PTEN Gene Promoter Research Articles

Depleting long noncoding RNA HOTAIR attenuates chronic myelocytic leukemia progression by binding to DNA methyltransferase 1 and inhibiting PTEN gene promoter methylation

Long noncoding RNAs (lncRNAs) are known to play a key role in chronic myelocytic leukemia (CML) development, and we aimed to identify the involvement of the lncRNA HOX antisense intergenic RNA (HOTAIR) in CML via binding to DNA methyltransferase 1 (DNMT1) to accelerate methylation of the phosphatase and tensin homolog (PTEN) gene promoter. Bone marrow samples from CML patients and normal bone marrow samples from healthy controls were collected. HOTAIR, DNMT1, DNMT3A, DNMT3B, and PTEN expression was detected. The biological characteristics of CML cells were detected. The relationship among HOTAIR, DNMT1, and PTEN was verified. Tumor volume and weight in mice injected with CML cells were tested. We found that HOTAIR and DNMT1 expression was increased and PTEN expression was decreased in CML. We also investigated whether downregulated HOTAIR or DNMT1 reduced proliferation, colony formation, invasion, and migration and increased the apoptosis rate of CML cells. Moreover, we tested whether low expression of HOTAIR or DNMT1 reduced the volume and weight of tumors in mice with CML. Collectively, the results of this studied showed that depleted HOTAIR demonstrated reduced binding to DNMT1 to suppress CML progression, which may be related to methylation of the PTEN promoter.

Exosome-transmitted lncRNA UFC1 promotes non-small-cell lung cancer progression by EZH2-mediated epigenetic silencing of PTEN expression

Long non-coding RNAs (LncRNAs) have been suggested as important regulators of cancer development and progression in non-small cell lung cancer (NSCLC). Nevertheless, the biological roles and clinical significance of lncRNA UFC1 in NSCLC remain unclear. We detected the expression of UFC1 in tumor tissues, serum, and serum exosomes of NSCLC patients by qRT-PCR. Gene overexpression or silencing were used to examine the biological roles of UFC1 in NSCLC. RNA immunoprecipitation and ChIP assays were performed to evaluate the interaction between UFC1 and enhancer of zeste homolog 2 (EZH2) and the binding of EZH2 to PTEN gene promoter. Rescue study was used to access the importance of PTEN regulation by UFC1 in NSCLC progression. UFC1 expression was upregulated in tumor tissues, serum, and serum exosomes of NSCLC patients and high level of UFC1 was associated with tumor infiltration. UFC1 knockdown inhibited NSCLC cell proliferation, migration and invasion while promoted cell cycle arrest and apoptosis. UFC1 overexpression led to the opposite effects. Mechanistically, UFC1 bound to EZH2 and mediated its accumulation at the promoter region of PTEN gene, resulting in the trimethylation of H3K27 and the inhibition of PTEN expression. UFC1 knockdown inhibited NSCLC growth in mouse xenograft tumor models while the simultaneous depletion of PTEN reversed this effect. NSCLC cells derived exosomes could promote NSCLC cell proliferation, migration and invasion through the transfer of UFC1. Moreover, Exosome-transmitted UFC1 promotes NSCLC progression by inhibiting PTEN expression via EZH2-mediated epigenetic silencing. Exosome-mediated transmit of UFC1 may represent a new mechanism for NSCLC progression and provide a potential marker for NSCLC diagnosis.

Genome-wide characterization of 5-hydoxymethylcytosine in melanoma reveals major differences with nevus.

Melanoma demonstrates altered patterns of DNA methylation that are associated with genetic instability and transcriptional repression of numerous genes. Active DNA demethylation is mediated by TET enzymes that catalyze conversion of 5‐methylcytosine (mC) to 5‐hydroxymethylcytosine (hmC). Loss of hmC occurs in melanoma and correlates with disease progression. Here we analyzed the genomic distribution of hmC along with mC in nevus and melanoma using oxidative bisulfite chemistry combined with high‐density arrays. HmC was enriched relative to mC at enhancers, 5′UTR regions and CpG shores in nevus and melanoma samples, pointing to specific TET enzyme activity. The proportion of interrogated CpG sites with high hmC levels was lower in melanoma (0.54%) than in nevus (2.0%). Depletion of hmC in melanoma was evident across all chromosomes and intragenic regions, being more pronounced in metastatic than in non‐metastatic tumors. The patterns of hmC distribution in melanoma samples differed significantly from those in nevus samples, exceeding differences in mC patterns. We identified specific CpG sites and regions with significantly lower hmC levels in melanoma than in nevus that might serve as diagnostic markers. Differentially hydroxymethylated regions localized to cancer‐related genes, including the PTEN gene promoter, suggesting that deregulated DNA hydroxymethylation may contribute to melanoma pathogenesis.

