MOLECULAR CARCINOGENESIS 54:E138–E147 (2015)
Zbed3 Contributes to Malignant Phenotype of Lung Cancer via Regulating b-catenin and P120-catenin 1 Chuifeng Fan,1,2 Guiyang Jiang,1, 2 Xiupeng Zhang,1,2 Yuan Miao,1,2 Xuyong Lin,1,2 Lan Luan,1,2 Zhonghai Xu,1,2 Yijun Zhang,1,2 Huanyu Zhao,1,2 Di Liu,1,2 and Enhua Wang1,2* 1
Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences of China Medical University, Shenyang, China 2 Institute of Pathology and Pathophysiology, China Medical University, Shenyang, China
Our previous studies indicate that abnormal expression of several Wnt signaling molecules including Axin, Dvl and b-catenin are involved in proliferation, invasion and metastasis of lung cancer. Zbed3 was found to inhibit function of AxinGSK3b complex and thus lead to accumulation of b-catenin in NIH3T3 and HEK293T cells. However its function in malignant tumors is largely unknown. Here we investigate the clinico-pathological significance of Zbed3 expression and its function in non-small cell lung cancer. We use immunohistochemistry and Western blotting to examine Zbed3 expression in non-small cell lung cancer and lung tissues. Transfection of siRNA and plasmid was used to study the function of Zbed3 in lung cancer cells in vitro. We found Zbed3 expression was elevated in cancer tissues compared to normal lung tissues. Increased Zbed3 expression is significantly associated with lymph node metastasis, advanced TNM stages, higher Ki67 status and patients' poor clinical outcome. Higher Zbed3 expression was also found in lung cancer cell lines compared to bronchial epithelial cell line HBE. Downregulation of Zbed3 by siRNA significantly inhibits cancer cell proliferation and invasion in vitro. Downregulation of Zbed3 also significantly inhibits expression of b-catenin, downstream molecules of Wnt signaling and P120ctn-1 in lung cancer cells. These results suggest that Zbed3 may contribute to lung cancer cell invasion through regulating b-catenin and p120ctn-1 and may be a promissing cancer marker in non-small cell lung cancer. © 2014 Wiley Periodicals, Inc.
Key words: b-catenin; NSCLC; p120ctn-1; Zbed3
INTRODUCTION Lung cancer is one of the most common malignant tumors all around the world today. To fully understand the mechanism involved in the invasion and metastasis of lung cancer is important for finding out new targets for clinical therapy and improving the clinical treatment. The Wnt/b-catenin signaling pathway regulates the expression of a number of genes essential for cell proliferation and differentiation [1–4]. Abnormal activation of the Wnt signaling pathway is closely related to tumor proliferation, invasion and metastasis [1,5–14]. The central denominator of Wnt signaling is the armadillo protein b-catenin which belongs to the armadillo protein family [1–5]. Cytoplasmic b-catenin associates with the destruction Axin-GSK3b complex in the absence of an active Wnt signal. The degradation processing of b-catenin is phosphorylation-dependent and regulated by a stepwise series of phosphorylations triggered by the kinases CK1 and GSK3 [1–5]. When Wnt binds to there receptors, function of Axin-GSK3b complex is blocked and b-catenin accumulates in the cytoplasm and then translocates into the nucleus, leading to activation of Wnt-responsive genes, including cyclin D1 [1–5]. Roles of the key molecules in this pathway (such as Axin and b-catenin) in lung cancer invasion, metastasis and prognosis have been frequently reported [1,15,16]. However, the factors that affect ß 2014 WILEY PERIODICALS, INC.
these molecules are very complicated and not fully discovered. Zbed3 (Zinc-finger BED Domaincontaining 3), a zinc finger protein, was recently found to combine Axin, a negative regulatory factor in the Wnt signaling pathway, inhibit b-catenin degradation, and eventually promote proliferation of NIH3T3 and HEK293T cells [17]. Zbed3 contains a zinc finger domain, and has a total of 234 amino acids in which the first 107–113 amino acid sequence, namely “PPPPSPT” was defined as the key site of the combination of Zbed3 with Axin [17]. However the function of Zbed3 in malignant tumors is largely unknown. The purpose of this study is to reveal Zbed3 expression, clinical significance, biological function and possible roles in regulating b-catenin in nonsmall cell lung cancer (NSCLC).
Conflict of interest: The authors declare that they have no conflict of interest. Grant sponsor: National Natural Science Foundation of China; Grant number: 81071905 *Correspondence to: Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences of China Medical University, 110001 Shenyang, China. Received 5 February 2014; Revised 2 July 2014; Accepted 30 July 2014 DOI 10.1002/mc.22216 Published online 27 September 2014 in Wiley Online Library (wileyonlinelibrary.com).
