Showing papers by "Carlo M. Croce published in 2010"

Targeting microRNAs in cancer: rationale, strategies and challenges.

Abstract: MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate gene expression. Early studies have shown that miRNA expression is deregulated in cancer and experimental data indicate that cancer phenotypes can be modified by targeting miRNA expression. Based on these observations, miRNA-based anticancer therapies are being developed, either alone or in combination with current targeted therapies, with the goal to improve disease response and increase cure rates. The advantage of using miRNA approaches is based on its ability to concurrently target multiple effectors of pathways involved in cell differentiation, proliferation and survival. In this Review, we describe the role of miRNAs in tumorigenesis and critically discuss the rationale, the strategies and the challenges for the therapeutic targeting of miRNAs in cancer.

Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis

Abstract: Summary Background Analyses of microRNA expression profiles have shown that many microRNAs are expressed aberrantly and correlate with tumorigenesis, progression, and prognosis of various haematological and solid tumours. We aimed to assess the relation between microRNA expression and progression and prognosis of gastric cancer. Methods 353 gastric samples from two independent subsets of patients from Japan were analysed by microRNA microarray. MicroRNA expression patterns were compared between non-tumour mucosa and cancer samples, graded by diffuse and intestinal histological types and by progression-related factors (eg, depth of invasion, metastasis, and stage). Disease outcome was calculated by multivariable regression analysis to establish whether microRNAs are independent prognostic factors. Findings In 160 paired samples of non-tumour mucosa and cancer, 22 microRNAs were upregulated and 13 were downregulated in gastric cancer; 292 (83%) samples were distinguished correctly by this signature. The two histological subtypes of gastric cancer showed different microRNA signatures: eight microRNAs were upregulated in diffuse-type and four in intestinal-type cancer. In the progression-related signature, miR-125b, miR-199a, and miR-100 were the most important microRNAs involved. Low expression of let-7g (hazard ratio 2·6 [95% CI 1·3–4·9]) and miR-433 (2·1 [1·1–3·9]) and high expression of miR-214 (2·4 [1·2–4·5]) were associated with unfavourable outcome in overall survival independent of clinical covariates, including depth of invasion, lymph-node metastasis, and stage. Interpretation MicroRNAs are expressed differentially in gastric cancers, and histological subtypes are characterised by specific microRNA signatures. Unique microRNAs are associated with progression and prognosis of gastric cancer. Funding National Cancer Institute.

miR-221 overexpression contributes to liver tumorigenesis

Abstract: MicroRNA (miRNAs) are negative regulators of gene expression and can function as tumor suppressors or oncogenes. Expression patterns of miRNAs and their role in the pathogenesis of hepatocellular carcinoma (HCC) are still poorly understood. We profiled miRNA expression in tissue samples (104 HCC, 90 adjacent cirrhotic livers, 21 normal livers) as well as in 35 HCC cell lines. A set of 12 miRNAs (including miR-21, miR-221/222, miR-34a, miR-519a, miR-93, miR-96, and let-7c) was linked to disease progression from normal liver through cirrhosis to full-blown HCC. miR-221/222, the most up-regulated miRNAs in tumor samples, are shown to target the CDK inhibitor p27 and to enhance cell growth in vitro. Conversely, these activities can be efficiently inhibited by an antagomiR specific for miR-221. In addition, we show, using a mouse model of liver cancer, that miR-221 overexpression stimulates growth of tumorigenic murine hepatic progenitor cells. Finally, we identified DNA damage-inducible transcript 4 (DDIT4), a modulator of mTOR pathway, as a bona fide target of miR-221. Taken together, these data reveal an important contribution for miR-221 in hepatocarcinogenesis and suggest a role for DDIT4 dysregulation in this process. Thus, the use of synthetic inhibitors of miR-221 may prove to be a promising approach to liver cancer treatment.

miR-15a and miR-16-1 in cancer: discovery, function and future perspectives

Abstract: MicroRNAs (miRNAs) encoded by the miR-15/16 cluster are known to act as tumor suppressors. Expression of these miRNAs inhibits cell proliferation, promotes apoptosis of cancer cells, and suppresses tumorigenicity both in vitro and in vivo. miR-15a and miR-16-1 function by targeting multiple oncogenes, including BCL2, MCL1, CCND1, and WNT3A. Down-regulation of these miRNAs has been reported in chronic lymphocytic lymphoma (CLL), pituitary adenomas, and prostate carcinoma. This review summarizes the discovery, functions, and clinical relevance of these miRNAs in cancer, particularly CLL.

