The transcription factor ZEB1 is normally not expressed in epithelial cells. When inappropriately expressed in carcinomas, ZEB1 initiates epithelial to mesenchymal transition due to its ability to repress E-cadherin and other genes involved in polarity. Recently, ZEB1 and ZEB2 have been identified as direct targets of the microRNA-200c family. We find that miR-200c levels are high in well-differentiated endometrial, breast, and ovarian cancer cell lines, but extremely low in poorly differentiated cancer cells. Low or absent miR-200c results in aberrant expression of ZEB1 and consequent repression of E-cadherin. Reinstatement of miR-200c to such cells restores E-cadherin and dramatically reduces migration and invasion. Microarray profiling reveals that in addition to ZEB1 and ZEB2, other mesenchymal genes (such as FN1, NTRK2, and QKI), which are also predicted direct targets of miR-200c, are indeed inhibited by addition of exogenous miR-200c. One such gene, class III β-tubulin (TUBB3), which encodes a tubulin isotype normally found only in neuronal cells, is a direct target of miR-200c. This finding is of particular significance because we show that restoration of miR-200c increases sensitivity to microtubule-targeting agents by 85%. Because expression of TUBB3 is a common mechanism of resistance to microtubule-binding chemotherapeutic agents in many types of solid tumors, the ability of miR-200c to restore chemosensitivity to such agents may be explained by its ability to reduce TUBB3. Because miR-200c is crucial for maintenance of epithelial identity, behavior, and sensitivity to chemotherapy, we propose that it warrants further investigation as a therapeutic strategy for aggressive, drug-resistant cancers.

Epithelial-mesenchymal (EMT) and mesenchymal-epithelial (MET) transitions occur in the development of human tumorigenesis and are part of the natural history of the process to adapt to the changing microenvironment. In this setting, the miR-200 family is recognized as a master regulator of the epithelial phenotype by targeting ZEB1 and ZEB2, two important transcriptional repressors of the cell adherence (E-cadherin) and polarity (CRB3 and LGL2) genes. Recently, the putative DNA methylation associated inactivation of various miR-200 members has been described in cancer. Herein, we show that the miR-200ba429 and miR-200c141 transcripts undergo a dynamic epigenetic regulation linked to EMT or MET phenotypes in tumor progression. The 5'-CpG islands of both miR-200 loci were found unmethylated and coupled to the expression of the corresponding miRNAs in human cancer cell lines with epithelial features, such as low levels of ZEB1/ZEB2 and high expression of E-cadherin, CRB3 and LGL2, while CpG island hypermethylation-associated silencing was observed in transformed cells with mesenchymal characteristics. The recovery of miR-200ba429 and miR-200c141 expression by stable transfection in the hypermethylated cells restored the epithelial markers and inhibited migration in cell culture and tumoral growth and metastasis formation in nude mice. We also discovered, using both cell culture and animal models, that the miR-200 epigenetic silencing is not an static and fixed process but it can be shifted to hypermethylated or unmethylated 5'-CpG island status corresponding to the EMT and MET phenotypes, respectively. In fact, careful laser microdissection in human primary colorectal tumorigenesis unveiled that in normal colon mucosa crypts (epithelia) and stroma (mesenchyma) already are unmethylated and methylated at these loci, respectively; and that the colorectal tumors undergo selective miR-200 hypermethylation of their epithelial component. These findings indicate that the epigenetic silencing plasticity of the miR-200 family contributes to the evolving and adapting phenotypes of human tumors.

