Multiomics Profiling Reveals Signatures of Dysmetabolism in Urban Populations in Central India.

BACKGROUND: Non-communicable diseases (NCDs) have become a major cause of morbidity and mortality in India. Perturbation of host-microbiome interactions may be a key mechanism by which lifestyle-related risk factors such as tobacco use, alcohol consumption, and physical inactivity may influence metabolic health. There is an urgent need to identify relevant dysmetabolic traits for predicting risk of metabolic disorders, such as diabetes, among susceptible Asian Indians where NCDs are a growing epidemic. METHODS: Here, we report the first in-depth phenotypic study in which we prospectively enrolled 218 adults from urban and rural areas of Central India and used multiomic profiling to identify relationships between microbial taxa and circulating biomarkers of cardiometabolic risk. Assays included fecal microbiota analysis by 16S ribosomal RNA gene amplicon sequencing, quantification of serum short chain fatty acids by gas chromatography-mass spectrometry, and multiplex assaying of serum diabetic proteins, cytokines, chemokines, and multi-isotype antibodies. Sera was also analysed for N-glycans and immunoglobulin G Fc N-glycopeptides. RESULTS: Multiple hallmarks of dysmetabolism were identified in urbanites and young overweight adults, the majority of whom did not have a known diagnosis of diabetes. Association analyses revealed several host-microbe and metabolic associations. CONCLUSIONS: Host-microbe and metabolic interactions are differentially shaped by body weight and geographic status in Central Indians. Further exploration of these links may help create a molecular-level map for estimating risk of developing metabolic disorders and designing early interventions.

Akt3 induces oxidative stress and DNA damage by activating the NADPH oxidase via phosphorylation of p47phox.

Akt activation up-regulates the intracellular levels of reactive oxygen species (ROS) by inhibiting ROS scavenging. Of the Akt isoforms, Akt3 has also been shown to up-regulate ROS by promoting mitochondrial biogenesis. Here, we employ a set of isogenic cell lines that express different Akt isoforms, to show that the most robust inducer of ROS is Akt3. As a result, Akt3-expressing cells activate the DNA damage response pathway, express high levels of p53 and its direct transcriptional target miR-34, and exhibit a proliferation defect, which is rescued by the antioxidant N-acetylcysteine. The importance of the DNA damage response in the inhibition of cell proliferation by Akt3 was confirmed by Akt3 overexpression in p53-/- and INK4a-/-/Arf-/- mouse embryonic fibroblasts (MEFs), which failed to inhibit cell proliferation, despite the induction of high levels of ROS. The induction of ROS by Akt3 is due to the phosphorylation of the NADPH oxidase subunit p47phox, which results in NADPH oxidase activation. Expression of Akt3 in p47phox-/- MEFs failed to induce ROS and to inhibit cell proliferation. Notably, the proliferation defect was rescued by wild-type p47phox, but not by the phosphorylation site mutant of p47phox In agreement with these observations, Akt3 up-regulates p53 in human cancer cell lines, and the expression of Akt3 positively correlates with the levels of p53 in a variety of human tumors. More important, Akt3 alterations correlate with a higher frequency of mutation of p53, suggesting that tumor cells may adapt to high levels of Akt3, by inactivating the DNA damage response.

Lysine methyltransferase 2D regulates pancreatic carcinogenesis through metabolic reprogramming.

OBJECTIVE: Despite advances in the identification of epigenetic alterations in pancreatic cancer, their biological roles in the pathobiology of this dismal neoplasm remain elusive. Here, we aimed to characterise the functional significance of histone lysine methyltransferases (KMTs) and demethylases (KDMs) in pancreatic tumourigenesis. DESIGN: DNA methylation sequencing and gene expression microarrays were employed to investigate CpG methylation and expression patterns of KMTs and KDMs in pancreatic cancer tissues versus normal tissues. Gene expression was assessed in five cohorts of patients by reverse transcription quantitative-PCR. Molecular analysis and functional assays were conducted in genetically modified cell lines. Cellular metabolic rates were measured using an XF24-3 Analyzer, while quantitative evaluation of lipids was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Subcutaneous xenograft mouse models were used to evaluate pancreatic tumour growth in vivo. RESULTS: We define a new antitumorous function of the histone lysine (K)-specific methyltransferase 2D (KMT2D) in pancreatic cancer. KMT2D is transcriptionally repressed in human pancreatic tumours through DNA methylation. Clinically, lower levels of this methyltransferase associate with poor prognosis and significant weight alterations. RNAi-based genetic inactivation of KMT2D promotes tumour growth and results in loss of H3K4me3 mark. In addition, KMT2D inhibition increases aerobic glycolysis and alters the lipidomic profiles of pancreatic cancer cells. Further analysis of this phenomenon identified the glucose transporter SLC2A3 as a mediator of KMT2D-induced changes in cellular, metabolic and proliferative rates. CONCLUSION: Together our findings define a new tumour suppressor function of KMT2D through the regulation of glucose/fatty acid metabolism in pancreatic cancer.

Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration.

Pleiotrophin (PTN) stimulates endothelial cell migration through binding to receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) and ανß3 integrin. Screening for proteins that interact with RPTPß/ζ and potentially regulate PTN signaling, through mass spectrometry analysis, identified cyclin-dependent kinase 5 (CDK5) activator p35 among the proteins displaying high sequence coverage. Interaction of p35 with the serine/threonine kinase CDK5 leads to CDK5 activation, known to be implicated in cell migration. Protein immunoprecipitation and proximity ligation assays verified p35-RPTPß/ζ interaction and revealed the molecular association of CDK5 and RPTPß/ζ. In endothelial cells, PTN activates CDK5 in an RPTPß/ζ- and phosphoinositide 3-kinase (PI3K)-dependent manner. On the other hand, c-Src, ανß3 and ERK1/2 do not mediate the PTN-induced CDK5 activation. Pharmacological and genetic inhibition of CDK5 abolished PTN-induced endothelial cell migration, suggesting that CDK5 mediates PTN stimulatory effect. A new pyrrolo[2,3-α]carbazole derivative previously identified as a CDK1 inhibitor, was found to suppress CDK5 activity and eliminate PTN stimulatory effect on cell migration, warranting its further evaluation as a new CDK5 inhibitor. Collectively, our data reveal that CDK5 is activated by PTN, in an RPTPß/ζ-dependent manner, regulates PTN-induced cell migration and is an attractive target for the inhibition of PTN pro-angiogenic properties.

A step-by-step microRNA guide to cancer development and metastasis.

BACKGROUND: Cancer is one of the leading causes of mortality. The neoplastic transformation of normal cells to cancer cells is caused by a progressive accumulation of genetic and epigenetic alterations in oncogenes, tumor suppressor genes and epigenetic regulators, providing cells with new properties, collectively known as the hallmarks of cancer. During the process of neoplastic transformation cells progressively acquire novel characteristics such as unlimited growth potential, increased motility and the ability to migrate and invade adjacent tissues, the ability to spread from the tumor of origin to distant sites, and increased resistance to various types of stresses, mostly attributed to the activation of genetic stress-response programs. Accumulating evidence indicates a crucial role of microRNAs (miRNAs or miRs) in the initiation and progression of cancer, acting either as oncogenes (oncomirs) or as tumor suppressors via several molecular mechanisms. MiRNAs comprise a class of small ~22 bp long noncoding RNAs that play a key role in the regulation of gene expression at the post-transcriptional level, acting as negative regulators of mRNA translation and/or stability. MiRNAs are involved in the regulation of a variety of biological processes including cell cycle progression, DNA damage responses and apoptosis, epithelial-to-mesenchymal cell transitions, cell motility and stemness through complex and interactive transcription factor-miRNA regulatory networks. CONCLUSIONS: The impact and the dynamic potential of miRNAs with oncogenic or tumor suppressor properties in each stage of the multistep process of tumorigenesis, and in the adaptation of cancer cells to stress, are discussed. We propose that the balance between oncogenic versus tumor suppressive miRNAs acting within transcription factor-miRNA regulatory networks, influences both the multistage process of neoplastic transformation, whereby normal cells become cancerous, and their stress responses. The role of specific tumor-derived exosomes containing miRNAs and their use as biomarkers in diagnosis and prognosis, and as therapeutic targets, are also discussed.

