Targeting a novel cancer-driving protein (LAPTM4B-35) by a small molecule (ETS) to inhibit cancer growth and metastasis

Abstract

Our previous studies demonstrated that LAPTM4B-35 is overexpressed in a variety of solid cancers including hepatocellular carcinoma (HCC), and is an independent factor for prognosis. LAPTM4B-35 overexpression causes carcinogenesis and enhances cancer growth, metastasis and multidrug resistance, and thus may be a candidate for therapeutic targeting. The present study shows ethylglyoxal bisthiosemicarbazon (ETS) has effective anticancer activity through LAPTM4B-35 targeting. Bel-7402 and HepG2 cell lines from human HCC were used as cell models in which LAPTM4B-35 is highly expressed, and a human fetal liver cell line was used as a control. The results showed ETS has a specific and pronounced lethal effect on HCC cells, but not on fetal liver cells in culture. ETS also attenuated growth and metastasis of human HCC xenograft in nude mice, and extended the life span of mice with HCC. ETS induced HCC cell apoptosis, and upregulated a large number of proapoptotic genes and downregulated antiapoptotic genes. When endogenous overexpression of LAPTM4B-35 was knocked down with RNAi, the killing effect of ETS on HepG2 cells was significantly attenuated. ETS also inhibited phosphorylation of LAPTM4B-35 Tyr285, which involves in activation of the PI3K/Akt signaling pathway induced by LAPTM4B-35 overexpression. In addition, the induction of alterations in quantity of c-Myc, Bcl-2, Bax, cyclinD1 and Akt-p molecules in HepG2 cells by LAPTM4B-35 overexpression could be reversed by ETS.

Conclusion: ETS is a promising candidate for treatment of HCC through LAPTM4B-35 protein targeting.

Keywords: LAPTM4B-35; apoptosis; cancer targeted therapy; ethylglyoxal bisthiosemicarbazon (ETS); hepatocellular-carcinoma.

Figure 1. Inhibitory and killing effects of ETS on HCC cells

a. Various cancer cell lines were cultured in the absence or presence of ETS at indicated concentrations for 48 h. b. HepG2 cells were cultured in the absence or presence of various drugs at indicated concentrations for 48 h. c. HepG2 cells were cultured in the presence and absence of ETS for indicated hours and then treated with LIVE/DEAD* Viability/Cytotoxicity Kit for detection of viable and dead cells. The killing effect of ETS is time-dependent with the majority of HepG2 cells were killed by ETS at 48 hours. d. The cells were fluorescently double-stained with Calcein-AM (1 μmol/L) and EthD-1 (2 μmol/L) at 37°C for 30 min and then surveyed under fluorescence microscope. Cells emitting green fluorescence were viable cells which were merely stained by Calcein-AM. Cells emitting red fluorescence were dead cells or apoptotic cells which merely stained by EthD-1. Upper panel: HepG2 HCC cells were treated with ETS at a concentration of 2 μmol/L for 48 h. The vast majority of HepG2 cells were killed by ETS. Lower panel: human fetal liver cells were treated with ETS at a concentration of 25 μmol/L for 48 h. None of fetal liver cells were killed by ETS. e. HepG2 cells were treated with ETS at a concentration of 2 μmol/L for 8, 16, 24 or 36 hours. The apoptotic cells were measured by Flow-cytometry. The dying cells including at early and late apoptotic phases were calculated together. The promoting effect of ETS on apoptosis of HepG2 cells was time-dependent. The experiment was repeated several times.

Figure 2. Inhibitory effect of ETS on growth and metastasis of human HCC xenograft in nude mice

Human HCC Bel-7402 cells (1 × 10⁶) were subcutaneously inoculated into each nude mouse. ETS (2.5, 7.5, 22.5 mg/kg/d), cisplatin (1.0 mg/kg/d), mitomycin (1.0 mg/kg/d), solvent and PBS (controls) were administered by intraperitoneal injection from day 9 when the xenograft grew out. Mitomycin and cisplatin were used as the positive controls, solvent and PBS were used as negative controls. The inhibitory effect of ETS on xenograft growth was observed to be dose-dependent as compared with the control groups of solvent and PBS. a. Tumor growth curves of human HCC xenograft in nude mice with variant treatments. b. Tumor photograph of human HCC xenograft grown in nude mice with variant treatments for 6 weeks. Left panel: Size of human HCC xenograft in variant groups. Right panel: Number of lymph nodes metastases in variant groups. c. The survival curves of mice with ascites HCC in variant groups. Mouse hepatocellular carcinoma H22 cells (1 X10⁶) were inoculated into peritoneal of each ICR mouse. ETS (0.5, 1.5 mg/kg/d) and solvent were intraperitoneally administered to each ICR mouse in various group (n = 10). The life span showed a significant prolongation with a dose-dependent manner in the ETS groups.

Figure 3. Molecular alterations induced by ETS

a. Western blot profiles of cyclin D1, Bcl-2, Bax, and phosphorylated p53 proteins from lysate of HepG2 cells incubated in the presence of ETS (2 μmol/L) for indicated times, indicating that proliferation- and apoptosis-related proteins are altered by ETS in a time-dependent manner. b. Western blot profile of procaspase 3 and cleaved caspase 3 from lysate of HepG2 cells incubated in the absence and presence of ETS (2 μmol/L) for indicated times, indicating the activation of key effector molecule by ETS in apoptotic pathway. c. Western blot profile of c-Myc protein from lysate of HepG2 cells incubated in the absence and presence of ETS at indicated concentrations for indicated hours, indicating remarkable decrease of c-Myc protein with ETS treatment in a dose- and time-dependent manner. d. cDNA array analysis shows the upregulated and downregulated genes that promote and inhibit apoptosis, respectively, by treatment of ETS. e. Schematic representation of antagonistic effects of ETS on gene expression that had been induced by overexpression of LAPTM4B-35 in HCC.

Figure 4. Inhibitory effects of ETS on phosphorylation of Akt and LAPTM4B-35

a. Western blot profile of phosphorylated Akt (Ser₄₇₃) from lysate of HepG2 cells incubated in the absence and presence of ETS (2 μmol/L), indicating the inhibitory effect of ETS on activation of PI3K/Akt signaling pathway either in the presence or absence of serum. b. Western blot profile demonstrated that Akt-p (Ser₄₇₃) is decreased in the mutated AF (PA) cells in which P12, 13, 15A mutated plasmids of LAPTM4B-35 were transfected, and also in the mutated AF (YF) cells in which Tyr (Y)₂₈₅ Phe (F) mutated plasmids of LAPTM4B-35 were transfected, as compared with wild-type LAPTM4B-35 (AF). These results indicate that the proline-rich domain (PPRP) in the N-terminal and the Tyr₂₈₅ in C-terminal tails of LAPTM4B-35 are necessary for Akt phosphorylation. c. Co-IP and Western blot profile shows that Tyr₂₈₅ in C-terminal tails of LAPTM4B-35 is the only one Tyrosine that can be phosphorylated. BEL-7402 HCC cells were transfected by AF plasmids which contains the wild type LAPTM4B-35 cDNA with a FLAG as a tag or by AF (YF) plasmid which contains a Tyr (Y)₂₈₅ Phe (F) mutation of LAPTM4B-35 cDNA with a FLAG as a tag. Cells were first serum-starved for 16 h, and then seeded onto laminin substrate for 15 minutes. The expressed exogenous LAPTM4B was immunoprecipitated by Anti-FLAG-mAb. After PAGE the Anti-phosphorylated Tyr mAb was used for blotting the Tyr-phosphorylated LAPTM4B. The Western blot profile shows that the phosphorylated Tyr appeared merely in the wild type BEL-7402 HCC-AF cells as one thick band, but no any Tyr-phosphoralated band appeared in the Tyr ₂₈₅ mutated BEL7402 HCC-YF cell lysate, indicating that LAPTM4B-35 Tyr₂₈₅ is the only single phosphorylation site. d. Co-IP and Western blot profile shows that ETS significantly decreased the phosphorylation of LAPTM4B-35 Tyr₂₈₅. HepG2 cells were first serum-starved for 16 h, then serum and ETS or PBS (control) were added for 15 min incubation. The cell lysate was first precipitated by anti-LAPTM4B-N10-pAb, which reacts specifically with LAPTM4B-35. Anti-phosphorylated Tyr-mAb was used as the blotting antibody. The profile shows that the phosphorylated LAPTM4B-35 is attenuated by ETS treatment compared with the control. e. HepG2 cells were transfected with LAPTM4B-shRNA or Mock plasmids. The expression of LAPTM4B-35 was significantly reduced in the LAPTM4B-shRNA stably transfected HepG2 cells (HepG2-RNAi) as compared with HepG2 Mock cells and the parent HepG2 cells. Cells in the three groups were respectively treated by ETS at indicated concentrations for 48 h. The HepG2-RNAi cells showed less sensitive to ETS. f. Schematic representation of molecular mechanism of ETS for targeting LAPTM4B-35.