The role of microRNAs (miRs), which are endogenous RNA oligonucleotides that regulate gene expression, in diabetic nephropathy is unknown. Here, we performed expression profiling of cultured proximal tubular cells (PTCs) under high-glucose and control conditions. We identified expression of 103 of 328 microRNAs but did not observe glucose-induced changes in expression. Next, we performed miR expression profiling in pooled RNA from formalin-fixed, paraffin-embedded tissue from renal biopsies. We studied three groups of patients with established diabetic nephropathy and detected 103 of 365 miRs. Two miRs differed by more than two-fold between progressors and nonprogressors, and 12 miRs differed between late presenters and other biopsies. We noted the greatest change in miR-192 expression, which was significantly lower in late presenters. Furthermore, in individual biopsies, low expression of miR-192 correlated with tubulointerstitial fibrosis and low estimated GFR. In vitro, treatment of PTCs with TGF-beta1 decreased miR-192 expression. Overexpression of miR-192 suppressed expression of the E-Box repressors ZEB1 and ZEB2, thereby opposing TGF-beta-mediated downregulation of E-cadherin. In summary, loss of miR-192 expression associates with increased fibrosis and decreased estimated GFR in diabetic nephropathy in vivo, perhaps by enhancing TGF-beta-mediated downregulation of E-cadherin in PTCs.

Recent studies reveal that long non-coding RNAs (lncRNAs) have been shown to have important regulatory roles in cancer biology, and lncRNA MALAT-1 expression is upregulated in some tumors. However, the contributions of MALAT-1 to bladder cancer metastasis remain largely unknown. In the present study we evaluated MALAT-1 expression in bladder cancer tissues by real-time PCR, and defined its biological functions. We verified that MALAT-1 levels were upregulated in bladder cancer tissues compared with adjacent normal tissues, and MALAT-1 expression was remarkably increased in primary tumors that subsequently metastasized, when compared to those primary tumors that did not metastasize. SiRNA-mediated MALAT-1 silencing impaired in vitro bladder cancer cell migration. Downregulation of MALAT-1 resulted in a decrease of the epithelial-mesenchymal transition (EMT)-associated ZEB1, ZEB2 and Slug levels, and an increase of E-cadherin levels. We further demonstrated that MALAT-1 promoted EMT by activating Wnt signaling in vitro. These data suggest an important role for MALAT-1 in regulating metastasis of bladder cancer and the potential application of MALAT-1 in bladder cancer therapy.

Aberrant expression of embryonic epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) in epithelial cells triggers EMT, neoplastic transformation, stemness, and metastatic dissemination. We found that regulation and functions of EMT-TFs are different in malignant melanoma. SNAIL2 and ZEB2 transcription factors are expressed in normal melanocytes and behave as tumor-suppressor proteins by activating an MITF-dependent melanocyte differentiation program. In response to NRAS/BRAF activation, EMT-TF network undergoes a profound reorganization in favor of TWIST1 and ZEB1. This reversible switch cooperates with BRAF in promoting dedifferentiation and neoplastic transformation of melanocytes. We detected EMT-TF reprogramming in late-stage melanoma in association with enhanced phospho-ERK levels. This switch results in E-cadherin loss, enhanced invasion, and constitutes an independent factor of poor prognosis in melanoma patients.

Aberrant DNA methylation and microRNA expression play important roles in the pathogenesis of pancreatic cancer. While interrogating differentially methylated CpG islands in pancreatic cancer, we identified two members of miR-200 family, miR-200a and miR-200b, that were hypomethylated and overexpressed in pancreatic cancer. We also identified prevalent hypermethylation and silencing of one of their downstream targets, SIP1 (ZFHX1B, ZEB2), whose protein product suppresses E-cadherin expression and contributes to epithelial mesenchymal transition. In a panel of 23 pancreatic cell lines, we observed a reciprocal correlation between miR-200, SIP1, and E-cadherin expression, with pancreatic cancer-associated fibroblasts showing the opposite expression pattern to most pancreatic cancers. In Panc-1 cells, which express SIP1, have low E-cadherin expression, and do not express miR-200a or miR-200b, treatment with miR-200a and miR-200b downregulated SIP1 mRNA and increased E-cadherin expression. However, most pancreatic cancers express miR-200a and miR-200b, but this expression does not affect SIP1 expression, as the SIP1 promoter is silenced by hypermethylation and in these cancers E-cadherin is generally expressed. Both miR-200a and miR-200b were significantly elevated in the sera of pancreatic cancer and chronic pancreatitis patients compared with healthy controls (P < 0.0001), yielding receiver operating characteristic curve areas of 0.861 and 0.85, respectively. In conclusion, most pancreatic cancers display hypomethylation and overexpression of miR-200a and miR-200b, silencing of SIP1 by promoter methylation, and retention of E-cadherin expression. The elevated serum levels of miR-200a and miR-200b in most patients with pancreatic cancer could have diagnostic utility.