A functional microRNA library screen reveals miR-410 as a novel anti-apoptotic regulator of cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma is characterized by late diagnosis and a poor survival rate. MicroRNAs have been involved in the pathogenesis of different cancer types, including cholangiocarcinoma. Our aim was to identify novel microRNAs regulating cholangiocarcinoma cell growth in vitro and in vivo. METHODS: A functional microRNA library screen was performed in human cholangiocarcinoma cells to identify microRNAs that regulate cholangiocarcinoma cell growth. Real-time PCR analysis evaluated miR-9 and XIAP mRNA levels in cholangiocarcinoma cells and tumors. RESULTS: The screen identified 21 microRNAs that regulated >50 % cholangiocarcinoma cell growth. MiR-410 was identified as the top suppressor of growth, while its overexpression significantly inhibited the invasion and colony formation ability of cholangiocarcinoma cells. Bioinformatics analysis revealed that microRNA-410 exerts its effects through the direct regulation of the X-linked inhibitor of apoptosis protein (XIAP). Furthermore, overexpression of miR-410 significantly reduced cholangiocarcinoma tumor growth in a xenograft mouse model through induction of apoptosis. In addition, we identified an inverse relationship between miR-410 and XIAP mRNA levels in human cholangiocarcinomas. CONCLUSIONS: Taken together, our study revealed a novel microRNA signaling pathway involved in cholangiocarcinoma and suggests that manipulation of the miR-410/XIAP pathway could have a therapeutic potential for cholangiocarcinoma.

Transcriptomic and CRISPR/Cas9 technologies reveal FOXA2 as a tumor suppressor gene in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with low survival rates and limited therapeutic options. Thus elucidation of signaling pathways involved in PDAC pathogenesis is essential for identifying novel potential therapeutic gene targets. Here, we used a systems approach to elucidate those pathways by integrating gene and microRNA profiling analyses together with CRISPR/Cas9 technology to identify novel transcription factors involved in PDAC pathogenesis. FOXA2 transcription factor was found to be significantly downregulated in PDAC relative to control pancreatic tissues. Functional experiments revealed that FOXA2 has a tumor suppressor function through inhibition of pancreatic cancer cell growth, migration, invasion, and colony formation. In situ hybridization analysis revealed miR-199a to be significantly upregulated in pancreatic cancer. Bioinformatics and luciferase analyses showed that miR-199a negatively but directly regulates FOXA2 expression through binding in its 3'-untranslated region (UTR). Evaluation of the functional importance of miR-199a on pancreatic cancer revealed that miR-199a acts as an inhibitor of FOXA2 expression, inducing an increase in pancreatic cancer cell proliferation, migration, and invasion. Additionally, gene ontology and network analyses in PANC-1 cells treated with a small interfering RNA (siRNA) against FOXA2 revealed an enrichment for cell invasion mechanisms through PLAUR and ERK activation. FOXA2 deletion (FOXA2Δ) by using two CRISPR/Cas9 vectors in PANC-1 cells induced tumor growth in vivo resulting in upregulation of PLAUR and ERK pathways in FOXA2Δ xenograft tumors. We have identified FOXA2 as a novel tumor suppressor in pancreatic cancer and it is regulated directly by miR-199a, thereby enhancing our understanding of how microRNAs interplay with the transcription factors to affect pancreatic oncogenesis.

Functional microRNA high throughput screening reveals miR-9 as a central regulator of liver oncogenesis by affecting the PPARA-CDH1 pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths, reflecting the aggressiveness of this type of cancer and the absence of effective therapeutic regimens. MicroRNAs have been involved in the pathogenesis of different types of cancers, including liver cancer. Our aim was to identify microRNAs that have both functional and clinical relevance in HCC and examine their downstream signaling effectors. METHODS: MicroRNA and gene expression levels were measured by quantitative real-time PCR in HCC tumors and controls. A TargetScan algorithm was used to identify miR-9 downstream direct targets. RESULTS: A high-throughput screen of the human microRNAome revealed 28 microRNAs as regulators of liver cancer cell invasiveness. MiR-9, miR-21 and miR-224 were the top inducers of HCC invasiveness and also their expression was increased in HCC relative to control liver tissues. Integration of the microRNA screen and expression data revealed miR-9 as the top microRNA, having both functional and clinical significance. MiR-9 levels correlated with HCC tumor stage and miR-9 overexpression induced SNU-449 and HepG2 cell growth, invasiveness and their ability to form colonies in soft agar. Bioinformatics and 3'UTR luciferase analyses identified E-cadherin (CDH1) and peroxisome proliferator-activated receptor alpha (PPARA) as direct downstream effectors of miR-9 activity. Inhibition of PPARA suppressed CDH1 mRNA levels, suggesting that miR-9 regulates CDH1 expression directly through binding in its 3'UTR and indirectly through PPARA. On the other hand, miR-9 inhibition of overexpression suppressed HCC tumorigenicity and invasiveness. PPARA and CDH1 mRNA levels were decreased in HCC relative to controls and were inversely correlated with miR-9 levels. CONCLUSIONS: Taken together, this study revealed the involvement of the miR-9/PPARA/CDH1 signaling pathway in HCC oncogenesis.

