Deniz Mahmut Özata

The role of microRNA deregulation in the pathogenesis of adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is an aggressive tumor showing frequent metastatic spread and poor survival. Although recent genome-wide studies of ACC have contributed to our understanding of the disease, major challenges remain for both diagnostic and prognostic assessments. The aim of this study was to identify specific microRNAs (miRNAs) associated with malignancy and survival of ACC patients. miRNA expression profiles were determined in a series of ACC, adenoma, and normal cortices using microarray. A subset of miRNAs showed distinct expression patterns in the ACC compared with adrenal cortices and adenomas. Among others, miR-483-3p, miR-483-5p, miR-210, and miR-21 were found overexpressed, while miR-195, miR-497, and miR-1974 were underexpressed in ACC. Inhibition of miR-483-3p or miR-483-5p and overexpression of miR-195 or miR-497 reduced cell proliferation in human NCI-H295R ACC cells. In addition, downregulation of miR-483-3p, but not miR-483-5p, and increased expression of miR-195 or miR-497 led to significant induction of cell death. Protein expression of p53 upregulated modulator of apoptosis (PUMA), a potential target of miR-483-3p, was significantly decreased in ACC, and inversely correlated with miR-483-3p expression. In addition, high expression of miR-503, miR-1202, and miR-1275 were found significantly associated with shorter overall survival among patients with ACC (P values: 0.006, 0.005, and 0.042 respectively). In summary, we identified additional miRNAs associated with ACC, elucidated the functional role of four miRNAs in the pathogenesis of ACC cells, demonstrated the potential involvement of the pro-apoptotic factor PUMA (a miR-483-3p target) in adrenocortical tumors, and found novel miRNAs associated with survival in ACC.

miR-185 and miR-133b deregulation is associated with overall survival and metastasis in colorectal cancer.

Colorectal cancer (CRC) is one of the most common and deadly forms of cancer. Despite improved treatment modalities, post-operative recurrence and metastasis remain the major problems for extending patient survival after surgery. This highlights the need to search for biomarkers for prognostication and treatment stratification of colorectal cancer patients. In this study, we applied the SYBR-green quantitative PCR-based array approach to screen for differentially expressed miRNAs between patients with short (<50 months, range 10-33 months) and long survival (≥ 50 months, range 50-152 months). The selected candidate prognostic miRNAs were validated in a cohort of 50 CRC patients by TaqMan quantitative PCR. We found that high expression of miR-185 and low expression of miR-133b were correlated with poor survival (p=0.001 and 0.028, respectively) and metastasis (p=0.007 and 0.036, respectively) in colorectal cancer. Our findings suggest the potential prognostic values of these miRNAs for predicting clinical outcome after surgery.

Clinical and functional impact of TARBP2 over-expression in adrenocortical carcinoma

Deregulation of microRNA (miRNA) expression in adrenocortical carcinomas (ACCs) has been documented to have diagnostic, prognostic, as well as functional implications. Here, we evaluated the mRNA expression of DROSHA, DGCR8, DICER (DICER1), TARBP2, and PRKRA, the core components in the miRNA biogenesis pathway, in a cohort of 73 adrenocortical tumors (including 43 adenomas and 30 carcinomas) and nine normal adrenal cortices using a RT-qPCR approach. Our results show a significant over-expression of TARBP2, DICER, and DROSHA in the carcinomas compared with adenomas or adrenal cortices (P<0.001 for all comparisons). Using western blot and immunohistochemistry analyses, we confirmed the higher expression of TARBP2, DICER, and DROSHA at the protein level in carcinoma cases. Furthermore, we demonstrate that mRNA expression of TARBP2, but not DICER or DROSHA, is a strong molecular predictor to discriminate between adenomas and carcinomas. Functionally, we showed that inhibition of TARBP2 expression in human NCI-H295R ACC cells resulted in a decreased cell proliferation and induction of apoptosis. TARBP2 over-expression was not related to gene mutations; however, copy number gain of the TARBP2 gene was observed in 57% of the carcinomas analyzed. In addition, we identified that miR-195 and miR-497 could directly regulate TARBP2 and DICER expression in ACC cells. This is the first study to demonstrate the deregulation of miRNA-processing factors in adrenocortical tumors and to show the clinical and biological impact of TARBP2 over-expression in this tumor type.