Dendritic cells (DC) are a class of bone-marrow-derived cells arising from lympho-myeloid haematopoiesis that form an essential interface between the innate sensing of pathogens and the activation of adaptive immunity. This task requires a wide range of mechanisms and responses, which are divided between three major DC subsets: plasmacytoid DC (pDC), myeloid/conventional DC1 (cDC1) and myeloid/conventional DC2 (cDC2). Each DC subset develops under the control of a specific repertoire of transcription factors involving differential levels of IRF8 and IRF4 in collaboration with PU.1, ID2, E2-2, ZEB2, KLF4, IKZF1 and BATF3. DC haematopoiesis is conserved between mammalian species and is distinct from monocyte development. Although monocytes can differentiate into DC, especially during inflammation, most quiescent tissues contain significant resident populations of DC lineage cells. An extended range of surface markers facilitates the identification of specific DC subsets although it remains difficult to dissociate cDC2 from monocyte-derived DC in some settings. Recent studies based on an increasing level of resolution of phenotype and gene expression have identified pre-DC in human blood and heterogeneity among cDC2. These advances facilitate the integration of mouse and human immunology, support efforts to unravel human DC function in vivo and continue to present new translational opportunities to medicine.

OBJECTIVE

Advanced metastatic prostate cancer (PCa) is a fatal disease, with only palliative therapeutic options. Though almost 80% of cases of metastatic PCa present bone metastasis, our current understanding of the molecular mechanisms that govern this metastatic dissemination remains fragmentary. The main objective of the present study was to identify microRNA (miRNA) genes that regulate metastatic PCa.

METHODS

miRNA expression profiling was done in human prostate cell lines to identify dysregulated miRNA components of advanced PCa. miR-203 expression was assessed in prostate carcinoma cell lines and clinical specimens by real-time PCR and in situ hybridization. To assess the biological significance of miR-203, miR-203 was reexpressed in bone metastatic PCa cell lines followed by in vitro and in vivo functional assays.

RESULTS

miR-203 expression is specifically attenuated in bone metastatic PCa suggesting a fundamental antimetastatic role for this miRNA. Reintroduction of miR-203 in bone metastatic PCa cell lines suppresses metastasis via inhibition of several critical steps of the metastatic cascade including epithelial-mesenchymal transition, invasion, and motility. Ectopic miR-203 significantly attenuated the development of metastasis in a bone metastatic model of PCa. Importantly, miR-203 regulates a cohort of pro-metastatic genes including ZEB2, Bmi, survivin, and bone-specific effectors including Runx2, a master regulator of bone metastasis.

CONCLUSIONS

miR-203 is an "antimetastatic" miRNA in PCa that acts at multiple steps of the PCa metastatic cascade via repression of a cohort of prometastatic targets. miR-203 may be an attractive target for therapeutic intervention in advanced PCa.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that induces growth arrest, tissue fibrosis, and epithelial-mesenchymal transition (EMT) through activation of Smad and non-Smad signaling pathways. EMT is the differentiation switch by which polarized epithelial cells differentiate into contractile and motile mesenchymal cells. Cell motility and invasive capacity are activated upon EMT. Multiple transcription factors, including deltaEF1/ZEB1, SIP1/ZEB2, and Snail/SNAI1, are induced by TGF-beta-Smad signaling and play critical roles in TGF-beta-induced EMT. In addition, both non-Smad signaling activated by TGF-beta and cross-talk with other signaling pathways play important roles in induction of EMT. Of these, Ras signaling synergizes with TGF-beta-Smad signaling, and plays an important role in the induction of EMT. TGF-beta inhibitors prevent invasion and metastasis of advanced cancer through multiple mechanisms, including inhibition of EMT. The discovery of molecules that inhibit TGF-beta-induced EMT but not TGF-beta-induced growth arrest may be an ideal strategy for treatment of invasion and metastasis of cancer.

