Loss of ZNF677 expression is a predictive biomarker for lymph node metastasis in Middle Eastern Colorectal Cancer

Abstract

Zinc-finger proteins are transcription factors with a "finger-like" domain that are widely involved in many biological processes. The zinc-finger protein 677 (ZNF677) belongs to the zinc-finger protein family. Previous reports have highlighted the tumor suppressive role of ZNF677 in thyroid and lung cancer. However, its role in colorectal cancer (CRC) has not been explored. ZNF677 protein expression was analyzed by immunohistochemistry in a large cohort of 1158 CRC patients. ZNF677 loss of expression was more frequent in CRC tissues (45.3%, 525/1158), when compared to that of normal tissue (5.1%, 11/214) (p < 0.0001) and was associated with mucinous histology (p = 0.0311), advanced pathological stage (p < 0.0001) and lymph node (LN) metastasis (p = 0.0374). Further analysis showed ZNF677 loss to be significantly enriched in LN metastatic CRC compared to overall cohort (p = 0.0258). More importantly, multivariate logistic regression analysis showed that ZNF677 loss is an independent predictor of LN metastasis in CRC (Odds ratio = 1.41; 95% confidence interval 1.05-1.87; p = 0.0203).The gain- and loss-of-function studies in CRC cell lines demonstrated that loss of ZNF677 protein expression prominently increased cell proliferation, progression of epithelial-mesenchymal transition and conferred chemoresistance, whereas its overexpression reversed the effect. In conclusion, loss of ZNF677 protein expression is common in Middle Eastern CRC and contributes to the prediction of biological aggressiveness of CRC. Therefore, ZNF677 could not only serve as a marker in predicting clinical prognosis in patient with CRC but also as a potential biomarker for personalized targeted therapy.

Figure 1

Immunohistochemical analysis of ZNF677 expression in colorectal carcinoma (CRC). Representative examples of tumors showing (A) loss of expression and (B) positive expression of ZNF677 in CRC tissues. Representative examples of normal colonic tissues showing (C) loss of expression and (D) positive expression of ZNF677. (20×/0.70 objective on an Olympus BX 51 microscope (Olympus America Inc, Center Valley, PA, USA).

Figure 2

Survival analysis of ZNF677 protein expression in colorectal carcinoma (CRC). Kaplan Meier survival plot showing no statistically significant difference between ZNF677 positive and negative tumors for (A) distant disease-free survival (p = 0.6931), (B) CRC-specific survival (p = 0.3770) and (C) overall survival (p = 0.8307).

Figure 3

Knockdown of ZNF677 increases the chemoresistance in CRC cell lines. (A) Basal expression of ZNF677 in a panel of CRC cell lines. Proteins were isolated from nine CRC cell lines and immunoblotted with antibodies against ZNF677 and β-actin. (B, C) Knockdown of ZNF677 increases clonogenicity. CRC cells were transfected with scrambled siRNA and two different ZNF677 siRNA’s (20 nM). After 48 h, cells were seeded at a density of 500 cells per well in 6-well plate, and grown for an additional 10 days, then stained with crystal violet and colonies were counted (n = 3), *p < 0.05, compared to control. (D) Knockdown of ZNF677 increases the chemoresistance. CRC cells were transfected with either scrambled siRNA or two different ZNF677 siRNA’s (20 nM) and subsequently treated with 50 and 100 μM 5-fluorouracil (FU) for 48 h. Following treatment, cells were analysed for apoptosis by flow cytometry (n = 3), *p < 0.05, compared to respective 5-FU alone treated control. (E) Knockdown of ZNF677 increase the markers of cell survival and EMT progression. After transfection with ZNF677 siRNA’s, cells were immuno-blotted with antibodies against ZNF677, pERK1/2, ERK1/2, E-cadherin, N-cadherin, TWIST, Zeb1 and β-actin as indicated.

Figure 4

Ectopic expression of ZNF677 decreases the chemoresistance in CRC cell lines. (A, B) Overexpression of ZNF677 decreases clonogenicity. CRC cells were transfected with either empty vector or ZNF677 cDNA. After 48 h, cells were seeded at a density of 500 cells per well in 6-well plate, and grown for an additional 10 days, then stained with crystal violet and colonies were counted (n = 3), *p < 0.05, compared to control. (C). Overexpression of ZNF677 decreases the chemoresistance. After transfection with ZNF677 cDNA, cells were treated with 50 and 100 μM 5-fluorouracil (FU) for 48 h and analysed for apoptosis by flow cytometry (n = 3), *p < 0.05, compared to respective 5-FU alone treated control. (D) Overexpression of ZNF677 decrease the markers of cell survival and EMT progression. After transfection with ZNF677 cDNA, cells were immuno-blotted with antibodies against ZNF677, pERK1/2, ERK1/2, E-cadherin, N-cadherin, TWIST, Zeb1 and β-actin as indicated.

Figure 5

Loss of ZNF677 protein expression is associated with ZNF677 promoter methylation. (A) Methylation status of CRC cell lines were assessed by methylation-specific PCR for the ZNF677 gene. MSP analyses of both methylated (M) and unmethylated (U) reactions were amplified from bisulfite-treated DNA and run in a 2% agarose gel. (B) Demethylation of the ZNF677 gene restored ZNF677 expression in COLO-320 and HT29 cells. COLO-320 and HT29 cell lines were treated with different doses (0.5, 1, and 2 µM) of 5-aza-2′deoxycytidine for 72 h before lysis. Equal amounts of proteins were immunoblotted with antibodies against ZNF677 and β-actin.