MORC2 promotes development of an aggressive colorectal cancer phenotype through inhibition of NDRG1

Abstract

MORC2 (microrchidia family CW-type zinc finger 2) is a newly identified chromatin remodeling protein that functions in diverse biological processes including gene transcription. NDRG1 is a metastasis suppressor and a prognostic biomarker for colorectal cancer (CRC). However, the relationship between MORC2 and NDRG1 transcriptional regulation and the roles of MORC2 in CRC remain elusive. Here, we showed that MORC2 downregulated NDRG1 mRNA, protein levels, and promoter activity in CRC cells. We also found that MORC2 bound to the -446 to -213 bp region of the NDRG1 promoter. Mechanistically, histone deacetylase sirtuin 1 (SIRT1) was involved in NDRG1 transcriptional regulation. MORC2 was able to interact with SIRT1 and inhibit NDRG1 promoter activity cumulatively with SIRT1. MORC2 overexpression led to a decrease of H3Ac and H4Ac of the NDRG1 promoter. Importantly, we showed that NDRG1 was essential in MORC2-mediated promotion of CRC cell migration and invasion in vitro, as well as lung metastasis of CRC cells in vivo. Moreover, MORC2 expression correlated negatively with NDRG1 expression in CRC patients. High expression of MORC2 was significantly associated with lymph node metastasis (P = 0.019) and poor pTNM stage (P = 0.02) and the expression of MORC2 correlated with poor prognosis in colon cancer patients. Our findings thus contribute to the knowledge of the regulatory mechanism of MORC2 in downregulating NDRG1, and suggest MORC2 as a potential therapeutic target for CRC.

Keywords: MORC2; NDRG1; SIRT1; colorectal cancer; transcriptional regulation.

Figure 1

MORC2 downregulates NDRG1 in colorectal cancer cells. A,B, MORC2 overexpression downregulated NDRG1 mRNA and protein expression. Flag‐MORC2 or vector control was stably transfected into HT‐29 cells (left panel) and SW‐620 cells (right panel). A, mRNA level was examined by quantitative RT‐PCR analysis. *P < 0.05. B, Protein level was analyzed by western blot. C‐F Specific knockdown of MORC2 upregulated NDRG1 mRNA and protein levels. C,E, mRNA level was estimated by quantitative RT‐PCR. D,F, Protein level was analyzed by western blot. NC, negative control

Figure 2

MORC2 binds to NDRG1 promoter and inhibits its activity. A,B, HEK‐293 and SW‐620 colon cancer cells were transiently transfected with pGL3‐NDRG1‐luc reporter plasmid, pRL‐TK vector along with Flag‐MORC2 as indicated. Luciferase activities were determined and normalized to pRL‐TK (Renilla) activity 24 hours after transfection. *P < 0.05, **P < 0.01. C, Left panel, schematic representation of a series of 5′‐deleted NDRG1 promoter/luciferase constructs. Bent arrow indicates transcription initiation site. Right panel, HEK‐293 cells were transiently transfected with various NDRG1 promoter deletion vectors indicated in the left panel with or without Flag‐MORC2 expression vector as indicated. Results are expressed as a percentage of the MORC2‐untransfected control that is taken as 100%. *P < 0.05 compared with control. D, ChIP assays were carried out using MORC2 Abs, and appropriate negative control Abs (IgG), in MORC2 overexpressed (Flag‐MORC2) SW‐480 cells, followed by quantitative PCR with primers amplifying the NDRG1 promoter region (−759 to −446 bp, −446 to −213 bp, and −213 to +69 bp). Data are plotted as fold‐enrichment of specific Ab binding over IgG control. E, ChIP assays were carried out using IgG and MORC2 Abs in SW‐480 cells, followed by quantitative PCR with primers amplifying the NDRG1 promoter region (−446 to −213 bp)

