miR-152 is a tumor suppressor microRNA that is silenced by DNA hypermethylation in endometrial cancer

The etiology and development of human cancers that remain little understood might be enlightened by defining tumor suppressor microRNAs (TS-miRNA). In this study, we identified TS-miRNAs silenced by aberrant DNA hypermethylation in endometrial cancer. Functional screening of 327 synthetic miRNAs in an endometrial cancer cell proliferation assay identified 103 miRNAs that inhibited cell growth. We then determined the sequence, DNA methylation status, and expression levels of these miRNAs in endometrial cancer cell lines and primary tumors. These determinations led to the identification of miR-152 as a candidate TS-miRNA gene in endometrial cancer. Epigenetic silencing documented in miR-152 was consistent with its location at 17q21.32 in intron 1 of the COPZ2 gene, which is also silenced often in endometrial cancer by DNA hypermethylation, and also with evidence that miR-152 targets the DNA methyltransferase DNMT1. Notably, restoration of miR-152 expression in endometrial cancer cell lines was sufficient to inhibit tumor cell growth in vitro and in vivo. We identified E2F3, MET, and Rictor as novel candidate targets of miR-152, suggesting how its epigenetic silencing can drive endometrial carcinogenesis. Our findings define a central role for miR-152 in endometrial cancer, and they also suggest its use in new therapeutic strategies to treat this cancer.     

miR-124a is frequently down-regulated in glioblastoma and is involved in migration and invasion

Glioblastoma (GBM) represents a formidable clinical challenge for both patients and treating physicians. Due to better local treatments and prolonged patient survival, remote recurrences are increasingly observed, underpinning the importance of targeting tumour migration and attachment. Aberrant expression of microRNA (miRNA) is commonly associated with cancer and loss of miR-124a has previously been implicated to function as a tumour suppressor. The assessment of miR-124a in clinical specimens has been limited and a potential role in migration and invasion has been unexplored until now. We measured the expression levels of mature miR-124a in a retrospective series of 119 cases of histologically confirmed GBM and found its expression was markedly lower in over 80% of the GBM clinical specimens compared to normal brain tissue. The level of reduction in the clinical cohort varied significantly and patients with lower than the average miR-124a expression levels displayed shorter survival times. Endogenous miR-124a expression and the protein expression of three of its targets; IQ motif containing GTPase activating protein 1 (IQGAP1), laminin γ1 (LAMC1) and integrin β1 (ITGB1) were significantly reciprocally associated in the majority of the clinical cases. We confirmed this association in our in vitro model. Functionally, the ectopic expression of mature miR-124a in a GBM cell line resulted in significant inhibition of migration and invasion, demonstrating a role for miR-124a in promoting tumour invasiveness. Our results suggest that miR-124a may play a role in GBM migration, and that targeted delivery of miR-124a may be a novel inhibitor of GBM invasion.     

WAVE3, an actin remodeling protein, is regulated by the metastasis suppressor microRNA, miR-31, during the invasion-metastasis cascade

WAVE3, an actin cytoskeleton remodeling protein, is highly expressed in advanced stages of breast cancer and influences tumor cell invasion. Loss of miR-31 has been associated with cancer progression and metastasis. Here, we show that the activity of WAVE3 to promote cancer cell invasion is regulated by miR-31. An inverse correlation was demonstrated between expression levels of WAVE3 and miR-31 in invasive versus noninvasive breast cancer cell lines. miR-31 directly targeted the 3'-UTR of the WAVE3 mRNA and inhibited its expression in the invasive cancer cells, i.e., miR-31-mediated down-regulation of WAVE3 resulted in a significant reduction in the invasive phenotype of cancer cells. This relationship was specific to the loss of WAVE3 expression because re-expression of a miR-31-resistant form of WAVE3 reversed miR-31-mediated inhibition of cancer cell invasion. Furthermore, expression of miR-31 correlates inversely with breast cancer progression in humans, where an increase in expression of miR-31 target genes was observed as the tumors progressed to more aggressive forms. In conclusion, a novel mechanism for the regulation of WAVE3 expression in cancer cells has been identified, which controls the invasive properties of cancer cells. The study also identifies a critical role for WAVE3, downstream of miR-31, in the invasion-metastasis cascade.     

