MicroRNAs (miRNAs) make up a novel class of gene regulators; they function as oncogenes or tumor suppressors by targeting tumor-suppressor genes or oncogenes. A recent study that analysed a large number of human cancer cell lines showed that miR-330 is a potential tumorsuppressor gene. However, the function and molecular mechanism of miR-330 in determining the aggressiveness of human prostate cancer has not been studied. Here, we show that miR-330 is significantly lower expressed in human prostate cancer cell lines than in nontumorigenic prostate epithelial cells. Bioinformatics analyses reveal a conserved target site for miR-330 in the 3 0 -untranslated region (UTR) of E2F1 at nucleotides 1018-1024. MiR-330 significantly suppressed the activity of a luciferase reporter containing the E2F1-3 0 -UTR in the cells. This activity could be abolished with the transfection of anti-miR-330 or mutated E2F1-3 0 -UTR. In addition, the expression level of miR-330 and E2F1 was inversely correlated in cell lines and prostate cancer specimens. After overexpressing of miR-330 in PC-3 cells, cell growth was suppressed by reducing E2F1-mediated Akt phosphorylation and thereby inducing apoptosis. Collectively, this is the first study to show that E2F1 is negatively regulated by miR-330 and also show that miR-330 induces apoptosis in prostate cancer cells through E2F1-mediated suppression of Akt phosphorylation.

Prostate cancer (PCa) is a leading cause of mortality and morbidity in men worldwide, and emerging evidence suggests that the CD44(high) prostate tumor-initiating cells (TICs) are associated with its poor prognosis. Although microRNAs are frequently dysregulated in human cancers, the influence of microRNAs on PCa malignancy and whether targeting TIC-associated microRNAs inhibit PCa progression remain unclear. In this study, we found that miR-320 is significantly downregulated in PCa. Overexpression of miR-320 in PCa cells decreases PCa tumorigenesis in vitro and in vivo. Global gene expression profiling of miR-320-overexpressing PCa cells reveals that downstream target genes of Wnt/β-catenin pathway and cancer stem cell markers are significantly decreased. MicroRNA-320 inhibits β-catenin expression by targeting the 3'-untranslated region of β-catenin mRNA. The reduction of miR-320 associated with increased β-catenin was also found in CD44(high) subpopulation of prostate cancer cells and clinical PCa specimens. Interestingly, knockdown of miR-320 significantly increases the cancer stem-like properties, such as tumorsphere formation, chemoresistance and tumorigenic abilities, although enriching the population of stem-like TICs among PCa cells. Furthermore, increased miR-320 expression in prostate stem-like TICs significantly suppresses stem cell-like properties of PCa cells. These results support that miR-320 is a key negative regulator in prostate TICs, and suggest developing miR-320 as a novel therapeutic agent may offer benefits for PCa treatment.

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In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacological activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKβ-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis.