Fig 2: ALK, FGFR2, and EphA5 are stringent client proteins of Hsp90. (A) Lysates of SMMC‐7721 cells were immunoprecipitated with ALK, FGFR2, or EphA5 antibodies. Their bindings to endogenous Hsp90 were detected by immunoblotting. Antibody against immunoglobulin G was used for internal control. (B) Lysates of ZIP177 and SMMC‐7721 cells were immunoprecipitated with Hsp90 or immunoglobulin G antibodies and subjected to immunoblotting analysis with indicated antibodies. (C) Lysates of ZIP177 and SMMC‐7721 cells were immunoprecipitated with PU‐H71 beads or CON beads and analyzed by immunoblotting. (D) SMMC‐7721 cells were treated with ganetespib at 1 μM for 4 hours in serum‐free medium, then the cell lysates were immunoprecipitated with ALK, FGFR2, or EphA5 antibodies and analyzed by immunoblotting. (E) ZIP177 and SMMC‐7721 cells were treated with ganetespib at 1 μM for 4 hours, followed by the immunoprecipitation with Hsp90 antibody and analysis with immunoblotting. (F) SMMC‐7721 cells were preincubated with MG‐132 at 10 μM for 6 hours, washed, and then treated with ganetespib at 0.1 μM for 24 hours. Cell lysates were subjected to immunoblotting. Abbreviations: IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitate.

Fig 3: Coactivation of ALK, FGFR2, and EphA5 is highly correlated with poor prognosis in a subpopulation of HCC patients. (A) The expression of p‐ALK, p‐FGFR2, and p‐EphA5 in HCC cell lines and QSG‐7701 was analyzed by immunoblotting. (B,C) Expression of p‐ALK, p‐FGFR2, and p‐EphA5 in 24 paired tumor tissues was detected with immunoblotting. Dot plots represent the protein levels normalized to β‐actin (B), and representative immunoblotting results are shown (C). (D) Representative immunochemical staining results for p‐ALK, p‐FGFR2, and p‐EphA5 with different abundance are shown (original magnification, ×200). (E) Scatter plot of the p‐ALK, p‐FGFR2, and p‐EphA5 scores generated from the image analysis intensity algorithm. Red plots depict the subtype of patients with triple strong staining. (F) Retrospective analyses for overall survival of HCC patients were conducted using the Kaplan‐Meier method. Abbreviations: CI, confidence interval; H, relative abundance higher than the median; H&E, hematoxylin and eosin; HR, hazard ratio; L, relative abundance lower than the median; N, nontumor; T, tumor.

Fig 4: ALK, FGFR2, and EphA5 collaborate to regulate the viability of HCC cells through AKT‐dependent, ERK‐dependent, and p38‐dependent pathways. (A) The basal phosphorylation of the 20 top‐ranked kinases in HCC cell lines was selected for cluster analysis. (B) ZIP177 and SMMC‐7721 cells were treated with ceritinib (1 μM), AZD4547 (1 μM), and dasatinib (1 μM) respectively or in combination, for 72 hours. Cell viability was assessed by sulforhodamine B assay. Bars represent means ± standard deviation. (C) Cells were treated with indicated inhibitors at 1 μM for 48 hours. Apoptosis was analyzed by flow cytometry and immunoblotting. Bars represent means ± standard deviation. (D) ZIP177 and SMMC‐7721 cells were transfected with siRNAs for each indicated dasatinib target with or without ceritinib and AZD4547 for 72 hours. Mean values were compared by two‐way analysis of variance. +, ceritinib+ AZD4547+; –, ceritinib– AZD4547–. (E) ZIP177 cells were transfected with indicated siRNAs for 72 hours and analyzed by immunoblotting. (Fi) ZIP177 and SMMC‐7721 cells were treated with inhibitors at 1 μM for 3 hours, followed by immunoblotting analysis. (Fii) Cells were treated with MK2206 (1 μM), trametinib (1 μM) and skepinone‐L (10 μM). Cell viability was assessed by sulforhodamine B assay after 72h, and cell lysates were subjected to immunoblotting after 3h or 48h. Abbreviation: PARP, poly(adenosine diphosphate ribose) polymerase.

Fig 5: Abrogating ALK, FGFR2, and EphA5 activity leads to HCC patient–derived xenograft growth suppression. (A) Mice bearing HCC patient–derived xenograft model LI0752 were treated with vehicle or ceritinib (25 mg/kg), AZD4547 (12.5 mg/kg), or dasatinib (25 mg/kg) individually or in combination once a day for a consecutive 3 weeks. Tumor volume was measured twice a week. Bars represent means ± SEM, n = 6. (B) Tumor lysates were prepared and subjected to immunoblotting analysis with indicated antibodies. (C) Mice bearing HCC patient–derived xenograft model LI0752 were treated with vehicle or ganetespib (30 mg/kg or 50 mg/kg, three times per week). Tumor volume was measured twice a week. Bars represent means ± SEM, n = 6. (D) Tumor lysates were prepared and subjected to immunoblotting analysis with indicated antibodies. (E) A proposed overview of ALK, FGFR2 and EphA5 co‐activation in HCC cells and therapeutic strategies to block their activities were illustrated. Abbreviations: FRS2, FGFR substrate 2; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; T/C, treatment/control.