Cancer progression is a complex series of events thought to incorporate the reversible developmental process of epithelial-to-mesenchymal transition (EMT). In vitro, the microRNA-200 family maintains the epithelial phenotype by posttranscriptionally inhibiting the E-cadherin repressors, ZEB1 and ZEB2. Here, we used in situ hybridization and immunohistochemistry to assess expression of miR-200 and EMT biomarkers in formalin-fixed paraffin-embedded human colorectal adenocarcinomas. In addition, laser capture microdissection and quantitative real-time polymerase chain reaction were employed to quantify levels of miR-200 in the normal epithelium, tumor core, invasive front, and stroma. We find that miR-200 is downregulated at the invasive front of colorectal adenocarcinomas that have destroyed and invaded beyond the basement membrane. However, regional lymph node metastases and vascular carcinoma deposits show strong expression of miR-200, suggesting this family of miRNAs is involved in the recapitulation of the primary tumor phenotype at metastatic sites. In contrast, adenomas and adenocarcinomas with intact basement membranes showed uniform miR-200 expression from the tumor core to the tumor-host interface. Taken together, these data support the involvement of EMT and mesenchymal-to-epithelial transition (MET) in the metastasis cascade and show that miR-200 is downregulated in the initial stages of stromal invasion but is restored at metastatic sites.

BACKGROUND

Congenital heart disease (CHD) is among the most common birth defects. Most cases are of unknown pathogenesis.

OBJECTIVE

To determine the contribution of de novo copy number variants (CNVs) in the pathogenesis of sporadic CHD.

RESULTS

We studied 538 CHD trios using genome-wide dense single nucleotide polymorphism arrays and whole exome sequencing. Results were experimentally validated using digital droplet polymerase chain reaction. We compared validated CNVs in CHD cases with CNVs in 1301 healthy control trios. The 2 complementary high-resolution technologies identified 63 validated de novo CNVs in 51 CHD cases. A significant increase in CNV burden was observed when comparing CHD trios with healthy trios, using either single nucleotide polymorphism array (P=7×10(-5); odds ratio, 4.6) or whole exome sequencing data (P=6×10(-4); odds ratio, 3.5) and remained after removing 16% of de novo CNV loci previously reported as pathogenic (P=0.02; odds ratio, 2.7). We observed recurrent de novo CNVs on 15q11.2 encompassing CYFIP1, NIPA1, and NIPA2 and single de novo CNVs encompassing DUSP1, JUN, JUP, MED15, MED9, PTPRE SREBF1, TOP2A, and ZEB2, genes that interact with established CHD proteins NKX2-5 and GATA4. Integrating de novo variants in whole exome sequencing and CNV data suggests that ETS1 is the pathogenic gene altered by 11q24.2-q25 deletions in Jacobsen syndrome and that CTBP2 is the pathogenic gene in 10q subtelomeric deletions.

CONCLUSIONS

We demonstrate a significantly increased frequency of rare de novo CNVs in CHD patients compared with healthy controls and suggest several novel genetic loci for CHD.

Fibroblasts can be reprogrammed to induced pluripotent stem cells (iPSCs) by application of transcription factors octamer-binding protein 4 (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), and c-Myelocytomatosis oncogene (c-Myc) (OSKM), but the underlying mechanisms remain unclear. Here, we report that exogenous Oct4 and Sox2 can bind at the promoter regions of mir-141/200c and mir-200a/b/429 cluster, respectively, and induce the transcription activation of miR-200 family during the OSKM-induced reprogramming. Functional suppression of miR-200s with specific inhibitors significantly represses the OSKM-caused mesenchymal-to-epithelial transition (MET, an early event in reprogramming of fibroblasts to iPSCs) and iPSC generation, whereas overexpression of miR-200s promotes the MET and iPSC generation. Mechanistic studies showed that miR-200s significantly repress the expression of zinc finger E-box binding homeobox 2 (ZEB2) through directly targeting its 3' UTR and direct inhibition of ZEB2 can mimic the effects of miR-200s on iPSC generation and MET process. Moreover, the effects of miR-200s during iPSC generation can be blocked by ZEB2 overexpression. Collectively, our findings not only reveal that members of the miR-200 family are unique mediators of the reprogramming factors Oct4/Sox2, but also demonstrate that the miR-200/ZEB2 pathway as one critical mechanism of Oct4/Sox2 to induce somatic cell reprogramming at the early stage.

