Showing papers in "Carcinogenesis in 2011"

(-)-Epigallocatechin-3-gallate reactivates silenced tumor suppressor genes, Cip1/p21 and p16INK4a, by reducing DNA methylation and increasing histones acetylation in human skin cancer cells.

Abstract: The anti-skin carcinogenic effects of green tea catechins have been studied extensively in vitro and in vivo models but the precise epigenetic molecular mechanisms are still unclear. Accumulating data suggest that dietary phytochemicals may alter cancer risk by modifications of epigenetic processes in the cells. The present study was designed to investigate whether tea catechins, particularly (-)-epigallocatechin-3-gallate (EGCG), would modify epigenetic events to regulate DNA methylation-silenced tumor suppressor genes in skin cancer cells. DNA methylation, histone modifications and tumor suppressor gene expressions were studied in detail using human epidermoid carcinoma A431 cells as an in vitro model after EGCG treatment using cytostaining, western blotting, dot blot analysis, real-time polymerase chain reaction and enzymatic activity assays. Our study shows that EGCG treatment decreased global DNA methylation levels in A431 cells in a dose-dependent manner. EGCG decreased the levels of 5-methylcytosine, DNA methyltransferase (DNMT) activity, messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b. EGCG decreased histone deacetylase activity and increased levels of acetylated lysine 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysine 5, 12 and 16 on histone H4 but decreased levels of methylated H3-Lys 9. Additionally, EGCG treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, p16INK4a and Cip1/p21. Together, our study provides new insight into the epigenetic mechanism of action of EGCG that may contribute to the chemoprevention of skin cancer and may have important implications for epigenetic therapy.

The EMT regulator slug and lung carcinogenesis

Abstract: Lung cancer is the leading cause of cancer death worldwide. Cancer metastasis and resistance to treatment (including radiotherapy, chemotherapy and targeted therapy) are two major causes for the poor survival of lung cancer patients. Epithelial-mesenchymal transition (EMT) is involved in cancer cell invasion, resistance to apoptosis and stem cell features. The process of EMT is controlled by a group of transcriptional factors, zinc finger proteins and basic helix-loop-helix factors. Signaling pathways activated by intrinsic or extrinsic stimuli converge on these transcriptional factors and regulated the phenotypic changes of cancer cells. These EMT regulators may play an important role in cancer progression. In lung cancer, Slug is the most thoroughly investigated EMT regulator. The expression of Slug is associated with lung cancer invasion and resistance to target therapy. In this review, we focus on the current understanding of the role of Slug in the carcinogenesis and progression of lung cancer.

The multiple roles of autophagy in cancer.

Abstract: Autophagy is an evolutionarily conserved, catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Autophagy is regulated via a group of genes called AuTophaGy-related genes and is executed at basal levels in virtually all cells as a homeostatic mechanism for maintaining cellular integrity. The levels and cargos of autophagy can be modulated in response to a variety of intra- and extracellular cues to bring about specific and selective events. Autophagy is a multifaceted process and alterations in autophagic signalling pathways are frequently found in cancer and many other diseases. During tumour development and in cancer therapy, autophagy has paradoxically been reported to have roles in promoting both cell survival and cell death. In addition, autophagy has been reported to control other processes relevant to the aetiology of malignant disease, including oxidative stress, inflammation and both innate and acquired immunity. It is the aim of this review to describe the molecular basis and the signalling events that control autophagy in mammalian cells and to summarize the cellular functions that contribute to tumourigenesis when autophagy is perturbed.

Green tea polyphenol EGCG suppresses lung cancer cell growth through upregulating miR-210 expression caused by stabilizing HIF-1α

Abstract: (-)-Epigallocatechin-3-gallate (EGCG) has been reported to affect many cellular regulatory pathways. This study aims to determine whether EGCG could target microRNA (miRNA), one of the mechanisms for cells to achieve subtle change in multiple targets. We found that, in both human and mouse lung cancer cells in culture, EGCG specifically upregulated the expression of miR-210, a major miRNA regulated by HIF-1α. Furthermore, we found that overexpression of miR-210 led to reduced cell proliferation rate and anchorage-independent growth as well as reduced sensitivity to EGCG. On the mechanisms of miR-210 regulation by EGCG, we demonstrated that the regulation was mediated through the hypoxia-response element in miR-210 promoter. Consistently, the upregulation of miR-210 was found to be correlated with the stabilized HIF-1α in lung cancer cell lines after EGCG treatment. This EGCG-induced stabilization of HIF-1α was further shown by the stabilization of HA-tagged HIF-1α but not the P402A/P564A-mutated HIF-1α by EGCG, suggesting that EGCG targets the oxygen-dependent degradation (ODD) domain. Direct evidence was obtained by affinity binding assay showing that EGCG specifically binds HIF-1α with a K(d) = 3.47 μM. This result suggests that EGCG binding interferes with the hydroxylation of key Pro residues in the ODD domain, preventing HIF-1α from the Pro hydroxylation-dependent ubiquitination and subsequent proteosome-mediated degradation. In summary, our results demonstrated, for the first time, the elevation of miR-210 by EGCG in lung cancer cell lines and this is mediated by the stabilization of HIF-1α. This event contributes to the anticancer activity of EGCG.

YAP is a candidate oncogene for esophageal squamous cell carcinoma

Abstract: Yes-associated protein (YAP), the nuclear effector of the Hippo pathway, is a key regulator of organ size and a candidate human oncogene located at chromosome 11q22. Since we previously reported amplification of 11q22 region in esophageal squamous cell carcinoma (ESCC), in this study we focused on the clinical significance and biological functions of YAP in this tumor. Frequent overexpression of YAP protein was observed in ESCC cells including those with a robust amplicon at position 11q22. Overexpression of the YAP protein was frequently detected in primary tumors of ESCC as well. Patients with YAP-overexpressing tumors had a worse overall rate of survival than those with non-expressing tumors, and YAP positivity was independently associated with a worse outcome in the multivariate analysis. Further analyses in cells in which YAP was either overexpressed or depleted confirmed that cell proliferation was promoted in a YAP isoform-independent but YAP expression level-dependent manner. YAP depletion inhibited cell proliferation mainly in the G(0)-G(1) phase and induced an increase in CDKN1A/p21 transcription but a decrease in BIRC5/survivin transcription. Our results indicate that YAP is a putative oncogene in ESCC and it represents a potential diagnostic and therapeutic target.

