RAB10 overexpression promotes tumor growth and indicates poor prognosis of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC), one of the most common and lethal cancers worldwide, has a high recurrence rate with current treatment modalities. Identifying biomarkers for early diagnosis and discovering new sufficient molecular targets for the development of targeted therapies are urgently needed. RAB10, a member of the RAS family, has been shown to be highly expressed in HCC. However, the function of RAB10 in HCC is less studied. Here we report that RAB10 acts as an oncogene in HCC. The shRNA-mediated knockdown of RAB10 significantly reduced the proliferation of HCC cells and colony formation, induced cell cycle arrest at G0/G1 phase and increased apoptosis in vitro. In addition, RAB10 knockdown suppressed HCC growth in nude mice. Moreover, RAB10 silencing decreased the phosphorylation of InsR, Met/HGFR, Ron/MST1R, Ret, c-Kit/SCFR, EphA3, EphB4, Tyro3/Dtk, Axl, Tie2/TEK, VEGFR2/KDR, Akt/PKB/Rac, S6 Ribosomal Protein and c-Abl, while the phosphorylation of HSP27, p38 MAPK, Chk2 and TAK1 increased significantly. These results suggest that RAB10 regulates cell survival and proliferation through multiple oncogenic, cell stress and apoptosis pathways. More importantly, high RAB10 expression levels in HCC cells correlated with a poor prognosis in HCC patients. Therefore, our findings revealed an oncogenic role for RAB10 in the pathogenesis of HCC and that RAB10 is a potential molecular target or a biomarker for HCC.

Keywords: RAB10; biomarker; hepatocellular carcinoma; oncogene; therapeutic target.

Figure 1. RAB10 knockdown suppressed cell proliferation in SMMC-7721 cells

NC, cells infected with non-targeting shRNA lentivirus; RAB10, cells infected with RAB10-targeting shRNA lentivirus. (A) Cell growth was measured via multiparametric high-content screening (HCS) every day for five days after lentivirus infection. (B) SMMC-7721 cell numbers were quantified by Cellomics ArrayScan VTI every day and the cell proliferation rate was analyzed. Data were shown as means ± SD (n = 3), **P < 0.01.

Figure 2. RAB10 knockdown induced cell cycle arrest and apoptosis and reduced colony formation in SMMC-7721 cells

Control-shRNA, cells infected with negative control shRNA lentivirus; shRAB10, cells infected with RAB10 shRNA lentivirus. (A) RAB10 mRNA expression levels were detected by qRT-PCR in human normal hepatic cell line (LO2) and four HCC cell lines (SMMC-7721, Huh-7, Hep 3B and Hep G2). (B) RAB10 protein levels in SMMC-7721 cells and RAB10 mRNA levels in SMMC-7721 cells were knocked down efficiently by shRNA. (C) Knockdown of RAB10 expression induced G0/G1 phase arrest. (D) Knockdown of RAB10 increased apoptosis. (E) Knockdown of RAB10 reduced colony formation. Data were shown as means ± SD (n = 3), *P < 0. 05, **P < 0.01, ***P < 0.001.

Figure 3. RAB10 knockdown induced global changes in SMMC-7721 gene expression

NC, cells infected with negative control shRNA lentivirus; KD, cells infected with RAB10 shRNA lentivirus. (A) Heatmap of 695 genes showed significant (P < 0.05) differential expression (fold change > 1) between cells transfected with RAB10 shRNA (green) and with control shRNA (red). Rows and columns represented genes and samples, respectively. A color scale for normalized expression data was shown in the upper left corner of the heatmap (green represented down-regulated genes and red represented up-regulated genes). (B) Functional pathway enrichment of differentially expressed genes was analyzed based on KEGG and BIOCARTA databases. Statistically significant modulations (P < 0.001) of the top 10 pathways were shown. The statistical significance shown on the X axis was represented by the inverse log of the P value. C-D. Networks were constructed between RAB10 and select genes (C) and pathway-related or down-stream genes (D), respectively. Green circles represented down-regulated genes, red circles represented up-regulated genes, and gray rhombuses represented linker genes. Solid arrows indicated confirmed regulatory relationships and dotted lines predicted regulatory relationships. Inhibitory relationships were indicated by “T” bars. (E) Differentially expressed genes (P < 0.05 and fold change > 1) in network (D). (F) Protein levels of select genes in SMMC-7721 cells transfected with negative control shRNA (NC) or RAB10 shRNA (KD) measured by Western blot.

Figure 4. Effects of RAB10 knockdown on stress, apoptosis and RTK signaling pathways in SMMC-7721 cells

shControl, cells infected with control shRNA lentivirus; shRAB10, cells infected with RAB10-targeting shRNA lentivirus. (A) Knockdown of RAB10 changed the expression levels of genes in stress and apoptosis signaling pathways. (B) Knockdown of RAB10 changed the expression levels of genes in the RTK signaling pathway. Data were shown as means ± SD (n = 6), *P < 0. 05, **P < 0.01, ***P < 0.001.

Figure 5. RAB10 knockdown inhibited tumorigenicity of hepatocarcinoma cells in vivo

BALB/c nude mice were subcutaneously injected with control shRNA cells or RAB10 shRNA cells. (A) RAB10 knockdown significantly suppressed the formation and growth of xenografts. (B) Tumor volumes were measured on the indicated days. Knockdown of RAB10 decreased the volumes of xenografts significantly. (C) RAB10 knockdown reduced the weight of xenografts. Data were shown as means ± SD (n = 10), ***P < 0.001.

Figure 6. Expression of RAB10 was elevated in tissues of HCC patients

(A) Representative pictures of immunohistochemistry analysis of RAB10 protein in each of the primary hepatocellular carcinoma tissue (T) and noncancerous tissues (NT) from the same patient. (B) Increased RAB10 expression is associated with shorter time to recurrence in patients with HCC. RAB10 protein expression in cytoplasm was analyzed by immunohistochemistry composed of 90 FFPE (Formalin-Fixed and Parrffin-Embedded) histologic tissue specimens. Differences in time to recurrence between patients stratified by RAB10 expression were statistically assessed by the log-rank test and Kaplan-Meier survival probability methods and depicted for TNM stage, age, gender.