Fig 2: 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.

Fig 3: The mechanism underlying 3D-GF-PADM-induced hyaluronan secretion. 3D-GF-PADM treatment increases the level of JAK2 phosphorylation, which leads to the increased expression of STAT3, and activated STAT3 enters the nucleus and binds to the promoter of the HAS2 gene to upregulate its expression at both RNA and protein levels. This eventually results in an increase in the synthesis of hyaluronic acid.

Fig 4: (A) HAS2 expression can be inhibited by blocking the JAK2/STAT3 pathway in HSFs. HSFs were grown to 70–80% confluence and treated with 3D-GF-PADM alone or with the following pathway inhibitors: 10 μM TGF-β receptor inhibitor SB431542, 10 μM PI3K inhibitor LY294002, 10 μM JAK-2 inhibitor AG490, and 10 μM ERK-specific inhibitor PD98059. Cells were then lysed at 24 h after treatment and western blot analysis was performed for HAS2. (B) Inhibition of the JAK2/STAT3 signalling attenuates 3D-GF-PADM-induced HAS2 expression in HSFs. (C) Immunofluorescent staining of the 3D-GF-PADM.

Fig 5: E2F1 or MTA1 knockdown suppresses metastases formation in vivo. (A) IB depicting E2F1 and MTA1 knockdown in SK-Mel-147 cells stably expressing sh.ctrl, sh.E2F1, or sh.MTA1 prior to i.v. injection in mice (left). Representative lung images (center top; arrow; metastases) and corresponding hematoxylin/eosin sections (center bottom). Metastases (top) and pulmonary nodules (bottom) on lungs of mice injected with sh.ctrl-expressing cells are depicted with arrows. Metastatic dissemination is measured as relative area of metastases versus total lung area (right; n=5/group, 2-tailed Student's t-test, *p< 0.05). Scale bar: 2 mm. (B, C) IHC of (B) HAS2 levels (brown staining, scale bar: 50 µm) and (C) CD206 marker of recruitment of tumor-associated macrophages (green staining) on metastasized pulmonary tissue (scale bar: 100 µm). (D) IB depicting E2F1, and MTA1 knockdown in PancTuI cells stably expressing sh.ctrl, sh.E2F1 or sh.MTA1, prior to their orthotopic injection into SCID-beige mice. Metastatic outcome was estimated via (E) occurrence of recurrent tumor and liver metastases, (F) recurrent tumor weight and (G) number of liver metastases, after resection of the primary tumors (Mann-Whitney-U test, * p= 0.00466; † p= 0.00124).