Clinical response and miR-29b predictive significance in older AML patients treated with a 10-day schedule of decitabine

Abstract: A phase II clinical trial with single-agent decitabine was conducted in older patients (>or=60 years) with previously untreated acute myeloid leukemia (AML) who were not candidates for or who refused intensive chemotherapy. Subjects received low-dose decitabine at 20 mg/m(2) i.v. over 1 h on days 1 to 10. Fifty-three subjects enrolled with a median age of 74 years (range, 60-85). Nineteen (36%) had antecedent hematologic disorder or therapy-related AML; 16 had complex karyotypes (>or=3 abnormalities). The complete remission rate was 47% (n = 25), achieved after a median of three cycles of therapy. Nine additional subjects had no morphologic evidence of disease with incomplete count recovery, for an overall response rate of 64% (n = 34). Complete remission was achieved in 52% of subjects presenting with normal karyotype and in 50% of those with complex karyotypes. Median overall and disease-free survival durations were 55 and 46 weeks, respectively. Death within 30 days of initiation of treatment occurred in one subject (2%), death within 8 weeks in 15% of subjects. Given the DNA hypomethylating effect of decitabine, we examined the relationship of clinical response and pretreatment level of miR-29b, previously shown to target DNA methyltransferases. Higher levels of miR-29b were associated with clinical response (P = 0.02). In conclusion, this schedule of decitabine was highly active and well tolerated in this poor-risk cohort of older AML patients. Levels of miR-29b should be validated as a predictive factor for stratification of older AML patients to decitabine treatment.

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

Abstract: 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. Cancer Res; 70(12); 5184–93. ©2010 AACR.

Reprogramming of miRNA networks in cancer and leukemia

Abstract: We studied miRNA profiles in 4419 human samples (3312 neoplastic, 1107 nonmalignant), corresponding to 50 normal tissues and 51 cancer types. The complexity of our database enabled us to perform a detailed analysis of microRNA (miRNA) activities. We inferred genetic networks from miRNA expression in normal tissues and cancer. We also built, for the first time, specialized miRNA networks for solid tumors and leukemias. Nonmalignant tissues and cancer networks displayed a change in hubs, the most connected miRNAs. hsa-miR-103/106 were downgraded in cancer, whereas hsa-miR-30 became most prominent. Cancer networks appeared as built from disjointed subnetworks, as opposed to normal tissues. A comparison of these nets allowed us to identify key miRNA cliques in cancer. We also investigated miRNA copy number alterations in 744 cancer samples, at a resolution of 150 kb. Members of miRNA families should be similarly deleted or amplified, since they repress the same cellular targets and are thus expected to have similar impacts on oncogenesis. We correctly identified hsa-miR-17/92 family as amplified and the hsa-miR-143/145 cluster as deleted. Other miRNAs, such as hsa-miR-30 and hsa-miR-204, were found to be physically altered at the DNA copy number level as well. By combining differential expression, genetic networks, and DNA copy number alterations, we confirmed, or discovered, miRNAs with comprehensive roles in cancer. Finally, we experimentally validated the miRNA network with acute lymphocytic leukemia originated in Mir155 transgenic mice. Most of miRNAs deregulated in these transgenic mice were located close to hsa-miR-155 in the cancer network.

Single-nucleotide polymorphisms inside microRNA target sites influence tumor susceptibility.