TMPRSS4 is a novel type II transmembrane serine protease found at the cell surface that is highly expressed in pancreatic, colon and gastric cancer tissues. However, the biological functions of TMPRSS4 in cancer are unknown. Here we show, using reverse transcription-PCR, that TMPRSS4 is highly elevated in lung cancer tissues compared with normal tissues and is also broadly expressed in a variety of human cancer cell lines. Knockdown of TMPRSS4 by small interfering RNA treatment in lung and colon cancer cell lines was associated with reduction of cell invasion and cell-matrix adhesion as well as modulation of cell proliferation. Conversely, the invasiveness, motility and adhesiveness of SW480 colon carcinoma cells were significantly enhanced by TMPRSS4 overexpression. Furthermore, overexpression of TMPRSS4 induced loss of E-cadherin-mediated cell-cell adhesion, concomitant with the induction of SIP1/ZEB2, an E-cadherin transcriptional repressor, and led to epithelial-mesenchymal transition events, including morphological changes, actin reorganization and upregulation of mesenchymal markers. TMPRSS4-overexpressing cells also displayed markedly increased metastasis to the liver in nude mice upon intrasplenic injection. Taken together, these studies suggest that TMPRSS4 controls the invasive and metastatic potential of human cancer cells by facilitating an epithelial-mesenchymal transition; TMPRSS4 may be a potential therapeutic target for cancer treatment.

Noncoding RNAs are emerging as central players in gene expression regulation through the targeting of a plethora of mRNAs. Several recent papers report on the regulation of the epithelial phenotype by the miR-200 (200 family of miRNAs) family and a natural antisense transcript, both acting on the E-cadherin repressors zinc-finger E-box-binding homeobox factors ZEB1 and ZEB2. These findings open new avenues for understanding the modulation of the epithelial-to-mesenchymal transition in physiological and pathological situations.

BACKGROUND

The microRNA-200 (miR-200) family has been reported to induce epithelial differentiation and suppress epithelial-mesenchymal transition (EMT) by inhibiting translation of zinc finger E-box-binding homeobox (ZEB) 1 and 2 mRNAs in several types of cancers. This study aimed to clarify the role of miR-200b in regulating EMT and promoting cellular proliferation, invasion, and migration in gastric cancer.

METHODS

The relationships among the expression levels of miR-200b, ZEB1 and ZEB2, and E-cadherin mRNAs were analyzed by quantitative real-time reverse transcription-polymerase chain reaction in frozen tissue samples from 40 gastric cancer patients who underwent gastrectomy from 2008 to 2010. The effects of miR-200b on EMT in gastric cancer cells in vitro were also analyzed.

RESULTS

Diffuse histologic type, depth of tumor, tumor size, lymph node metastasis, and lymphatic invasion were significantly higher in the low-miR-200b expression group compared with the high expression group. There was a strong correlation between the levels of miR-200b, and ZEB2 and E-cadherin mRNAs in gastric cancer patients. Upregulation of miR-200b in gastric cancer cells changed the cell morphology from fibroblast- to epithelial-like, associated with localization of E-cadherin to the plasma membrane. ZEB2 mRNA levels fell, while E-cadherin expression levels increased in gastric cells overexpressing miR-200b, associated with significantly reduced cellular proliferation, and inhibition of cellular migration and invasion.

CONCLUSIONS

miR-200b regulates ZEB2 expression and thus controls metastasis in gastric cancer.

