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. 2019 Feb 20;9(5):1490–1509. doi: 10.7150/thno.29546

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© Ivyspring International Publisher

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The E2F1:MTA1 complex induces HAS2 expression to promote a malignant phenotype. (A) A scheme for array-based prediction of common targets of E2F1 and MTA1. Downregulated genes were analyzed for E2F-binding sites (E2F-BS) and categorized into GO-term based subgroups. A pair of bars represents one target gene (a ranked list of those genes based on weighted sum of their fold changes in both microarrays is given as Supplemental Table S4). The asterisk (*) marks HAS2. (B) Association of E2F1, MTA1, and HAS2 levels with the metastatic potential of prostate (P: n=10, M: n=21), melanoma (P: n=16, M: n=40) and pancreatic cancer (P: n=22, M: n=6); obtained from the Oncomine™ database. In each graph, the solid lines within the boxes represent the median value and boxes show the 25th to 75th percentile range. Bars represent 90% confidence intervals with circles representing outliers. P: primary tumor; M: metastasis. (C) Semi-quantitative RT-PCR for HAS2 and HAS3 mRNA in PC-3 cells with knockdown of E2F1 (sh.E2F1) or MTA1 (sh.MTA1). (D) Semi-quantitative RT-PCR for HAS2 and HAS3 mRNA in PC-3 cells with E2F1 overexpression (left) or E2F1 overexpression plus MTA1 knockdown (sh.MTA1) (right). (E) A scheme of putative E2F1 binding site on the HAS2 promoter (top). ChIP assay in PC-3.ER-E2F1 cells (bottom). IB for HAS2 in PC-3.ER-E2F1 cells upon 4-OHT induction. (F) Relative luciferase activities after cotransfection of HAS2 promoter construct with E2F1, MTA1, or E2F1 and MTA1 expression plasmids in depicted cells. (G) Relative cell invasion in SK-Mel-147 cells with knockdown for E2F1 (sh.E2F1), MTA1 (sh.MTA1), or both. Corresponding protein levels of HAS2 after E2F1 and MTA1 knockdown were monitored by IB, using actin as loading control. (H) ELISA for HA release on cell culture supernatants of SK-Mel-147 stably transduced with either sh.E2F1 or sh.MTA1. (I, J) Kaplan-Meier analyses of (I) melanoma patients and (J) the Pan-Cancer cohort showing that patients with high E2F1/MTA1 (black) split into a subgroup with high HAS2 (red) and one with low HAS2 levels (cyan), versus all other patients (blue). Log-rank test p-values are depicted on the survival curves. Bar graphs represent means ± SD of three independent experiments; 2-tailed Student's t-test, *p< 0.05.