Abstract: Single-nucleotide polymorphisms (SNP) associated with polygenetic disorders, such as breast cancer (BC), can create, destroy, or modify microRNA (miRNA) binding sites; however, the extent to which SNPs interfere with miRNA gene regulation and affect cancer susceptibility remains largely unknown. We hypothesize that disruption of miRNA target binding by SNPs is a widespread mechanism relevant to cancer susceptibility. To test this, we analyzed SNPs known to be associated with BC risk, in silico and in vitro, for their ability to modify miRNA binding sites and miRNA gene regulation and referred to these as target SNPs. We identified rs1982073-TGFB1 and rs1799782-XRCC1 as target SNPs, whose alleles could modulate gene expression by differential interaction with miR-187 and miR-138, respectively. Genome-wide bioinformatics analysis predicted ∼64% of transcribed SNPs as target SNPs that can modify (increase/decrease) the binding energy of putative miRNA::mRNA duplexes by >90%. To assess whether target SNPs are implicated in BC susceptibility, we conducted a case-control population study and observed that germline occurrence of rs799917-BRCA1 and rs334348-TGFR1 significantly varies among populations with different risks of developing BC. Luciferase activity of target SNPs, allelic variants, and protein levels in cancer cell lines with different genotypes showed differential regulation of target genes following overexpression of the two interacting miRNAs (miR-638 and miR-628-5p). Therefore, we propose that transcribed target SNPs alter miRNA gene regulation and, consequently, protein expression, contributing to the likelihood of cancer susceptibility, by a novel mechanism of subtle gene regulation. Cancer Res; 70(7); 2789–98

MicroRNA-21 induces resistance to 5-fluorouracil by down-regulating human DNA MutS homolog 2 (hMSH2)

Abstract: The overexpression of microRNA-21 (miR-21) is linked to a number of human tumors including colorectal cancer, where it appears to regulate the expression of tumor suppressor genes including p21, phosphatase and tensin homolog, TGFβ receptor II, and B-cell leukemia/lymphoma 2 -associated X protein. Here we demonstrate that miR-21 targets and down-regulates the core mismatch repair (MMR) recognition protein complex, human mutS homolog 2 (hMSH2) and 6 (hMSH6). Colorectal tumors that express a high level of miR-21 display reduced hMSH2 protein expression. Cells that overproduce miR-21 exhibit significantly reduced 5-fluorouracil (5-FU)-induced G2/M damage arrest and apoptosis that is characteristic of defects in the core MMR component. Moreover, xenograft studies demonstrate that miR-21 overexpression dramatically reduces the therapeutic efficacy of 5-FU. These studies suggest that the down-regulation of the MMR mutator gene associated with miR-21 overexpression may be an important clinical indicator of therapeutic efficacy in colorectal cancer.

Modulation of mismatch repair and genomic stability by miR-155

Abstract: Inactivation of mismatch repair (MMR) is the cause of the common cancer predisposition disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), as well as 10–40% of sporadic colorectal, endometrial, ovarian, gastric, and urothelial cancers. Elevated mutation rates (mutator phenotype), including simple repeat instability [microsatellite instability (MSI)] are a signature of MMR defects. MicroRNAs (miRs) have been implicated in the control of critical cellular pathways involved in development and cancer. Here we show that overexpression of miR-155 significantly down-regulates the core MMR proteins, hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and MSI. An inverse correlation between the expression of miR-155 and the expression of MLH1 or MSH2 proteins was found in human colorectal cancer. Finally, a number of MSI tumors with unknown cause of MMR inactivation displayed miR-155 overexpression. These data provide support for miR-155 modulation of MMR as a mechanism of cancer pathogenesis.

A network connecting Runx2, SATB2, and the miR-23a~27a~24-2 cluster regulates the osteoblast differentiation program.