Overexpression of MBD2 in glioblastoma maintains epigenetic silencing and inhibits the antiangiogenic function of the tumor suppressor gene BAI1

Brain angiogenesis inhibitor 1 (BAI1) is a putative G protein-coupled receptor with potent antiangiogenic and antitumorigenic properties that is mutated in certain cancers. BAI1 is expressed in normal human brain, but it is frequently silenced in glioblastoma multiforme. In this study, we show that this silencing event is regulated by overexpression of methyl-CpG-binding domain protein 2 (MBD2), a key mediator of epigenetic gene regulation, which binds to the hypermethylated BAI1 gene promoter. In glioma cells, treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-Aza-dC) was sufficient to reactivate BAI1 expression. Chromatin immunoprecipitation showed that MBD2 was enriched at the promoter of silenced BAI1 in glioma cells and that MBD2 binding was released by 5-Aza-dC treatment. RNA interference-mediated knockdown of MBD2 expression led to reactivation of BAI1 gene expression and restoration of BAI1 functional activity, as indicated by increased antiangiogenic activity in vitro and in vivo. Taken together, our results suggest that MBD2 overexpression during gliomagenesis may drive tumor growth by suppressing the antiangiogenic activity of a key tumor suppressor. These findings have therapeutic implications because inhibiting MBD2 could offer a strategy to reactivate BAI1 and suppress glioma pathobiology.     

CD47 expression regulated by the miR-133a tumor suppressor is a novel prognostic marker in esophageal squamous cell carcinoma

CD47 inhibits phagocytosis and its overexpression is correlated with poor prognosis in patients with several types of cancer. It has also been reported that CD47 expression in multiple sclerosis is regulated by microRNAs. However, the regulatory mechanism of CD47 in cancer tissues has not been yet clarified. Re-analysis of a public microarray database revealed that miR-133a is downregulated in esophageal squamous cell carcinoma (ESCC). Moreover, in?silico algorithms predicted that miR-133a is a regulator of CD47. The purpose of this study was to clarify the clinical significance of CD47 and its regulatory mechanism by miR-133a in ESCC. Quantitative real-time RT-PCR was used to evaluate CD47 and miR-133a expression in 102 cases of curative resected ESCC and adjacent non-cancerous tissue. The regulation of CD47 by miR-133a was examined with precursor miR-133a-transfected cells. A mouse xenograft model was used to investigate the ability of miR-133a to suppress tumor progression. High expression levels of CD47 were associated with lymph node metastasis (P=0.049). Multivariate analysis showed that CD47 expression was an independent prognostic factor (P=0.045). miR-133a expression was significantly lower in cancer tissues compared to adjacent non-cancerous tissues (P<0.001). In?vitro assays showed that miR-133a is a direct regulator of CD47. miR?133a significantly inhibited tumorigenesis and growth in?vivo. CD47 expression is a novel prognostic marker in ESCC that is directly inhibited by the miR-133a tumor suppressor. This correlation could provide new insight into the mechanism of cancer progression and a promising candidate for target therapy in ESCC.     

Frequent silencing of the candidate tumor suppressor PCDH20 by epigenetic mechanism in non-small-cell lung cancers

Protocadherins are a major subfamily of the cadherin superfamily, but little is known about their functions and intracellular signal transduction. We identified a homozygous loss of protocadherin 20 (PCDH20, 13q21.2) in the course of a program to screen a panel of non-small-cell lung cancer (NSCLC) cell lines (1 of 20 lines) for genomic copy number aberrations using an in-house array-based comparative genomic hybridization. PCDH20 mRNA was expressed in normal lung tissue but was not expressed in the majority of NSCLC cell lines without a homozygous deletion of this gene (10 of 19 lines, 52.6%). Expression of PCDH20 mRNA was restored in gene-silenced NSCLC cells after treatment with 5-aza 2'-deoxycytidine. The DNA methylation status of the PCDH20 CpG-rich region correlated inversely with the expression of the gene and a putative target region for methylation showed clear promoter activity in vitro. Methylation of this PCDH20 promoter was frequently observed in primary NSCLC tissues (32 of 59 tumors, 54.2%). Among our primary NSCLC cases, the methylated PCDH20 seemed to be associated with a shorter overall survival (P = 0.0140 and 0.0211 in all and stage I tumors, respectively; log-rank test), and a multivariate analysis showed that the PCDH20 methylation status was an independent prognosticator. Moreover, restoration of PCDH20 expression in NSCLC cells reduced cell numbers in colony formation and anchorage-independent assays. These results suggest that epigenetic silencing by hypermethylation of the CpG-rich promoter region of PCDH20 leads to loss of PCDH20 function, which may be a factor in the carcinogenesis of NSCLC.     

The hypoxia-related microRNA miR-199a-3p displays tumor suppressor functions in ovarian carcinoma

During the dissemination of ovarian cancer cells, the cells float in the peritoneal cavity without access to a vascular supply and so are exposed to hypoxic conditions, which may cause the ovarian cancer cells to acquire a more aggressive and malignant phenotype. In this study, we screened microRNAs (miRNAs) to identify those that displayed altered expression patterns under hypoxic conditions and then analyzed their functional roles in ovarian cancer progression. miRNA PCR arrays performed on cells from 2 ovarian cancer cell lines (CaOV3 and RMUG-S) revealed miR-199a-3p as one of the miRNAs that are downregulated under hypoxia. In silico analyses indicated that MET is one of the target genes for miR-199a-3p; subsequently, miR-199a-3p expression was found to be inversely correlated with c-Met expression in ovarian cancer. Transfection of precursor miR-199a-3p into ovarian cancer cells reduced c-Met expression and inhibited the phosphorylation of c-Met, extracellular signal-regulated kinase, and AKT; in addition, proliferation, adhesion, and invasiveness were inhibited. Moreover, overexpression of miR-199a-3p in cancer cells significantly suppressed peritoneal dissemination in a xenograft model. In summary, the hypoxia-related microRNA miR-199a-3p drastically inhibits ovarian cancer progression through the downregulation of c-Met expression. Therefore, miR-199a-3p is a potential target for treating ovarian cancer dissemination.     

PTEN, NHERF1 and PHLPP form a tumor suppressor network that is disabled in glioblastoma

The phosphatidylinositol-3-OH kinase (PI3K)-Akt pathway is activated in cancer by genetic or epigenetic events and efforts are under way to develop targeted therapies. phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is the major brake of the pathway and a common target for inactivation in glioblastoma, one of the most aggressive and therapy-resistant cancers. To achieve potent inhibition of the PI3K-Akt pathway in glioblastoma, we need to understand its mechanism of activation by investigating the interplay between its regulators. We show here that PTEN modulates the PI3K-Akt pathway in glioblastoma within a tumor suppressor network that includes Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) and pleckstrin-homology domain leucine-rich repeat protein phosphatases 1 (PHLPP1). The NHERF1 adaptor, previously characterized by our group as a PTEN ligand and regulator, shows also PTEN-independent Akt-modulating effects that led us to identify the PHLPP1/PHLPP2 Akt phosphatases as NHERF1 ligands. NHERF1 interacts via its PDZ domains with PHLPP1/PHLPP2 and scaffolds heterotrimeric complexes with PTEN. Functionally, PHLPP1 requires NHERF1 for membrane localization and growth-suppressive effects. PHLPP1 loss boosts Akt phosphorylation only in PTEN-negative cells and cooperates with PTEN loss for tumor growth. In a panel of low-grade and high-grade glioma patient samples, we show for the first time a significant disruption of all three members of the PTEN-NHERF1-PHLPP1 tumor suppressor network in high-grade tumors, correlating with Akt activation and patient's abysmal survival. We thus propose a PTEN-NHERF1-PHLPP PI3K-Akt pathway inhibitory network that relies on molecular interactions and can undergo parallel synergistic hits in glioblastoma.     

