Maddalena Milazzo

MiR-199a-3p Regulates mTOR and c-Met to Influence the Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

MicroRNAs (miRNA) have rapidly emerged as modulators of gene expression in cancer in which they may have great diagnostic and therapeutic import. MicroRNA-199a-3p (miR-199a-3p) is downregulated in several human malignancies including hepatocellular carcinoma (HCC). Here, we show that miR-199a-3p targets mammalian target of rapamycin (mTOR) and c-Met in HCC cells. Restoring attenuated levels of miR-199a-3p in HCC cells led to G(1)-phase cell cycle arrest, reduced invasive capability, enhanced susceptibility to hypoxia, and increased sensitivity to doxorubicin-induced apoptosis. These in vitro findings were confirmed by an analysis of human HCC tissues, which revealed an inverse correlation linking miR-199a-3p and mTOR as well as a shorter time to recurrence after HCC resection in patients with lower miR-199a-3p expression. These results suggest that tactics to regulate mTOR and c-Met by elevating levels of miR-199a-3p may have therapeutic benefits in highly lethal cancers such as HCC.

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)

Significance of serum and hepatic microRNA-122 levels in patients with non-alcoholic fatty liver disease.

Non‐alcoholic fatty liver disease (NAFLD) is believed to be a type of metabolic syndrome. MicroRNA‐122 (miR‐122) is the most abundant microRNA in the liver and is an important factor for the metabolism of glucose and lipids. In the present study, we examined the correlation between the hepatic and serum miR‐122 expression levels and the clinicopathological factors of patients with NAFLD.

Circulating microRNAs, miR-939, miR-595, miR-519d and miR-494, Identify Cirrhotic Patients with HCC

The performance of circulating biomarkers for the diagnosis of hepatocellular carcinoma (HCC) is sub-optimal. In this study we tested circulating microRNAs as biomarkers for HCC in cirrhotic patients by performing a two stage study: a discovery phase conducted by microarray and a validation phase performed by qRT-PCR in an independent series of 118 patients. Beside miRNAs emerged from the discovery phase, miR-21, miR-221, miR-519d were also tested in the validation setting on the basis of literary and tissue findings. Deregulated microRNAs were assayed in HCC-derived cells in the intracellular compartment, cell culture supernatant and exosomal fraction. Serum and tissue microRNA levels were compared in 14 patients surgically treated for HCC. From the discovery study, it emerged that seven circulating microRNAs were differentially expressed in cirrhotic patients with and without HCC. In the validation set, miR-939, miR-595 and miR-519d were shown to differentiate cirrhotic patients with and without HCC. MiR-939 and miR-595 are independent factors for HCC. ROC curves of miR-939, miR-595 and miR-519d displayed that AUC was higher than AFP. An exosomal secretion of miR-519d, miR-21, miR-221 and miR-1228 and a correlation between circulating and tissue levels of miR-519d, miR-494 and miR-21 were found in HCC patients. Therefore, we show that circulating microRNAs deserve attention as non-invasive biomarkers in the diagnostic setting of HCC and that exosomal secretion contributes to discharging a subset of microRNAs into the extracellular compartment.

Anti-Tumor Activity of a miR-199-dependent Oncolytic Adenovirus

The down-regulation of miR-199 occurs in nearly all primary hepatocellular carcinomas (HCCs) and HCC cell lines in comparison with normal liver. We exploited this miR-199 differential expression to develop a conditionally replication-competent oncolytic adenovirus, Ad-199T, and achieve tumor-specific viral expression and replication. To this aim, we introduced four copies of miR-199 target sites within the 3’ UTR of E1A gene, essential for viral replication. As consequence, E1A expression from Ad-199T virus was tightly regulated both at RNA and protein levels in HCC derived cell lines, and replication controlled by the level of miR-199 expression. Various approaches were used to asses in vivo properties of Ad-199T. Ad-199T replication was inhibited in normal, miR-199 positive, liver parenchyma, thus resulting in reduced hepatotoxicity. Conversely, the intrahepatic delivery of Ad-199T in newborn mice led to virus replication and fast removal of implanted HepG2 liver cancer cells. The ability of Ad-199T to control tumor growth was also shown in a subcutaneous xenograft model in nude mice and in HCCs arising in immune-competent mice. In summary, we developed a novel oncolytic adenovirus, Ad-199T, which could demonstrate a therapeutic potential against liver cancer without causing significant hepatotoxicity.

p53/mdm2 feedback loop sustains miR-221 expression and dictates the response to anticancer treatments in hepatocellular carcinoma.