MicroRNA214 Is Associated With Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC). METHODS: We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214). RESULTS: A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN. CONCLUSIONS: Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

Pleiotrophin-induced endothelial cell migration is regulated by xanthine oxidase-mediated generation of reactive oxygen species.

Pleiotrophin (PTN) is a heparin-binding growth factor that induces cell migration through binding to its receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) and integrin alpha v beta 3 (ανß3). In the present work, we studied the effect of PTN on the generation of reactive oxygen species (ROS) in human endothelial cells and the involvement of ROS in PTN-induced cell migration. Exogenous PTN significantly increased ROS levels in a concentration and time-dependent manner in both human endothelial and prostate cancer cells, while knockdown of endogenous PTN expression in prostate cancer cells significantly down-regulated ROS production. Suppression of RPTPß/ζ through genetic and pharmacological approaches, or inhibition of c-src kinase activity abolished PTN-induced ROS generation. A synthetic peptide that blocks PTN-ανß3 interaction abolished PTN-induced ROS generation, suggesting that ανß3 is also involved. The latter was confirmed in CHO cells that do not express ß3 or over-express wild-type ß3 or mutant ß3Y773F/Y785F. PTN increased ROS generation in cells expressing wild-type ß3 but not in cells not expressing or expressing mutant ß3. Phosphoinositide 3-kinase (PI3K) or Erk1/2 inhibition suppressed PTN-induced ROS production, suggesting that ROS production lays down-stream of PI3K or Erk1/2 activation by PTN. Finally, ROS scavenging and xanthine oxidase inhibition completely abolished both PTN-induced ROS generation and cell migration, while NADPH oxidase inhibition had no effect. Collectively, these data suggest that xanthine oxidase-mediated ROS production is required for PTN-induced cell migration through the cell membrane functional complex of ανß3 and RPTPß/ζ and activation of c-src, PI3K and ERK1/2 kinases.

XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway.

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)--a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)--is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44(high)CD24(low) population. Hypoxia-inducing factor 1α (HIF1α) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.

Phosphoproteomics screen reveals akt isoform-specific signals linking RNA processing to lung cancer.

The three Akt isoforms are functionally distinct. Here we show that their phosphoproteomes also differ, suggesting that their functional differences are due to differences in target specificity. One of the top cellular functions differentially regulated by Akt isoforms is RNA processing. IWS1, an RNA processing regulator, is phosphorylated by Akt3 and Akt1 at Ser720/Thr721. The latter is required for the recruitment of SETD2 to the RNA Pol II complex. SETD2 trimethylates histone H3 at K36 during transcription, creating a docking site for MRG15 and PTB. H3K36me3-bound MRG15 and PTB regulate FGFR-2 splicing, which controls tumor growth and invasiveness downstream of IWS1 phosphorylation. Twenty-one of the twenty-four non-small-cell-lung carcinomas we analyzed express IWS1. More importantly, the stoichiometry of IWS1 phosphorylation in these tumors correlates with the FGFR-2 splicing pattern and with Akt phosphorylation and Akt3 expression. These data identify an Akt isoform-dependent regulatory mechanism for RNA processing and demonstrate its role in lung cancer.

Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling.