Cancer stem-like cells (CSCs) are a highly tumorigenic cell type present as a minority population in developmentally diverse tumors and cell lines. Using a genetic screen in an inducible model of CSC formation in a breast cell line, we identify microRNAs (miRNAs) that inhibit CSC growth and are down-regulated in CSCs. Aside from the previously identified miR-200 family, these include the miR-15/16 (miR-16, miR-15b) and miR-103/107 (miR-103, miR-107) families as well as miR-145, miR-335, and miR-128b. Interestingly, these miRNAs affect common target genes that encode the Bmi1 and Suz12 components of the polycomb repressor complexes as well as the DNA-binding transcription factors Zeb1, Zeb2, and Klf4. Conversely, expression of the CSC-modulating miRNAs is inhibited by Zeb1 and Zeb2. There is an inverse relationship between the levels of CSC-regulating miRNAs and their respective targets in samples from triple-negative breast cancer patients, providing evidence for the relevance of these interactions in human cancer. In addition, combinatorial overexpression of these miRNAs progressively attenuates the growth of CSCs derived from triple-negative breast cancers. These observations suggest that CSC formation and function are reinforced by an integrated regulatory circuit of miRNAs, transcription factors, and chromatin-modifying activities that can act as a bistable switch to drive cells into either the CSC or the nonstem state within the population of cancer cells.

The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms) expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1) expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133high/CD44high) were more resistant to gamma radiation than the ten percent with lowest expression (CD133low/CD44low). The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2) did not differ. Our results demonstrate that CD133high/CD44high expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.

EBV-associated gastric carcinoma is a distinct gastric carcinoma subtype with characteristic morphologic features similar to those of cells that undergo epithelial-to-mesenchymal transition. The effect of microRNA abnormalities in carcinogenesis was investigated by measuring the expression of the epithelial-to-mesenchymal transition-related microRNAs, miR-200a and miR-200b, in 36 surgically resected gastric carcinomas using quantitative reverse transcription-PCR analysis. MiR-200 family expression was decreased in EBV-associated gastric carcinoma, as compared with that in EBV-negative carcinoma. Downregulation of the miR-200 family was found in gastric carcinoma cell lines infected with recombinant EBV (MKN74-EBV, MKN7-EBV, and NUGC3-EBV), accompanied by the loss of cell adhesion, reduction of E-cadherin expression, and upregulation of ZEB1 and ZEB2. E-cadherin expression was partially restored by transfection of EBV-infected cells with miR-200 family precursors. Reverse transcription-PCR analysis of primary precursors of miR-200 (pri-miR-200) revealed that the transcription of pri-miR-200 was decreased in EBV-infected cells. Transfection of MKN74 cells with BARF0, EBNA1, and LMP2A resulted in a decrease of pri-miR-200, whereas transfection with EBV-encoded small RNA (EBER) did not. These four latent genes contributed to the downregulation of the mature miR-200 family and the subsequent upregulation of ZEB1/ZEB2, resulting in the reduction of E-cadherin expression. These findings indicate that all the latency type I genes have a synergetic effect on the downregulation of the miR-200 family that leads to reduced E-cadherin expression, which is a crucial step in the carcinogenesis of EBV-associated gastric carcinoma.

Epithelial-mesenchymal transition (EMT) is a physiological process that plays important roles in tumor metastasis, "stemness," and drug resistance. EMT is typically characterized by the loss of the epithelial marker E-cadherin and increased expression of EMT-associated transcriptional repressors, including ZEB1 and ZEB2. The miR-200 family and miR-205 prevent EMT through suppression of ZEB1/2. p53 has been implicated in the regulation of miR-200c, but the mechanisms controlling miR-205 expression remain elusive. Here we report that the p53 family member and p63 isoform, ΔNp63α, promotes miR-205 transcription and controls EMT in human bladder cancer cells. ΔNp63α, E-cadherin and miR-205 were coexpressed in a panel of bladder cancer cell lines (n = 28) and a cohort of primary bladder tumors (n = 98). Stable knockdown of ΔNp63α in the "epithelial" bladder cancer cell line UM-UC6 decreased the expression of miR-205 and induced the expression of ZEB1/2, effects that were reversed by expression of exogenous miR-205. Conversely, overexpression of ΔNp63α in the "mesenchymal" bladder cancer cell line UM-UC3 induced miR-205 and suppressed ZEB1/2. ΔNp63α knockdown reduced the expression of the primary and mature forms of miR-205 and the miR-205 "host" gene (miR-205HG) and decreased binding of RNA Pol II to the miR-205HG promoter, inhibiting miR-205HG transcription. Finally, high miR-205 expression was associated with adverse clinical outcomes in bladder cancer patients. Together, our data demonstrate that ΔNp63α-mediated expression of miR-205 contributes to the regulation of EMT in bladder cancer cells and identify miR-205 as a molecular marker of the lethal subset of human bladder cancers.

Recent observations that aberrant expression of tissue transglutaminase (TG2) promotes growth, survival, and metastasis of multiple tumor types is of great significance and could yield novel therapeutic targets for improved patient outcomes. To accomplish this, a clear understanding of how TG2 contributes to these phenotypes is essential. Using mammary epithelial cell lines (MCF10A, MCF12A, MCF7 and MCF7/RT) as a model system, we determined the impact of TG2 expression on cell growth, cell survival, invasion, and differentiation. Our results show that TG2 expression promotes drug resistance and invasive functions by inducing epithelial-mesenchymal transition (EMT). Thus, TG2 expression supported anchorage-independent growth of mammary epithelial cells in soft-agar, disrupted the apical-basal polarity, and resulted in disorganized acini structures when grown in 3D-culture. At molecular level, TG2 expression resulted in loss of E-cadherin and increased the expression of various transcriptional repressors (Snail1, Zeb1, Zeb2 and Twist1). Tumor growth factor-beta (TGF-β) failed to induce EMT in cells lacking TG2 expression, suggesting that TG2 is a downstream effector of TGF-β-induced EMT. Moreover, TG2 expression induced stem cell-like phenotype in mammary epithelial cells as revealed by enrichment of CD44(+)/CD24(-/low) cell populations. Overall, our studies show that aberrant expression of TG2 is sufficient for inducing EMT in epithelial cells and establish a strong link between TG2 expression and progression of metastatic breast disease.

Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro-inflammatory mediators and undergoing epithelial-to-mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation-induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation-induced pneumonitis and pulmonary fibrosis, and increased the expression of IL-1β, Ccl2, Cxcl5, Snail1, Zeb1, Zeb2 and Foxf1. Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation-induced pulmonary fibrosis and prevented the increase in EMT-associated gene expression. siRNA-mediated inhibition of Foxm1 prevented TGF-β-induced EMT in vitro. Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF-β-induced loss of E-cadherin in Foxm1-deficient cells in vitro. Lineage-tracing studies demonstrated that Foxm1 increased EMT during radiation-induced pulmonary fibrosis in vivo. Foxm1 is required for radiation-induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT.

Stem cell pluripotency, angiogenesis and epithelial-mesenchymal transition (EMT) have been shown to be significantly upregulated in pancreatic ductal adenocarcinoma (PDAC) and many other aggressive cancers. The dysregulation of these processes is believed to play key roles in tumor initiation, progression, and metastasis, and is contributory to PDAC being the fourth leading cause of cancer-related deaths in the US. The tumor suppressor miRNA miR-145 downregulates critical pluripotency factors and oncogenes and results in repressed metastatic potential in PDAC. Additionally, the miR-200 family regulates several angiogenic factors which have been linked to metastasis in many solid tumors. We have previously demonstrated that downregulation of DCLK1 can upregulate critical miRNAs in both in vitro and in vivo cancer models and results in downregulation of c-MYC, KRAS, NOTCH1 and EMT-related transcription factors. A recent report has also shown that Dclk1 can distinguish between normal and tumor stem cells in Apc (min/+) mice and that ablation of Dclk1(+) cells resulted in regression of intestinal polyps without affecting homeostasis. Here we demonstrate that the knockdown of DCLK1 using poly(lactide-co-glycolide)-encapsulated-DCLK1-siRNA results in AsPC1 tumor growth arrest. Examination of xenograft tumors revealed, (a) increased miR-145 which results in decreased pluripotency maintenance factors OCT4, SOX2, NANOG, KLF4 as well as KRAS and RREB1; (b) increased let-7a which results in decreased pluripotency factor LIN28B; and (c) increased miR-200 which results in decreased VEGFR1, VEGFR2 and EMT-related transcription factors ZEB1, ZEB2, SNAIL and SLUG. Specificity of DCLK1 post-transcriptional regulation of the downstream targets of miR-145, miR-200 and let-7a was accomplished utilizing a luciferase-based reporter assay. We conclude that DCLK1 plays a significant master regulatory role in pancreatic tumorigenesis through the regulation of multiple tumor suppressor miRNAs and their downstream pro-tumorigenic pathways. This novel concept of targeting DCLK1 alone has several advantages over targeting single pathway or miRNA-based therapies for PDAC.

Long non-coding RNA (lncRNAs) play a critical role in the development of cancers. LncRNA metastasis-associated lung adenocarcinoma transcript 1(MALAT1) has recently been identified to be involved in tumorigenesis of several cancers such as lung cancer, bladder cancer and so on. Here, we found that MALAT1 exist a higher fold change (Tumor/Normal) in clear cell kidney carcinoma (KIRC) from The Cancer Genome Atlas (TCGA) Data Portal and a negative correlation with miR-200s family. We further demonstrated MALAT1 promote KIRC proliferation and metastasis through sponging miR-200s in vitro and in vivo. In addition, miR-200c can partly reverse the MALAT1's stimulation on proliferation and metastasis in KIRC. In summary we unveil a branch of the MALAT1/miR-200s/ZEB2 pathway that regulates the progression of KIRC. The inhibition of MALAT1 expression may be a promising strategy for KIRC therapy.

BACKGROUND

MicroRNAs (miRNAs) are non-coding, single-stranded RNA molecules that play important roles in a number of physiological and pathological processes. Previous studies showed that miRNAs targeting transcription factors ZEB1 and ZEB2 may repress epithelial-mesenchymal transition (EMT).

METHODS

We studied 34 consecutive patients with biopsy-proven hypertensive nephrosclerosis. Intrarenal expression of miR-200 family, miR-205, and miR-192 were determined. We also studied normal renal tissue from 20 patients with nephrectomy for kidney cancer as controls.

RESULTS

The level of intrarenal of miR-200a, miR-200b, miR-141, miR-429, miR-205, and miR-192 were significantly higher in patients with hypertensive nephrosclerosis than controls. Proteinuria correlated with intrarenal expression of miR-200a (r = 0.594, P < 0.001), miR-200b (r = 0.395, P = 0.004), miR-141 (r = 0.377, P = 0.007), miR-429 (r = 0.346, P = 0.013), miR-205 (r = 0.636, P < 0.001), and miR-192 (r = 0.306, P = 0.029). Estimated glomerular filtration rate (GFR) correlated with intrarenal expression of miR-200a (r = -0.374, P = 0.007) and miR-205 (r = -0.400, P = 0.005). Intrarenal expression of ZEB1 inversely correlated with intrarenal expression of miR-429, whereas expression of ZEB2 inversely correlated with miR-200a, miR-200b, and miR-429.

CONCLUSIONS

The results show that intrarenal expression of miR-200a, miR-200b, miR-141, miR-429, miR-205, and miR-192 were increased in hypertensive nephrosclerosis, and the degree of upregulation correlated with disease severity. The results suggested that these miRNA species may play important roles in the pathogenesis of hypertensive nephrosclerosis.

Epithelial to mesenchymal transition (EMT) is a process during which cells lose their epithelial characteristics, for instance cell polarity and cell-cell contact, and gain mesenchymal properties, such as increased motility. In colorectal cancer (CRC), EMT is associated with an invasive or metastatic phenotype. In this review, we discuss recent studies exploring novel regulation mechanisms of EMT in CRC, including the identification of new CRC EMT regulators. Upregulation of inducers can promote EMT, leading to increased invasiveness and metastasis in CRC. These inducers can downregulate E-cadherin and upregulate N-cadherin and vimentin (VIM) through modulating EMT-related signaling pathways, for instance WNT/β-catenin and TGF-β, and EMT transcription factors, such as zinc finger E-box binding homeobox 1 (ZEB1) and ZEB2. In addition, several microRNAs (miRNAs), including members of the miR-34 and miR-200 families, are found to target mRNAs of EMT-transcription factors, for example ZEB1, ZEB2, or SNAIL. Downregulation of these miRNAs is associated with distant metastasis and advanced stage tumors. Furthermore, the role of EMT in circulating tumor cells (CTCs) is also discussed. Mesenchymal markers on the surface of EMT CTCs were found to be associated with metastasis and could serve as potential biomarkers for metastasis. Altogether, these studies indicate that EMT is orchestrated by a complicated network, involving regulators of different signaling pathways. Further studies are required to understand the mechanisms underlying EMT in CRC.

BACKGROUND

Esophageal squamous cell carcinoma (ESCC) is often diagnosed at later stages until they are incurable. MicroRNA (miR) is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC.

METHODS

Total RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT)-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR.

RESULTS

Based on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold) in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2, accompanied by reduction of E-cadherin, a regulator of epithelial mesenchymal transition. The miR-205 expression levels were not associated with histological differentiation of human ESCC.

CONCLUSIONS

These results imply that miR-205 is an ESCC-specific miR that exerts tumor-suppressive activities with EMT inhibition by targeting ZEB2.

Osteosarcoma typically arises in tissues of mesenchymal origin, and is the most malignant bone tumor characterized by high local aggressiveness, with poor therapeutic outcome. Busulfan has been widely used to treat CML. So far, there are no reports on the therapeutic effect of busulfan on osteosarcoma. Here, we showed that busulfan dose-dependently reduced the cell viability and proliferation, and induced cell apoptosis, senescence, and reactive oxygen species levels in two osteosarcoma cell lines. Moreover, a series of loss-of-function and gain-of-function experiments further indicated that busulfan may have its anti-osteosarcoma effect by upregulating the microRNA-200 (miR-200) family which subsequently downregulated its target genes ZEB1 and ZEB2. Furthermore, treatment with busulfan potentially inhibited the growth of implanted osteosarcoma in nude mice. Taken together, our data suggest that busulfan may have an anti-osteosarcoma effect through downregulating ZEB1 and ZEB2 through activating the miR-200 family, highlighting a possibility of using busulfan as a novel therapy for osteosarcoma.

BACKGROUND

The development of effective drug delivery systems capable of transporting small interfering RNA (siRNA) has been elusive. We have previously reported that colorectal cancer tumor xenograft growth was arrested following treatment with liposomal preparation of siDCAMKL-1. In this report, we have utilized Nanoparticle (NP) technology to deliver DCAMKL-1 specific siRNA to knockdown potential key cancer regulators. In this study, mRNA/miRNA were analyzed using real-time RT-PCR and protein by western blot/immunohistochemistry. siDCAMKL-1 was encapsulated in Poly(lactide-co-glycolide)-based NPs (NP-siDCAMKL-1); Tumor xenografts were generated in nude mice, treated with NP-siDCAMKL-1 and DAPT (γ-secretase inhibitor) alone and in combination. To measure let-7a and miR-144 expression in vitro, HCT116 cells were transfected with plasmids encoding the firefly luciferase gene with let-7a and miR-144 miRNA binding sites in the 3'UTR.

RESULTS

Administration of NP-siDCAMKL-1 into HCT116 xenografts resulted in tumor growth arrest, downregulation of proto-oncogene c-Myc and Notch-1 via let-7a and miR-144 miRNA-dependent mechanisms, respectively. A corresponding reduction in let-7a and miR-144 specific luciferase activity was observed in vitro. Moreover, an upregulation of EMT inhibitor miR-200a and downregulation of the EMT-associated transcription factors ZEB1, ZEB2, Snail and Slug were observed in vivo. Lastly, DAPT-mediated inhibition of Notch-1 resulted in HCT116 tumor growth arrest and down regulation of Notch-1 via a miR-144 dependent mechanism.

CONCLUSIONS

These findings demonstrate that nanoparticle-based delivery of siRNAs directed at critical targets such as DCAMKL-1 may provide a novel approach to treat cancer through the regulation of endogenous miRNAs.