MiR-152 functions as a tumor suppressor in glioblastoma stem cells by targeting Krüppel-like factor 4

Glioblastoma (GBM) is the most common central nervous system tumor and the molecular mechanism driving its development is still largely unknown, limiting the treatment of this disease. In the present study, we explored the potential role of miR-152 in glioblastoma stem cells (GSCs) as well as the possible molecular mechanisms. Our results proved that miR-152 was down-regulated in human GSCs. Restoring the expression of miR-152 dramatically reduced the cell proliferation, cell migration and invasion as well as inducing apoptosis. Mechanistic investigations defined Krüppel-like factor 4 (KLF4) as a direct and functional downstream target of miR-152, which was involved in the miR-152-mediated tumor-suppressive effects in GSCs. Meanwhile, this process was coincided with the down-regulated LGALS3 that could be bound and promoted by KLF4, leading to attenuate the activation of MEK1/2 and PI3K signal pathways. Moreover, the in vivo study showed that miR-152 over-expression and KLF4 knockdown produced the smallest tumor volume and the longest survival in nude mice. Taken together, these results elucidated the function of miR-152 in GSCs progression and suggested a promising application of it in glioma treatment.     

Neuromedin U is regulated by the metastasis suppressor RhoGDI2 and is a novel promoter of tumor formation, lung metastasis and cancer cachexia

Most deaths from urinary bladder cancer are owing to metastatic disease. A reduction in Rho GDP Dissociation Inhibitor 2 (RhoGDI2) protein has been associated with increased risk of metastasis in patients with locally advanced bladder cancer, whereas in animal models, RhoGDI2 reconstitution in cells without expression results in lung metastasis suppression. Recently, we noted an inverse correlation between tumor RhoGDI2 and Neuromedin U (NMU) expression, suggesting that NMU might be a target of the lung metastasis suppressor effect of RhoGDI2. Here we evaluated whether NMU is regulated by RhoGDI2 and is functionally important in tumor progression. We used small interfering RNA knockdown of endogenous RhoGDI2 in poorly tumorigenic and non-metastatic human bladder cancer T24 cells and observed increased NMU RNA expression. Although NMU overexpression did not increase the monolayer growth of T24 or related T24T poorly metastatic human bladder cancer cells, it did augment anchorage-independent growth for the latter. Overexpression of NMU in T24 and T24T cells significantly promoted tumor formation of both cell lines in nude mice, but did not alter the growth rate of established tumors. Furthermore, NMU-overexpressing xenografts were associated with lower animal body weight than control tumors, indicating a possible role of NMU in cancer cachexia. NMU overexpression in T24T cells significantly enhanced their lung metastatic ability. Bioluminescent in vivo imaging revealed that lung metastases in T24T grew faster than the same tumors in the subcutaneous microenvironment. In conclusion, NMU is a RhoGDI2-regulated gene that appears important for tumorigenicity, lung metastasis and cancer cachexia, and thus a promising therapeutic target in cancer.     

