Fig 2: Vemurafenib enhances RASSF1A-mediated LATS1-SMAC interaction in A375 melanoma cells.A LATS1 phosphorylation of A375 melanoma cells upon increasing amounts of RASSF1A expression in growing conditions or after 2 µM of Vemurafenib treatment for 1 hour. Levels of transfected HA-RASSF1A and endogenous LATS1 and phospho-LATS1 (pT1079-LATS1) are shown. Values correspond to densitometric quantification of pT1079 signal vs total LATS1 levels. B Left: Apoptosis of A375 melanoma cells transfected with RASSF1A and treated with 2 µM of Vemurafenib for 18 hours. Percentage of apoptotic cells was assayed by flow cytometry after YoPro-PI staining. Data shows fold change of apoptosis from 4 independent experiments and bars represent averages. Right: Percentage of A375 melanoma cells showing Caspase 3 activation upon RASSF1A (R1A) overexpression and treated with 2 µM of Vemurafenib for 18 hours determined by flow cytometry after FITC-VAD-FMK staining. Statistical analysis: t test p = 0.1*; p = 0.05**; p = 0.01***. C LATS1-SMAC interaction in A375 melanoma cells transfected with HA-RASSF1A upon 2 µM of Vemurafenib treatment for 10 hours assayed by co-immunoprecipitation. Levels of immunoprecipitated LATS1 and SMAC are shown (upper panel). Normal IgG was used as negative control. Total levels of LATS1, XIAP, SMAC and transfected HA-RASSF1A are shown (lower panel). Actin levels were used as loading control. D Effect of SMAC knockdown over IAP protein levels upon RASSF1A expression and Vemurafenib treatment. A375 melanoma cells were co-transfected with HA-RASSF1A and siRNA for SMAC (or the corresponding empty vector/non-target siRNA) as indicated and treated with 2 µM of Vemurafenib for 18 hours. Total levels of LATS1, C-IAP1, XIAP, transfected HA-RASSF1A and downregulation of SMAC levels are shown. Actin levels were used as loading control (upper panel). Fold change of XIAP total levels normalised vs actin levels are shown after densitometric quantification of 2 independent experiments ±SD (lower panel). E Effect of LATS1 knockdown over IAP protein levels upon RASSF1A expression and Vemurafenib treatment. A375 melanoma cells were co-transfected with GFP-RASSF1A and siRNA for SMAC (or the corresponding empty vector/non-target siRNA) as indicated and treated with 2 µM of Vemurafenib for 18 hours. Total levels of SMAC, C-IAP1, XIAP, transfected GFP-RASSF1A and downregulation of LATS1 levels are shown. Actin levels were used as loading control (upper panel). Fold change of XIAP total levels normalised vs actin levels are shown after densitometric quantification of 3 independent experiments ±SD (lower panel).

Fig 3: LATS1-SMAC interaction correlates with XIAP degradation and caspase cleavage.A Simplified scheme depicting regulation dynamics between IAPs, SMAC and caspases upon survival or apoptotic conditions. B Exogenous LATS1-SMAC interaction in A375 cells transfected with FLAG-LATS1 and GFP-SMAC assessed by co-immunoprecipitation assay followed by western blot analysis upon knocking down XIAP levels using specific siRNA. Cells were also transfected with HA-RASSF1A or the corresponding empty vector and serum-deprived overnight. FLAG-LATS1 was immunoprecipitated using anti-FLAG beads. Levels of immunoprecipitated FLAG-LATS1, GFP-SMAC and XIAP are shown. C Total lysates of A375 cells transfected with the indicated plasmids and siRNAs or their respective controls corresponding to B) assayed by western blot. Levels of transfected FLAG-LATS1, GFP-SMAC and HA-RASSF1A and endogenous C-IAP and XIAP are shown. Actin was used as loading control. Samples run in two separate gels which were transferred together to the same membrane to compare protein levels between two different gels. D XIAP ubiquitination approach measured by western blot. A375 cells were transfected with FLAG-LATS1, GFP-SMAC and GFP-RASSF1A as indicated and endogenous XIAP was immunoprecipitated after serum deprivation. HA-Ub was co-transfected and XIAP ubiquitination was measured by detecting HA. Immunoprecipitated XIAP levels are shown. E Caspase 3 cleavage of A375 cells transfected with FLAG-LATS1, HA-RASSF1A and GFP-SMAC as indicated upon serum deprivation. Caspase 3 cleavage was measured by western blot. Actin was used as loading control. F Exogenous LATS1-SMAC interaction in A375 cells transfected with FLAG-LATS1 and GFP-SMAC assessed by co-immunoprecipitation assay followed by western blot analysis upon knocking down C-IAP1 levels using specific siRNA. Cells were also transfected with HA-RASSF1A or the corresponding empty vector and serum-deprived overnight. FLAG-LATS1 was immunoprecipitated using an anti-FLAG beads. Levels of immunoprecipitated FLAG-LATS1 and GFP-SMAC are shown. G Total lysates of A375 cells transfected with the indicated plasmids and siRNAs or their respective controls corresponding to F) assayed by western blot. Levels of transfected FLAG-LATS1, GFP-SMAC, HA-RASSF1A and endogenous C-IAP1 and XIAP are shown. Actin was used as loading control. Samples run in two separate gels which were transferred together to the same membrane to compare protein levels between two different gels.