Fresh red raspberry phytochemicals suppress the growth of hepatocellular carcinoma cells by PTEN/AKT pathway.

The red raspberry (Rubus idaeus L.) is a common fruit worldwide and its extract has been found to inhibit the growth of many types of tumors, mainly because it is rich in bioactive phytochemicals. However, the mechanism underlying its anticancer activity in hepatocellular carcinoma (HCC) is not well understood. Herein, the aim of this study was to determine the effects of red raspberry phytochemicals on the proliferation of hepatocellular carcinoma cells and to elucidate its biochemical and molecular targets. CCK8 and colony formation, as well as flow cytometry assays, were employed to determine the effects of red raspberry extract (RRE) on cell proliferation and cell cycle distribution in HCC cells. Our results showed that RRE significantly inhibited cell proliferation and arrested cell cycle progression at the S phase in HCC cells. RRE increased the expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) by reducing the methylation status of the PTEN gene promoter and inhibiting DNMT1 expression and regulated AKT signaling pathway. These findings show that red raspberry phytochemicals inhibit the proliferation of HCC cells by regulating PTEN/AKT signaling pathway, providing evidence that RRE may be used as a potential auxiliary therapy for patients with HCC.

The Synthesis of 1,3,5-triazine Derivatives and JNJ7777120 Analogues with Histamine H4 Receptor Affinity and Their Interaction with PTEN Promoter.

The involvement of histamine and H4 receptor (H4 R) in cancer has been investigated recently using the H4 R agonists and antagonists. The scope of the research project was synthesis and exploration of the consequences of a group of compounds with histamine H4 receptor (H4 R) affinity on the promoter of PTEN gene encoding the antitumor PTEN protein. The series of novel compounds based either on H4 R antagonists JNJ7777120 structure or 1,3,5-triazine scaffold were synthesized, evaluated for histamine H4 R affinity and used in this study. Compounds 5 and 7 belonging to the group of JNJ7777120 analogues showed the highest interaction with the promoter of PTEN gene and weak affinity against H4 R with Ki value >100 μm. These compounds showed no significant effect on neuroblastoma IMR-32 cells viability indicating no correlation between PTEN gene promoter affinity and antitumor activity. Compound 6, another JNJ7777120 analogue, showed the highest effect on IMR-32 viability with calculated IC50 = 23.27 μm. The 1,3,5-triazine derivatives exhibited generally low or medium interaction with PTEN gene promoter. However, the 1,3,5-triazine derivative 11 with the para-bromo substituent showed the highest affinity against H4 R with Ki value of 520 nm and may be considered as a new lead structure.

Piwil1 causes epigenetic alteration of PTEN gene via upregulation of DNA methyltransferase in type I endometrial cancer

Piwil1, a member of the Piwi family, has been well demonstrated to mediate tumorigenesis associated with DNA hypermethylation. It has been reported that Piwil1 is overexpressed in various types of cancer, including endometrial cancer. However, the underlying mechanism of Piwil1 in endometrial cancer remains largely unclear. PTEN exerts an important tumor suppressor role in endometrial carcinogenesis. The present study aimed to investigate whether Piwil1 could regulate the expression of PTEN. Herein, we found that Piwil1 could promote the loss of PTEN expression and increase aberrant hypermethylation of PTEN gene promoter in Ishikawa cells. We also found that Piwil1 could regulate the expression of DNA methyltransferase 1 (DNMT1). Silencing DNMT1 gene could upregulate the PTEN gene expression and change the methylation status of PTEN gene promoter in Ishikawa cells. These results suggested that Piwil1 caused the loss of PTEN expression through DNMT1-mediated PTEN hypermethylation. Taken together, these data provide a novel regulatory mechanism of Piwil1 in endometrial cancer.