G-quadruplex structures in TP53 intron 3: role in alternative splicing and in production of p53 mRNA isoforms

Abstract: The tumor suppressor gene TP53, encoding p53, is expressed asseveral transcripts. The fully spliced p53 (FSp53) transcript enco-desthecanonicalp53protein.The alternativelysplicedp53I2 tran-script retains intron 2 andencodesD40p53 (or DNp53), an isoformlacking first 39 N-terminal residues corresponding to the maintransactivation domain. We demonstrate the formation of G-quad-ruplex structures (G4) in a GC-rich region of intron 3 that modu-lates the splicing ofintron 2. First, we show the formation of G4 insynthetic RNAs encompassing intron 3 sequences by ultravioletmelting, thermal difference spectra and circular dichroism spec-troscopy. These observations are confirmed by detection of G4-in-duced reverse transcriptase elongation stops in synthetic RNA ofintron 3. In this region, p53 pre-messenger RNA (mRNA) containsa succession of short exons (exons 2 and 3) and introns (introns 2and 4) covering a total of 333 bp. Site-directed mutagenesis of G-tracts putatively involved in G4 formation decreased by #30% theexcision ofintron 2 in a green fluorescent protein-reporter splicingassay. Moreover, treatment of lymphoblastoid cells with 360A,a synthetic ligand that binds to single-strand G4 structures, in-creasestheformation ofFSp53mRNAanddecreasesp53I2mRNAexpression. These results indicate that G4 structures in intron 3regulate the splicing of intron 2, leading to differential expressionof transcripts encoding distinct p53 isoforms.IntroductionThe tumor suppressor p53 protein controls antiproliferative responsesto various forms of stress (1). Its function is impaired in .50% ofhuman cancer, mainly by mutation (2). TP53 gene expression iscomplex, with different transcripts encoding isoforms carrying dis-tinctN- andC-termini (3,4).Todate,10isoformshavebeenidentifiedresulting from the usage of alternative promoters, splice sites and/ortranslational initiation sites (5). Several of these isoforms differ intheir N-terminal region. The N-terminus of p53 contains the maintransactivation domain (residues 1–42, transactivation domain I) aswell as the binding site of Hdm2, which targets p53 for proteasomedegradation and regulates p53 stability (1). Transcription of p53 mes-senger RNA (mRNA) from the proximal promoter generates twoproteins with distinct N-terminal domain. The first corresponds tothe canonical p53 protein, assembled from the fully spliced p53(FSp53) mRNA that retains 11 exons. This protein induces p53-me-diated growth suppression in response to stress. The second isoform,D40p53 (or DNp53), is assembled from an alternatively splicedmRNA retaining intron 2 (p53I2) and lacks the first 39 residues,corresponding to transactivation domain I, as well as Hdm2-bindingsite (3,6). The use of an internal promoter located in a region betweenintron 1 and exon 5 generates a third N-terminal isoform, D133p53,which lacks the first 132 residues (5).When expressed in excess to p53, D40p53 inhibits transcriptionalactivity and interferes in the control of cell cycle progression and apo-ptosisbyexertinganegativeeffectontheexpressionofp53-targetgenes(3,6,7). However, the biological circumstances and the molecularmechanisms regulating D40p53 expression are still poorly known. Re-tentionofintron2inp53I2mRNAintroducesseveralstopcodonsinthereading frame of AUG 1, thus preventing the synthesis of a full-lengthp53protein.However,p53I2mRNAcanbetranslatedusingAUG40asinitiation site, generating a protein isoform which differs from thecanonical p53 by the lack of the first 39 residues. Expression ofp53I2 transcript has been reported in cell lines, such as MCF-7, innormallymphocytesandinprimarymelanomaisolates(7,8).However,themechanismthatregulatesthesplicingofp53pre-mRNAintoFSp53or p53I2 is not understood. D40p53 protein isoform can also be pro-duced by internal ribosomal entry site-regulated internal initiation oftranslation using FSp53 mRNA (9,10).In recent years, it has been proposed that tridimensional RNA struc-tures such as G-quadruplexes may play important roles in regulatingsplicing (11). These structures result of the propensity of G-rich se-quences to fold into four-stranded cation-dependent structures (12).They are formed by the interaction of four guanines organized in acyclic Hoogsteen hydrogen bonding arrangement termed a G-quartetand by the stacking of several G-quartets (Figure 1A). It is estimatedthatover376 000sequencesinthehumangenomehavethepotentialtoadopt G-quadruplex structures, most of them located in non-codingregions (13,14).Atthe RNA level, G-quadruplexes may play a numberof roles.Innon-coding RNAs,they can affecttheir structuresand func-tions (15,16). In 5#-untranslated region of mRNAs, G-quadruplexeshave been shown to modulate translation (17,18). When present inintrons, G-quadruplexes can affect the splicing and expression patternsof genessuchashTERT (humantelomerasereversetranscriptase), Bcl-xL or FMRP (Fragile X mental retardation protein) (11,19,20).The sequence of intron 3 in TP53 contains tracts of G bases orga-nized in a pattern similar to the one of regions forming G-quadruplexstructures. Since exon 3 in TP53 is extremely short (22 bp), we rea-soned that motifs located in intron 3 might have an effect on theregulation of the splicing of intron 2. In this study, we provide anevidence for the formation of G-quadruplex in TP53 intron 3 and thatthese G-quadruplex structures may affect the splicing of intron 2,modulating the synthesis of either FSp53 (intron 2 spliced out) orp53I2 (intron 2 retained) mRNAs, which encode different p53 proteinisoforms.Materials and methods

Pathologically decreased miR-26a antagonizes apoptosis and facilitates carcinogenesis by targeting MTDH and EZH2 in breast cancer

Abstract: The role of miR-26a in carcinogenesis appears to be a complicated one, in the sense that both oncogenic and tumor suppressive effects were reported in cancers such as glioblastoma and hepatocellular carcinoma, respectively. Here, we report for the first time that miR-26a is downregulated in breast cancer specimens and cell lines and its transient transfection initiates apoptosis of breast cancer cell line MCF7 cells. Furthermore, retrovirus-delivered miR-26a impairs the in vitro colony forming and in vivo tumor-loading ability of MCF7 cells. Subsequently, MTDH and EZH2 are identified as two direct targets of miR-26a and they are significantly upregulated in breast cancer. MCF7 xenografts with exogenous miR-26a show that a decrease in expression of both MTDH and EZH2 is accompanied by an increase in apoptosis. Moreover, knockdown of MTDH causes apoptosis while reexpression of MTDH partially reverses the proapoptotic effect of miR-26a in MCF7 cells. Our findings suggest that miR-26a functionally antagonizes human breast carcinogenesis by targeting MTDH and EZH2.

Withaferin A induces p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells.

Abstract: Cervical cancer is caused by human papilloma virus (HPV) expressing E6 and E7 oncoproteins, which are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Repression of HPV oncoproteins would therefore result in reactivation of tumor suppressor pathways and cause apoptosis in cancer cells. Withaferin A (WA), the active component of the medicinal plant Withania Somnifera, has exhibited inhibitory effects against several different cancers. We examined the activity of WA on human cervical cancer cells in vitro and in vivo. WA potently inhibited proliferation of the cervical cancer cells, CaSki (IC(50) 0.45 ± 0.05 μM). Mechanistically, WA was found to (i) downregulate expression of HPV E6 and E7 oncoproteins, (ii) induce accumulation of p53, (iii) increase levels of p21(cip1/waf1) and its interaction with proliferating cell nuclear antigen (PCNA), (iv) cause G(2)/M cell cycle arrest, associated with modulation of cyclin B1, p34(cdc2) and PCNA levels, (v) decrease the levels of STAT3 and its phosphorylation at Tyr(705) and Ser(727) and (vi) alter expression levels of p53-mediated apoptotic markers-Bcl2, Bax, caspase-3 and cleaved PARP. In vivo, WA resulted in reduction of nearly 70% of the tumor volume in athymic nude mice with essentially similar trend in the modulation of molecular markers as in vitro. This is the first demonstration indicating that WA significantly downregulates expression of HPV E6/E7 oncogenes and restores the p53 pathway, resulting in apoptosis of cervical cancer cells. Together, our data suggest that WA can be exploited as a potent therapeutic agent for the treatment and prevention of cervical cancer without deleterious effects.

MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer.

Abstract: MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2'-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer.

COX-2 inhibition alters the phenotype of tumor-associated macrophages from M2 to M1 in ApcMin/+ mouse polyps

Abstract: Macrophages are a major component of tumor stroma. Tumor-associated macrophages (TAMs) show anti- (M1) or protumor (M2) functions depending on the cytokine milieu of the tumor microenvironment. Cyclooxygenase-2 (COX-2) is constitutively expressed in a variety of tumors including colorectal cancer. TAMs are known to be a major source of COX-2 in human and mice intestinal tumors. COX-2 inhibitor reduces the number and size of intestinal adenomas in familial adenomatous polyposis patients and Apc(Min/+) mice. Although COX-2 inhibitor is thought to regulate cancer-related inflammation, its effect on TAM phenotype remains unknown. Here, we examined the effects of COX-2 inhibition on TAM phenotype and cytokine expression both in vivo and in vitro. Firstly, the selective COX-2 inhibitor celecoxib changed the TAM phenotype from M2 to M1, in proportion to the reduction in number of Apc(Min/+) mouse polyps. Concomitantly, the expression of M1-related cytokine interfron (IFN)-γ was significantly upregulated by celecoxib, although the M2-related cytokines interleukin (IL)-4, IL-13 and IL-10 were not significantly altered. Secondly, IFN-γ treatment attenuated M2 phenotype of mouse peritoneal macrophages and oriented them to M1 even in the presence of M2-polarizing cytokines such as IL-4, IL-13 and IL-10. Thus, our results suggest that COX-2 inhibition alters TAM phenotype in an IFN-γ-dependent manner and subsequently may reduce intestinal tumor progression.

Th17 cells in cancer: help or hindrance?

Abstract: The role of CD4+ T helper (Th) 17 cells in malignancy is currently under debate. However, upon closer scrutiny, it becomes apparent that this discussion includes not only evaluations of Th17 cells but also IL-17+ cells from other immune populations, the cytokine interleukin (IL)-17 itself (both endogenous and exogenous) and IL-23. Further complicating the matter are occasionally conflicting results of studies in humans versus those in mice and contradictory data from immunocompetent versus immunodeficient mice. To better understand the role of Th17 cells in the tumor-bearing host, we focus first upon those studies investigating Th17 cells in patients and then those in mice, all the while keeping in mind that variables such as tumor-initiating agents, a pre-existing inflammatory environment and the immune competence of the host may have direct effects upon this T-cell subset. In this review, we will describe the phenotype of tumor-associated Th17 cells, review those studies that have examined the population directly, and finally, briefly discuss the studies involving Th17-associated signature cytokines.

MicroRNA-342 inhibits colorectal cancer cell proliferation and invasion by directly targeting DNA methyltransferase 1.

Abstract: Overexpressed DNA methyltransferase 1 (DNMT1) strongly contributes to tumor suppressor gene silencing in colorectal cancer (CRC). However, the underlying mechanism of DNMT1 overexpression is still unclear. MicroRNAs (miRNA) have been implicated as gene regulators controlling diverse biological processes, including carcinogenesis. In this study, we investigated whether some miRNA is involved in the regulation of DNMT1 and thus play a functional role in CRC. Our results showed that miR-342 was downregulated in CRC tissues and cell lines. Restoration of miR-342 resulted in a dramatic reduction of the expression of DNMT1 at both messenger RNA and protein levels by directly targeting its 3' untranslated region. This in turn reactivated ADAM23, Hint1, RASSF1A and RECK genes via promoter demethylation. Furthermore, the enhanced expression of miR-342 could significantly inhibit SW480 cell proliferation in vitro (P = 0.006). Further investigation demonstrated G(0)/G(1) cell cycle arrest in SW480 cells, which was associated with an upregulation of p21 and downregulation of cyclinE and CDK2. Overexpression of miR-342 also inhibited SW480 cell invasion. The in vivo antitumor effect was evaluated in SW480 cells with lentivirus-mediated expression of miR-342. Results showed that overexpression of miR-342 significantly inhibited tumor growth and lung metastasis in nude mice (P = 0.034). Our findings describe a new mechanism for the regulation of DNMT1 and aberrant DNA hypermethylation in CRC. This is also the first report to demonstrate that miR-342 may act as a tumor suppressor gene in CRC development. The newly identified miR-342/DNMT1 link provides a new, potential therapeutic target for the treatment of CRC.