Role of microRNAs and microRNA machinery in the pathogenesis of diffuse large B-cell lymphoma

Deregulation of microRNA (miRNA) expression has been documented in diffuse large B-cell lymphoma (DLBCL). However, the impact of miRNAs and their machinery in DLBCL is not fully determined. Here, we assessed the role of miRNA expression and their processing genes in DLBCL development. Using microarray and RT-qPCR approaches, we quantified global miRNAs and core components of miRNA-processing genes expression in 75 DLBCLs (56 de novo and 19 transformed) and 10 lymph nodes (LN). Differential miRNA signatures were identified between DLBCLs and LNs, or between the de novo and transformed DLBCLs. We also identified subsets of miRNAs associated with germinal center B-cell phenotype, BCL6 and IRF4 expression, and clinical staging. In addition, we showed a significant over-expression of TARBP2 in de novo DLBCLs as compared with LNs, and decreased expression of DROSHA, DICER, TARBP2 and PACT in transformed as compared with de novo cases. Interestingly, cases with high TARBP2 and DROSHA expression had a poorer chemotherapy response. We further showed that TARBP2 can regulate miRNA-processing efficiency in DLBCLs, and its expression inhibition decreases cell growth and increases apoptosis in DLBCL cell lines. Our findings provide new insights for the understanding of miRNAs and its machinery in DLBCL.

miRNA-214 is related to invasiveness of human non-small cell lung cancer and directly regulates alpha protein kinase 2 expression.

The prognosis of non‐small cell lung cancer (NSCLC) is poor, since it has often metastasized to distant organs by the time of diagnosis. Therefore, biomarkers predicting metastasis are crucial. miRNAs play important roles in the regulation of different tumor cell processes, including metastasis. We recently showed that miRNA‐214 is linked to a radioresistant phenotype of NSCLC. miRNA‐214 has been linked to metastasis in other tumor types. Therefore, we examined the role of miRNA‐214 in the metastatic potential of NSCLC. We showed that downregulation of miRNA‐214 increased invasive potential, and conversely, overexpression of miRNA‐214 decreased invasiveness of NSCLC cells in vitro. Gene expression and bioinformatic analyses of NSCLC cells with ablated miRNA‐214, identified a number of metastasis‐related target genes, including pregnancy‐associated plasma protein A (PAPP‐A), alpha protein kinase 2 (ALPK2), cyclin‐dependent kinase 6 (CDK6) and tumor necrosis‐factor alpha‐induced protein 3 (TNFAIP3). These were validated on mRNA and protein level to be regulated by miRNA‐214. Through immunoprecipitation we showed that only ALPK2 is directly regulated by miRNA‐214. We also examined the protein expression of these four genes in NSCLC tumors with respect to metastatic potential. These results showed that NSCLC tumors express these proteins at moderate‐high levels in the nucleus, cytoplasm and/or plasma membrane although with no significant correlation to the overall survival or the metastatic potential of the patients. However, we also showed that the membrane‐localized PAPP‐A had a higher expression level compared to the cytoplasm‐localized. In conclusion, we show that low miRNA‐214 expression is linked to a higher invasive potential of NSCLC cells.

MicroRNA expression patterns associated with hyperfunctioning and non-hyperfunctioning phenotypes in adrenocortical adenomas

