Fig. 6

SNS-032 inhibits RhoA activity, actin polymerization, migration/invasion, and liver metastasis of UM cells. a SNS-032 suppressed RhoA activity. Mel270 cells were treated with SNS-032 for 24 h, active GTP-bound forms of RhoA, Cdc42 and Rac1 in cell lysates were enriched by pull-down process and detected by Western blot analysis with the indicated antibodies, respectively. b SNS-032 inhibited the RhoA-ROCK-LIMK1/2-cofilin pathway. Mel270 and Omm2.3 cells were treated with increasing concentrations of SNS-032 for 48 h, and then subjected to Western blot analysis with the indicated antibodies. c SNS-032 inhibited actin polymerization and invadopodia formation in UM cells. Twenty-four hours after treatment with SNS-032, Mel270 and Omm2.3 cells were subjected to immunofluorescence staining with phalloidin. Representative images for each group are shown (scale bar, 10 μm) (left); the fluorescence intensities of F-actin were quantified by Image J and normalized to control cells (right). *, P < 0.05; ***, P < 0.001, Student’s t test. d SNS-032 inhibited the expression of c-Myc and RhoA in UM cells. The protein and mRNA levels of c-Myc and RhoA were detected by Western blot and qRT-PCR analysis, respectively. ***, P < 0.001, one-way ANOVA, post hoc intergroup comparisons, Tukey’s test. e SNS-032 reduced c-Myc binding to the gene promoter of RhoA detected by ChIP assay. IgG was used as negative control. **, P < 0.01, Student’s t test. f and g Overexpression of c-Myc attenuated SNS-032-mediated decrease of RhoA. Mel270 cells transduced with retroviral c-Myc were exposed to SNS-032, the protein and mRNA levels of RhoA were assessed by Western blot and qRT-PCR analysis, respectively. **, P < 0.01, Student’s t test. h and i Overexpression of RhoA and its constitutively activated mutant (Q63L) partially rescued the SNS-032-induced decrease in capacities of migration and invasion. Mel270 cells stably expressing RhoA, RhoA (Q63L) or empty vector were exposed to SNS-032 (0.5 μM). H, the protein levels of RhoA were examined by Western blot analysis. I, the cells were underwent migration and invasion assay. j NOG mice were intrasplenically injected with Mel270-luc or Omm2.3-luc cells and administered with vehicle or SNS-032 (15 mg/kg/day, i.p) for 28 days, liver metastasis was measured by bioluminescent imaging (n = 5 per group for Mel270-luc or n = 3 per group for Omm2.3-luc, respectively). Representative images and quantitative analysis of photon flux on day 28 are shown. ***, P < 0.001, Student’s t test. k Representative images of metastatic livers for Mel270-luc cells (left) are shown and surface metastatic nodules in the livers are counted (right). Error bars represent mean ± SD. ***, P < 0.001, Student’s t test. l H&E-stained sections of representative livers from each group are shown. m A proposed working model is shown. The selective CDK7/9 inhibitor SNS-032 inhibits gene transcription by reducing the CTD phosphorylation of RNA pol II, thereby blocks YAP signaling and inhibits cellular proliferation; lowers Survivin to induce intrinsic apoptosis in UM cells. SNS-032 abolishes KLF4-dependent CSCs properties, suppresses the activation of RhoA GTPase, actin polymerization, the invasive phenotypes (e.g., migration and invasion), and ultimately diminishes liver colonization and metastasis in UM