Fig 4: SM-164 induces apoptosis of breast cancer cells in combination with TNFa released by tumor-associated macrophages. (A) The parental MDA-MB-231 cells were treated with vehicle, TNFa (1 ng/ml) or TNFa + the indicated doses of SM-164 or BV6 overnight. Annexin V+PI+/- apoptotic cells were analyzed by flow cytometry. (B) Parental MDA-MB-231 cells were treated with the indicated doses of SM-164, BV6 or AT-406 alone, or with a combination of them plus TNFa (1 ng/ml) for 8 hours. Cell lysates were used to test protein levels of cIAP1/cIAP2 and GAPDH. (C) 1 × 104 GFP+ MDA-MB-231 cells were cultured alone or together with WT mouse BM cells in the presence of M-CSF +/- PBS (P) or IL-4 for 3 d, during which 3 nM SM-164 and 1 µg/ml TNFR:Fc (R:Fc) were added for the last 16 hr (last group on right +R:Fc). Annexin V+PI+/- apoptotic cells in the GFP+ population were analyzed by flow cytometry (left panel). The total number of GFP+ cells (left graph) was calculated based on % of GFP+ cells in the total cell number, and % of Annexin V+PI+/- apoptotic cells in the GFP+ population (right graph) was calculated. *p < 0.05 and **p < 0.01, one-way ANOVA +/Dunnett test.

Fig 5: SM-164 promotes osteoblast and inhibits osteoclast formation and reduces RANKL+ cells in bone and lung by preventing degradation of TRAF3. (A) Bone-derived mesenchymal progenitor cells (BdMPCs) from C57Bl6 mice were treated with 25 µg/ml L-ascorbic acid and 5 mM ß-glycerophosphate to induce OB differentiation in the presence of the indicated doses of SM-164 for 7 d. ALP staining was performed to evaluate ALP+ OB area. *p < 0.05 and **p < 0.01 vs. vehicle. (B) Effects of SM-164 on ALP+ OB differentiation in the presence of TGFß1, evaluated as in (A). *p < 0.05 and **p < 0.01 vs. TGFß1 alone; one-way ANOVA +/Dunnett test. (C) BdMPCs were treated with TGFß1 (Tß1), 1 ng/ml, or TNFa (T), 3 ng/ml, +/- SM-164. TRAF3, cIAP1/2 and ß-actin protein levels were assessed in cell lysates by Western blot (WB). Each gel image was cropped from same gel, and full-length gel images are presented in Supplementary Fig. 5A. (D) WT mouse BM cells were cultured with M-CSF for 2 d to generate macrophages, which were then treated with PBS (P), RANKL (R) or TNFa (T) in the presence of vehicle or SM-164 for 8 hr. cIAP1/2 and TRAF3 protein levels were assessed by WB. Each gel image was cropped from the same gel, and full-length gel images are presented in Supplementary Fig. 5B. (E) WT mouse macrophages, generated as in (D), were treated with 10 ng/ml RANKL in the presence of the indicated doses of SM-164 for an additional 2 d. TRAP staining was performed to evaluate OC numbers. **p < 0.01 vs. culture without SM-164, one-way ANOVA +/Dunnett test. (F) Sections from paraffin-processed vertebral bones without metastasis (upper panel) and lungs (lower panel) from vehicle- and SM-164-treated mice, as in Figs. 1 and 2, immunostained for RANKL (green) with DAPI counterstaining (blue). RANKL+ cells were counted on captured fluorescent images (40X). **p < 0.01 vs. vehicle, unpaired Student t test. All in vitro experiments were repeated 3 times with similar results.