OBJECTIVE

Increased deposition of extracellular matrix (ECM) within the kidney is driven by profibrotic mediators including transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF). We investigated whether some of their effects may be mediated through changes in expression of certain microRNAs (miRNAs).

METHODS

Proximal tubular cells, primary rat mesangial cells, and human podocytes were analyzed for changes in the expression of key genes, ECM proteins, and miRNA after exposure to TGF-beta (1-10 ng/microl). Tubular cells were also infected with CTGF-adenovirus. Kidneys from diabetic apoE mice were also analyzed for changes in gene expression and miRNA levels.

RESULTS

TGF-beta treatment was associated with morphologic and phenotypic changes typical of epithelial-mesenchymal transition (EMT) including increased fibrogenesis in all renal cell types and decreased E-cadherin expression in tubular cells. TGF-beta treatment also modulated the expression of certain miRNAs, including decreased expression of miR-192/215 in tubular cells, mesangial cells, which are also decreased in diabetic kidney. Ectopic expression of miR-192/215 increased E-cadherin levels via repressed translation of ZEB2 mRNA, in the presence and absence of TGF-beta, as demonstrated by a ZEB2 3'-untranslated region luciferase reporter assay. However, ectopic expression of miR-192/215 did not affect the expression of matrix proteins or their induction by TGF-beta. In contrast, CTGF increased miR-192/215 levels, causing a decrease in ZEB2, and consequently increased E-cadherin mRNA.

CONCLUSIONS

These data demonstrate the linking role of miRNA-192/215 and ZEB2 in TGF-beta/CTGF-mediated changes in E-cadherin expression. These changes appear to occur independently of augmentation of matrix protein synthesis, suggesting that a multistep EMT program is not necessary for fibrogenesis to occur.

BACKGROUND

The epithelial to mesenchymal transition (EMT) is a molecular process through which an epithelial cell undergoes transdifferentiation into a mesenchymal phenotype. The role of EMT in embryogenesis is well-characterized and increasing evidence suggests that elements of the transition may be important in other processes, including metastasis and drug resistance in various different cancers.

METHODS

Agilent 4 × 44 K whole human genome arrays and selected reaction monitoring mass spectrometry were used to investigate mRNA and protein expression in A2780 cisplatin sensitive and resistant cell lines. Invasion and migration were assessed using Boyden chamber assays. Gene knockdown of snail and slug was done using targeted siRNA. Clinical relevance of the EMT pathway was assessed in a cohort of primary ovarian tumours using data from Affymetrix GeneChip Human Genome U133 plus 2.0 arrays.

RESULTS

Morphological and phenotypic hallmarks of EMT were identified in the chemoresistant cells. Subsequent gene expression profiling revealed upregulation of EMT-related transcription factors including snail, slug, twist2 and zeb2. Proteomic analysis demonstrated up regulation of Snail and Slug as well as the mesenchymal marker Vimentin, and down regulation of E-cadherin, an epithelial marker. By reducing expression of snail and slug, the mesenchymal phenotype was largely reversed and cells were resensitized to cisplatin. Finally, gene expression data from primary tumours mirrored the finding that an EMT-like pathway is activated in resistant tumours relative to sensitive tumours, suggesting that the involvement of this transition may not be limited to in vitro drug effects.

CONCLUSIONS

This work strongly suggests that genes associated with EMT may play a significant role in cisplatin resistance in ovarian cancer, therefore potentially leading to the development of predictive biomarkers of drug response or novel therapeutic strategies for overcoming drug resistance.

Forkhead-box (FOX) family proteins, involved in cell growth and differentiation as well as embryogenesis and longevity, are DNA-binding proteins regulating transcription and DNA repair. The focus of this review is on the mechanisms of FOX-related human carcinogenesis. FOXA1 is overexpressed as a result of gene amplification in lung cancer, esophageal cancer, ER-positive breast cancer and anaplastic thyroid cancer and is point-mutated in prostate cancer. FOXA1 overexpression in breast cancer and prostate cancer is associated with good or poor prognosis, respectively. Single nucleotide polymorphism (SNP) within the 5'-UTR of the FOXE1 (TTF2) gene is associated with thyroid cancer risk. FOXF1 overexpression in breast cancer is associated with epithelial-to-mesenchymal transition (EMT). FOXM1 is overexpressed owing to gene amplification in basal-type breast cancer and diffuse large B-cell lymphoma (DLBCL), and it is transcriptionally upregulated owing to Hedgehog-GLI, hypoxia-HIF1α or YAP-TEAD signaling activation. FOXM1 overexpression leads to malignant phenotypes by directly upregulating CCNB1, AURKB, MYC and SKP2 and indirectly upregulating ZEB1 and ZEB2 via miR-200b downregulation. Tumor suppressor functions of FOXO transcription factors are lost in cancer cells as a result of chromosomal translocation, deletion, miRNA-mediated repression, AKT-mediated cytoplasmic sequestration or ubiquitination-mediated proteasomal degradation. FOXP1 is upregulated as a result of gene fusion or amplification in DLBCL and MALT lymphoma and also repression of miRNAs, such as miR-1, miR-34a and miR-504. FOXP1 overexpression is associated with poor prognosis in DLBCL, gastric MALT lymphoma and hepatocellular carcinoma but with good prognosis in breast cancer. In neuroblastoma, the entire coding region of the FOXR1 (FOXN5) gene is fused to the MLL or the PAFAH1B gene owing to interstitial deletions. FOXR1 fusion genes function as oncogenes that repress transcription of FOXO target genes. Whole-genome sequencing data from tens of thousands of human cancers will uncover the mutational landscape of FOX family genes themselves as well as FOX-binding sites, which will be ultimately applied for cancer diagnostics, prognostics, and therapeutics.

MicroRNAs (miRNAs) are single-stranded, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Genes encoding miRNAs are located in regions of the genome that are commonly amplified, deleted or rearranged. They are commonly dysregulated in human cancers and known to act as oncogenes or tumor suppressors. Members of the miR-200 miRNA family are downregulated in human cancer cells and tumors due to aberrant epigenetic gene silencing and play a critical role in the suppression of epithelial-to-mesenchymal transition (EMT), tumor cell adhesion, migration, invasion and metastasis, by targeting and repressing the expression of key mRNAs that are involved in EMT (ZEB1 and ZEB2), β-catenin/Wnt signaling (β-catenin), EGFR inhibitor resistance (ERRFI-1) and chemoresistance to therapeutic agents (TUBB3). Since the miR-200 family functions as putative tumor suppressors and represent biomarkers for poorly differentiated and aggressive cancers, restoration of miR-200 expression may have therapeutic implications for the treatment of metastatic and drug-resistant tumors.

BACKGROUND

miR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Loss of miR-200c is often observed in carcinoma cells that have undergone epithelial to mesenchymal transition (EMT). Restoration of miR-200c to such cells leads to a reduction in stem cell-like characteristics, reduced migration and invasion, and increased sensitivity to taxanes. Here we investigate the functional role of novel targets of miR-200c in the aggressive behavior of breast and endometrial cancer cells.

METHODS

Putative target genes of miR-200c identified by microarray profiling were validated as direct targets using dual luciferase reporter assays. Following restoration of miR-200c to triple negative breast cancer and type 2 endometrial cancer cell lines that had undergone EMT, levels of endogenous target mRNA and respective protein products were measured. Migration and sensitivity to anoikis were determined using wound healing assays or cell-death ELISAs and viability assays respectively.

RESULTS

We found that restoration of miR-200c suppresses anoikis resistance, a novel function for this influential miRNA. We identified novel targets of miR-200c, including genes encoding fibronectin 1 (FN1), moesin (MSN), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and Rho GTPase activating protein 19 (ARHGAP19). These targets all encode proteins normally expressed in cells of mesenchymal or neuronal origin; however, in carcinoma cells that lack miR-200c they become aberrantly expressed and contribute to the EMT phenotype and aggressive behavior. We showed that these targets are inhibited upon restoration of miR-200c to aggressive breast and endometrial cancer cells. We demonstrated that inhibition of MSN and/or FN1 is sufficient to mediate the ability of miR-200c to suppress cell migration. Lastly, we showed that targeting of TrkB mediates the ability of miR-200c to restore anoikis sensitivity.

CONCLUSIONS

miR-200c maintains the epithelial phenotype not only by targeting ZEB1/2, which usually facilitates restoration of E-cadherin expression, but also by actively repressing a program of mesenchymal and neuronal genes involved in cell motility and anoikis resistance.

LncRNAs have critical roles in various biological processes ranging from embryonic development to human diseases, including cancer progression, although their detailed mechanistic functions remain illusive. The lncRNA linc-ROR has been shown to contribute to the maintenance of induced pluripotent stem cells and embryonic stem cells. In this study, we discovered that linc-ROR was upregulated in breast tumor samples, and ectopic overexpression of linc-ROR in immortalized human mammary epithelial cells induced an epithelial-to-mesenchymal transition (EMT) program. Moreover, we showed that linc-ROR enhanced breast cancer cell migration and invasion, which was accompanied by generation of stem cell properties. Contrarily, silencing of linc-ROR repressed breast tumor growth and lung metastasis in vivo. Mechanistically, our data revealed that linc-ROR was associated with miRNPs and functioned as a competing endogenous RNA to mi-205. Specifically, linc-ROR prevented the degradation of mir-205 target genes, including the EMT inducer ZEB2. Thus our results indicate that linc-ROR functions as an important regulator of EMT and can promote breast cancer progression and metastasis through regulation of miRNAs. Potentially, the findings of this study implicate the relevance of linc-ROR as a possible therapeutic target for aggressive and metastatic breast cancers.

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence, and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity, and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b, and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein.

The basal-like subtype of breast cancer has an aggressive clinical behavior compared to that of the luminal subtype. We identified the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype-specific miRNAs and showed that expression of miR-221/222 decreased expression of epithelial-specific genes and increased expression of mesenchymal-specific genes, and increased cell migration and invasion in a manner characteristic of the epithelial-to-mesenchymal transition (EMT). The transcription factor FOSL1 (also known as Fra-1), which is found in basal-like breast cancers but not in the luminal subtype, stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of the epidermal growth factor receptor (EGFR) or MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. Furthermore, miR-221/222-mediated reduction in E-cadherin abundance depended on their targeting the 3' untranslated region of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1), which inhibited EMT by decreasing ZEB2 (zinc finger E-box-binding homeobox2) expression. We conclude that by promoting EMT, miR-221/222 may contribute to the more aggressive clinical behavior of basal-like breast cancers.

Most chronic kidney injuries inevitably progress to irreversible renal fibrosis. Tubular epithelial-to-mesenchymal transition (EMT) is recognized to play pivotal roles in the process of renal fibrosis. However, a comprehensive understanding of the pathogenesis of renal scar formation and progression remains an urgent task for renal researchers. The endogenously produced microRNAs (miRNAs), proved to play important roles in gene regulation, probably regulate most genes involved in EMT. In this study, we applied microarray analysis to investigate the expression profiles of miRNA in murine interstitial fibrotic kidneys induced by unilateral ureteral obstruction (UUO). It was found that miR-200a and miR-141, two members of the miR-200 family, were downregulated at the early phase of UUO. In TGF-β1-induced tubular EMT in vitro, it was also found that the members of the miR-200 family were downregulated in a Smad signaling-dependent manner. It was demonstrated that the miR-200 family was responsible for protecting tubular epithelial cells from mesenchymal transition by target suppression of zinc finger E-box-binding homeobox (ZEB) 1 and ZEB2, which are E-cadherin transcriptional repressors. The results suggest that downregulation of the miR-200 family initiates the dedifferentiation of renal tubules and progression of renal fibrosis, which might provide important targets for novel therapeutic strategies.