Hepatocellular carcinoma (HCC) is a slowly developing malignancy postulated to evolve from premalignant lesions in chronically damaged livers. However, it was never established that premalignant lesions actually contain tumor progenitors that give rise to cancer. Here, we describe isolation and characterization of HCC progenitor cells (HcPCs) from different mouse HCC models. Unlike fully malignant HCC, HcPCs give rise to cancer only when introduced into a liver undergoing chronic damage and compensatory proliferation. Although HcPCs exhibit a similar transcriptomic profile to bipotential hepatobiliary progenitors, the latter do not give rise to tumors. Cells resembling HcPCs reside within dysplastic lesions that appear several months before HCC nodules. Unlike early hepatocarcinogenesis, which depends on paracrine IL-6 production by inflammatory cells, due to upregulation of LIN28 expression, HcPCs had acquired autocrine IL-6 signaling that stimulates their in vivo growth and malignant progression. This may be a general mechanism that drives other IL-6-producing malignancies.

Tpl2 kinase regulates FcγR signaling and immune thrombocytopenia in mice.

The MAPK3 Tpl2 controls innate and adaptive immunity by regulating TLR, TNF-α, and GPCR signaling in a variety of cell types. Its ablation gives rise to an anti-inflammatory phenotype characterized by resistance to LPS-induced endotoxin shock, DSS-induced colitis, and TNF-α-induced IBD. Here, we address the role of Tpl2 in autoimmunity. Our data show that the ablation and the pharmacological inhibition of Tpl2 protect mice from antiplatelet antibody-induced thrombocytopenia, a model of ITP. Thrombocytopenia in this model and in ITP is caused by phagocytosis of platelets opsonized with antiplatelet antibodies and depends on FcγR activation in splenic and hepatic myeloid cells. Further studies explained how Tpl2 inhibition protects from antibody-induced thrombocytopenia, by showing that Tpl2 is activated by FcγR signals in macrophages and that its activation by these signals is required for ERK activation, cytoplasmic Ca(2+) influx, the induction of cytokine and coreceptor gene expression, and phagocytosis.

miRNAs link metabolic reprogramming to oncogenesis.

The most profound biochemical phenotype of cancer cells is their ability to metabolize glucose to lactate, even under aerobic conditions. This alternative metabolic circuitry is sufficient to support the biosynthetic and energy requirements for cancer cell proliferation and metastasis. Alterations in oncogenes and tumor-suppressor genes are involved in the metabolic switch of cancer cells to aerobic glycolysis, increased glutaminolysis, and fatty acid biosynthesis. miRNAs mediate fine-tuning of genes involved directly or indirectly in cancer metabolism. In this review we discuss the regulatory role of miRNAs on enzymes, signaling pathways, and transcription factors involved in glucose and lipid metabolism. We further consider the therapeutic potential of metabolism-related miRNAs in cancer.

Salutary effects of adiponectin on colon cancer: in vivo and in vitro studies in mice.

BACKGROUND: Obesity and a high-fat diet are associated with the risk and progression of colon cancer. Low adiponectin levels may play an important role in the development of colon and other obesity-related malignancies. No previous studies have directly investigated the mechanistic effects of adiponectin on colon cancer in the settings of obesity, a high-fat diet and/or adiponectin deficiency. OBJECTIVE: To investigate the effects of adiponectin on the growth of colorectal cancer in adiponectin-deficient or wild-type-C57BL/6 mice fed a low-fat or high-fat diet. RESULTS: Mice fed a high-fat-diet gained more weight and had larger tumours than mice fed a low-fat-diet. Adiponectin administration suppressed implanted tumour growth, causing larger central necrotic areas. Adiponectin treatment also suppressed angiogenesis assessed by CD31 staining and VEGFb and VEGFd mRNA expression in tumours obtained from mice fed a high-fat-diet and from adiponectin-deficient mice. Adiponectin treatment decreased serum insulin levels in mice on a high-fat-diet and increased serum-interleukin (IL)-12 levels in adiponectin-deficient mice. In vitro, it was found that adiponectin directly controls malignant potential (cell proliferation, adhesion, invasion and colony formation) and regulates metabolic (AMPK/S6), inflammatory (STAT3/VEGF) and cell cycle (p21/p27/p53/cyclins) signalling pathways in both mouse MCA38 and human HT29, HCT116 and LoVo colon cancer cell lines in a LKB1-dependent way. CONCLUSION: These new mechanistic and pathophysiology studies provide evidence for an important role of adiponectin in colon cancer. The data indicate that adiponectin or analogues might be useful agents in the management or chemoprevention of colon cancer.

Therapeutically targeting microRNAs in liver cancer.

MicroRNAs are small non-coding RNAs that negatively regulate gene expression through binding on the 3' untranslated region (UTR) of genes. Although microRNAs constitute a small fraction of the human genome, multiple studies have indicated their involvement in the pathogenesis of different types of cancer. Hepatocellular carcinoma (liver) is one of the most aggressive types of cancer with very few therapeutic options. Several studies have revealed that microRNAs are deregulated during liver cancer development and affect central oncogenic and anti-apoptotic liver cancer signaling pathways. Furthermore, the expression levels of specific microRNAs have been identified to be correlated with clinicopathological parameters and treatment responses in liver cancer patients. Here, we review how different epidemiological and liver cancer risk factors, such as the hepatitis B and C viruses, deregulate microRNA-gene circuits in the liver, contributing to liver cancer development. Furthermore, we describe how the most frequently deregulated microRNAs identified in liver cancer patients control their down-stream signaling pathways in liver cancer cells. In addition, we provide examples of microRNAs or microRNA inhibitors that have been used as liver cancer therapeutics and describe novel delivery technologies that could be potentially used in order to optimize the delivery of microRNAs in the liver without having any toxicity or side effects in other major organs. Taken together, there is ample evidence suggesting the deregulation of microRNA-gene circuits in liver, promising that the development of microRNA-based therapeutics could be a clinically viable approach for liver cancer patients.

Implications of pleiotrophin in human PC3 prostate cancer cell growth in vivo.

Pleiotrophin (PTN) is a heparin-binding growth factor with diverse functions related to tumor growth, angiogenesis, and metastasis. Pleiotrophin seems to have a significant role in prostate cancer cell growth and to mediate the stimulatory actions of other factors that affect prostate cancer cell functions. However, all studies carried out up to date are in vitro, using different types of human prostate cancer cell lines. The aim of the present work was to study the role of endogenous PTN in human prostate cancer growth in vivo. For this purpose, human prostate cancer PC3 cells were stably transfected with a plasmid vector, bearing the antisense PTN sequence, in order to inhibit PTN expression (AS-PC3). Migration, apoptosis, and adhesion on osteoblastic cells were measured in vitro. In vivo, PC3 cells were s.c. injected into male NOD/SCID mice, and tumor growth, survival rates, angiogenesis, apoptosis, and the number of metastasis were estimated. Pleiotrophin depletion resulted in a decreased migration capability of AS-PC3 cells compared with the corresponding mock-transfected or the non-transfected PC3 cells, as well as increased apoptosis and decreased adhesiveness to osteoblastic cells in vitro. In prostate cancer NOD/SCID mouse xenografts, PTN depletion significantly suppressed tumor growth and angiogenesis and induced apoptosis of cancer cells. In addition, PTN depletion decreased the number of metastases, providing a survival benefit for the animals bearing AS-PC3 xenografts. Our data suggest that PTN is implicated in human prostate cancer growth in vivo and could be considered a potential target for the development of new therapeutic approaches for prostate cancer.

The protein kinase Akt1 regulates the interferon response through phosphorylation of the transcriptional repressor EMSY.

The protein kinases Akt1, Akt2, and Akt3 possess nonredundant signaling properties, few of which have been investigated. Here, we present evidence for an Akt1-dependent pathway that controls interferon (IFN)-regulated gene expression and antiviral immunity. The target of this pathway is EMSY, an oncogenic interacting partner of BRCA2 that functions as a transcriptional repressor. Overexpression of EMSY in hTERT-immortalized mammary epithelial cells, and in breast and ovarian carcinoma cell lines, represses IFN-stimulated genes (ISGs) in a BRCA2-dependent manner, whereas its knockdown has the opposite effect. EMSY binds to the promoters of ISGs, suggesting that EMSY functions as a direct transcriptional repressor. Akt1, but not Akt2, phosphorylates EMSY at Ser209, relieving EMSY-mediated ISG repression. The Akt1/EMSY/ISG pathway is activated by both viral infection and IFN, and it inhibits the replication of HSV-1 and vesicular stomatitis virus (VSV). Collectively, these data define an Akt1-dependent pathway that contributes to the full activation of ISGs by relieving their repression by EMSY and BRCA2.

An HNF4α-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis.

Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24, and miR-629. Moreover, we show that, once this circuit is activated, it maintains suppression of HNF4α and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.