Fig 2: HDLBP promotes the MEKK1-mediated RAF1-MAPK signaling pathway.A, Immunoblotting and qRT-PCR analyses of HDLBP and RAF1 expression in the indicated HCC cells transfected with Flag-CTL or Flag-HBP. B, Immunoblots showing HDLBP and RAF1 levels in Hep3B and SNU387 cells infected with Lv-HBP or Lv-CTL. C–E, Immunoblots showing HDLBP and RAF1 levels in Huh7 and HepG2 cells transfected or infected with the indicated sequence. F, Immunoblots showing HDLBP, p-ERK, ERK, p-MEK, and MEK levels in Hep3B and SNU387 cells transfected with Flag-CTL or Flag-HBP. G, Hep3B and SNU387 cells were transfected with Flag-HBP or the empty Flag vector for 12 hours and then cotransfected with HBP-sh, HBP-res, or the corresponding empty vector for 24 hours as indicated. The protein levels of HDLBP, p-ERK, ERK, p-MEK, and MEK were measured using immunoblotting. H, Hep3B and SNU387 cells were transfected with Flag-HBP or the empty Flag vector for 24 hours and then treated with the indicated Ras inhibitor for 12 hours. The protein levels of HDLBP, p-ERK, ERK, p-MEK, and MEK were measured using immunoblotting. I, Huh7 lysates were immunoprecipitated with the anti-IgG, anti-HDLBP, or anti-RAF1 antibody and immunoblotted with the indicated antibody. J, Immunoblot s showing the levels of MEKK1, p-ERK, ERK, p-MEK, and MEK in Huh7 and HepG2 cells transfected with CTL-sh or MEKK1-sh. K, Immunoblots showing the levels of the indicated proteins in HCC cells treated with DMSO or AMG510 for 12 hours. L, Immunoblots showing the levels of the indicated proteins in Huh7 cells transfected with the indicated doses of HBP-sh1 for 12 h and then treated with AMG510 for 12 hours. M, Hep3B cells were transfected with Myc-MEKK1 or the empty Myc vector (CTL) for 12 hours and then transfected with HA-RAF1, HA-CTL, or HA fusion mutant vector for 24 hours. The levels of the indicated proteins were measured using immunoblotting. N, Hep3B cells were transfected with Flag-HBP or the empty Flag vector for 12 hours and transfected with HA-RAF or HA fusion mutant vector for 24 hours, and then transfected Huh7 cells were treated with AMG510 for 12 hours. The levels of the indicated proteins were measured using immunoblotting. O, Immunoblots showing the levels of HDLBP, and RAF1 in parental HCC cells and corresponding SR HCC cells. For all the experiments described above, the data are presented as the means ± standard deviations (SDs), and 3 independent experiments (N = 3) were performed in triplicate. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. ns, Not significant.

Fig 3: HDLBP causes sorafenib resistance in HCC in vivo.A, Hep3B cells infected with Lv-HBP or Lv-CTL were transplanted into the right flanks of mice (n = 7 mice per group), and the differences in tumor growth after sorafenib treatment are shown (upper panel). The xenografted tumors in the mice were weighed, and the growth of tumors was measured (lower panel). B, Representative images of hematoxylin and eosin staining and IHC staining for HDLBP, RAF1, and Ki67 expression in serial sections. C, Huh7-SR cells infected with CTL-sg or HBP-sg were transplanted into the right flanks of mice (n = 7 mice per group), and the differences in tumor growth after sorafenib treatment are shown (upper panel). The xenografted tumors in the mice were weighed, and the growth of tumors was measured (upper panel). D, Representative images of hematoxylin and eosin staining and IHC staining for HDLBP, RAF1, and Ki67 expression in serial sections. E, Representative images of IHC staining for HDLBP and RAF1 in HCC samples from patient cohort 2 with different HDLBP expression levels. Scale bars, 100 μm. F, HDLBP expression was correlated with RAF1 expression in HCC samples from patient cohort 2. G, HDLBP expression was correlated with different sorafenib efficacies in patients with HCC from patient cohort 2. In H and I, the expression levels of HDLBP and RAF1 were classified as low if the immunoreactivity score was less than 5 and high if the immunoreactivity score was ≥5. H, The Pearson correlation analysis between HDLBP and RAF1 expression in GSE109211. I, Box plot of RAF1 expression in HCC with different HDLBP expression levels. J, HDLBP expression was correlated with different sorafenib sensitivities in patients with HCC from GSE109211. For all the aforementioned experiments, the data are presented as the means ± standard deviations (SDs). ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001.

