Silencing homeobox C6 inhibits colorectal cancer cell proliferation

Abstract

Homeobox C6 (HOXC6), a member of the homeobox family that encodes highly conserved transcription factors, plays a vital role in various carcinomas. In this study, we used a tissue microarray (TMA) consisting of 462 CRC samples to demonstrate that HOXC6 is more abundantly expressed in colorectal cancer (CRC) tissues than adjacent normal mucosa. Clinicopathological data indicated that higher HOXC6 expression correlated with poor overall survival and was associated with primary tumor location in the right colon, primary tumor (pT) stage 3/4 and primary node (pN) stage 1/2. Multivariate analysis showed that high HOXC6 expression was an independent risk factor for poor CRC patient prognosis. HOXC6 downregulation via lentivirus-mediated expression of HOXC6-targeting shRNA reduced HCT116 cell viability and colony formation in vitro, and reduced growth of subcutaneous xenografts in nude mouse. HOXC6 thus appears to promote CRC cell proliferation and tumorigenesis through autophagy inhibition and mTOR pathway activation.

Keywords: HOXC6; autophagy; colorectal cancer; mTOR; proliferation.

Figure 1. HOXC6 was highly expressed in CRC tumor tissues

Representative images of HOXC6 IHC staining intensity: (−) no staining; (+) weak staining; (++) moderate staining; (+++) strong staining (A) HOXC6 IHC staining score was significantly higher in tumor tissues than in normal tissues (B) ***P < 0.001 via Wilcoxon signed rank test (matched pairs).

Figure 2. High HOXC6 expression in tumor tissues indicated poor OS

Kaplan–Meier survival curve with HOXC6 expression in tumor tissues for CRC patients after primary tumor resection. HR: hazard ratio; CI: confidence interval; P value: log-rank test.

Figure 3. HOXC6 downregulation inhibited tumor cell proliferation in vitro

Western blotting analysis for HOXC6 in untreated (CON) and Lv-shCON- or Lv-shHOXC6-infected HCT116 cells, with GAPDH as the control (A) RT-PCR analysis of HOXC6 mRNA levels with GAPDH as the control (B) CON, Lv-shCON and Lv-shHOXC6 group growth curves as measured by MTT assay (C) Colony counts (D) and images (E) for CON cells and Lv-shCON- or Lv-shHOXC6-infeced cells. Apoptosis was assayed by flow cytometry (F) **P < 0.01; ***p < 0.001.

Figure 4. HOXC6 downregulation inhibited tumor growth in vivo

Representative photos of mice (A) and tumors (B) were taken after sacrifice on day 18, and tumor size was measured (C) *P < 0.05 compared with control group. Ki-67 expression in Lv-shHOXC6-infected tumors was less than in controls as shown by H&E and IHC staining (D).

Figure 5. HOXC6 downregulation inhibited autophagy

Following HCT116 cell infection with LV-shHOXC6 or LV-shCON, autophagy genes were analyzed by western blotting (A) mTOR pathway activity was detected by western blotting (B) Representative immunofluorescent images showing LC3 staining in cells infected with mRFP-GFP-LC3 adenovirus (nuclei are stained with DAPI) (C) Scale bar, 10 μm. Autophagy-related gene mRNA levels were detected by RT-PCR (D) The viability of HOXC6-downregulated cells treated with an autophagy activator was measured via CCK-8 assay (E).