Osteopontin promotes CCL5-mesenchymal stromal cell-mediated breast cancer metastasis

Abstract: The interaction between cancer and its local microenvironment can determine properties of growth and metastasis. A critical component of the tumor microenvironment in this context is the cancer-associated fibroblast (CAF), which can promote tumor growth, angiogenesis and metastasis. It has been hypothesized that CAF may be derived from mesenchymal stromal cells (MSC), derived from local or distant sources. However, the signaling mechanisms by which tumors and MSCs interact to promote CAF-dependent cancer growth are largely unknown. In this study with in vitro and in vivo models using MDA-MB231 human breast cancer cells, we demonstrate that tumor-derived osteopontin (OPN) induces MSC production of CCL5; the mechanism involves OPN binding to integrin cell surface receptors and activator protein-1 c-jun homodimer transactivation. In a murine xenograft model, concomitant inoculation of MSC with MDA-MB231 cells induces: (i) significantly increased growth and metastasis of MB231 cells and (ii) increased MSC migration to metastatic sites in lung and liver; this mechanism is both OPN and CCL5 dependent. MSCs retrieved from sites of metastases exhibit OPN-dependent expression of the CAF markers, α-smooth muscle actin, tenascin-c, CXCL12 (or stromal cell-derived factor 1) and fibroblast-specific protein-1 and the matrix metalloproteinases (MMP)-2 and MMP-9. Based upon these results, we propose that tumor-derived OPN promotes tumor progression via the transformation of MSC into CAF.

Neuropilin-1 exerts co-receptor function for TGF-beta-1 on the membrane of cancer cells and enhances responses to both latent and active TGF-beta.

Abstract: Neuropilin (Nrp)-1 and Nrp-2 are multifunctional proteins frequently expressed by cancer cells and contribute to tumor progression by mechanisms that are not well understood. They are co-receptors for vascular endothelial growth factor and class 3 semaphorins, but recently we found that Nrp1 also binds latent and active transforming growth factor (TGF)-β1, and activates the latent form latency-associated peptide (LAP)-TGF-β1. Here, we report that Nrp1 has affinity for TGF-β receptors TβRI and TβRII, the signaling TGF-β receptors, as well as TβRIII (betaglycan), as determined in binding assays, pull down assays and confocal microscopy. Nrp1 had a higher affinity for TβRI than TβRII and could form a complex with these receptors. In breast cancer cells, Nrp1 and TβRI cointernalized in the presence of TGF-β1. Nrp1 acted as a TGF-β co-receptor by augmenting canonical Smad2/3 signaling. Importantly, Nrp-positive cancer cells, unlike negative cells, were able to activate latent TGF-β1 and respond. We examined two other membrane proteins that bind LAP-TGF-β, i.e. an RGD-binding integrin (αvβ3) and Glycoprotein A repetitions predominant (CLRRC32). RGD-binding integrins are frequently expressed by cancer cells, and glycoprotein A repetitions predominant is expressed by activated regulatory T cells that appear linked to poor tumor immunity. In vitro, these receptors did not activate LAP-TGF-β1, but subsequent addition of Nrp1 activated the cytokine. Thus, Nrp1 might collaborate with other latent TGF-β receptors in TGF-β capture and activation. We also show that Nrp2 has activities similar to Nrp1. We conclude that Nrp1 is a co-receptor for TGF-β1 and augments responses to latent and active TGF-β. Since TGF-β promotes metastasis this is highly relevant to cancer biology.

MicroRNA-150 directly targets MUC4 and suppresses growth and malignant behavior of pancreatic cancer cells

Abstract: Pancreaticcancer (PC)has theworstprognosisamongallcancersdue to its late diagnosis and lack of effective therapies. Therefore,identification of novel gene targets, which are differentially ex-pressed in PC and functionally involved in malignant phenotypes,is critical to achieve early diagnosis and development of effectivetherapeutic strategies. We have shown previously that MUC4,an aberrantly overexpressed transmembrane mucin, promotesgrowth, invasion and metastasis of PC cells, thus underscoringits potential as a clinical target. Here, we report a novel micro-RNA(miRNA)-mediatedmechanismunderlyingaberrantexpres-sion of MUC4 in PC. We demonstrate that the 3# untranslatedregion of MUC4 contains a highly conserved miRNA-150 (miR-150) binding motif and its direct interaction with miR-150 down-regulates endogenous MUC4 protein levels. We also show thatmiR-150-mediated MUC4 downregulation is associated witha concomitant decrease in human epidermal growth factor recep-tor 2 and its phosphorylated form, leading to reduced activationof downstream signaling. Furthermore, our findings demonstratethat miR-150 overexpression inhibits growth, clonogenicity, mi-gration and invasion and enhances intercellular adhesion in PCcells. Finally, our data reveal a downregulated expression of miR-150 in malignant pancreatic tissues, which is inversely associatedwith MUC4 protein levels. Altogether, these findings establishmiR-150 as a novel regulator of MUC4 and a tumor suppressormiRNA in PC.IntroductionPancreatic cancer (PC) is a highly lethal malignancy and has the worstprognosis among all cancers. Currently, it is the fourth leading causeof cancer-related deaths in the USA (1). The collective median sur-vival for all patients with PC is 2–8 months, and only 1–4% of allpatients survive 5 years after diagnosis (2). Such a grim prognosis ofPC is explained by the fact that at the time of diagnosis, majority ofpatients have already developed an aggressive form of the disease thuslimiting the potential for therapeutic intervention (3). Even smalladenocarcinoma of pancreas at diagnosis is genetically advancedand carries numerous genetic and epigenetic aberrations that co-operatively act to confer aggressive malignant phenotypes (4,5).Recent years have witnessed important advances in our understandingof the molecular progression of PC, and several important targets havebeen identified and experimentally tested for their functional participa-tion in the disease processes (4,6,7).MUC4 is a high-molecular weight glycoprotein that belongs to thefamily of membrane-bound mucins (8). It is overexpressed in pancre-atic adenocarcinomas and tumor cell lines while remains undetectablein the normal pancreas (9). Expression analysis of MUC4 in increas-ing grade pancreatic intraepithelial neoplasias and malignant lesionsdemonstrated a positive correlation of MUC4 with disease progres-sion (10). Importantly, in our earlier studies, we have shown a patho-genic role of MUC4 in promoting pancreatic tumor growth andmetastasis (11,12). Furthermore, aberrant MUC4 expression is alsoreported in other malignancies indicating its clinical relevance asa target for therapeutic intervention (8,13). However, there is still littleknown about the molecular mechanisms that regulate MUC4 expres-sion and whose perturbation ultimately leads to its aberrant expressionduring cancer initiation and progression (8,14).Recently, a novel class of endogenous small non-coding gene reg-ulatory RNAs, termed as microRNAs (miRNAs or miRs), has gainedsignificant attention (15). These small molecules exert their regulatoryeffects by base pairing with partially complementary messengerRNAs (mRNAs) and function by two mechanisms: degrading targetmRNA or inhibiting their translation (16,17). It is now well estab-lished that miRNAs play critical roles in the development of cancer byaltering the expression of oncogenes and tumor suppressor genes(15,16). In the present study, we have investigated the role of micro-RNA-150 (miR-150) in the regulation of MUC4 expression in PCcells. Our findings demonstrate that 3# untranslated region (UTR)of MUC4 contains putative binding site for miR-150, which is highlyconserved across several mammalian species. Furthermore, we exper-imentally show that miR-150 directly targets the 3# UTR of MUC4 tosuppress its expression. Downregulation of MUC4 by miR-150 alsoleads to a concomitant decrease in human epidermal growth factorreceptor 2 (HER2), an interacting partner of MUC4 (18), and itsphosphorylated form leading to reduced activation of downstreamsignaling molecules. Our findings also demonstrate that miR-150overexpression leads to reduced growth, clonogenicity, migrationand invasion in PC cells. Finally, our data reveal a discordant expres-sion of MUC4 at the transcript and protein levels, which is inverselyassociated with miR-150 expression in malignant clinical specimens.Altogether, our study characterizes a novel miRNA-mediated mech-anism of MUC4 regulation and suggests tumor suppressive actions ofmiR-150 in PC cells.Materials and methods

