Flt-4, a VEGF receptor, is activated by its specific ligand, VEGF-C. The resultant signaling pathway promotes angiogenesis and/or lymphangiogenesis. This report provides evidence that the VEGF-C/Flt-4 axis enhances cancer cell mobility and invasiveness and contributes to the promotion of cancer cell metastasis. VEGF-C/Flt-4-mediated invasion and metastasis of cancer cells were found to require upregulation of the neural cell adhesion molecule contactin-1 through activation of the Src-p38 MAPK-C/EBP-dependent pathway. Examination of tumor tissues from various types of cancers revealed high levels of Flt-4 and VEGF-C expression that correlated closely with clinical metastasis and patient survival. The VEGF-C/Flt-4 axis, through upregulation of contactin-1, may regulate the invasive capacity in different types of cancer cells.

Connective tissue growth factor (CTGF) expression is elevated in advanced stages of breast cancer, but the regulatory role of CTGF in invasive breast cancer cell phenotypes is unclear. Presently, overexpression of CTGF in MCF-7 cells (MCF-7/CTGF cells) enhanced cellular migratory ability and spindle-like morphological alterations, as evidenced by actin polymerization and focal-adhesion-complex aggregation. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) impaired cellular migration and promoted a change to an epithelial-like morphology. A neutralizing antibody against integrin αvβ3 significantly attenuated CTGF-mediated ERK1/2 activation and cellular migration, indicating that the integrin-αvβ3–ERK1/2 signaling pathway is crucial in mediating CTGF function. Moreover, the cDNA microarray analysis revealed CTGF-mediated regulation of the prometastatic gene S100A4. Transfection of MCF-7/CTGF cells with AS-S100A4 reversed the CTGF-induced cellular migratory ability, whereas overexpression of S100A4 in MDA231/AS cells restored their high migratory ability. Genetic and pharmacological manipulations suggested that the CTGF-mediated S100A4 upregulation was dependent on ERK1/2 activation, with expression levels of CTGF and S100A4 being closely correlated with human breast tumors. We conclude that CTGF plays a crucial role in migratory/invasive processes in human breast cancer by a mechanism involving activation of the integrin-αvβ3–ERK1/2–S100A4 pathway.

Hypoxia-inducible factor-1α (HIF-1α) is widely considered to be one of the key regulators of tumor angiogenesis. The upstream regulation is complex and involves several growth factors, cytokines, and hypoxia. Herein, we have identified miR-519c as a hypoxia-independent regulator of HIF-1α, acting through direct binding to the HIF-1α 3′ untranslated region and leading to reduced tumor angiogenesis. Overexpression of miR-519c resulted in a significant decrease of HIF-1α protein levels and reduced the tube formation of human umbilical vein endothelial cells; similarly, antagomir inhibition of miR-519c increased the level of HIF-1α protein and enhanced angiogenic activity, suggesting an important role of miR-519c in HIF-1α-mediated angiogenesis. Consistent with the overexpression of miR-519c in cancer patients with better prognosis, mice injected with miR-519c-overexpressing cells exhibited dramatically reduced HIF-1α levels, followed by suppressed tumor angiogenesis, growth, and metastasis. In addition, we found that hepatocyte growth factor (HGF), a known HIF-1α inducer, reduced the miR-519c levels through an Akt-dependent pathway. This regulation was posttranscriptional and may be mediated by suppression of miR-519c maturation. Taken together, our findings provide the first evidence that miR-519c is a pivotal regulator of tumor angiogenesis and that microenvironmental HGF contributes to regulating miR-519c biogenesis in cancer cells. Cancer Res; 70(7); 2675-85. ©2010 AACR.

MicroRNAs (miRNAs) influence many biological processes, including cancer. They do so by posttranscriptionally repressing target mRNAs to which they have sequence complementarity. Although it has been postulated that miRNAs can regulate other miRNAs, this has never been shown experimentally to our knowledge. Here, we demonstrate that miR-107 negatively regulates the tumor suppressor miRNA let-7 via a direct interaction. miR-107 was found to be highly expressed in malignant tissue from patients with advanced breast cancer, and its expression was inversely correlated with let-7 expression in tumors and in cancer cell lines. Ectopic expression of miR-107 in human cancer cell lines led to destabilization of mature let-7, increased expression of let-7 targets, and increased malignant phenotypes. In contrast, depletion of endogenous miR-107 dramatically increased the stability of mature let-7 and led to downregulation of let-7 targets. Accordingly, miR-107 expression increased the tumorigenic and metastatic potential of a human breast cancer cell line in mice via inhibition of let-7 and upregulation of let-7 targets. By mutating individual sites within miR-107 and let-7, we found that miR-107 directly interacts with let-7 and that the internal loop of the let-7/miR-107 duplex is critical for repression of let-7 expression. Altogether, we have identified an oncogenic role for miR-107 and provide evidence of a transregulational interaction among miRNAs in human cancer development.