miR-193a-3p is a potential tumor suppressor in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an asbestos-induced cancer with poor prognosis that displays characteristic alterations in microRNA expression. Recently it was reported that the expression of a subset of microRNAs can distinguish between MPM and adenocarcinoma of the lung. However, the functional importance of these changes has yet to be investigated. We compared expression of miR-192, miR-193a-3p and the miR-200 family in normal pleura and MPM tumor specimens and found a statistically significant reduction in the levels of miR-193a-3p (3.1-fold) and miR-192 (2.8-fold) in MPM. Transfection of MPM cells with a miR-193a-3p mimic resulted in inhibition of growth and an induction of apoptosis and necrosis in vitro. The growth inhibitory effects of miR-193a-3p were associated with a decrease in MCL1 expression and were recapitulated by RNAi-mediated MCL1 silencing. Targeted delivery of miR-193a-3p mimic using EDV minicells inhibited MPM xenograft tumour growth, and was associated with increased apoptosis. In conclusion, miR-193a-3p appears to have importance in the biology of MPM and may represent a target for therapeutic intervention.     

Multi-gene epigenetic silencing of tumor suppressor genes in T-cell lymphoma cells; delayed expression of the p16 protein upon reversal of the silencing

To understand better T-cell lymphomagenesis, we examined promoter CpG methylation and mRNA expression of closely related genes encoding p16, p15, and p14 tumor suppressor genes in cultured malignant T-cells that were derived from cutaneous, adult type, and anaplastic lymphoma kinase (ALK)-expressing T-cell lymphomas. p16 gene was epigenetically silenced in all but one of the 10 malignant T-cell lines examined, p15 gene silenced in roughly half of the lines, and p14 was the least frequently affected. Extensive methylation of the p16 promoter was seen in six out of 10 cutaneous T-cell lymphoma patient samples and corresponded with lack of p16 protein expression in the cases examined. Treatment of cultured T-cells with the DNA methyltransferase inhibitor, 5-aza-2-deoxy-cytidine, resulted in reversal of the p16 gene silencing. However, expression of p16 protein was delayed in relationship to p16 promoter demethylation and required up to 3 weeks to occur, seemingly reflecting late activation of the p16 gene. These findings indicate that epigenetic silencing affects in T-cell malignancies, often simultaneously, several tumor suppressor genes that impact on key cell functions. The observed differential silencing of p16 and p14, and to a lesser degree p15 gene, indicates that the silencing is governed by precise, promoter region-specific mechanisms. The study provides also further rationale for treatment of at least some types of T-cell lymphomas with DNA methyltransferase inhibitors to target the epigenetically silenced tumor suppressor genes.     

Epigenetic silencing of the kinase tumor suppressor WNK2 is tumor-type and tumor-grade specific

Both genetic and epigenetic mechanisms contribute to meningioma development by altering gene expression and protein function. To determine the relative contribution of each mechanism to meningioma development, we used an integrative approach measuring copy number and DNA methylation changes genomewide. We found that genetic alterations affected 1.9%, 7.4%, and 13.3% of the 691 loci studied, whereas epigenetic mechanisms affected 5.4%, 9.9%, and 10.3% of these loci in grade I, II, and III meningiomas, respectively. Genetic and epigenetic mechanisms rarely involved the same locus in any given tumor. The predilection for epigenetic rather than genetic silencing was exemplified at the 5′ CpG island of WNK2, a serine-threonine kinase gene on chromosome 9q22.31. WNK2 is known to negatively regulate epidermal growth factor receptor signaling via inhibition of MEK1 (mitogen-activated protein kinase kinase 1), and point mutations have been reported in WNK1, WNK2, WNK3, and WNK4. In meningiomas, WNK2 was aberrantly methylated in 83% and 71% of grade II and III meningiomas, respectively, but rarely in a total of 209 tumors from 13 other tumor types. Aberrant methylation of the CpG island was associated with decreased expression in primary tumors. WNK2 could be reactivated with a methylation inhibitor in IOMM-Lee, a meningioma cell line with a densely methylated WNK2 CpG island and lack of WNK2 expression. Expression of exogenous WNK2 inhibited colony formation, implicating it as a potential cell growth suppressor. These findings indicate that epigenetic mechanisms are common across meningiomas of all grades and that for specific genes such as WNK2, epigenetic alteration may be the dominant, grade-specific mechanism of gene inactivation.