Hypoxia-increased RAGE and P2X7R expression regulates tumor cell invasion through phosphorylation of Erk1/2 and Akt and nuclear translocation of NF-κB

Abstract: The role of hypoxia in regulating tumor progression is still controversial. Here, we demonstrate that, similarly to what previously observed by us in human prostate and breast tumor samples, hypoxia increases expression of the receptor for advanced glycation end products (RAGE) and the purinergic receptor P2X7 (P2X7R). The role of hypoxia was shown by the fact that hypoxia-inducible factor (HIF)-1α silencing downregulated RAGE and P2X7R protein levels as well as nuclear factor-kappaB (NF-κB) expression. In contrast, NF-κB silencing reduced P2X7R expression without affecting RAGE protein levels or nuclear accumulation of HIF-1α. Treatment of hypoxic tumor cells with HMGB1 and BzATP ligands, respectively, of RAGE and P2X7R, activated a signaling pathway that, through Akt and Erk phosphorylation, determines nuclear accumulation of NF-κB and increases cell invasion. Inhibition of Akt by SH5 and Erk by INH1 prevented both nuclear translocation of NF-κB and cell invasion. Moreover, silencing RAGE and P2X7R abolished nuclear accumulation of NF-κB as well as cell invasion without affecting HIF-1α stabilization. Once in the nucleus, NF-κB would contribute to cell survival and invasion under hypoxia, by maintaining RAGE and P2X7R expression levels and matrix metalloproteinases 2 and 9 synthesis. These results show that, hypoxia can upregulate expression levels of membrane receptors that, by binding extracellular molecules eventually released by necrotic cells, contribute to the increased invasiveness of transformed tumor cells. Moreover, these observations strengthen our working hypothesis that upregulation of damage-associated molecular patterns receptors by HIF-1α represents the crucial event bridging hypoxia and inflammation in obtaining the malignant phenotype.

Downregulation of miR-132 by promoter methylation contributes to pancreatic cancer development

Abstract: MicroRNAs (miRNAs), which regulate gene expression by partial complementarity to the 3' untranslated region of their target genes, have been implicated in cancer initiation and progression. However, the molecular mechanism underlying the regulation of miRNA expression during pancreatic tumorigenesis has not been extensively reported. In this study, we first compared the miRNA expression in human pancreatic cancers and adjacent normal tissues by miRNA array and identified 12 differentially expressed miRNAs. miR-132, which is downregulated in tumors, was further studied in greater detail. Decreased expression of miR-132 was confirmed in 16 of 20 pancreatic carcinomas (P < 0.0001), compared with their respective benign tissues by TaqMan miRNA assays. miR-132 expression was remarkably influenced by promoter methylation in PANC1 and SW1990 cells. Promoter hypermethylation was observed in tumor samples but not in the normal counterparts, and the expression of miR-132 negatively correlated with its methylation status (P = 0.013). miR-132 was transcribed by RNA polymerase II, and Sp1 played a major role in miR-132 transcription. The expression of Sp1 correlated with that of miR-132 in tissues. Moreover, cancerous tissues showed significantly lower Sp1-binding affinity to the miR-132 promoter, relative to non-tumor samples. Proliferation and colony formation of pancreatic cancer cells were suppressed in cells transfected with miR-132 mimics and enhanced in cells transfected with miR-132 inhibitor by negatively regulating the Akt-signaling pathway. Our present findings illustrate the mechanism driving miR-132 downregulation and the important role of miR-132 in pancreatic cancer development.

NF-κB targets miR-16 and miR-21 in gastric cancer: involvement of prostaglandin E receptors

Abstract: Cigarette smoke is one of the risk factors for gastric cancer and nicotine has been reported to promote tumor growth. Deregulation ofmicroRNA (miRNA) and cyclooxygenase-2 (COX-2) expressions are hallmarks of many cancers including gastric cancer. Here, we used an miRNA array platform covering a panel of 95 human miRNAs to examine the expression profile in nicotine-treated gastric cancer cells. We found that miR-16 and miR-21 were upregulated upon nicotine stimulation, transfection with anti-miR-16 or anti-miR-21 significantly abrogated cell proliferation. In contrast, ectopic miR-16 or miR-21 expression exhibited a similar stimulatory effect on cell proliferation as nicotine. Nicotine-mediated IkappaBα degradation and nuclear factor-kappa B (NF-κB) translocation dose-dependently. Knockdown of NF-κB by short interfering RNA (siRNA) or specific inhibitor (Bay-11-7085) markedly suppressed nicotine-induced cell proliferation and upregulation of miR-16 and miR-21. Interestingly, NF-κB-binding sites were located in both miR-16 and miR-21 gene transcriptional elements and we showed that nicotine enhanced the binding of NF-κB to the promoters of miR-16 and miR-21. Furthermore, activation of COX-2/prostagiandin E 2 (PGE 2 ) signaling in response to nicotine was mediated by the action of prostaglandin E receptors (EP2 and EP4). EP2 or EP4 siRNA or antagonists impaired the nicotine-mediated NF-κB activity, upregulation of miR-16 and miR-21 and cell proliferation. Taken together, these results suggest that miR-16 and miR-21 are directly regulated by the transcription factor NF-κB and yet nicotine-promoted cell proliferation is mediated via EP2/4 receptors. Perhaps this study may shed light on the development of anticancer drugs to improve the chemosensitivity in smokers.

MicroRNA in colorectal cancer: from benchtop to bedside

Abstract: Colon carcinogenesis represents a stepwise progression from benign polyps to invasive adenocarcinomas and distant metastasis. It is believed that these pathologic changes are contributed by aberrant activation or inactivation of protein-coding proto-oncogenes and tumor suppressor genes. However, recent discoveries in microRNA (miRNA) research have reshaped our understanding of the role of non-protein-coding genes in carcinogenesis. In this regard, a remarkable number of miRNAs exhibit differential expression in colon cancer tissues. These miRNAs alter cell proliferation, apoptosis and metastasis through their interactions with intracellular signaling networks. From a clinical perspective, polymorphisms within miRNA-binding sites are associated with the risk for colon cancer, whereas miRNAs isolated from feces or blood may serve as biomarkers for early diagnosis. Altered expression of miRNA or polymorphisms in miRNA-related genes have also been shown to correlate with patient survival or treatment outcome. With further insights into miRNA dysregulation in colon cancer and the advancement of RNA delivery technology, it is anticipated that novel miRNA-based therapeutics will emerge.

Breast cancer stem cells: treatment resistance and therapeutic opportunities

Abstract: The clinical and pathologic heterogeneity of human breast cancer has long been recognized Now, molecular profiling has enriched our understanding of breast cancer heterogeneity and yielded new prognostic and predictive information Despite recent therapeutic advances, including the HER2-specific agent, trastuzumab, locoregional and systemic disease recurrence remain an ever-present threat to the health and well being of breast cancer survivors By definition, disease recurrence originates from residual treatment-resistant cells, which regenerate at least the initial breast cancer phenotype The discovery of the normal breast stem cell has re-ignited interest in the identity and properties of breast cancer stem-like cells and the relationship of these cells to the repopulating ability of treatment-resistant cells The cancer stem cell model of breast cancer development contrasts with the clonal evolution model, whereas the mixed model draws on features of both Although the origin and identity of breast cancer stem-like cells is contentious, treatment-resistant cells survive and propagate only because aberrant and potentially druggable signaling pathways are recruited As a means to increase the rates of breast cancer cure, several approaches to specific targeting of the treatment-resistant cell population exist and include methods for addressing the problem of radioresistance in particular

Berberine, an isoquinoline alkaloid, inhibits melanoma cancer cell migration by reducing the expressions of cyclooxygenase-2, prostaglandin E2 and prostaglandin E2 receptors

Abstract: Melanoma is the leading cause of death from skin disease due, in large part, to its propensity to metastasize. We have examined the effect of berberine, an isoquinoline alkaloid, on human melanoma cancer cell migration and the molecular mechanisms underlying these effects using melanoma cell lines, A375 and Hs294. Using an in vitro cell migration assay, we show that over expression of cyclooxygenase (COX)-2, its metabolite prostaglandin E2 (PGE2) and PGE2 receptors promote the migration of cells. We found that treatment of A375 and Hs294 cells with berberine resulted in concentration-dependent inhibition of migration of these cells, which was associated with a reduction in the levels of COX-2, PGE2 and PGE2 receptors (EP2 and EP4). Treatment of cells with celecoxib, a COX-2 inhibitor, or transient transfection of cells with COX-2 small interfering RNA, also inhibited cell migration. Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of COX-2 or PGE2, enhanced cell migration, whereas berberine inhibited TPA- or PGE2-promoted cell migration. Berberine reduced the basal levels as well as PGE2-stimulated expression levels of EP2 and EP4. Treatment of the cells with the EP4 agonist stimulated cell migration and berberine blocked EP4 agonist-induced cell migration activity. Moreover, berberine inhibited the activation of nuclear factor-kappa B (NF-κB), an upstream regulator of COX-2, in A375 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, inhibited cell migration. Together, these results indicate for the first time that berberine inhibits melanoma cell migration, an essential step in invasion and metastasis, by inhibition of COX-2, PGE2 and PGE2 receptors.

MicroRNA-499-5p promotes cellular invasion and tumor metastasis in colorectal cancer by targeting FOXO4 and PDCD4

Abstract: MicroRNAs (miRNAs) regulate tumor progression and invasion via direct interaction with target messenger RNAs (mRNAs). We defined miRNAs involved in cancer metastasis (metastamirs) using an established in vitro colorectal cancer (CRC) model of minimally metastatic cells (SW480 line) from a colon adenocarcinoma primary lesion and highly metastatic cells (SW620 line) from a metastatic lymph node from the same patient 1 year later. We used microarray analysis to identify miRNAs differentially expressed in SW480 and SW620 cells, focusing on miR-499-5p as a novel candidate prometastatic miRNA whose functions in cancer had not been studied. We confirmed increased miR-499-5p levels in highly invasive CRC cell lines and lymph node-positive CRC specimens. Furthermore, enhancing the expression of miR-499-5p promoted CRC cell migration and invasion in vitro and lung and liver metastasis in vivo, while silencing its expression resulted in reduced migration and invasion. Additionally, we identified FOXO4 and PDCD4 as direct and functional targets of miR-499-5p. Collectively, these findings suggested that miR-499-5p promoted metastasis of CRC cells and may be useful as a new potential therapeutic target for CRC.

Suppressed liver tumorigenesis in fat-1 mice with elevated omega-3 fatty acids is associated with increased omega-3 derived lipid mediators and reduced TNF-α.