BACKGROUND The adrenocortical adenoma (ACA) entity includes aldosterone-producing adenoma (APA), cortisol-producing adenoma (CPA), and non-hyperfunctioning adenoma (NHFA) phenotypes. While gene mutations and mRNA expression profiles have been partly characterized, less is known about the alterations involving microRNA (miRNA) expression. AIM To characterize miRNA expression profile in relation to the subtypes of ACAs. SUBJECTS AND METHODS miRNA expression profiles were determined in 26 ACAs (nine APAs, ten CPAs, and seven NHFAs) and four adrenal references using microarray-based screening. Significance analysis of microarrays (SAM) was carried out to identify differentially expressed miRNAs between ACA and adrenal cortices or between tumor subtypes. Selected differentially expressed miRNAs were validated in an extended series of 43 ACAs and ten adrenal references by quantitative RT-PCR. RESULTS An hierarchical clustering revealed separate clusters for APAs and CPAs, while the NHFAs were found spread out within the APA/CPA clusters. When NHFA was excluded, the clustering analysis showed a better separation between APA and CPA. SAM analysis identified 40 over-expressed and three under-expressed miRNAs in the adenomas as compared with adrenal references. Fourteen miRNAs were common among the three ACA subtypes. Furthermore, we found specific miRNAs associated with different tumor phenotypes. CONCLUSION The results suggest that miRNA expression profiles can distinguish different subtypes of ACA, which may contribute to a deeper understanding of ACA development and potential therapeutics.

Loss of miR-514a-3p regulation of PEG3 activates the NF-kappa B pathway in human testicular germ cell tumors

Deregulation of microRNAs (miRNAs) contributes to the development and progression of many cancer types; however, their functions in the pathogenesis of testicular germ cell tumor (TGCT) remain unclear. Here, we determined miRNA expression profiles of TGCTs and normal testes using small RNA sequencing, and identified several deregulated miRNAs in TGCTs, including the miR-506~514 cluster. In functional studies in vitro we demonstrated that miR-514a-3p induced apoptosis through direct regulation of the paternally expressed gene 3 (PEG3), and ectopically expressed PEG3 could rescue the apoptotic effect of miR-514a-3p overexpression. Silencing of PEG3 or miR-514a-3p overexpression reduced nuclear accumulation of p50 and NF-κB reporter activity. Furthermore, PEG3 was co-immunoprecipitated with tumor necrosis factor receptor-associated factor 2 (TRAF2) in TGCT cell lysates. We propose a model of PEG3-mediated activation of NF-κB in TGCT. Loss of miR-514a-3p expression in TGCT increases PEG3 expression that recruits TRAF2 and activates the NF-kappa B pathway, which protects germ cells from apoptosis. Importantly, we observed strong expression of PEG3 and nuclear p50 in the majority of TGCTs (83% and 78%, respectively). In conclusion, our study describes a novel function for miR-514a-3p in TGCT and highlights an unrecognized mechanism of PEG3 regulation and NF-κB activation in TGCT.

Abstract 4940: Microrna expression profiling in adrenocortical neoplasia: Implications on tumor size and functionality

Background: Tumors arising from the adrenal cortex (ACT) can be hormonally hyperfunctioning or silent. This functional phenotype, as well as their size, is of clinical importance. MicroRNAs are a class of small non-coding RNAs that negatively regulates gene expression. There is increasing evidence for their significant role in tumorigenesis. Their relevance on the functional phenotype and size of these tumors has not been previously documented. Aim: To characterize differential expression patterns of microRNAs regarding the increasing size and the functional phenotypes of ACT. Methods: Seventy frozen tissue samples were included: 16 aldosterone-producing adenomas (APA), 14 cortisol producing adenomas (CPA), 14 non-hyperfunctioning adenomas (NhFA), 19 adrenocortical carcinomas (ACC) and 7 normal adrenal cortices as controls. Global microRNA expression profiles were determined using the Agilent Human microRNA microarray, and the results were verified by quantitative real-time PCR. Results: We observed distinct microRNA expression patterns among different ACT phenotypes: majority of the APA was clustered separately from NhFA and CPA, and the ACC was independently clustered apart from all benign tumors. A larger number of miRNAs were differentially expressed between APA and CPA, while a smaller subset of differently expressed microRNAs was found between functional and non-hyperfunctional adenomas. Among the benign tumors, we found miR-21 expression was significantly correlated with increasing tumor size. Conclusion: Deregulation of microRNA expression is associated with the functional phenotype and size in ACT, and these microRNAs may have implication related to the development of adrenocortical tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4940. doi:10.1158/1538-7445.AM2011-4940