Cadherin Related Family Member 2 Acts As A Tumor Suppressor By Inactivating AKT In Human Hepatocellular Carcinoma

Abstract

Cadherin related family member 2 (CDHR2) belongs to the protocadherin family and is abundant in normal liver, kidney, and colon tissues, but weakly expressed in cancers arising from these tissues. In this study, we demonstrated that CDHR2 was highly expressed in para-cancer tissues of human hepatocellular carcinoma (HCC), but significantly downregulated or silenced in 85.7% (6/7) of HCC cell lines by both semi-quantitative PCR and western blot, and 79.1% (19/24) and 80.2% (89/111) of tumor tissues from patients with HCC by semi-quantitative PCR, and immunohistochemistry, respectively. Interestingly, CpG islands in the promoter of CDHR2 gene were hypermethylated in HCC cell lines and tissues compared with the para-cancer tissues by methylation-specific PCR analysis, leading to transcriptional repression and silencing of CDHR2 in HCC. In addition, CDHR2 overexpression by lentiviral vectors had suppressive effects on HCC cell growth and proliferation, as evidenced by prolonged cell doubling time and reduced colony-forming ability in vitro, as well as by decreased tumorigenicity in vivo. Mechanistically, CDHR2 overexpression resulted in AKT dephosphorylation along with downregulation of cyclooxygenase-2 (COX2), a downstream target of AKT. This effect was reversed by myristoylated AKT, a constitutively active form of AKT, suggesting an involvement of CDHR2-AKT-COX2 axis in the suppression of HCC growth. Taken together, our study identified CDHR2 as a novel tumor suppressor in HCC and provided a new therapeutic target for HCC.

Keywords: AKT; CDHR2; COX2; hepatocarcinoma; methylation; proliferation.

Figure 1

Expression of CDHR2 in HCC cell lines and tissues. Semi-quantitative RT-PCR was performed to examine mRNA expression of CDHR2 in 7 HCC cell lines (A) and 24 pairs of primary HCC tissues and adjacent normal tissues (B). (C) Western blot was performed to detect the protein expression levels of CDHR2 in 7 HCC cell lines. T, HCC tissue; S, Tumor-surrounding tissue.

Figure 2

Representative IHC staining for CDHR2 in 3 pairs of primary HCC tissues and adjacent non-neoplastic liver tissues. T, HCC tissue; S, Tumor-surrounding tissue.

Figure 3

Validation of ectopic expression and subcellular localization of CDHR2 protein in HCC cell lines. (A) Western blot detection of ectopic CDHR2 expression in SMMC-7721 (left panel) cells and PLC/PRF/5 cells (right panel) infected with LV-CDHR2 or LV-Luc. (B) Immunofluorescence staining shows the over-expression and localization of CDHR2 (red) in SMMC-7721 cells infected with LV-CDHR2, compared with LV-Luc group.

Figure 4

The effect of CDHR2 on HCC cell growth and proliferation. (A) The effect of CDHR2 over-expression on doubling time of HCC cell lines, SMMC-7721 and PLC/PRF/5 cells, respectively. (B) Edu incorporation assay was performed on LV-CDHR2-infected SMMC-7721 and PLC/PRF/5 cells to evaluate the proliferation rate of sparse and confluent cultures. (C) Cells were plated in 10-cm Petri dishes at densities varying from 1,000 to 5,000 cells per dish in fresh medium for colony formation. Colonies were stained with crystal violet after 14 days, and counted as indicated. (D) Cells were plated in semisolid soft agar medium to monitor anchorage-independent growth. The numbers represent the mean±SD of 3 independent experiments. Photographs are representative images from each group as indicated under the microscope. The experiments were repeated 3 times. *p<0.05 vs. control lentivirus group.

Figure 5

Overexpression of CDHR2 inhibited tumorigenicity HCC xenografts in nude mice. (A and B) Photographs of mice with primary tumors derived from CDHR2-overexpressing SMMC-7721 (left panel) and PLC/PRF/5 (right panel) cells. (C and D) Tumor volumes. *p<0.05, **p<0.01, n=6.

Figure 6

Involvement of CDHR2 in dephosphorylation of AKT and COX2 downregulation of downstream. High-throughput protein microarray analysis (A) and Western blot detection (B) of p-AKT and total AKT in LV-CDHR2 or LV-luc infected SMMC-7721 cells. (C) The effect of myr-Akt on doubling time of SMMC-7721 cells stably expressing CDHR2. (D) Real-time PCR analysis of mRNA expression of COX2 in SMMC-7721 cells stably expressing CDHR2. (E) The effect of myr-Akt on mRNA expression of COX2 in CDHR2-expressing SMMC-7721 cells.

Figure 7

CpG island methylation of CDHR2 gene promoter. Methylation-Specific PCR analysis in 5 HCC cell lines (A) and some primary HCC tissues (B). (C) RT-PCR detection of CDHR2 expression in HCC cell lines treated with DNA and TSA methylation inhibitor AZA individually or in combination. UT: untreated; AZA: 5-Aza-2'-deoxycytidine; TSA: Trichostatin A.