Abstract: Liver tumors, particularly hepatocellular carcinoma (HCC), are a major cause of morbidity and mortality worldwide. The development of HCC is mostly associated with chronic inflammatory liver disease of various etiologies. Previous studies have shown that omega-3 (n-3) polyunsaturated fatty acids (PUFAs) dampen inflammation in the liver and decrease formation of tumor necrosis factor (TNF)-α. In this study, we used the fat-1 transgenic mouse model, which endogenously forms n-3 PUFA from n-6 PUFA to determine the effect of an increased n-3 PUFA tissue status on tumor formation in the diethylnitrosamine (DEN)-induced liver tumor model. Our results showed a decrease in tumor formation, in terms of size and number, in fat-1 mice compared with wild-type littermates. Plasma TNF-α levels and liver cyclooxygenase-2 expression were markedly lower in fat-1 mice. Furthermore, there was a decreased fibrotic activity in the livers of fat-1 mice. Lipidomics analyses of lipid mediators revealed significantly increased levels of the n-3 PUFA-derived 18-hydroxyeicosapentaenoic acid (18-HEPE) and 17-hydroxydocosahexaenoic acid (17-HDHA) in the livers of fat-1 animals treated with DEN. In vitro experiments showed that 18-HEPE and 17-HDHA could effectively suppress lipopolysacharide-triggered TNF-α formation in a murine macrophage cell line. The results of this study provide evidence that an increased tissue status of n-3 PUFA suppresses liver tumorigenesis, probably through inhibiting liver inflammation. The findings also point to a potential anticancer role for the n-3 PUFA-derived lipid mediators 18-HEPE and 17-HDHA, which can downregulate the important proinflammatory and proproliferative factor TNF-α.

Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways

Abstract: Cholangiocarcinoma (CCA) is a tumor with poor prognosis that is resistant to all currently available treatments. Whether curcumin, a nutraceutical derived from turmeric (Curcuma longa), has potential therapeutic activity against human CCA was investigated using three CCA cell lines (KKU100, KKU-M156 and KKU-M213). Examination of mitochondrial dehydrogenase activity, phosphatidylserine externalization, esterase staining, caspase activation and poly-adenosine diphosphate ribose polymerase cleavage demonstrated that curcumin inhibited proliferation of and induced apoptosis in these biliary cancer cells. Colony-formation assay confirmed the growth-inhibitory effect of curcumin on CCA cells. When examined for the mechanism, curcumin was found to activate multiple cell signaling pathways in these cells. First, all CCA cells exhibited constitutively active nuclear factor (NF)-κB, and treatment with curcumin abolished this activation as indicated by DNA binding, nuclear translocation and p65 phosphorylation. Second, curcumin suppressed activation of signal transducer and activator of transcription-3 as indicated by decreased phosphorylation at both tyrosine705 and serine727 and inhibition of janus kinase-1 phosphorylation. Third, curcumin induced expression of peroxisome proliferator-activated receptor gamma. Fourth, curcumin upregulated death receptors, DR4 and DR5. Fifth, curcumin suppressed the Akt activation pathway. Sixth, curcumin inhibited expression of cell survival proteins such as B-cell lymphoma-2, B-cell leukemia protein xL, X-linked inhibitor of apoptosis protein, c-FLIP, cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2 and survivin and proteins linked to cell proliferation, such as cyclin D1 and c-Myc. Seventh, the growth inhibitory effect of curcumin was enhanced in the IκB kinase-deficient cells, the enzyme required for nuclear factor-kappaB activation. Overall, our results indicate that curcumin mediates its antiproliferative and apoptotic effects through activation of multiple cell signaling pathways, and thus, its activity against CCA should be further investigated.

Human polymorphisms at long non-coding RNAs (lncRNAs) and association with prostate cancer risk

Abstract: Long non-coding RNAs (lncRNAs), representing a large proportion of non-coding transcripts across the human genome, are evolutionally conserved and biologically functional. At least one-third of the phenotype-related loci identified by genome-wide association studies (GWAS) are mapped to non-coding intervals. However, the relationships between phenotype-related loci and lncRNAs are largely unknown. Utilizing the 1000 Genomes data, we compared single-nucleotide polymorphisms (SNPs) within the sequences of lncRNA and protein-coding genes as defined in the Ensembl database. We further annotated the phenotype-related SNPs reported by GWAS at lncRNA intervals. Because prostate cancer (PCa) risk-related loci were enriched in lncRNAs, we then performed meta-analysis of two existing GWAS for discovery and an additional sample set for replication, revealing PCa risk-related loci at lncRNA regions. The SNP density in regions of lncRNA was similar to that in protein-coding regions, but they were less polymorphic than surrounding regions. Among the 1998 phenotype-related SNPs identified by GWAS, 52 loci were located directly in lncRNA intervals with a 1.5-fold enrichment compared with the entire genome. More than a 5-fold enrichment was observed for eight PCa risk-related loci in lncRNA genes. We also identified a new PCa risk-related SNP rs3787016 in an lncRNA region at 19q13 (per allele odds ratio = 1.19; 95% confidence interval: 1.11-1.27) with P value of 7.22 × 10(-7). lncRNAs may be important for interpreting and mining GWAS data. However, the catalog of lncRNAs needs to be better characterized in order to fully evaluate the relationship of phenotype-related loci with lncRNAs.

Anti-inflammatory effects of freeze-dried black raspberry powder in ulcerative colitis

Abstract: Ulcerative colitis (UC) is a chronic inflammatory disease of the colonic mucosa that can dramatically increase the risk of colon cancers. In the present study, we evaluated the effects of a dietary intervention of freeze-dried black raspberries (BRB), a natural food product with antioxidant and anti-inflammatory bioactivities, on disease severity in an experimental mouse model of UC using 3% dextran sodium sulfate (DSS). C57BL/6J mice were fed either a control diet or a diet containing BRB (5 or 10%) for 7-14 days and then the extent of colonic injury was assessed. Dietary BRB markedly reduced DSS-induced acute injury to the colonic epithelium. This protection included better maintenance of body mass and reductions in colonic shortening and ulceration. BRB treatment, however, did not affect the levels of either plasma nitric oxide or colon malondialdehyde, biomarkers of oxidative stress that are otherwise increased by DSS-induced colonic injury. BRB treatment for up to 7 days suppressed tissue levels of several key pro-inflammatory cytokines, including tumor necrosis factor α and interleukin 1β. Further examination of the inflammatory response by western blot analysis revealed that 7 day BRB treatment reduced the levels of phospho-IκBα within the colonic tissue. Colonic cyclooxygenase 2 levels were also dramatically suppressed by BRB treatment, with a concomitant decrease in the plasma prostaglandin E₂ (276 versus 34 ng/ml). These findings demonstrate a potent anti-inflammatory effect of BRB during DSS-induced colonic injury, supporting its possible therapeutic or preventive role in the pathogenesis of UC and related neoplastic events.

β-catenin-independent WNT signaling in basal-like breast cancer and brain metastasis

Abstract: A role of WNT signaling for primary breast cancers of the basal-like subtype and as a predictor of brain metastasis has been described. However, a responsible WNT ligand has not been identified. To further clarify this question, we comparatively investigated 22 human breast cancer brain metastases as well as the highly invasive human breast cancer cell line MDA-MB-231 and the weakly motile MCF-7 as models for the basal-like and the luminal A subtype. WNT5A and B were found overexpressed in MDA-MB-231 cells as compared with MCF-7. This corresponded to reduction of MDA-MB-231 invasiveness by WNT inhibitors, whereas MCF-7 invasion was enhanced by recombinant WNT5B and abolished by WNT and Jun-N-terminal kinase antagonists. Expression and subcellular distribution of β-catenin remained uninfluenced. Consistently, β-catenin was not localized in the nuclei of brain metastases while there was strong nuclear c-Jun staining. Similar to MDA-MB-231, metastases showed expression of WNT5A/B and the alternative WNT receptors ROR1 and 2. These findings were validated using external gene expression datasets (Gene Expression Omnibus) of different breast cancer subtypes and brain metastases. Hierarchical cluster analysis yielded a close relation between basal-like cancers and brain metastases. Gene set enrichment analyses confirmed WNT pathway enrichment not only in basal-like primaries but also in cerebral metastases of all subtypes. In conclusion, WNT signaling seems highly relevant for basal-like and other subtypes of breast cancers metastasizing into the brain. β-catenin-independent WNT signaling, presumably via ROR1-2, plays a major role in this context.

Nicotinamide N-methyltransferase induces cellular invasion through activating matrix metalloproteinase-2 expression in clear cell renal cell carcinoma cells

Abstract: Nicotinamide N-methyltransferase (NNMT) was recently identified as one clear cell renal cell carcinoma (ccRCC)-associated gene by analyzing full-length complementary DNA-enriched libraries of ccRCC tissues. The aim of this study is to investigate the potential role of NNMT in cellular invasion. A strong NNMT expression is accompanied with a high invasive activity in ccRCC cell lines, and small interfering RNA-mediated NNMT knockdown effectively suppressed the invasive capacity of ccRCC cells, whereas NNMT overexpression markedly enhanced that of human embryonic kidney 293 (HEK293) cells. A positive correlation between the expression of NNMT and matrix metallopeptidase (MMP)-2 was found in ccRCC cell lines and clinical tissues. The treatment of blocking antibody or inhibitor specific to MMP-2 significantly suppressed NNMT-dependent cellular invasion in HEK293 cells. Furthermore, SP-1-binding region of MMP-2 promoter was found to be essential in NNMT-induced MMP-2 expression. The specific inhibitors of PI3K/Akt signaling markedly decreased the binding of SP1 to MMP-2 promoter as shown by chromatin immunoprecipitation assay. We also demonstrated that PI3K/Akt pathway plays a role in NNMT-dependent cellular invasion and MMP-2 activation. Moreover, short hairpin RNA-mediated knockdown of NNMT expression efficiently inhibited the growth and metastasis of ccRCC cells in non-obese diabetic severe combined immunodeficiency mice. Taken together, the present study suggests that NNMT has a crucial role in cellular invasion via activating PI3K/Akt/SP1/MMP-2 pathway in ccRCC.

Hedgehog signaling and therapeutics in pancreatic cancer

Abstract: Objective To conduct a systematic review of the role that the hedgehog signaling pathway has in pancreatic cancer tumorigenesis. Method PubMed search (2000-2010) and literature based references. Results Firstly, in 2009 a genetic analysis of pancreatic cancers found that a core set of 12 cellular signaling pathways including hedgehog were genetically altered in 67-100% of cases. Secondly, in vitro and in vivo studies of treatment with cyclopamine (a naturally occurring antagonist of the hedgehog signaling pathway component; Smoothened) has shown that inhibition of hedgehog can abrogate pancreatic cancer metastasis. Thirdly, experimental evidence has demonstrated that sonic hedgehog (Shh) is correlated with desmoplasia in pancreatic cancer. This is important because targeting the Shh pathway potentially may facilitate chemotherapeutic drug delivery as pancreatic cancers tend to have a dense fibrotic stroma that extrinsically compresses the tumor vasculature leading to a hypoperfusing intratumoral circulation. It is probable that patients with locally advanced pancreatic cancer will derive the greatest benefit from treatment with Smoothened antagonists. Fourthly, it has been found that ligand dependent activation by hedgehog occurs in the tumor stromal microenvironment in pancreatic cancer, a paracrine effect on tumorigenesis. Finally, in pancreatic cancer, cells with the CD44+CD24+ESA+ immunophenotype select a population enriched for cancer initiating stem cells. Shh is increased 46-fold in CD44+CD24+ESA+ cells compared with normal pancreatic epithelial cells. Medications that destruct pancreatic cancer initiating stem cells are a potentially novel strategy in cancer treatment. Conclusions Aberrant hedgehog signaling occurs in pancreatic cancer tumorigenesis and therapeutics that target the transmembrane receptor Smoothened abrogate hedgehog signaling and may improve the outcomes of patients with pancreatic cancer.

MicroRNA-423 promotes cell growth and regulates G(1)/S transition by targeting p21Cip1/Waf1 in hepatocellular carcinoma.

Abstract: MicroRNAs (miRNAs) are small non-coding RNA molecules that are often located in genomic breakpoint regions and can act as oncogenes or tumor suppressor genes in human cancer. Our previous study showed that microRNA-423 (miR-423), which localized to the frequently amplified region of chromosome 17q11, was upregulated in hepatocellular carcinoma (HCC). However, the potential functions and exact mechanistic roles of miR-423 in hepatic carcinogenesis remain unknown. Here, we demonstrated that miR-423 significantly promotes cell growth and cell cycle progression at the G(1)/S transition in HCC cells. In particular, we found that miR-423-3p contributes to these effects, whereas miR-423-5p does not. Further studies revealed that p21Cip1/Waf1 is a downstream target of miR-423 in HCC cells, as miR-423 bound directly to its 3' untranslated region and reduced both the messenger RNA and protein levels of p21Cip1/Waf1. Moreover, enforced expression of p21Cip1/Waf1 abrogated miR-423-induced effects on HCC cell proliferation and cell cycle progression. These findings indicate that miR-423 exerts growth-promoting effects in hepatic carcinogenesis through the suppression of tumor suppressor p21Cip1/Waf1 expression. The results of this study define miR-423 as a new oncogenic miRNA in HCC.