Elevated p53 expression is associated with several kidney diseases including diabetic nephropathy (DN). However, the mechanisms are unclear. We report that expression levels of transforming growth factor-β1 (TGF-β), p53, and microRNA-192 (miR-192) are increased in the renal cortex of diabetic mice, and this is associated with enhanced glomerular expansion and fibrosis relative to nondiabetic mice. Targeting miR-192 with locked nucleic acid-modified inhibitors in vivo decreases expression of p53 in the renal cortex of control and streptozotocin-injected diabetic mice. Furthermore, mice with genetic deletion of miR-192 in vivo display attenuated renal cortical TGF-β and p53 expression when made diabetic, and have reduced renal fibrosis, hypertrophy, proteinuria, and albuminuria relative to diabetic wild-type mice. In vitro promoter regulation studies show that TGF-β induces reciprocal activation of miR-192 and p53, via the miR-192 target Zeb2, leading to augmentation of downstream events related to DN. Inverse correlation between miR-192 and Zeb2 was observed in glomeruli of human subjects with early DN, consistent with the mechanism seen in mice. Our results demonstrate for the first time a TGF-β-induced feedback amplification circuit between p53 and miR-192 related to the pathogenesis of DN, and that miR-192-knockout mice are protected from key features of DN.

Urinary bladder cancer (UBC) patients at muscle invasive stage have poor clinical outcome, due to high propensity for metastasis. Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor stroma, play an important role in tumor development. However, it is unclear whether CAFs from UBC induce cell invasion and which signaling pathway is involved. Herein, we found that conditional medium from UBC CAFs (CAF-CM) enhanced the invasion of UBC cells. CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells. Higher concentration of TGFβ1 in CAF-CM, comparing with the CM from adjacent normal fibroblast, led to phosphorylation of Smad2 in UBC cells. Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion. Interestingly, a long non-coding RNA, ZEB2NAT, was demonstrated to be essential for this TGFβ1-dependent process. ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level. Consistently, TGFβ1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens. Our data revealed a novel mechanism that CAFs induces EMT and invasion of human UBC cells through the TGFβ1-ZEB2NAT-ZEB2 axis.

The invasion and metastasis of tumors are triggered by an epithelial to mesenchymal transition (EMT), which is regulated by microRNAs (miRNAs). EMT also promotes malignant tumor progression and the maintenance of the stem cell property, which endows cancer cells with the capabilities of self-renewal and immortalized proliferation. The transcriptional repressor zinc-finger E-box binding homeobox 2 (ZEB2), as an EMT activator, might be an important promoter of metastasis in some tumors. Here, we report that ZEB2 directly represses the transcription of miR-145, which is a strong repressor of EMT. In turn, ZEB2 is also a direct target of miR-145. Further, our findings show that the downregulation of ZEB2 not only represses invasion, migration, EMT, and the stemness of prostate cancer (PCa) cells, but also suppresses the capability of PC-3 cells to invade bone in vivo. Importantly, the expression level of ZEB2 as revealed by immunohistochemical analysis is positively correlated to bone metastasis, the serum free PSA level, the total PSA level, and the Gleason score in PCa patients and is negatively correlated with miR-145 expression in primary PCa specimens. Thus, our findings demonstrate a double-negative feedback loop between ZEB2 and miR-145 and indicate that the ZEB2/miR-145 double-negative feedback loop plays a significant role in the control of EMT and stem cell properties during the bone metastasis of PCa cells. These results suggest that the double-negative feedback loop between ZEB2 and miR-145 contributes to PCa progression and metastasis and might have therapeutic relevance for the bone metastasis of PCa.

Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in TGF-β-mediated senescence, epithelial-to-mesenchymal transition (EMT), and cancer stem cell functions. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here, we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like 1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1-enriched EMT-competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage-independent growth and tumor formation in nude mice. RNA interference experiments suggested the involvement of ZEBs in anchorage-independent colony formation, invasion, and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation were recapitulated upon Notch inhibition by DNMAML1 in organotypic three-dimensional culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of esophageal squamous cancers.

Mammalian pallial (cortical and hippocampal) and striatal interneurons are both generated in the embryonic subpallium, including the medial ganglionic eminence (MGE). Herein we demonstrate that the Zfhx1b (Sip1, Zeb2) zinc finger homeobox gene is required in the MGE, directly downstream of Dlx1&2, to generate cortical interneurons that express Cxcr7, MafB, and cMaf. In its absence, Nkx2-1 expression is not repressed, and cells that ordinarily would become cortical interneurons appear to transform toward a subtype of GABAergic striatal interneurons. These results show that Zfhx1b is required to generate cortical interneurons, and suggest a mechanism for the epilepsy observed in humans with Zfhx1b mutations (Mowat-Wilson syndrome).