miRNA-200 Family Research Articles

Molecular regulation of ovarian cancer cell invasion.

The molecular mechanism underlying ovarian cancer invasiveness and metastasis remains unclear. Since significant downregulation in microRNA 200 (miRNA200) family (miR200a, miR200b, and miR200c) has been reported in the invasive ovarian cancer cells, here, we used two human ovarian cancer cell lines, OVCAR3 and SKOV3, to study the molecular basis of miR200, matrix metalloproteinase 3 (MMP3) activation, and cancer invasiveness. We found that overexpression of either miR200 family member in OVCAR3 or SKOV3 cells significantly inhibited production and secretion of MMP3 and cancer invasiveness. Moreover, forced MMP3 expression abolished miR200-induced inhibition of ovarian cancer cell invasiveness, suggesting that miR200 family inhibited ovarian cell invasiveness via downregulating MMP3. Furthermore, ZEB1, a major target of miR200, was inhibited by miR200 overexpression. Forced ZEB1 expression abolished miR200-induced inhibition of ovarian cancer cell invasiveness, suggesting that ZEB1 is a direct target of miR200 for inhibiting ovarian cell invasiveness. Finally, phosphorylated SMAD3 (pSMAD3), a major partner of ZEB1, was efficiently inhibited by miR200, which could be restored by forced expression of ZEB1, but not by forced expression of MMP3, suggesting that ZEB1/pSMAD3 is signaling cascade upstream of MMP3 in this model. Taken together, our data suggest that miR200 family inhibited ovarian cancer cell invasiveness and metastasis by downregulating MMP3, possibly through ZEB1/pSMAD3.

Glabridin attenuates the migratory and invasive capacity of breast cancer cells by activating microRNA-200c.

Current treatments for breast cancer, a common malignancy in human females, are less than satisfactory because of high rates of metastasis. Glabridin (GLA), which acts through the FAK/ROS signaling pathway, has been used as an antioxidant and anti-metastatic agent. However, little is known regarding the effect of microRNA (miRNA) on GLA's anti-metastatic activity. The miRNA-200 family, which is frequently expressed at low levels in triple negative breast cancers, inhibits metastasis by blocking the epithelial–mesenchymal transition. Here, we found that GLA attenuated the migratory and invasive capacity of breast cancer cells by activating miR-200c. GLA induced the mesenchymal–epithelial transition in vitro and in vivo, as determined by increased expression of the epithelial marker, E-cadherin, and decreased expression of the mesenchymal marker, vimentin. Overexpression of miR-200c enhanced the expression of E-cadherin and decreased the expression of vimentin. Furthermore, in MDA-MB-231 and BT-549 breast cancer cells exposed to GLA, knockdown of miR-200c blocked the GLA-induced mesenchymal–epithelial transition and alleviated the GLA-induced inhibition of migration and invasion. Thus, elevation of miR-200c by GLA has considerable therapeutic potential for anti-metastatic therapy for breast cancer patients.

Cripto-1 as a novel therapeutic target for triple negative breast cancer.

Triple-negative breast cancer (TNBC) presents the poorest prognosis among the breast cancer subtypes and no current standard therapy. Here, we performed an in-depth molecular analysis of a mouse model that establishes spontaneous lung metastasis from JygMC(A) cells. These primary tumors resembled the triple-negative breast cancer (TNBC) both phenotypically and molecularly. Morphologically, primary tumors presented both epithelial and spindle-like cells but displayed only adenocarcinoma-like features in lung parenchyma. The use of laser-capture microdissection combined with Nanostring mRNA and microRNA analysis revealed overexpression of either epithelial and miRNA-200 family or mesenchymal markers in adenocarcinoma and mesenchymal regions, respectively. Cripto-1, an embryonic stem cell marker, was present in spindle-like areas and its promoter showed activity in primary tumors. Cripto-1 knockout by the CRISPR-Cas9 system inhibited tumor growth and pulmonary metastasis. Our findings show characterization of a novel mouse model that mimics the TNBC and reveal Cripto-1 as a TNBC target hence may offer alternative treatment strategies for TNBC.

