Silvia Sabbioni

MicroRNA Gene Expression Deregulation in Human Breast Cancer

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.

Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

We investigated the role of microRNAs (miRNAs) in the pathogenesis of human hepatocellular carcinoma (HCC). A genome-wide miRNA microarray was used to identify differentially expressed miRNAs in HCCs arisen on cirrhotic livers. Thirty-five miRNAs were identified. Several of these miRNAs were previously found deregulated in other human cancers, such as members of the let-7 family, mir-221, and mir-145. In addition, the hepato-specific miR-122a was found down-regulated in approximately 70% of HCCs and in all HCC-derived cell lines. Microarray data for let-7a, mir-221, and mir-122a were validated by Northern blot and real-time PCR analysis. Understanding the contribution of deregulated miRNAs to cancer requires the identification of gene targets. Here, we show that miR-122a can modulate cyclin G1 expression in HCC-derived cell lines and an inverse correlation between miR-122a and cyclin G1 expression exists in primary liver carcinomas. These results indicate that cyclin G1 is a target of miR-122a and expand our knowledge of the molecular alterations involved in HCC pathogenesis and of the role of miRNAs in human cancer.

MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma

The identification of target mRNAs is a key step for assessing the role of aberrantly expressed microRNAs in human cancer. MiR-221 is upregulated in human hepatocellular carcinoma (HCC) as well as in other malignancies. One proven target of miR-221 is CDKN1B/p27, whose downregulation affects HCC prognosis. Here, we proved that the cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 is also a direct target of miR-221. Indeed, downregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to miR-221 transfection into HCC-derived cells and a significant upregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to antimiR-221 transfection. A direct interaction of miR-221 with a target site on the 3′ UTR of CDKN1C/p57 mRNA was also demonstrated. By controlling these two CDKIs, upregulation of miR-221 can promote growth of HCC cells by increasing the number of cells in S-phase. To assess the relevance of these studies in primary tumors, matched HCC and cirrhosis samples were assayed for miR-221, for CDKN1B/p27 and CDKN1C/p57 expression. MiR-221 was upregulated in 71% of HCCs, whereas CDKN1B/p27 and CDKN1C/p57 proteins were downregulated in 77% of cases. A significant inverse correlation between miR-221 and both CDKN1B/p27 and CDKN1C/p57 was found in HCCs. In conclusion, we suggest that miR-221 has an oncogenic function in hepatocarcinogenesis by targeting CDKN1B/p27 and CDKN1C/p57, hence promoting proliferation by controlling cell-cycle inhibitors. These findings establish a basis toward the development of therapeutic strategies aimed at blocking miR-221 in HCC.

MiR-122/cyclin G1 interaction modulates p53 activity and affects doxorubicin sensitivity of human hepatocarcinoma cells

The identification of target genes is a key step for assessing the role of aberrantly expressed microRNAs (miRNA) in human cancer and for the further development of miRNA-based gene therapy. MiR-122 is a liver-specific miRNA accounting for 70% of the total miRNA population. Its down-regulation is a common feature of both human and mouse hepatocellular carcinoma (HCC). We have previously shown that miR-122 can regulate the expression of cyclin G1, whose high levels have been reported in several human cancers. We evaluated the role of miR-122 and cyclin G1 expression in hepatocarcinogenesis and in response to treatment with doxorubicin and their relevance on survival and time to recurrence (TTR) of HCC patients. We proved that, by modulating cyclin G1, miR-122 influences p53 protein stability and transcriptional activity and reduces invasion capability of HCC-derived cell lines. In addition, in a therapeutic perspective, we assayed the effects of a restored miR-122 expression in triggering doxorubicin-induced apoptosis and we proved that miR-122, as well as cyclin G1 silencing, increases sensitivity to doxorubicin challenge. In patients resected for HCC, lower miR-122 levels were associated with a shorter TTR, whereas higher cyclin G1 expression was related to a lower survival, suggesting that miR-122 might represent an effective molecular target for HCC. Our findings establish a basis toward the development of combined chemo- and miRNA-based therapy for HCC treatment.

MicroRNA-221 Targets Bmf in Hepatocellular Carcinoma and Correlates with Tumor Multifocality

Deregulated cell proliferation and apoptosis play a major role in hepatocellular carcinoma (HCC). MicroRNAs participate in the modulation of key molecules linked to hepatocarcinogenesis. Purpose: This study aims to investigate the role of miR-221 in the modulation of Bmf, a proapoptotic BH3-only protein, and to characterize miR-221 contribution to hepatocarcinogenesis through modulation of apoptosis. Experimental Design: Transfection of miR-221 and anti-miR-221 in HCC-derived cell lines and luciferase reporter assay were used to assess Bmf as a target of miR-221. Modulation of miR-221 and Bmf expression contributed to characterize their role in anoikis. Primary HCC tissues were analyzed to assess the clinical relevance of in vitro findings. Results: Enforced miR-221 expression caused Bmf down-regulation, whereas anti-miR-221 induced its up-regulation. A luciferase reporter assay confirmed Bmf as a target of miR-221. Following matrix detachment, miR-221 silencing led to increased apoptotic cell death. The analysis of HCC tissues revealed an inverse correlation between miR-221 and Bmf expression and a direct correlation between Bmf and activated caspase-3, as a marker of apoptosis. High miR-221 levels were associated with tumor multifocality and reduced time to recurrence after surgery. Conclusions: Our results indicate that miR-221, by targeting Bmf, inhibits apoptosis. Moreover, in HCC, miR-221 overexpression is associated with a more aggressive phenotype. These findings, together with the previously reported modulation of CDKN1B/p27 and CDKN1C/p57, show that miR-221 simultaneously affects multiple pro-oncogenic pathways and suggest miR-221 as a potential target for nonconventional treatment against HCC. (Clin Cancer Res 2009;15(16):5073–81)

MicroRNA involvement in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third cause of cancer‐related death worldwide. Curative options for HCC are limited and exclusively available for patients carrying an early stage HCC. In advanced stages, traditional chemotherapy proved to be only marginally effective or even toxic. Thus, the identification of new treatment options is needed. New targets for non‐conventional treatment will necessarily take advantage of progresses on the molecular pathogenesis of HCC. MicroRNAs (miRNAs) are a group of tiny RNAs with a fundamental role in the regulation of gene expression. Aberrant expression of several miRNAs was found to be involved in human hepatocarcinogenesis. miRNA expression signatures were correlated with bio‐pathological and clinical features of HCC. In some cases, aberrantly expressed miRNAs could be linked to cancer‐associated pathways, indicating a direct role in liver tumourigenesis. For example, up‐regulation of mir‐221 and mir‐21 could promote cell cycle progression, reduce cell death and favour angiogenesis and invasion. These findings suggest that miRNAs could become novel molecular targets for HCC treatment. The demonstration of in vivo efficacy and safety of anti‐miRNA compounds has opened the way to their use in clinical trials.

MicroRNAs in human cancer: from research to therapy.

Numerous miRNAs are deregulated in human cancers, and experimental evidence indicates that they can play roles as oncogenes or tumor suppressor genes. Similarly to cancer genes that encode proteins, deregulation of miRNA-encoding genes is associated with genetic or epigenetic alterations, such as deletions, amplifications, point mutations and aberrant DNA methylation. The discovery that miRNAs interact with known oncogenes has established further links with molecular pathways implicated in malignant transformation. Finally, miRNAs can be used as diagnostic markers, and their potential as therapeutic molecules has moved miRNAs from the area of basic research to the field of cancer biotechnology.

In hepatocellular carcinoma miR-519d is up-regulated by p53 and DNA hypomethylation and targets CDKN1A/p21, PTEN, AKT3 and TIMP2.

MiR‐519d belongs to the chromosome 19 miRNA cluster (C19MC), the largest human miRNA cluster. One of its members, miR‐519d, is over‐expressed in hepatocellular carcinoma (HCC) and we characterized its contribution to hepatocarcinogenesis. In HCC cells, the over‐expression of miR‐519d promotes cell proliferation, invasion and impairs apoptosis following anticancer treatments. These functions are, at least in part, exerted through the direct targeting of CDKN1A/p21, PTEN, AKT3 and TIMP2. The mechanisms underlying miR‐519d aberrant expression in HCC were assayed by genomic DNA amplification, methylation analysis and ChIP assay. The aberrant hypomethylation of C19MC and TP53 were respectively identified as an epigenetic change allowing the aberrant expression of miR‐519d and one of the factors able to activate its transcription. In conclusion, we assessed the oncogenic role of miR‐519d in HCC by characterizing its biological functions, including the modulation of response to anticancer treatments and by identifying CDKN1A/p21, PTEN, AKT3 and TIMP2 among its targets. Copyright

Liver tumorigenicity promoted by microRNA‐221 in a mouse transgenic model

MicroRNA‐221 (miR‐221) is one of the most frequently and consistently up‐regulated microRNAs (miRNAs) in human cancer. It has been hypothesized that miR‐221 may act as a tumor promoter. To demonstrate this, we developed a transgenic (TG) mouse model that exhibits an inappropriate overexpression of miR‐221 in the liver. Immunoblotting and immunostaining confirmed a concomitant down‐regulation of miR‐221 target proteins. This TG model is characterized by the emergence of spontaneous nodular liver lesions in approximately 50% of male mice and by a strong acceleration of tumor development in 100% of mice treated with diethylnitrosamine. Similarly to human hepatocellular carcinoma, tumors are characterized by a further increase in miR‐221 expression and a concomitant inhibition of its target protein‐coding genes (i.e., cyclin‐dependent kinase inhibitor [Cdkn]1b/p27, Cdkn1c/p57, and B‐cell lymphoma 2–modifying factor). To validate the tumor‐promoting effect of miR‐221, we showed that in vivo delivery of anti‐miR‐221 oligonucleotides leads to a significant reduction of the number and size of tumor nodules. Conclusions: This study not only establishes that miR‐221 can promote liver tumorigenicity, but it also establishes a valuable animal model to perform preclinical investigations for the use of anti‐miRNA approaches aimed at liver cancer therapy. (HEPATOLOGY 2012;56:1025–1033)

Frequent aberrant methylation of the CDH4 gene promoter in human colorectal and gastric cancer

Gene promoter methylation causes loss of tumor suppressor genes function in human cancer. Here, we show that the CDH4 gene, a member of the cadherin family encoding for R-cadherin, contains a CpG island located at the 5′ of the first exon, which functions as a promoter element and is frequently affected by methylation in human cancer. By using methylation-specific PCR and reverse transcription-PCR in human cancer cell lines, promoter methylation could be directly linked to loss of gene expression. After treatment with the demethylating agent 5-aza-2-deoxycytidine, expression could be restored. Analysis of human primary tumors revealed that the CDH4 gene is methylated in 78% (38 of 49) of colorectal and 95% (20 of 21) of gastric carcinomas. CDH4 methylation was not detected in nonneoplastic colonic (0 of 10) and stomach (0 of 10) tissues or in peripheral blood (0 of 17). CDH4 methylation was detected in histologically normal tissues located in proximity of the neoplasms, indicating that CDH4 methylation is an early event in gastrointestinal tumor progression. We also proved that CDH4 methylation can be revealed in the peripheral blood of cancer patients. Our results indicate that CDH4 may act as a tumor suppressor gene in human gastrointestinal tumors and can potentially be used as an early diagnostic marker for gastrointestinal tumorigenesis.