Abstract: Induced osteogenesis includes a program of microRNAs (miRs) to repress the translation of genes that act as inhibitors of bone formation How expression of bone-related miRs is regulated remains a compelling question Here we report that Runx2, a transcription factor essential for osteoblastogenesis, negatively regulates expression of the miR cluster 23a∼27a∼24-2 Overexpression, reporter, and chromatin immunoprecipitation assays established the presence of a functional Runx binding element that represses expression of these miRs Consistent with this finding, exogenous expression of each of the miRs suppressed osteoblast differentiation, whereas antagomirs increased bone marker expression The biological significance of Runx2 repression of this miR cluster is that each miR directly targets the 3′ UTR of SATB2, which is known to synergize with Runx2 to facilitate bone formation The findings suggest Runx2-negative regulation of multiple miRs by a feed-forward mechanism to cause derepression of SATB2 to promote differentiation We find also that miR-23a represses Runx2 in the terminally differentiated osteocyte, representing a feedback mechanism to attenuate osteoblast maturation We provide direct evidence for an interdependent relationship among transcriptional inhibition of the miR cluster by Runx2, translational repression of Runx2 and of SATB2 by the cluster miRs during progression of osteoblast differentiation Furthermore, miR cluster gain of function (ie, inhibition of osteogenesis) is rescued by the exogenous expression of SATB2 Taken together, we have established a regulatory network with a central role for the miR cluster 23a∼27a∼24-2 in both progression and maintenance of the osteocyte phenotype

MicroRNA Cluster 221-222 and Estrogen Receptor α Interactions in Breast Cancer

Abstract: Background Several lines of evidence have suggested that estrogen receptor α (ERα)–negative breast tumors, which are highly aggressive and nonresponsive to hormonal therapy, arise from ERα-positive precursors through different molecular pathways. Because microRNAs (miRNAs) modulate gene expression, we hypothesized that they may have a role in ER-negative tumor formation.

Oncogenic Role of miR-483-3p at the IGF2/483 Locus

Abstract: hsa-mir-483 is located within intron 2 of the IGF2 locus. We found that the mature microRNA (miRNA) miR-483-3p is overexpressed in 100% of Wilms9 tumors. In addition, colon, breast, and liver cancers exhibit high or even extremely high levels of miR-483-3p in ∼30% of the cases. A coregulation with IGF2 mRNA was detected, although some tumors exhibited high expression of miR-483-3p without a concomitant increase of IGF2. These findings suggested that miR-483-3p could cooperate with IGF2 or act as an autonomous oncogene. Indeed, here we prove that an anti-miRNA oligonucleotide against miR-483-3p could inhibit the miRNAs without affecting IGF2 mRNA and it could suppress tumorigenicity of HepG2 cells, a cell line that overexpresses miR-483-3p and IGF2. Conversely, no antitumor effect was elicited by inhibition of IGF2. The oncogenic mechanism of miR-483-3p was at least partially clarified by the finding that it could modulate the proapoptotic protein BBC3/PUMA and miR-483-3p enforced expression could protect cells from apoptosis. Our results indicate that miR-483-3p could function as an antiapoptotic oncogene in various human cancers and reveal a new, potentially important target for anticancer therapy. Cancer Res; 70(8); 3140–9. ©2010 AACR.

Epigenetically Deregulated microRNA-375 Is Involved in a Positive Feedback Loop with Estrogen Receptor α in Breast Cancer Cells

Abstract: Estrogen receptor α (ERα) upregulation causes abnormal cell proliferation in about two thirds of breast cancers, yet understanding of the underlying mechanisms remains incomplete. Here, we show that high expression of the microRNA miR-375 in ERα-positive breast cell lines is a key driver of their proliferation. miR-375 overexpression was caused by loss of epigenetic marks including H3K9me2 and local DNA hypomethylation, dissociation of the transcriptional repressor CTCF from the miR-375 promoter, and interactions of ERα with regulatory regions of miR-375. Inhibiting miR-375 in ERα-positive MCF-7 cells resulted in reduced ERα activation and cell proliferation. A combination of expression profiling from tumor samples and miRNA target prediction identified RASD1 as a potential miR-375 target. Mechanistic investigations revealed that miR-375 regulates RASD1 by targeting the 3′ untranslated region in RASD1 mRNA. Additionally, we found that RASD1 negatively regulates ERα expression. Our findings define a forward feedback pathway in control of ERα expression, highlighting new strategies to treat ERα-positive invasive breast tumors. Cancer Res; 70(22); 9175–84. ©2010 AACR.

MiR-145 participates with TP53 in a death-promoting regulatory loop and targets estrogen receptor-α in human breast cancer cells

Abstract: Understanding the consequences of miR-145 reintroduction in human breast cancer (BC) could reveal its tumor-suppressive functions and may disclose new aspects of BC biology. Therefore, we characterized the effects of miR-145 re-expression in BC cell lines by using proliferation and apoptosis assays. As a result, we found that miR-145 exhibited a pro-apoptotic effect, which is dependent on TP53 activation, and that TP53 activation can, in turn, stimulate miR-145 expression, thus establishing a death-promoting loop between miR-145 and TP53. We also found that miR-145 can downregulate estrogen receptor-α (ER-α) protein expression through direct interaction with two complementary sites within its coding sequence. In conclusion, we described a tumor suppression function of miR-145 in BC cell lines, and we linked miR-145 to TP53 and ER-α. Moreover, our findings support a view that miR-145 re-expression therapy could be mainly envisioned in the specific group of patients with ER-α-positive and/or TP53 wild-type tumors.

Resveratrol decreases the levels of miR-155 by upregulating miR-663, a microRNA targeting JunB and JunD.

Abstract: An inflammatory component is present in the microenvironment of most neoplastic tissues, including those not causally related to an obvious inflammatory process. Several microRNAs, and especially miR-155, play an essential role in both the innate and adaptative immune response. Resveratrol (trans-3,4',5-trihydroxystilbene) is a natural antioxidant with anti-inflammatory properties that is currently at the stage of preclinical studies for human cancer prevention. Here, we establish that, in human THP-1 monocytic cells as well as in human blood monocytes, resveratrol upregulates miR-663, a microRNA potentially targeting multiple genes implicated in the immune response. In THP-1 cells, miR-663 decreases endogenous activator protein-1 (AP-1) activity and impairs its upregulation by lipopolysaccharides (LPS), at least in part by directly targeting JunB and JunD transcripts. We further establish that the downregulation of AP-1 activity by resveratrol is miR-663 dependent and that the effects of resveratrol on both AP-1 activity and JunB levels are dose dependent. Finally, we show that resveratrol impairs the upregulation of miR-155 by LPS in a miR-663-dependent manner. Given the role of miR-155 in the innate immune response and the fact that it is upregulated in many cancers, our results suggest that manipulating miR-663 levels may help to optimize the use of resveratrol as both an anti-inflammatory and anticancer agent against malignancies associated with high levels of miR-155.

CpG island hypermethylation-associated silencing of non-coding RNAs transcribed from ultraconserved regions in human cancer

Abstract: Although only 1.5% of the human genome appears to code for proteins, much effort in cancer research has been devoted to this minimal fraction of our DNA. However, the last few years have witnessed the realization that a large class of non-coding RNAs (ncRNAs), named microRNAs, contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also shown that epigenetic silencing of microRNAs with tumor suppressor features by CpG island hypermethylation is a common hallmark of human tumors. Thus, we wondered whether there were other ncRNAs undergoing aberrant DNA methylation-associated silencing in transformed cells. We focused on the transcribed-ultraconserved regions (T-UCRs), a subset of DNA sequences that are absolutely conserved between orthologous regions of the human, rat and mouse genomes and that are located in both intra- and intergenic regions. We used a pharmacological and genomic approach to reveal the possible existence of an aberrant epigenetic silencing pattern of T-UCRs by treating cancer cells with a DNA-demethylating agent followed by hybridization to an expression microarray containing these sequences. We observed that DNA hypomethylation induces release of T-UCR silencing in cancer cells. Among the T-UCRs that were reactivated upon drug treatment, Uc.160+, Uc283+A and Uc.346+ were found to undergo specific CpG island hypermethylation-associated silencing in cancer cells compared with normal tissues. The analysis of a large set of primary human tumors (n=283) demonstrated that hypermethylation of the described T-UCR CpG islands was a common event among the various tumor types. Our finding that, in addition to microRNAs, another class of ncRNAs (T-UCRs) undergoes DNA methylation-associated inactivation in transformed cells supports a model in which epigenetic and genetic alterations in coding and non-coding sequences cooperate in human tumorigenesis.

Stem cell-like micro-RNA signature driven by Myc in aggressive liver cancer

Abstract: Myc activation has been implicated in the pathogenesis of hepatoblastoma (HB), a rare embryonal neoplasm derived from liver progenitor cells. Here, microRNA (miR) expression profiling of 65 HBs evidenced differential patterns related to developmental stage and Myc activity. Undifferentiated aggressive HBs overexpressed the miR-371–3 cluster with concomitant down-regulation of the miR-100/let-7a-2/miR-125b-1 cluster, evoking an ES cell expression profile. ChIP and Myc inhibition assays in hepatoma cells demonstrated that both miR clusters are regulated by Myc in an opposite manner. We show that the two miR clusters exert antagonistic effects on cell proliferation and tumorigenicity. Moreover, their combined deregulation cooperated in modulating the hepatic tumor phenotype, implicating stem cell-like regulation of Myc-dependent miRs in poorly differentiated HBs. Importantly, a four-miR signature representative of these clusters efficiently stratified HB patients, and when applied to 241 hepatocellular carcinomas (HCCs), it identified invasive tumors with a poor prognosis. Our data argue that Myc-driven reprogramming of miR expression patterns contributes to the aggressive phenotype of liver tumors originating from hepatic progenitor cells.

miR-212 Increases Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Sensitivity in Non–Small Cell Lung Cancer by Targeting the Antiapoptotic Protein PED

Abstract: PED/PEA-15 (PED) is a death effector domain family member of 15 kDa with a broad antiapoptotic function found overexpressed in a number of different human tumors, including lung cancer. To date, the mechanisms that regulate PED expression are unknown. Therefore, we address this point by the identification of microRNAs that in non-small cell lung cancer (NSCLC) modulate PED levels. In this work, we identify miR-212 as a negative regulator of PED expression. We also show that ectopic expression of this miR increases tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death in NSCLC cells. In contrast, inhibition of endogenous miR-212 by use of antago-miR results in increase of PED protein expression and resistance to TRAIL treatment. Besides, in NSCLC, we show both in vitro and in vivo that PED and miR-212 expressions are inversely correlated, that is, PED is upregulated and miR-212 is rarely expressed. In conclusion, these findings suggest that miR-212 should be considered as a tumor suppressor because it negatively regulates the antiapoptotic protein PED and regulates TRAIL sensitivity.

Disrupted microRNA expression caused by Mecp2 loss in a mouse model of Rett syndrome

Abstract: MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate post-transcriptional gene expression. They influence a wide range of physiological functions, including neuronal processes, and are regulated by various mechanisms, such as DNA methylation. This epigenetic mark is recognized by transcriptional regulators such as the methyl CpG binding protein Mecp2. Rett syndrome is a complex neurological disorder that has been associated with mutations in the gene coding for Mecp2. Thus, we examined the possible miRNA misregulation caused by Mecp2 absence in a mouse model of Rett syndrome. Using miRNA expression microarrays, we observed that the brain of Rett syndrome mice undergoes a disruption of the expression profiles of miRNAs. Among the significantly altered miRNAs (26%, 65 of 245), overall downregulation of these transcripts was the most common feature (71%), whilst the remaining 30% were upregulated. Further validation by quantitative RT-PCR demonstrated that the most commonly disrupted miRNAs we...

Chemoprevention of Cigarette Smoke–Induced Alterations of MicroRNA Expression in Rat Lungs

Abstract: We previously showed that exposure to environmental cigarette smoke (ECS) for 28 days causes extensive downregulation of microRNA expression in the lungs of rats, resulting in the overexpression of multiple genes and proteins. In the present study, we evaluated by microarray the expression of 484 microRNAs in the lungs of either ECS-free or ECS-exposed rats treated with the orally administered chemopreventive agents N-acetylcysteine, oltipraz, indole-3-carbinol, 5,6-benzoflavone, and phenethyl isothiocyanate (as single agents or in combinations). This is the first study of microRNA modulation by chemopreventive agents in nonmalignant tissues. Scatterplot, hierarchical cluster, and principal component analyses of microarray and quantitative PCR data showed that none of the above chemopreventive regimens appreciably affected the baseline microRNA expression, indicating potential safety. On the other hand, all of them attenuated ECS-induced alterations but to a variable extent and with different patterns, indicating potential preventive efficacy. The main ECS-altered functions that were modulated by chemopreventive agents included cell proliferation, apoptosis, differentiation, Ras activation, P53 functions, NF-kappaB pathway, transforming growth factor-related stress response, and angiogenesis. Some microRNAs known to be polymorphic in humans were downregulated by ECS and were protected by chemopreventive agents. This study provides proof-of-concept and validation of technology that we are further refining to screen and prioritize potential agents for continued development and to help elucidate their biological effects and mechanisms. Therefore, microRNA analysis may provide a new tool for predicting at early carcinogenesis stages both the potential safety and efficacy of cancer chemopreventive agents.

MicroRNAs as regulators of death receptors signaling

Abstract: Death receptors, belonging to the TNF receptor superfamily, induce apoptosis through two different pathways, one involving the effector caspases directly (type I cells or mitochondria-independent death), the other one amplifying the death signal through the mitochondrial pathway (type II cells or mitochondria-dependent death). MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate the stability or translational efficiency of targeted messenger RNAs. MiRNAs are involved in many cellular processes that are altered in cancer, such as differentiation, proliferation and apoptosis. In this review we will discuss recent findings implicating miRNAs as regulators of death receptors and pro- and antiapoptotic genes involved in programmed cell death pathways.

Hepatitis C Virus Proteins Modulate MicroRNA Expression and Chemosensitivity in Malignant Hepatocytes

Abstract: Purpose: Hepatocellular cancer (HCC) is highly resistant to chemotherapy and is associated with poor prognosis. Chronic hepatitis C virus (HCV) infection is a major cause of HCC. However, the effect of viral proteins in mediating chemosensitivity in tumor cells is unknown. We postulated that HCV viral proteins could modulate therapeutic responses by altering host cell microRNA (miRNA) expression. Experimental Design: HepG2 malignant hepatocytes were stably transfected with full-length HCV genome (Hep-394) or an empty vector (Hep-SWX). MiRNA profiling was done by using a custom microarray, and the expression of selected miRNAs was validated by real-time PCR. Protein expression was assessed by Western blotting, whereas caspase activation was assessed by a luminometric assay. Results: The IC 50 to sorafenib was lower in Hep-394 compared with Hep-SWX control cells. Alterations in miRNA expression occurred with 10 miRNAs downregulated >2-fold and 23 miRNAs upregulated >2-fold in Hep-394 cells compared with controls. Of these, miR-193b was overexpressed by 5-fold in Hep-394 cells. miR-193b was predicted to target Mcl-1, an antiapoptotic protein that can modulate the response to sorafenib. The expression of Mcl-1 was decreased, and basal caspase-3/7 activity and poly ADP ribose polymerase cleavage were increased in Hep-394 cells compared with controls. Moreover, transfection with precursors to miR-193b decreased both Mcl-1 expression and the IC 50 to sorafenib. Conclusions : Cellular expression of full-length HCV increases sensitivity to sorafenib by the miRNA-dependent modulation of Mcl-1 and apoptosis. Modulation of miRNA responses may be a useful strategy to enhance response to chemotherapy in HCC. Clin Cancer Res; 16(3); 957–66