The overexpression of microRNA-221 (miR-221) is reported in several human cancers including hepatocellular carcinoma, and its targeting by tailored treatments has been proposed. The evidence supporting the role of miR-221 in cancer is growing and has been mainly focused on the discovery of miR-221 targets as well as on its possible therapeutic exploitations. However, the mechanism sustaining miR-221 aberrant expression remains to be elucidated. In this study, MDM2 (E3 ubiquitin-protein ligase homolog), a known p53 (TP53) modulator, is identified as a direct target of miR-221, and a feed-forward loop is described that sustains miR-221 aberrant expression. Interestingly, miR-221 can activate the p53/mdm2 axis by inhibiting MDM2 and, in turn, p53 activation contributes to miR-221 enhanced expression. Moreover, by modulating the p53 axis, miR-221 impacts cell-cycle progression and apoptotic response to doxorubicin in hepatocellular carcinoma–derived cell lines. Finally, CpG island methylation status was assessed as a causative event associated with miR-221 upregulation in hepatocellular carcinoma cells and primary tumor specimens. In hepatocellular carcinoma–derived cell lines, pharmacologically induced DNA hypomethylation potentiated a significant increase in miR-221 expression. These data were confirmed in clinical specimens of hepatocellular carcinoma in which elevated miR-221 expression was associated with the simultaneous presence of wild-type p53 and DNA hypomethylation. Implications: These findings reveal a novel miR-221–sustained regulatory loop that determines a p53-context-specific response to doxorubicin treatment in hepatocellular carcinoma. Mol Cancer Res; 12(2); 203–16. ©2013 AACR.

Inflammation increases NOTCH1 activity via MMP9 and is counteracted by Eicosapentaenoic Acid-free fatty acid in colon cancer cells

Aberrant NOTCH1 signalling is critically involved in multiple models of colorectal cancer (CRC) and a prominent role of NOTCH1 activity during inflammation has emerged. Epithelial to Mesenchymal Transition (EMT), a crucial event promoting malignant transformation, is regulated by inflammation and Metalloproteinase-9 (MMP9) plays an important role in this process. Eicosapentaenoic Acid (EPA), an omega-3 polyunsaturated fatty acid, was shown to prevent colonic tumors in different settings. We recently found that an extra-pure formulation of EPA as Free Fatty Acid (EPA-FFA) protects from colon cancer development in a mouse model of Colitis-Associated Cancer (CAC) through modulation of NOTCH1 signalling. In this study, we exposed colon cancer cells to an inflammatory stimulus represented by a cytokine-enriched Conditioned Medium (CM), obtained from THP1-differentiated macrophages. We found, for the first time, that CM strongly up-regulated NOTCH1 signalling and EMT markers, leading to increased invasiveness. Importantly, NOTCH1 signalling was dependent on MMP9 activity, upon CM exposure. We show that a non-cytotoxic pre-treatment with EPA-FFA antagonizes the effect of inflammation on NOTCH1 signalling, with reduction of MMP9 activity and invasiveness. In conclusion, our data suggest that, in CRC cells, inflammation induces NOTCH1 activity through MMP9 up-regulation and that this mechanism can be counteracted by EPA-FFA.

Molecular and proteomic insight into Notch1 characterization in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) ranks fifth in frequency worldwide amongst all human cancers causing one million deaths annually. Despite many promising treatment options, long-term prognosis remains dismal for the majority of patients who develop recurrence or present with advanced disease. Notch signaling is an evolutionarily conserved pathway crucial for the development and homeostasis of many organs including liver. Herein we showed that aberrant Notch1 is linked to HCC development, tumor recurrence and invasion, which might be mediated, at least in part, through the Notch1-E-Cadherin pathway. Collectively, these findings suggest that targeting Notch1 has important therapeutic value in hepatocellular carcinoma. In this regard, comparative analysis of the secretome of HepG2 and HepG2 Notch1 depleted cells identified novel secreted proteins related to Notch1 expression. Soluble E-Cadherin (sE-Cad) and Thrombospondin-1 (Thbs1) were further validated in human serum as potential biomarkers to predict response to Notch1 inhibitors for a tailored individualized therapy.

Early prediction of treatment response to sorafenib with elastosonography in a mice xenograft model of hepatocellular carcinoma: a proof-of-concept study.

PURPOSE Sorafenib is the reference therapy for advanced hepatocellular carcinoma (HCC). There is no method for predicting in the early period subsequent individual response. Starting from the clinical experience in humans that subcutaneous metastases may rapidly change consistency under sorafenib and that elastosonography allows assessment of tissue elasticity, we investigated the role of this ultrasound-based technique in the early prediction of tumor response to sorafenib in a HCC mice model. MATERIALS AND METHODS HCC subcutaneous xenografting in mice was utilized. Mice were randomized to vehicle (17 mice) or treatment with sorafenib (19 mice). Strain elastosonography (Esaote, Italy) of the tumor mass was performed at different time points (day 0, + 2 and + 14 from treatment start) until the mice were sacrificed (day + 14). At the same time points, the volume was calculated with ultrasonography. RESULTS Sorafenib-treated mice showed a smaller increase in tumor size on day + 14 in comparison to vehicle-treated mice (tumor volume increase + 175 % vs. + 382 %, p = 0.009). The median tumor elasticity increased in both groups on day + 2 (+ 5.65 % and + 3.86 %, respectively) and decreased on day + 14 (-3.86 % and -3.63 %, respectively). However, among Sorafenib-treated tumors, 13 mice with a growth percentage delta < 200 % (considered as good treatment response) showed an increase in elasticity on day + 2 (+ 8.9 %, range -12.6 - + 64) while the other 6 with a growth percentage delta > 300 % (considered as poor treatment response) showed a decrease in elasticity (-17 %, range -30.2 - + 15.3) (p = 0.044). CONCLUSION Elastosonography appears to be a promising noninvasive new technique for the early treatment prediction of HCC tumor response to sorafenib. Specifically, an early increase in tumor elasticity (corresponding to tumors becoming softer) is associated with a good response.