JARID1B promotes metastasis and epithelial-mesenchymal transition via PTEN/AKT signaling in hepatocellular carcinoma cells.

JARID1B is a member of the family of JmjC domain-containing proteins that removes methyl residues from methylated lysine 4 on histone H3 lysine 4 (H3K4). JARID1B has been proposed as an oncogene in many types of tumors; however, its role and underlying mechanisms in hepatocellular carcinoma (HCC) remain unknown. Here we show that JARID1B is elevated in HCC and its expression level is positively correlated with metastasis. In addition Kaplan-Meier survival analysis showed that high expression of JARID1B was associated with decreased overall survival of HCC patients. Overexpression of JARID1B in HCC cells increased proliferation, epithelial-mesenchymal transition, migration and invasion in vitro, and enhanced tumorigenic and metastatic capacities in vivo. In contrast, silencing JARID1B in aggressive and invasive HCC cells inhibited these processes. Mechanistically, we found JARID1B exerts its function through modulation of H3K4me3 at the PTEN gene promoter, which was associated with inactive PTEN transcription. PTEN overexpression blocked JARID1B-driven proliferation, EMT, and metastasis. Our results, for the first time, portray a pivotal role of JARID1B in stimulating metastatic behaviors of HCC cells. Targeting JARID1B may thus be a useful strategy to impede HCC cell invasion and metastasis.

A novel functional interplay between Progesterone Receptor-B and PTEN,viaAKT, modulates autophagy in breast cancer cells.

The tumour suppressor activity of the phosphatase and tensin homologue on chromosome 10 (PTEN) is subject of intense investigative efforts, although limited information on its regulation in breast cancer is available. Herein, we report that, in breast cancer cells, progesterone (OHPg), through its cognate receptor PR-B, positively modulates PTEN expression by inducing its mRNA and protein levels, and increasing PTEN-promoter activity. The OHPg-dependent up-regulation of PTEN gene activity requires binding of the PR-B to an Sp1-rich region within the PTEN gene promoter. Indeed, ChIP and EMSA analyses showed that OHPg treatment induced the occupancy of PTEN promoter by PR and Sp1 together with transcriptional coactivators such as SRC1 and CBP. PR-B isoform knockdown abolished the complex formation indicating its specific involvement. The OHPg/PR-B dependent induction of PTEN causes the down-regulation of PI3K/AKT signal, switching on the autophagy process through an enhanced expression of UVRAG and leading to a reduced cell survival. Altogether these findings highlight a novel functional connection between OHPg/PR-B and tumour suppressor pathways in breast cancer.

Promoter region methylation and loss of protein expression of PTEN and significance in cervical cancer.

The genetic basis underlying cervical tumorigenesis and progression are largely unknown. Phosphatase and tensin homologue (PTEN) is a tumor suppressor gene, and genetic changes of PTEN occurs in various types of cancer suggesting that the inactivation of PTEN may play an important role in the pathogenesis of a variety of human malignancies. In the present study, 102 cervical cancer specimens were examined for the expression of the PTEN gene and promoter methylation using methylation-specific-polymerase chain reaction and immunohistochemistry. The PTEN gene mutation was also assessed using PCR single-strand conformational polymorphism. We examined the correlation between PTEN expression and its associated methylation status and the clinical characteristics of cervical cancer. The results showed that there was one case of an A to G point mutation on exon 9 of the PTEN gene in the cervical cancer tissues. This mutation caused the change of aspartic acid to glycine, and the rate of mutation was 1%. The PTEN gene methylation rate of cervical cancer was 62% (63/102) and the rate was associated with the International Federation of Gynecology and Obstetrics stage, cell differentiation, tumor size and lymph node metastasis (P<0.05). The positive rate of PTEN expression was 49% (50/102) in cervical carcinoma and the PTEN expression between stage I-II and III-IV [60 (27/45) vs. 40% (23/57)] was statistically significant (P<0.01). The PTEN gene expression between the metastasis and no lymph node metastasis groups [26 (10/38) vs. 63% (40/64)] was significantly different (P<0.01). The PTEN gene promoter methylation and its protein expression had a significant correlation (P=0.042). These results suggest that hypermethylation can inactivate the transcription of PTEN and reduce its protein expression. Downregulated PTEN expression is involved in the pathogenesis, invasion and metastasis of cervical cancer, possibly by regulating the balance between apoptosis and proliferation. Therefore, the PTEN expression may be a good marker for the prognosis of cervical cancer.

