GPR126 regulates colorectal cancer cell proliferation by mediating HDAC2 and GLI2 expression

Abstract

The G-protein-coupled receptor 126 (GPR126) may play an important role in tumor development, although its role remains poorly understood. We found that GPR126 had higher expression in most colorectal cancer cell lines than in normal colon epithelial cell lines, and higher expression levels in colorectal cancer tissues than in normal adjacent colon tissues. GPR126 knockdown induced by shRNA inhibited cell viability and colony formation in HT-29, HCT116, and LoVo cells, decreased BrdU incorporation into newly synthesized proliferating HT-29 cells, led to an arrest of cell cycle progression at the G1 phase in HCT-116 and HT-29 cells, and suppressed tumorigenesis of HT-29, HCT116, and LoVo cells in nude mouse xenograft models. GPR126 knockdown engendered decreased transcription and translation of histone deacetylase 2 (HDAC2), previously implicated in the activation of GLI1 and GLI2 in the Hedgehog signaling pathway. Ectopic expression of HDAC2 in GPR126-silenced cells restored cell viability and proliferation, GLI2 luciferase reporter activity, partially recovered GLI2 expression, and reduced the cell cycle arrest. HDAC2 regulated GLI2 expression and, along with GLI2, it bound to the PTCH1 promoter, as evidenced by a chip assay with HT-29 cells. Purmorphamine, a hedgehog agonist, largely restored the cell viability and expression of GLI2 proteins in GPR126-silenced HT-29 cells, whereas GANT61, a hedgehog inhibitor, further enhanced the GPR126 knockdown-induced inhibitory effects. Our findings demonstrate that GPR126 regulates colorectal cancer cell proliferation by mediating the expression of HDAC2 and GLI2, therefore it may represent a suitable therapeutic target for colorectal cancer treatment.

Keywords: GPR126; RNA-Seq; cell proliferation; colorectal cancer; xenografts.

FIGURE 1

GPR126 is enriched in colorectal cancer cell lines and clinical colorectal cancer tissues. A, Relative GPR126 mRNA and protein expression levels evaluated by qRT‐PCR and western blotting, with the β‐actin as the internal control, in the indicated colorectal cancer cell lines. B, Representative immunohistochemistry staining of GPR126 (brown) in matched human colorectal cancer tissue and adjacent normal tissue. Scale bar = 50 µm. C, Statistical analysis of immunohistochemistry staining scores of GPR126 from 50 pairs of human colorectal cancer tissue (green) and matched adjacent normal colorectal tissue (grey) in the tissue microarray. The number on each column top represents the percentage of tissues per indicated grade. Chi square test was used for comparisons. *, P <.05

FIGURE 2

GPR126 knockdown attenuates colorectal cancer cell growth in vitro. A‐C, Cell viability and growth of the indicated cells after GPR126 knockdown. The effect of two shRNAs targeting GPR126 (sh1, sh2) on GPR126 mRNA expression normalized to β‐actin mRNA in HT‐29 cells (A), cell viability of the indicated cells (B), and the growth of HT‐29 cells (C). NC, control vector. D, E, Cell colony formation in soft agar after shRNA (sh1)‐mediated GPR126 knockdown. NC, control vector. Representative images of the stained cell colonies (triplicate plates for each indicated cell line were used) (D). Quantification of cell colony numbers in (D) represented as a bar plot (E). Values are presented as the mean ± SEM. ***P <.001

FIGURE 3

GPR126 knockdown attenuates colorectal cancer cell growth in vivo. A‐C, GPR126 knockdown in HT‐29, HCT116, and LoVo cells inhibited tumorigenesis in nude mice xenografts. Top panels, pictures of five representative tumors grown from each cell line. Bottom panels, statistical analysis of the volumes of 10 tumors formed from HCT‐116 (A), 15 tumors from HT‐29 (B), and eight tumors from LoVo (C). The volume curves were calculated on day 29 for HCT‐116 tumors (A), day 27 for HT‐29 (B), and day 35 for LoVo (C). Values are presented as the mean ± SEM. ***P <.001

FIGURE 4

GPR126 knockdown leads to DNA synthesis inhibition and cell arrest in G1 phase in HT‐29 and HCT‐116 cells. A, B, Representative images of BrdU (red) and DAPI (blue) staining in NC‐ and GPR126‐sh1‐infected HT‐29 cells; scale bars = 20 µm (A). Ratio of BrdU‐positive cells to the total number of DAPI‐positive cells; more than 10 fields were counted per group (B). C, Cell cycle analysis of NC‐ and GPR126‐sh1‐infected HCT‐116 and HT‐29 cells; three independent experiments were performed. The cell percentages in the G1, S, and G2 phases are indicated. Values are presented as the mean ± SEM. ***P <.001

FIGURE 5

GPR126 knockdown decreases DNA synthesis and expression of cell cycle‐related genes. A, Top 15 signaling pathways affected by GPR126 knockdown in gene ontology annotation analysis according to RNA‐Seq. B, Representative genes regulated by GPR126 participating in DNA replication and cell cycle regulation in RNA‐Seq. C, qRT‐PCR, with the β‐actin as the internal control was used to analyze mRNA levels of the indicated genes, which were found to be regulated by GPR126 via RNA‐Seq. D, Western blotting of the indicated proteins in NC‐ and GPR126‐sh1‐infected HT‐29 and HCT‐116 cells. β‐actin was used as loading control. E, Western blotting of the indicated proteins in HT‐29‐ and HCT‐116‐xenograft tumors

FIGURE 6

Ectopic expression of HDAC2 in GPR126‐silenced cells restores cell viability, BrdU incorporation into DNA, and decreases cell accumulation in the G1 phase. A, B, Ectopic expression of HDAC2 in GPR126‐silenced HT‐29 cells. Relative cell viability (A) and western blotting of protein expression (B1) and the quantification of protein levels (B2) of the indicated groups. NC, cells infected with control vector; sh1, cells infected with GPR126 shRNA; NC + Zs, cells infected with control vector and empty overexpression control vector control (Zs); sh1 + Zs, cells infected with GPR126 shRNA and empty overexpression control vector control; sh1 + HDAC2, cells infected with GPR126 shRNA and HDAC2 overexpression vector. β‐actin was used as loading control. C‐F, Brdu incorporation assay and cell cycle analysis of ectopic expression of HDAC2 in GPR126‐silenced HT‐29 cells. Representative immunostaining images of BrdU (red) and DAPI (blue) staining of the indicated groups; scale bars = 20 μm (C). Ratio of BrdU‐positive cells to the total number of DAPI‐positive cells; more than 10 fields were counted per groups (D). Cell cycle analysis of the indicated groups in HT‐29 cells (E) and the quantification of cell percentages in the G1, S, and G2 phases (F); three independent experiments were performed. Values are presented as the mean ± SEM. **P <.01

FIGURE 7

GLI2 binding with HDAC2 to the PTCH1 promoter is regulated by HDAC2 and its expression recovery restores cell viability in GPR126‐silenced cells. A, Western blotting of HDAC2 and GLI2 protein levels in NC‐ and HDAC2‐shRNA‐infected HT‐29 cells. β‐actin was used as a loading control. B, Chromatin from HT‐29 cells was immunoprecipitated with anti‐HDAC2 antibodies and then eluted and immunoprecipitated again with normal mouse IgG(IgG) or anti‐Gli2(GLI2 Ab). Eluted DNA was PCR‐amplified using PTCH1 and GAPDH primers (B1). Quantitative PCR(qPCR) was used to examine the abundance of eluted DNA from B1, with the DNAs from input control as the internal control (B2). C, Ectopic expression of HDAC2 in GPR126‐silenced cells restores transcription activity of GLI2 promoter (Gli2‐Luc). NC or Sh1, cell infected by control virus or GPR126 shRNA virus; pGL3, empty luciferase vector; Gli2‐Luc, luciferase vector with Gli2 promoter; zs‐HDAC2, HDAC2 ectopic expression construct; zs, empty vector for cloning HDAC2 expression vector; ‘‐’, not used in co‐transfection; ‘+’, used in co‐transfection. D, E, the effect of Purmorphamine (hedgehog agonist) and GANT61 (GLI2 inhibitor), at indicated concentration, on cell viability and GLI2 protein expression in indicate colorectal cancer cells. DMSO, the solvent of Purmorphamine and GANT61. β‐actin was used as a loading control. F, Diagram illustrating the putative mechanisms of GPR126 function in colorectal cancer cells. GPR126 regulates SMO, GLI1, and HDAC2 expression (GSE106696). GLI1 and GLI2, as the downstream components of GPR126 signaling, regulate the expression of hedgehog (HH) target genes, including PTCH1, regulating tumor growth. HDAC2 is regulated by GPR126 regulated HDAC2, mediating GLI2 expression, and binds with GLI2 to the PTCH1 promoter or other promoters of HH target genes. The dotted line indicating ‘Not reported in this article’; solid line indicating ‘Has support in this article’; ‘?’ meaning ‘through unknown mechanism’