HOXD10 acts as a tumor-suppressive factor via inhibition of the RHOC/AKT/MAPK pathway in human cholangiocellular carcinoma

Abstract

HOXD10, a key regulator of cell-differentiated phenotype maintainence, has been demonstrated to be involved in the tumorigenesis of many human malignacies. However, the status of HOXD10 expression and its biological function in cholangiocellular carcinoma (CCC) remain to be clarified. In the present study, we investigated the clinical significance and biological functions of HOXD10 in CCC and found that the expression of HOXD10 and its downstream effector RHOC was significantly different in well-differentiated CCC tissues compared with poorly-differentiated lesions. We also observed a significant correlation between low HOXD10 and high RHOC expression levels and worse prognosis. The stable overexpression of HOXD10 by lentivirus vector significantly inhibited cell invasion partly by downregulating the expression of MMP2 and MMP9, and significantly increased early apoptosis in CCC cell lines and induced G1 phase cell cycle arrest, contributing to the inhibition of cell proliferation in vitro. Additionally, we demonstrated that the inactivation of the RHOC/AKT/MAPK pathway was involved in the tumor-suppressive functions of HOXD10 in CCC. These results suggested that HOXD10 may be a putative suppressor gene and can act as a prognostic marker and potentially a novel therapeutic target for CCC.

Figure 1

Decreased HOXD10 and increased RHOC expression in CCC and the negative association of the dysregulated expressions with prognosis. (A) Immunohistochemical stainings for HOXD10 and RHOC expression. The positive HOXD10 staining was primarily localized in the nucleus, while the positive RHOC staining was localized in the cytoplasm. The HOXD10 stainings are strong, strong, moderate and weak in para-carcinoma (×100), well- (×400), moderately- (×400) and poorly-differentiated carcinoma (×400), respectively, while the RHOC stainings are weak, weak, moderate and strong in para-carcinoma (×200), well- (×200), moderately- (×400) and poorly-differentiated carcinoma (×400), respectively. (B) Kaplan-Meier plots of the overall survival rate of CCC patients based on nuclear HOXD10-positive or -negative expression. (C) Kaplan-Meier plots of the overall survival rate of CCC patients based on RHOC-positive or -negative expression. CCC, cholangiocellular carcinoma.

Figure 2

Effect of HOXD10 overexpression on RBE and HCCC-9810 cell proliferation. (A and B) Low endogenous HOXD10 mRNA and protein expression levels in RBE and HCCC-9810 cell lines. (C) Lentiviral transduction efficiency. Compared with the control cell under a light microscope (×100), a fluorescence microscopy (×100) shows >95% RBE and HCCC-9810 cells are effectively transfected with negative HOXD10 lentivirus and lentivirus-HOXD10 4 days after transfection at a MOI of 30. (D and E) Relative HOXD10 mRNA and proteins expression 5 days after lentivirus transfection. (F and G) Cell viability of untransfected and stably transfected RBE and HCCC-9810 cells by an MTT assay, ^***P<0.001. (H) Colony formation assay of RBE and HCCC-9810 cells. (I) A statistical plot of the average number of RBE and HCCC-9810 cells forming colonies in each group. The graph shows the mean ± SD; ^***P<0.001. CTRL, blank control; Ne-CTRL, cells transduced with negative HOXD10 lentivirus; Lv-HOXD10, cells transduced with positive HOXD10 lentivirus; CCC, cholangiocellular carcinoma.

Figure 3

Effects of HOXD10 overexpression on cell cycle progression, apoptosis and cell invasion. (A) G1 phase cell cycle arrest induced by overexpression of HOXD10 in RBE and HCCC-9810 cells. (B and C) A statistical plot of cell cycle distributions of RBE and HCCC-9810 cells in each group, ^**P=0.003. (D) Population of early apoptosis in RBE and HCCC-9810 cells. (E) Statistical plot of percentages of early apoptosis in RBE and HCCC-9810 cells, ^*P<0.05. (F) Representative photos of crystal violet-stained RBE and HCCC-9810 cells that invaded through Matrigel of the invasion chamber. (G) Statistical plot of the average number of invaded RBE and HCCC-9810 cells in each group. The graph shows the mean ± SD, ^***P<0.001. CTRL, blank control; Ne-CTRL, cells transduced with negative HOXD10 lentivirus; Lv-HOXD10, cells transfected with positive HOXD10 lentivirus; CCC, cholangiocellular carcinoma.

Figure 4

Expression of MMP2, MMP9 and MMP14 and RHOC/AKT/ERK activities in RBE and HCCC-9810 cells. (A and B) Decreased relative mRNA and protein expressions of MMP2, MMP9 and the unchanged expression of MMP14 in RBE and HCCC-9810 cells. (C) Suppressed protein expression levels of RHOC, phosphorylated AKT, and phosphorylated ERK (1/2), and unchanged expression of AKT and ERK (1/2) in RBE and HCCC-9810 cells compared with the control groups. CTRL, blank control; Ne-CTRL, cells transfected with negative HOXD10 lentivirus; Lv-HOXD10, cells transfected with positive HOXD10 lentivirus; CCC, cholangiocellular carcinoma.