MicroRNA define the glycocode: identification of critical glycogenes that shape the cell (788.6)

Cell surface glycans create an intricate source of biological information which is dynamically altered by cells in developmental processes and used by pathogens during host invasion. Mapping of the glycocode and identification of critical glycan altering enzymes in biological processes is confounded by the complexity and redundancy of glycan biosynthetic machinery. Recently, we integrated microRNA (miRNA) expression data with our glycomics analysis for the NCI‐60 cancer cell line panel, and showed that miRNA act as a major regulator of the human glycome by targeting critical nodes within the glycan biosynthesis pathway. Among others, our analysis revealed a strong association of fucose signature and miRNA‐200 family (miR‐200f), known regulators of epithelial to mesenchymal transition (EMT) a process during which miR‐200f is downregulated. Within the NCI‐60, expression pattern of miR‐200f maps onto fucose signature: both are high in epithelial and low in mesenchymal cells, indicating that fucose might be a marker of epithelial/mesenchymal cell state. In line with our findings we utilize miRNA target prediction software and identify a secreted α‐fucosidase (FUCA2) and O‐linked fucose β‐1,3‐glucosyltransferase (B3GALTL) as most common predicted targets. Both enzymes have the potential to reduce availability of terminal fucose and are downregulated by miRNA‐200f. We show that B3GALTL has a direct role in EMT as B3GALTL knockout induced a change in morphology from mesenchymal to epithelial in mesenchymal breast (MB‐231) and melanoma (M14) cell lines. Taken together our data validates miRNA based mapping of the glycome and identifies B3GALTL as an important regulator of EMT.Grant Funding Source: Sponsored by NIH

Loss of microRNA-200a expression correlates with tumor progression in breast cancer

MicroRNAs (miRNAs) are 19∼22 nucleotide-long, noncoding, small RNAs, involved in post-transcriptional regulation of many target genes. The miRNA-200 family has been shown to play a crucial role in the epithelial to mesenchymal transition in human cancers. In situ hybridization (ISH) was used to investigate the expression level of miRNA-200a in breast cancers. Formalin-fixed, paraffin embedded (FFPE) tissues from normal breast, ductal carcinoma in situ (DCIS), primary cancers, and metastatic lymph nodes were achieved and constructed to tissue microarrays. MiRNA-200a expression was demonstrated in 95.2% of normal breast tissue samples and 80.4% of DCIS, whereas 178 (58.0%) of 307 breast cancers and 83.3% of metastatic lymph node samples lacked miRNA-200a expression (P<0.001). Moreover, loss of MiRNA-200a expression correlated with high histologic grade (P=0.017) and perinodal tumor extension (P=0.026). However, miRNA-200a expression did not predict tumor recurrence or patient survival. In conclusion, loss of miRNA-200a is frequently observed in breast cancers, especially tumors with high grade histology. These findings suggest that miRNA-200a may play an important role in breast cancer initiation and progression. ISH can be used to detect miRNAs in FFPE sections, and should permit the validation of miRNAs as biomarkers in large clinical samples.

Down-Regulation of the miRNA-200 Family at the Invasive Front of Colorectal Cancers with Degraded Basement Membrane Indicates EMT Is Involved in Cancer Progression

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.

The miRNA-200 family and miRNA-9 exhibit differential expression in primary versus corresponding metastatic tissue in breast cancer

Metastases are the major cause of cancer-related deaths, but the mechanisms of the metastatic process remain poorly understood. In recent years, the involvement of microRNAs (miRNAs) in cancer has become apparent, and the objective of this study was to identify miRNAs associated with breast cancer progression. Global miRNA expression profiling was performed on 47 tumor samples from 14 patients with paired samples from primary breast tumors and corresponding lymph node and distant metastases using LNA-enhanced miRNA microarrays. The identified miRNA expression alterations were validated by real-time PCR, and tissue distribution of the miRNAs was visualized by in situ hybridization. The patients, in which the miRNA profile of the primary tumor and corresponding distant metastasis clustered in the unsupervised cluster analysis, showed significantly shorter intervals between the diagnosis of the primary tumor and distant metastasis (median 1.6 years) compared to those that did not cluster (median 11.3 years) (p<0.003). Fifteen miRNAs were identified that were significantly differentially expressed between primary tumors and corresponding distant metastases, including miR-9, miR-219-5p and four of the five members of the miR-200 family involved in epithelial-mesenchymal transition. Tumor expression of miR-9 and miR-200b were confirmed using in situ hybridization, which also verified higher expression of these miRNAs in the distant metastases versus corresponding primary tumors. Our results demonstrate alterations in miRNA expression at different stages of disease progression in breast cancer, and suggest a direct involvement of the miR-200 family and miR-9 in the metastatic process.

Non-vesicle-bound free microRNAs could enter cells and affect gene expression

Background: Cell-free microRNAs (miRNAs) exist in body fluid. Previous studies showed that cell-free mi-RNAs are partly bound in microvesicles, and could transfer between cells via fusion with cell membrane. Methods: We quantified the amount of a panel of mi-RNA targets in and outside microvesicles in human proximal tubular epithelial cell (HK2) medium by microarray and real-time quantitative polymerase chain reaction (RT-QPCR). Intercellular miRNA transfer was explored by medium transfer experiments. Results: We identified a portion of cell-free miRNAs that exists as non-vesicle bound, truly naked form. More importantly, these non-vesicle bound free miRNA could transfer between cells and exert biological effects. By miRNA microarray, we showed that the expression of many miRNA targets in HK-2 cells were altered, either up- or down-regulated, after exposure to extrinsic free miRNAs. The miRNA-200 family was the most affected in our model, with a corresponding alteration in the messenger RNA expression of down-stream targets including ZEB1 and vimentin. Conclusion: Our results suggest that free miRNA may serve as an intercellular messenger, a phenomenon that needs further exploration.

The miRNA-200 family and miRNA-9 exhibit differential expression in primary versus corresponding metastatic tissue in breast cancer

Metastases are the major cause of cancer-related deaths, but the mechanisms of the metastatic process remain poorly understood. In recent years, the involvement of microRNAs (miRNAs) in cancer has become apparent, and the objective of this study was to identify miRNAs associated with breast cancer progression.

Abstract B148: The miRNA-200 family regulates cell migration and metastasis in lung adenocarcinoma.

Abstract miRNAs are post-transcriptional regulators of genes involved in such canonical signaling pathways as the TGF-β and EGF pathways, both of which are frequently altered in tumor cells. miRNA downregulation results in upregulation of its target genes and ultimately aberrant signaling of pathways involved in invasion and migration, for example. To better understand how alterations in miRNA expression could lead to metastasis, we used a lung adenocarcinoma mouse model in which a lentivirus expressing the Cre recombinase activates oncogenic K-Ras and deletes p53, giving rise to primary lung tumors (T) and metastases (M). We performed miRNA expression profiling on 30 T and M derived cell lines and identified three members of the miR-200 family - miR-200b, miR-200c, miR-429 - to be significantly downregulated in the M lines compared to their related T line. To explore the functional contribution of the miR-200s to metastasis, we designed a miRNA sponge to stably inhibit endogenous miR-200 levels in T cell lines. We found miR-200 depletion promoted wound healing and cell migration towards either serum or EGF. Transplantation of the sponge-expressing lines via tail vein led to in vivo formation of more invasive, metastatic tumors compared to controls. Previously, the miR-200 family has been shown to inhibit the TGF-β target gene and EMT mediator, Zeb1. We have identified another miR-200 target, Epidermal Growth Factor Receptor Substrate 8 (Eps8), which mediates actin based cell motility. In miR-200 low (M) cells, Eps8 is expressed three-fold higher than in corresponding miR-200 high (T) cells. Additionally, exogenous expression of Eps8 in T lines promotes wound healing and cell migration, relative to control cells. Thus, our findings implicate the miR-200s as a class of metastasis regulators that modulate expression of genes functionally relevant in tumor cell dissemination and provide new targets for therapeutic intervention of late stage lung adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B148.

The expression of the miRNA-200 family in endometrial endometrioid carcinoma

Objective. Recent reports suggest that targeting the unique miRNAs highly expressed in several cancers may be a promising approach in the development of new cancer therapeutic tools. The purpose of this study was to evaluate the roles of miRNAs as therapeutic targets in human endometrial endometrioid carcinomas (EECs). Methods. We evaluated the differential expressions of miRNAs in EECs and normal endometrial tissues using microarrays and cluster analysis. After validation of differentially expressed miRNAs in another set of EECs and normal endometrial tissues, we performed the in vitro experiment using endometrial cancer cells with anti-miRNA (anti-miR) to evaluate the roles of miRNAs that are highly expressed in EECs for cell proliferation and chemosensitivity. Results. A miRNA microarray showed that the miR-200 family, including hsa-miR-141, hsa-miR-200a, hsa-miR-200b, hsa-miR-200c, and hsa-miR-429, was up-regulated in EECs as compared with that in normal endometrial tissues. When we treated endometrial cancer cells with specific anti-miRs, including anti-miR-141, -200a, -200b, -200c, or -429, we found that anti-miR-200a, -200b, -200c, and -429 significantly inhibited the growth of HEC-1A cells and anti-miR-141, -200c, and -429 significantly inhibited the growth of Ishikawa cells. Moreover, transfection with anti-miR-429 enhanced the cytotoxic effect of cisplatin in HEC-1A cells. Conclusions. These results indicate that the miR-200 family is highly expressed in EECs compared with that of normal endometrial tissues and could play an important role in cancer growth. Specifically, anti-miR-429 could enhance the cytotoxic activity with cisplatin in EECs. Therefore, the miR-200 family may offer new candidate targets to be exploited in therapeutic strategies for patients with these carcinomas.

Abstract IA-20: EMT in tumor invasion and metastasis

Abstract Most colorectal adenocarcinomas (CRC) have mutations in the APC gene leading to overexpression of the Wnt-pathway effector β-catenin. By acting as a transcriptional activator, nuclear β-catenin is involved in two fundamental processes in embryonic development: epithelial to mesenchymal transition (EMT) and generation of stem cells. We have previously shown, that in particular tumor cells at the invasive front of CRCs accumulate nuclear β-catenin, undergo an EMT and aberrantly express EMT-associated transcriptional repressors, like ZEB1. The amount of such cells strongly correlates with clinical outcome and metastasis formation. We further showed that ZEB1 represses target genes involved in basement membrane formation and epithelial cell polarity. Moreover we report that ZEB1 directly suppresses transcription of members of the miRNA-200 family, which strongly activate epithelial differentiation in pancreatic, colorectal and breast cancer cells. Notably, the EMT-activators TGFβ2 and ZEB1 itself are predominant targets downregulated by these miRNAs. These results indicate that ZEB1 triggers a microRNA-mediated positive feedbackloop, which stabilizes EMT and promotes invasion of cancer cells. In contrast tumor cells in central tumor areas are differentiated and often show membranous, E-cadherin-bound β-catenin. Strikingly, metastases show again a differentiated phenotype and lack nuclear beta-catenin, indicating a mesenchymo-epithelial re-transition (MET) and a regulatory role of the tumor environment during malignant tumor progression. Based on the developmental functions of beta-catenin and our data we propose, that the EMT-associated tumor cells at the invasive front act as “migrating cancer stem cells” which can re-differentiate and, depending on the range of dissemination, give raise to the primary carcinoma and metastases. We suggest that both primary tumors and metastases are derived from a pool of EMT-associated “migrating cancer stem cells” at the tumor host interface. They are defined by strong nuclear beta-catenin accumulation and EMT-phenotype, which gives these epithelial tumor cells a feature driving malignant tumor progression including metastasis: the unusual combination of a migratory and stem cell phenotype. Citation Information: Cancer Res 2009;69(23 Suppl):IA-20.

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

The microRNA-200 Family Regulates Epithelial to Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is implicated in metastasis initiation and has recently been shown to be regulated by the miRNA-200 family and miR-205. Expression of these miRNAs was lost in invasive breast cancer cell lines displaying mesenchymal-like morphology suggesting these microRNAs may play a role in cancer metastasis.

Members of the miRNA-200 Family Regulate Olfactory Neurogenesis

SummaryMicroRNAs (miRNAs) are highly expressed in vertebrate neural tissues, but the contribution of specific miRNAs to the development and function of different neuronal populations is still largely unknown. We report that miRNAs are required for terminal differentiation of olfactory precursors in both mouse and zebrafish but are dispensable for proper function of mature olfactory neurons. The repertoire of miRNAs expressed in olfactory tissues contains over 100 distinct miRNAs. A subset, including the miR-200 family, shows high olfactory enrichment and expression patterns consistent with a role during olfactory neurogenesis. Loss of function of the miR-200 family phenocopies the terminal differentiation defect observed in absence of all miRNA activity in olfactory progenitors. Our data support the notion that vertebrate tissue differentiation is controlled by conserved subsets of organ-specific miRNAs in both mouse and zebrafish and provide insights into control mechanisms underlying olfactory differentiation in vertebrates.