SHH, IHH, and DHH are lipid-modified secreted proteins binding to Patched receptors, and CDON, BOC or GAS1 co-receptors. In the absence of Hedgehog signaling, GLI1 is transcriptionally repressed, GLI2 is phosphorylated by GSK3 and CK1 for the FBXW11 (betaTRCP2)-mediated degradation, and GLI3 is processed to a cleaved repressor. In the presence of Hedgehog signaling, Smoothened is relieved from Patched-mediated suppression due to the Hedgehog-dependent internalization of Patched, which leads to MAP3K10 (MST) activation and SUFU inactivation for the stabilization and nuclear accumulation of GLI family members. GLI activators then upregulate CCND1, CCND2 for cell cycle acceleration, FOXA2, FOXC2, FOXE1, FOXF1, FOXL1, FOXP3, POU3F1, RUNX2, SOX13, TBX2 for cell fate determination, JAG2, INHBC, and INHBE for stem cell signaling regulation. Hedgehog signals also upregulate SFRP1 in mesenchymal cells for WNT signaling regulation. Epithelial-to-mesenchymal transition (EMT) during embryogenesis, adult tissue homeostasis and carcinogenesis is characterized by class switch from E-cadherin to N-cadherin. SNAI1 (Snail), SNAI2 (Slug), SNAI3, ZEB1, ZEB2 (SIP1), KLF8, TWIST1, and TWIST2 are EMT regulators repressing CDH1 gene encoding E-cadherin. Hedgehog signals induce JAG2 upregulation for Notch-CSL-mediated SNAI1 upregulation, and also induce TGFbeta1 secretion for ZEB1 and ZEB2 upregulation via TGFbeta receptor and NF-kappaB. TGFbeta-mediated downregulation of miR-141, miR-200a, miR-200b, miR-200c, miR-205, and miR-429 results in upregulation of ZEB1 and ZEB2 proteins. Hedgehog signaling activation indirectly leads to EMT through FGF, Notch, TGFbeta signaling cascades, and miRNA regulatory networks. miRNAs targeted to stem cell signaling components or EMT regulators are potent drug targets; however, off-target effects should be strictly controlled before clinical application of synthetic miRNA. Peptide mimetic and RNA aptamer could also be utilized as Hedgehog signaling inhibitors or EMT suppressors.

Down-regulation of miR-138 (microRNA-138) has been frequently observed in various cancers, including HNSCC (head and neck squamous cell carcinoma). Our previous studies suggest that down-regulation of miR-138 is associated with mesenchymal-like cell morphology and enhanced cell migration and invasion. In the present study, we demonstrated that these miR-138-induced changes were accompanied by marked reduction in E-cad (E-cadherin) expression and enhanced Vim (vimentin) expression, characteristics of EMT (epithelial-mesenchymal transition). On the basis of a combined experimental and bioinformatics analysis, we identified a number of miR-138 target genes that are associated with EMT, including VIM, ZEB2 (zinc finger E-box-binding homeobox 2) and EZH2 (enhancer of zeste homologue 2). Direct targeting of miR-138 to specific sequences located in the mRNAs of the VIM, ZEB2 and EZH2 genes was confirmed using luciferase reporter gene assays. Our functional analyses (knock-in and knock-down) demonstrated that miR-138 regulates the EMT via three distinct pathways: (i) direct targeting of VIM mRNA and controlling the expression of VIM at a post-transcriptional level, (ii) targeting the transcriptional repressors (ZEB2) which in turn regulating the transcription activity of the E-cad gene, and (iii) targeting the epigenetic regulator EZH2 which in turn modulates its gene silencing effects on the downstream genes including E-cad. These results, together with our previously observed miR-138 effects on cell migration and invasion through targeting RhoC (Rho-related GTP-binding protein C) and ROCK2 (Rho-associated, coiled-coil-containing protein kinase 2) concurrently, suggest that miR-138 is a multi-functional molecular regulator and plays major roles in EMT and in HNSCC progression.

OBJECTIVE

Our objective was to characterize the expression and function of the miR-200 family of microRNAs (miRNA) in ovarian carcinogenesis.

METHODS

We used qRT-PCR to examine expression of the miR-200 miRNA family and its predicted targets, the ZEB1 and ZEB2 transcriptional repressors, in primary cultures of normal cells from the surface of the ovary and in a panel of 70 ovarian cancer tissues and 15 ovarian cancer cell lines. We studied the mechanisms of regulation of miR-200 miRNAs and ZEB transcription factors in ovarian cells using 3' UTR luciferase reporters, promoter luciferase reporters and siRNAs.