Fig 4: HDLBP abrogates TRIM71-mediated RAF1 ubiquitination.A–C, Huh7 cells were transfected with CTL-sh or HBP sh1 for 24 hours. Then, the cells were treated with 1 μmol sorafenib and the proteasome inhibitor MG132 (30 mmol), chloroquine (Chl, 30 mmol), or lactacystin (Lac, 10 mmol) for 12 hours, and immunoblotting was performed with anti-RAF1 and anti-HDLBP antibodies. D, HEK293T cells were transfected with the indicated constructs and treated with MG132 for 12 hours. Lysates were immunoprecipitated with an anti-IgG or anti-HA antibody and detected with an anti-His antibody. E, Huh7 cells were transfected with the indicated constructs and treated with MG132 for 12 hours. Lysates were immunoprecipitated with an anti-IgG or anti-HA antibody and detected with an anti-His antibody. F, Huh7 cells were transfected with the indicated constructs and treated with MG132 for 12 hours. Lysates were immunoprecipitated with an anti-HA antibody and detected with an anti-His antibody. G–H, SNU387 (G) and Huh7 (H) cells were transfected and treated as indicated. Lysates were immunoprecipitated with an anti-HA antibody and detected with an anti-His antibody. I, Schematic diagram of the screening process for E3 ligases mediating RAF1 degradation. J, Huh7 cells were transfected with Flag-CTL or Flag-HBP for 48 hours. Cell lysates were immunoprecipitated with the indicated primary antibody and immunoblotted as indicated. K, Huh7 cells were transfected with Flag-CTL or Flag-HBP for 48 hours. Cell lysates were immunoprecipitated with anti-IgG or anti-RAF1 and immunoblotted as indicated (upper panel). Huh7 cells were transfected with NEDD4L si or CTL-si for 24 hours and were treated with 1 μmol of sorafenib (Sor) for 12 hours, and then immunoblot was performed with anti-NEDD4L and anti-RAF1 (lower panel). L–M, Immunoblots showing the levels of HDLBP, RAF1 and TRIM71 in Huh7 and HepG2 cells transfected with the indicated constructs. N–O, Huh7 cells were transfected as indicated, and then cell lysates were immunoprecipitated with anti-RAF1 antibody and detected with anti-His antibody. P, HEK293T cells were transfected with HA-RAF1 for 24 hours and then cotransfected with Myc-TRIM71, TRIM71-sh, or the corresponding empty vector for 24 hours as indicated. CHX was added for the indicated times, and the cell lysates were subjected to immunoblotting for TRIM71 and RAF1 (left panel). The relative quantification of RAF1 protein levels is shown (right panel). Q, HEK293T cells were cotransfected with HA-RAF1 and Myc-TRIM71 for 24 hours and then treated with DMSO or MG132 for 12 hours. CHX was added for the indicated times, and the cell lysates were subjected to immunoblotting for RAF1 and Myc (left panel). The relative quantification of RAF1 protein levels is shown (right panel). R, HEK293T cells were cotransfected with HA-RAF1 and Myc-TRIM71 for 12 hours and then transfected with Flag-HBP, HBP sh, or the corresponding empty vector for 24 hours as indicated. CHX was added for the indicated times, and the cell lysates were subjected to immunoblotting for RAF1, Flag, and Myc (left panel). The relative quantification of RAF1 protein levels is shown (right panel). S, Huh7 cells were transfected with Myc-TRIM71 or Myc-CTL for 12 hours, and then were cotransfected with HBP sh, HBP-res, or the corresponding empty vector for 24 hours as indicated. CHX (10 μmol) was added for the indicated time, and the cell lysates were subjected to immunoblotting of RAF1, HDLBP, and Myc (left). The relative quantification of RAF1 protein levels (right). For all the experiments shown above, the data are presented as the means ± standard deviations (SDs), and 3 independent experiments (N = 3) were performed in triplicate. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001.

Fig 5: RAF1 is critical for the regulatory effect of HDLBP on proliferation and sorafenib resistance in HCC.A, Immunoblots showing the levels of HDLBP and RAF1 in Huh7 and HepG2 cells cotransfected with Flag-HBP, RAF1 sh1, and the corresponding empty vector for 24 hours as indicated. B, A CCK-8 assay was performed in transfected Huh7 and HepG2 cells at the indicated time points. C, A CCK-8 assay was performed in Huh7 and HepG2 cells transfected with the indicated vectors and treated with a range of concentrations of sorafenib. Cell viability was assessed 3 days after sorafenib treatment. D, Immunoblots showing the levels of HDLBP and RAF1 in Huh7 and HepG2 cells cotransfected with HBP sh, HA-RAF1 and the corresponding empty vector for 24 hours as indicated. E, A CCK-8 assay was performed in transfected Huh7 and HepG2 cells at the indicated time points. F, A CCK-8 assay was performed in Huh7 and HepG2 cells transfected with the indicated vectors and treated with a range of concentrations of sorafenib. Cell viability was assessed 3 days after sorafenib treatment. G, Huh7 cells were stably infected with lentivirues containing HBP-sg1, Lv-RAF1, or the corresponding control sequence as indicated. Transfected cells were injected subcutaneously into the right flanks of male BALB/c nude mice (n = 5 mice per group). Representative images of xenografts are displayed (upper panel), and tumor weights and volumes were measured (lower panel). H, Representative images of hematoxylin and eosin staining and IHC staining for HDLBP, RAF1 and Ki67 expression in serial sections. I, Huh7 cells were stably infected with lentivirus containing HBP-sg1, Lv-RAF1, or the corresponding control sequence as indicated. Transfected cells were injected subcutaneously into the right flanks of male BALB/c nude mice (n = 7 mice per group), and the differences in tumor growth after sorafenib treatment are shown (upper panel). The tumor weights and volumes were measured (lower panel). J, Representative images of hematoxylin and eosin staining and IHC staining for HDLBP, RAF1, and Ki67 expression in serial sections. For all the experiments described above, the data are presented as the means ± standard deviations (SDs), and 3 independent experiments (N = 3) were performed in triplicate. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001.