Figure 3

MORC2 inhibits NDRG1 expression by decreasing the histone acetylation level of the NDRG1 promoter region. A,B, SW‐620 cells stably transfected with Flag‐MORC2 or vector control were treated with 50 μmol/L sirtinol for 24 hours. A, NDRG1 mRNA level was measured by quantitative (q)RT‐PCR. B, Protein levels of MORC2 and NDRG1 were measured by western blot. C,D, Endogenous sirtuin 1 (SIRT1) in SW‐620 cells was knocked down by shRNA targeting SIRT1 and lentivirus infection. C, NDRG1 mRNA level was measured by qRT‐PCR. *P < 0.05. D, Protein levels of SIRT1 and NDRG1 were measured by western blot. E, SW‐620 cells were transfected with pGL3‐NDRG1‐luc reporter construct, pRL‐TK plasmid, and SIRT1 expression vector as indicated. Luciferase activities were determined and normalized to Renilla activity 24 hours after transfection. *P < 0.05, **P < 0.01. F, For the immunoprecipitation (IP), cell lysates were immunoprecipitated by anti‐Flag Ab, and precipitates were immunoblotted (IB) with anti‐SIRT1 and anti‐Flag Abs. G, MORC2 and SIRT1 were transiently transfected into SW‐620 cells as indicated, and the promoter activity was estimated by luciferase assays. *P < 0.05, **P < 0.01. H, ChIP‐qPCR was carried out using H3Ac and H4Ac Abs, and negative control Abs (IgG) in control and MORC2 overexpressed SW‐480 cells, followed by qPCR with primers amplifying the NDRG1 promoter region (−446 to −213 bp). *P < 0.05, **P < 0.01

Figure 4

NDRG1 is essential in MORC2‐mediated promotion of colorectal cancer cell migration and invasion. A, Migratory capacities of HT‐29 cells was measured by Transwell assay after MORC2 overexpression. Representative images and quantitative data of 3 independent experiments are shown. *P < 0.05. B, Wound healing assays were undertaken to detect the migratory capacity of HT‐29 cells after MORC2 overexpression. Representative images of wound healing assays are presented from 3 independent experiments. Histograms represent the wound closure rates at the indicated times. *P < 0.05. C,D, Migratory and invasive capacities of SW‐480‐shNC (negative control), SW‐480‐shMORC2, SW‐480‐shNDRG1, and SW‐480‐shMORC2 + shNDRG1 were measured by Transwell assay. Representative images and quantitative data of 3 independent experiments are shown. *P < 0.05. Ctrl, control

Figure 5

NDRG1 is required for MORC2‐mediated promotion of CRC cell pulmonic metastasis. 5 × 10⁶ SW‐480‐shNC, SW‐480‐shMORC2, and SW‐480‐shMORC2 + shNDRG1 cells were injected into nude mice through the tail vein. A, After 8 weeks, mice were killed and lungs were macroscopically photographed. B, Graphical representation of the number of metastatic nodules in the lungs of each mouse (mean ± SD), *P < 0.05. C, Bar charts show the pulmonic metastasis ratio. D, Metastatic tumors were stained by H&E and evaluated for MORC2 and NDRG1 expression by immunohistochemistry

Figure 6

Negative correlation of MORC2 and NDRG1 expression in colorectal cancer (CRC) samples. A, Representative images of immunohistochemical staining for CRC specimens incubated with MORC2 or NDRG1. Intensity value is expressed as histological score (HSCORE). Data were analyzed by Mann‐Whitney U test. B, Spearman's rank test was used to analyze the correlation between MORC2 relative expression and NDRG1 relative expression in 119 CRC samples. C, Kaplan‐Meier overall survival curve of colon cancer patients based on MORC2 expression using The Cancer Genome Atlas database; P = 0.033. D, Proposed schematic model showing the roles of MORC2 and SIRT1 in regulation of the NDRG1 gene transcription. In our model, MORC2 binds the −446 to −213 bp region of NDRG1 promoter, and interacts with sirtuin 1 (SIRT1), leading to the a decrease of histone H3 and H4 acetylation levels of the NDRG1 promoter and thus the transcriptional repression of the NDRG1 gene. TSS, Transcription start site