RESULTS

miR-200 family members are expressed at low or negligible levels in normal ovarian surface cells and substantially increase in expression in ovarian cancer, whereas expression of ZEB1 and ZEB2 shows the opposite pattern. There is reciprocal repression between miR-200 family members and ZEB transcription factors, creating a double negative regulatory feedback loop resembling that reported in other cancer cell types. In contrast to epithelial cells from other sites, expression levels of miR-200 miRNAs and ZEB1/2 in cells from the ovarian surface are more consistent with a mesenchymal cell phenotype, potentially reflecting the mesothelial origin of the ovarian surface.

CONCLUSIONS

Analysis of ovarian cancer tissues suggests that ovarian surface cells acquire a more epithelial miR-200-ZEB1/2 phenotype as they undergo transformation, switching from a miR-200 familyLOW and ZEB1/2HIGH state to a miR-200 familyHIGH and ZEB1/2LOW phenotype. Collectively, our data support the mesothelial-to-epithelial (Meso-E-T) model for development of ovarian cancers that arise from ovarian surface cells, as has been proposed previously on the basis of studies of protein markers.

The conversion of early stage tumors into invasive malignancies with an aggressive phenotype has been associated with the irreversible loss of E-cadherin expression. The loss of E-cadherin expression in human tumors, including breast cancer, has been attributed to promoter CpG island hypermethylation and direct inhibition by transcriptional repressors. Recent evidence demonstrates that up-regulation of E-cadherin by microRNA-200b (miR-200b) and miR-200c through direct targeting of transcriptional repressors of E-cadherin, ZEB1, and ZEB2, inhibits epithelial-to-mesenchymal transition (EMT), a crucial process in the tumor progression. We demonstrate that microRNA miR-200 family-mediated transcriptional up-regulation of E-cadherin in mesenchymal MDA-MB-231 and BT-549 cells is associated directly with translational repression of ZEB1 and indirectly with increased acetylation of histone H3 at the E-cadherin promoter. The increase in histone H3 acetylation may be attributed to the disruption of repressive complexes between ZEB1 and histone deacetylases and to the inhibition of SIRT1, a class III histone deacetylase. These events inhibit EMT and reactivate a less aggressive epithelial phenotype in cancer cells. Additionally, disruption of ZEB1-histone deacetylase repressor complexes and down-regulation of SIRT1 histone deacetylase up-regulate proapoptotic genes in the p53 apoptotic pathway resulting in the increased sensitivity of cancer cells to the chemotherapeutic agent doxorubicin.

Throughout most of pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) transcriptional activity, whereas spontaneous labor is initiated/facilitated by a concerted series of biochemical events that activate inflammatory pathways and have a negative impact on PR function. In this study, we uncovered a previously undescribed regulatory pathway whereby micro-RNAs (miRNAs) serve as hormonally modulated and conserved mediators of contraction-associated genes in the pregnant uterus in the mouse and human. Using miRNA and gene expression microarray analyses of uterine tissues, we identified a conserved family of miRNAs, the miR-200 family, that is highly induced at term in both mice and humans as well as two coordinately down-regulated targets, zinc finger E-box binding homeobox proteins ZEB1 and ZEB2, which act as transcriptional repressors. We also observed up-regulation of the miR-200 family and down-regulation of ZEB1 and ZEB2 in two different mouse models of preterm labor. We further demonstrated that ZEB1 is directly up-regulated by the action of progesterone (P(4))/PR at the ZEB1 promoter. Excitingly, we observed that ZEB1 and ZEB2 inhibit expression of the contraction-associated genes, oxytocin receptor and connexin-43, and block oxytocin-induced contractility in human myometrial cells. Together, these findings implicate the miR-200 family and their targets, ZEB1 and ZEB2, as unique P(4)/PR-mediated regulators of uterine quiescence and contractility during pregnancy and labor and shed light on the molecular mechanisms involved in preterm birth.

Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M 'gate-keeper' mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC.