via proteasome-mediated degradation. Epigenetic modifying drugs in conjunction with enzymatic modulators are able to restore the normal functions of sirtuin 1 and might constitute relevant tools for targeted therapy of prostate cancer patients
- Histone posttranslational modifications and chromatin remodelers in prostate cancerPublication . Quintela Vieira, Ana Filipa; Almeida-Rios, Diogo; Pinho dos Santos Graça, Maria Inês; Henrique, Rui; Jerónimo, CarmenProstate cancer (PCa) is one of the most incident and prevalent cancers in men worldwide and a leading cause of cancer-related morbidity and mortality. Epigenetics plays an important role in prostate carcinogenesis, namely abnormal expression of histone modifier enzymes and related chromatin modifications. Notwithstanding, the limited knowledge of the specific role of deregulated activity/expression of histone modifiers and respective histone modifications hinders their use in clinical management, particularly for PCa detection and prognostication. Due to the limitations in the analysis of global patterns of histone modifications, few studies reported on the biomarker potential of these epigenetic marks. Importantly, the reversible nature of histone modifications makes them a promising therapeutic option for this malignancy. Indeed, over the past years, molecules with inhibitory activity upon the epigenetic machinery have been developed and are currently under evaluation in clinical trials to test their effectiveness.