p53-responsive miR-194 inhibits thrombospondin-1 and promotes angiogenesis in colon cancers.

Thrombospondin-1 (TSP-1) is an endogenous inhibitor of angiogenesis encoded by the THBS1 gene, whose promoter is activated by p53. In advanced colorectal cancers (CRC), its expression is sustained or even slightly increased despite frequent loss of p53. Here, we determined that in HCT116 CRC cells, p53 activates the THBS1 primary transcript, but fails to boost THBS1 mRNA or protein levels, implying posttranscriptional regulation by microRNAs (miRNA). In a global miRNA gain-of-function screen done in the Dicer-deficient HCT116 variant, several miRNAs negatively regulated THBS1 mRNA and protein levels, one of them being miR-194. Notably, in agreement with published data, p53 upregulated miR-194 expression in THBS1 retrovirus-transduced HCT116 cells, leading to decreased TSP-1 levels. This negative effect was mediated by a single miR-194 complementary site in the THBS1 3'-untranslated region, and its elimination resulted in TSP-1 reactivation, impaired angiogenesis in Matrigel plugs, and reduced growth of HCT116 xenografts. Conversely, transient overexpression of miR-194 in HCT116/THBS1 cells boosted Matrigel angiogenesis, and its stable overexpression in Ras-induced murine colon carcinomas increased microvascular densities and vessel sizes. Although the overall contribution of miR-194 to neoplastic growth is context dependent, p53-induced activation of this GI tract-specific miRNA during ischemia could promote angiogenesis and facilitate tissue repair.

The myc-miR-17~92 axis blunts TGF{beta} signaling and production of multiple TGF{beta}-dependent antiangiogenic factors.

c-Myc stimulates angiogenesis in tumors through mechanisms that remain incompletely understood. Recent work indicates that c-Myc upregulates the miR-17∼92 microRNA cluster and downregulates the angiogenesis inhibitor thrombospondin-1, along with other members of the thrombospondin type 1 repeat superfamily. Here, we show that downregulation of the thrombospondin type 1 repeat protein clusterin in cells overexpressing c-Myc and miR-17∼92 promotes angiogenesis and tumor growth. However, clusterin downregulation by miR-17∼92 is indirect. It occurs as a result of reduced transforming growth factor-ß (TGFß) signaling caused by targeting of several regulatory components in this signaling pathway. Specifically, miR-17-5p and miR-20 reduce the expression of the type II TGFß receptor and miR-18 limits the expression of Smad4. Supporting these results, in human cancer cell lines, levels of the miR-17∼92 primary transcript MIR17HG negatively correlate with those of many TGFß-induced genes that are not direct targets of miR-17∼92 (e.g., clusterin and angiopoietin-like 4). Furthermore, enforced expression of miR-17∼92 in MIR17HG(low) cell lines (e.g., glioblastoma) results in impaired gene activation by TGFß. Together, our results define a pathway in which c-Myc activation of miR-17∼92 attenuates the TGFß signaling pathway to shut down clusterin expression, thereby stimulating angiogenesis and tumor cell growth.

The Caenorhabditis elegans rsd-2 and rsd-6 genes are required for chromosome functions during exposure to unfavorable environments.

In Caenorhabditis elegans, exogenous dsRNA can elicit systemic RNAi, a process that requires the function of many genes. Considering that the activities of many of these genes are also required for normal development, it is surprising that exposure to high concentrations of dsRNA does not elicit adverse consequences to animals. Here, we report inducible phenotypes in attenuated C. elegans strains reared in environments that include nonspecific dsRNA and elevated temperature. Under these conditions, chromosome integrity is compromised in RNAi-defective strains harboring mutations in rsd-2 or rsd-6. Specifically, rsd-2 mutants display defects in transposon silencing, while meiotic chromosome disjunction is affected in rsd-6 mutants. RSD-2 proteins localize to multiple cellular compartments, including the nucleolus and cytoplasmic compartments that, in part, are congruent with calreticulin and HAF-6. We considered that the RNAi defects in rsd-2 mutants might have relevance to membrane-associated functions; however, endomembrane compartmentalization and endocytosis/exocytosis markers in rsd-2 and rsd-6 mutants appear normal. The mutants also possess environmentally sensitive defects in cell-autonomous RNAi elicited from transgene-delivered dsRNAs. Thus, the ultimate functions of rsd-2 and rsd-6 in systemic RNAi are remarkably complex and environmentally responsive.

Caenorhabditis elegans ABCRNAi transporters interact genetically with rde-2 and mut-7.

RNA interference (RNAi) mechanisms are conserved and consist of an interrelated network of activities that not only respond to exogenous dsRNA, but also perform endogenous functions required in the fine tuning of gene expression and in maintaining genome integrity. Not surprisingly, RNAi functions have widespread influences on cellular function and organismal development. Previously, we observed a reduced capacity to mount an RNAi response in nine Caenorhabditis elegans mutants that are defective in ABC transporter genes (ABC(RNAi) mutants). Here, we report an exhaustive study of mutants, collectively defective in 49 different ABC transporter genes, that allowed for the categorization of one additional transporter into the ABC(RNAi) gene class. Genetic complementation tests reveal functions for ABC(RNAi) transporters in the mut-7/rde-2 branch of the RNAi pathway. These second-site noncomplementation interactions suggest that ABC(RNAi) proteins and MUT-7/RDE-2 function together in parallel pathways and/or as multiprotein complexes. Like mut-7 and rde-2, some ABC(RNAi) mutants display transposon silencing defects. Finally, our analyses reveal a genetic interaction network of ABC(RNAi) gene function with respect to this part of the RNAi pathway. From our results, we speculate that the coordinated activities of ABC(RNAi) transporters, through their effects on endogenous RNAi-related mechanisms, ultimately affect chromosome function and integrity.

ATP-binding cassette transporters are required for efficient RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a conserved gene-silencing phenomenon that can be triggered by delivery of double-stranded RNA (dsRNA) to cells and is a widely exploited technology in analyses of gene function. Although a number of proteins that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here, we report that the Caenorhabditis elegans gene haf-6 is required for efficient RNAi. HAF-6 is a member of the ATP-binding cassette (ABC) transporter gene superfamily. ABC transporters use ATP to translocate small molecule substrates across the membranes in which they reside, often against a steep concentration gradient. Collectively, ABC transporters are involved in a variety of activities, including protective or barrier mechanisms that export drugs or toxins from cells, organellar biogenesis, and mechanisms that protect against viral infection. HAF-6 is expressed predominantly in the intestine and germline and is localized to intracellular reticular organelles. We further demonstrate that eight additional ABC genes from diverse subfamilies are each required for efficient RNAi in C. elegans. Thus, the ability to mount a robust RNAi response to dsRNA depends upon the deployment of two ancient systems that respond to environmental assaults: RNAi mechanisms and membrane transport systems that use ABC proteins.