DNA demethylation in the PTEN gene promoter induced by 5-azacytidine activates PTEN expression in the MG-63 human osteosarcoma cell line.

This study used the MG-63 osteosarcoma cell line to investigate the demethylation of the phosphate and tension homolog (PTEN) gene promoter and the change in PTEN gene expression levels, which are caused by the methylation inhibitor 5-azacytidine (5-Zac), and the association between the two. Different concentrations of 5-Zac (0, 5 and 10 μmol/l) were added into the MG-63 cell culture medium and the cells were cultured for 72 h. The following techniques were performed on the cells: Western blot analysis to detect the PTEN protein; reverse transcription-polymerase chain reaction (PCR) to detect the mRNA transcription levels of the PTEN gene; flow cytometry to detect the cell apoptotic rate; and sodium bisulfate to deal with the DNA of each group. The genes of the PTEN promoter and the transcription factors specificity protein 1 (Sp1) and Myc were PCR amplified and transformed into Escherichia coli, then a number of clones were selected for sequencing and the methylation status of the amplified PTEN promoter fragment was detected. Following culture of the MG-63 cells with 5-Zac at concentrations of 0, 5 and 10 μmol/l for 72 h, the expression levels of PTEN protein in each group were gradually increased, presenting a concentration-dependent effect: Group 0 μmol/l compared with groups 5 and 10 μmol/l, P<0.05; and group 5 μmol/l compared with group 10 μmol/l, P=0.007. The mRNA expression levels of the PTEN gene significantly increased. The apoptotic rates of groups 0, 5 and 10 μmol/l were 0.69±0.42, 2.50±0.30 and 6.59±0.62%, and significant differences (P<0.01) were observed between every two groups. The bisulfate DNA sequencing results of three groups showed that, following the treatment with 5-Zac, the binding of the CG site to transcription factors was affected by demethylation. The average rate of demethylation indicated a statistical difference among the three groups. In conclusion, the methylation inhibitor 5-Zac leads to a significant increase in the expression levels of the tumor suppressor gene PTEN in the MG-63 osteosarcoma cell line in vitro. The expression levels of mRNA and the cellular apoptotic rate were also increased. The elevated activation and expression levels of the PTEN gene may be associated with the low methylation levels of the CG site that binds to the transcription factors Sp1 and Myc in the PTEN gene promoter, and they promote the combination of the transcription factors and the gene promoter.

Methylation of the pseudogene PTENP1 5′-terminal region in endometrial cancer and hyperplasia

Genetic mutations in tumor suppressor gene PTEN are often detected in malignant human cells and these genomic changes are especially characteristic ofendometrial cancer. In our previous researches we assumed that alternative epigenetic mechanism of PTEN inactivation trough promoter methylation may exist in endometrial cancer. Moreover, PTENP1 pseudogene has recently been shown to play a role in positive regulation of PTENgene expression. Taking into account these facts, we analyzed PTEN and PTENP1 methylation status in endometrial hyperplasia and cancer. It was demonstrated that PTENgene promoter was not methylated but PTENP1 was methylated in 11 of 18 endometrial cancers and in 5 of9 endometrial hyperplasias. We can assume that PTENP1 methylation may inhibit transcription of this gene and also PTEN gene transcription trough RNA interference in accordance with ceRNA theory. Thus, aberrant suppression of PTENP1 transcription can play a role in endometrial cancer pathogenesis.

A novel PTEN gene promoter mutation and untypical Cowden syndrome.

Cowden syndrome (CS), an autosomal dominant disorder, is one of a spectrum of clinical disorders that have been linked to germline mutations in the phosphatase and tensin homolog (PTEN) gene. Although 70-80% of patients with CS have an identifiable germline PTEN mutation, the clinical diagnosis presents many challenges because of the phenotypic and genotypic variations. In the present study, we sequenced the exons and the promoter of PTEN gene, mutations and variations in the promoter and exons were identified, and a PTEN protein expression negative region was determined by immunohistochemistry (IHC). In conclusion, a novel promoter mutation we found in PTEN gene may turn off PTEN protein expression occasionally, leading to the disorder of PTEN and untypical CS manifestations.

Epigenetic Regulation of PTEN in Hepatic Cancer

The pathogenesis of hepatocellular carcinoma (HCC) remains misunderstood offering limited therapeutic options for patients with hepatic cancer.Treatment of HCC cell line (Huh‐7) with DNMT inhibitor, Decitabine, and HDAC inhibitor, Trichostatin A, increased the expression of PTEN protein. Methylation‐specific qPCR assay showed Decitabine/TSA treatment resulted in hypermethylation of CpG islands located on the PTEN gene promoter. Furthermore, miR‐185 targets DNMT1, thus upregulating the expression of PTEN in Huh‐7. Forced overexpression of miR‐185 abolished the expression of DNMT1 protein and enhanced the level of PTEN protein in Huh‐7 stable cells. MiR‐185 xenograft tumor volume from SCID mice was significantly decreased versus control. Likewise, Ki‐67 immunostaining in Huh‐7 control stable cell line showed a higher proliferative. Xenograft protein samples showed increased PTEN expression and decreased DNMT1 activity. DNMT‐1 3'UTR reporter plasmids were constructed/cloned showing the wild type‐3'UTR of DNMT1 was significantly depleted in Huh‐7 miR‐185 stable cell line versus control in luciferase assay experiments. Co‐transfection of Huh‐7 miR‐185 ORF versus control showed DNMT1 restoration via western blot, Invasion and Cell Proliferation Assay. In‐situ hybridization showed a substantial decrease of staining expression in human HCC tumor samples versus normal liver tissue probed for miR‐185.Our results demonstrate a novel epigenetic regulation of PTEN by miR‐185, which is critical in hepatic carcinogenesis.

Estrogen receptor beta (ERβ) produces autophagy and necroptosis in human seminoma cell line through the binding of the Sp1 on the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) promoter gene

Testicular germ cell tumors are the most common tumor in male and the least studied. We focused on human seminoma using the TCAM2 cell line. Through ERβ, 10 nM estradiol (E2) was able to induce PTEN gene expression and promoter transactivation. Transient transfections, ChIP and EMSA assays evidenced the 5′-flanking region of PTEN gene promoter E2-responsive. The ERβ binding to the Sp1 on PTEN promoter decreased cell survival. The presence of ERβ or PTEN is necessary to induce the loss of cell survival upon E2, addressing their cooperation in this action. pAKT and AKT expression decreased under E2 and DPN, while known apoptotic markers appeared to be unchanged. The PI3K/AKT pathway inhibition also leads to autophagy: E2 and DPN enhanced the expression of autophagy-related markers such as PI3III, Beclin 1, AMBRA and UVRAG. MDC and TEM assays confirmed E2-induced autophagy. The absence of DNA fragmentation, caspase 9 and PARP1 cleavages suggested that necroptosis and/or parthanatos may occur. FACS analysis, LDH assay and RIP1 expression attested this hypothesis. Our study reveals a unique mechanism through which ERβ/PTEN signaling induces cell death in TCAM2 by autophagy and necroptosis. These data, supporting estrogen-dependency of human seminoma, propose ERβ ligands for therapeutic use in the treatment of this pathological condition.

DNMT1-mediated PTEN hypermethylation confers hepatic stellate cell activation and liver fibrogenesis in rats

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN), a tumor suppressor, is a negative regulator of this process. PTEN promoter hypermethylation is a major epigenetic silencing mechanism in tumors. The present study aimed to investigate whether PTEN promoter methylation was involved in HSC activation and liver fibrosis. Treatment of activated HSCs with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) decreased aberrant hypermethylation of the PTEN gene promoter and prevented the loss of PTEN expression that occurred during HSC activation. Silencing DNA methyltransferase 1 (DNMT1) gene also decreased the PTEN gene promoter methylation and upregulated the PTEN gene expression in activated HSC-T6 cells. In addition, knockdown of DNMT1 inhibited the activation of both ERK and AKT pathways in HSC-T6 cells. These results suggest that DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the activation of the PI3K/AKT and ERK pathways, resulting in HSC activation.

Association of epigenetic alteration in PTEN gene with colorectal cancer progression among Indian population

Dear Editor: Cancer is a complex genetic disease which gradually progresses in relation to the downregulated expression of tumor suppressor genes (TSGs). Inactivation of TSGs has been known to contribute to the abnormal proliferation, transformation, and progression in human cancers. It has been widely demonstrated that both the genetic and epigenetic events, together, play a crucial role in tumor progression. Epigenetic is defined as the heritable changes in gene expression that are not caused by alterations in the DNA sequence. Several studies have demonstrated the epigenetic alterations in colorectal carcinoma. Colorectal cancer (CRC) is the third most common malignancy worldwide and a leading cause of cancer death in women and men in the USA. In contrast, the frequency of colorectal carcinoma is less in India. However, 20,159 cases of CRC among men and 16,317 cases among women were reported in India. The 14,134 and 11,556 mortality due to CRC was reported among men and women, respectively, in India. Most of the CRC cases originate from adenomas. The malignant potential of adenomas increases with size, grade of dysplasia, and degree of villous components, along with the number and order of genetic and epigenetic aberrations. Previous studies have also highlighted the hypermethylation in several TSGs in colorectal carcinoma. Recent reports suggest that PTEN has an important tumor suppressor role in colorectal carcinogenesis. However, more attentive study is needed to explore the association of PTEN loss with CRC development. PTEN, a tumor suppressor gene, has been recently isolated from chromosome 10q23.3. The involvement of PTEN promoter methylation has been reported in breast cancer, cervical cancer, endometrial cancer, colorectal cancer, and glioblastoma. However, no such study has been made to evaluate the role of the PTEN tumor suppressor gene in colorectal cancer in colorectal cancer in Indian population. The correlation of the progression of disease with that of the promoter hypermethylation seems to be an interesting phenomenon. Therefore, we aimed to analyze the PTEN gene promoter methylation in fresh colorectal cancer tissues and their adjacent normal tissues along with the colorectal metastases specimens and also to evaluate the association of PTEN hypermethylation with CRC progression. The major outcome of the study is that the PTEN hypermethylation is a frequent event in sporadic CRCs and colorectal metastases in the patients from Indian population. Promoter methylation of PTEN gene was determined by methylationspecific PCR. We also performed chi-square test to correlate the PTEN hypermethylation with the clinicobiological parameters of colorectal cancer and metastases specimens in order to evaluate the role of PTEN hypermethylation with the disease progression. Of the CRC specimens, 114 (51 %) have shown PTEN hypermethylation among the total of 223 CRC specimens. Thirty-eight specimens have been characterized as metastatic among 223 specimens. Thirty-one (82 %) specimens out of 38 colorectal metastases samples have also demonstrated PTEN hypermethylation. We would like to highlight the high rate of hypermethylation in advanced stage colorectal tumors than early stages of colorectal tumors. To examine the M. M. A. Rizvi (*) :A. Ali : S. J. Mehdi Genome Biology Lab, Department of Biosciences, Jamia Millia Islamia, Maulana Mohammad Ali Jauhar Marg, New Delhi 110025, India e-mail: rizvijmi@gmail.com

Abstract 3143: Epigenetic regulation of PTEN by miR-185 and miR-200b in liver cancer

Abstract PTEN is one of the most commonly lost tumor suppressors in human cancers. Deletion of PTEN in mice has been shown to induce steatohepatitis and the development of primary liver cancers (hepatocellular carcinoma and cholangiocarcinoma). This study provides novel evidence for epigenetic regulation of PTEN in both hepatocellular carcinoma and cholagniocarcinoma cells. Treatment of the hepatocellular carcinoma cell line (Huh-7) and cholangiocarcinoma cells line (TFK-1) with the DNA methyltransferase (DNMT) inhibitor, Decitabine (1 mM), and the histone deacetylase inhibitor, Trichostatin A (TSA, 10mM), increased the expression of PTEN protein. Methylation specific qPCR assay shows that Decitabine/TSA treatment significantly reduced the methylation of the CpG islands located at the PTEN gene promoter. Furthermore, we have shown that miR-185 and miR-200b target DNMT-1 and -3A/3B, respectively, thus upregulating the expression of PTEN in hepatic cancer cells. Forced overexpression of miR-185 or treatment with mimic miR-185 abolished the expression of DNMT1 protein and enhanced the level of PTEN protein in both Huh-7 and TFK-1 cells. Accordingly, miR-200b overexpression or mimic treatment significantly decreased DNMT3A and DNMT3B protein levels and enhanced the expression of PTEN. These results demonstrate a novel epigenetic regulation of PTEN by miR-185 and -200b which is critical in hepatic carcinogenesis Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3143. doi:1538-7445.AM2012-3143

Progesterone receptor B through Sp1 induces the phosphatase and tensin homologue deleted from chromosome 10 (PTEN) gene promoter activity and cell death in breast cancer cell lines

Phosphatase and tensin homolog on chromosome 10 (PTEN) is a tumor suppressor attracting considerable attention as new target for effective therapeutic strategies in breast cancer. The antitumors effect of progesterone were envisaged, however the mechanism thorough which this occurs was not clearly established. In the present study we report that OHPg, through the PR‐B isoform, produces a significant inhibition of MCF‐7 breast cancer cell survival with a concomitant increase of PTEN protein, mRNA and gene promoter activity. By using different deleted constructs of the PTEN promoter, we found that the OHPg failed to enhance the transcriptional activity of the PTEN promoter lacking the region between −1398 bp to −1118 bp. This DNA sequence contains several DNA motifs known to bind the transcription factors Sp1that has been shown to interact with PRs. In our experimental conditions mithramycin, prevented the PTEN promoter transactivation on OHPg treatment. ChIP and EMSA analyses showed specific recruitment of PR, Sp1 and RNA polymerase II upon OHPg treatment at PTEN promoter. The persistence of such effects in T47 cells which express high PR levels, addresses how OHPg/PR signalling may be considered as useful pharmacological tool to be exploited for innovative adjuvant strategies in breast cancer treatment.

DNA methylation of PTEN gene promoter region is not correlated with urocystic tumorigenesis

Tumor suppressor gene PTEN plays an important role in cell cycle. Disorder of PTEN protein can cause cell growth and division in an uncontrolled way, which can lead to the formation of tumors. It has been proven that epigenetic mechanisms, such as promoter hypermethylation, may account for inactivation of PTEN in a subset of tumors. PTEN promoter hypermethylation has been found to be involved in many kinds of cancers. Up to date, no report about the relationships between methylation of PTEN promoter region and bladder cancer has been found. To investigate the methylation pattern of PTEN gene transcriptional regulation region (TRR), bisulfite-specific (BSP) polymerase chain reaction (PCR)- based sequencing analysis was performed among 15 bladder cancer tissues and five normal bladder tissues. Analysis of PTEN gene TRR methylation showed that the methylation level in bladder cancer had no significant difference with that of normal (P = 0.4307, by unpaired Student’s t test). Key words: PTEN, promoter methylation, bladder cancer.

Abstract C239: Progesterone receptor B through Sp1 induces the phosphatase and tensin homologue deleted from chromosome 10 (PTEN) gene promoter activity and cell death in breast cancer cell lines.

Abstract Phosphatase and tensin homolog on chromosome 10 (PTEN) is a tumor suppressor attracting considerable attention as new target for effective therapeutic strategies in breast cancer. While recent studies suggest the down-regulation of PTEN by Estrogen Receptor alpha, the role of Progesterone Receptor (PR) is not clearly established. In the present study we report that PR produces a significant inhibition of MCF-7 breast cancer cell survival with a concomitant increase of PTEN protein, mRNA and gene promoter activity. On the contrary PRs failed to enhance the transcriptional activity of the PTEN promoter lacking the region between −1398 bp to −1118 bp. By analyzing this DNA sequence to search potential binding sites for transcription factors, we observed that this region contains several DNA motifs known to bind the transcription factors Sp1that has been shown to interact with PRs, activating specific gene transcription. In our experimental conditions mithramycin, prevented the PTEN promoter transactivation on OHPg treatment. ChIP analyses showed specific recruitment of PR, Sp1 and RNA polymerase II upon progesterone treatment at PTEN promoter. The persistence of such effects in T47 cells which express high PR levels, addresses how PR/PTEN signalling may be considered as useful pharmacological tool to be exploited for innovative adjuvant strategies in breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C239.