Zbed3 EXPRESSION IN LUNG CANCER
MATERIALS AND METHODS Tissue Samples Tumor specimens including NSCLC tissues and paired non-tumor portion (with >5 cm distance from the primary tumor’s edge) from 105 patients with NSCLC were obtained between 2003 and 2009 following surgical resection at the First Affiliated Hospital of China Medical University. Of the 105 lung cancer cases, 70 contained complete follow-up data. None of the patients had received radiotherapy, chemotherapy, or immunotherapy prior to tumor excision. The tumors were classified according to the TNM stage revised by the International Union Against Cancer (UICC) [18]. All specimens were re-evaluated for diagnosis following the criteria for classification of lung cancer by the World Health Organization (WHO) [19] and the revised edition for lung carcinoma in 2010 [20], and 49 squamous cell carcinomas (SCCs) and 56 adenocarcinomas were confirmed. This study was conducted under the regulations of the Institutional Review Board of China Medical University. Informed consent was obtained from all enrolled patients prior to surgery. Immunohistochemistry Formalin-fixed, paraffin-embedded specimens were cut into 4-mm-thick sequential sections. The sections were dewaxed in xylene and rehydrated stepwise in descending ethanol series. Endogenous peroxidase activity and non-specific binding were blocked with 3% H2O2 and non-immune sera, respectively. The sections were then incubated with primary rabbit anti-human polyclonal antibody Zbed3 (ab106383, Abcam, Hongkong; dilution 1:50), Ki67 (Ab-4, Lab Vision, Rockford; dilution, 1:100), mouse anti-human monoclonal antibody p120 (6H11) (sc-23873, Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:50) and P53 (Ab-2, Lab Vision; dilution, 1:00) overnight at 48C. Thereafter, the catalyzed signal amplification system (Maixin Biotechnology, Fuzhou, Fujian, China) was used for staining of these proteins according to the manufacturer’s instructions. The antibodies were detected by a standard avidin-biotin complex method with biotinylated secondary antibodies (Maixin) and an avidin-biotin complex (Maixin), and developed with diaminobenzidine. Counterstaining was done lightly with hematoxylin, and the sections were dehydrated in alcohol before mounting. The sections were assessed by at least three observers independently from each other. Scoring of IHC was based on two parameters. The proportion of immunopositive cells was categorized as follows: 0:
Zbed3 Contributes to Malignant Phenotype of Lung Cancer via Regulating b-catenin and P120-catenin 1 Chuifeng Fan,1,2 Guiyang Jiang,1, 2 Xiupeng Zhang,1,2 Yuan Miao,1,2 Xuyong Lin,1,2 Lan Luan,1,2 Zhonghai Xu,1,2 Yijun Zhang,1,2 Huanyu Zhao,1,2 Di Liu,1,2 and Enhua Wang1,2* 1
Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences of China Medical University, Shenyang, China 2 Institute of Pathology and Pathophysiology, China Medical University, Shenyang, China
Our previous studies indicate that abnormal expression of several Wnt signaling molecules including Axin, Dvl and b-catenin are involved in proliferation, invasion and metastasis of lung cancer. Zbed3 was found to inhibit function of AxinGSK3b complex and thus lead to accumulation of b-catenin in NIH3T3 and HEK293T cells. However its function in malignant tumors is largely unknown. Here we investigate the clinico-pathological significance of Zbed3 expression and its function in non-small cell lung cancer. We use immunohistochemistry and Western blotting to examine Zbed3 expression in non-small cell lung cancer and lung tissues. Transfection of siRNA and plasmid was used to study the function of Zbed3 in lung cancer cells in vitro. We found Zbed3 expression was elevated in cancer tissues compared to normal lung tissues. Increased Zbed3 expression is significantly associated with lymph node metastasis, advanced TNM stages, higher Ki67 status and patients' poor clinical outcome. Higher Zbed3 expression was also found in lung cancer cell lines compared to bronchial epithelial cell line HBE. Downregulation of Zbed3 by siRNA significantly inhibits cancer cell proliferation and invasion in vitro. Downregulation of Zbed3 also significantly inhibits expression of b-catenin, downstream molecules of Wnt signaling and P120ctn-1 in lung cancer cells. These results suggest that Zbed3 may contribute to lung cancer cell invasion through regulating b-catenin and p120ctn-1 and may be a promissing cancer marker in non-small cell lung cancer. © 2014 Wiley Periodicals, Inc.
Key words: b-catenin; NSCLC; p120ctn-1; Zbed3
INTRODUCTION Lung cancer is one of the most common malignant tumors all around the world today. To fully understand the mechanism involved in the invasion and metastasis of lung cancer is important for finding out new targets for clinical therapy and improving the clinical treatment. The Wnt/b-catenin signaling pathway regulates the expression of a number of genes essential for cell proliferation and differentiation [1–4]. Abnormal activation of the Wnt signaling pathway is closely related to tumor proliferation, invasion and metastasis [1,5–14]. The central denominator of Wnt signaling is the armadillo protein b-catenin which belongs to the armadillo protein family [1–5]. Cytoplasmic b-catenin associates with the destruction Axin-GSK3b complex in the absence of an active Wnt signal. The degradation processing of b-catenin is phosphorylation-dependent and regulated by a stepwise series of phosphorylations triggered by the kinases CK1 and GSK3 [1–5]. When Wnt binds to there receptors, function of Axin-GSK3b complex is blocked and b-catenin accumulates in the cytoplasm and then translocates into the nucleus, leading to activation of Wnt-responsive genes, including cyclin D1 [1–5]. Roles of the key molecules in this pathway (such as Axin and b-catenin) in lung cancer invasion, metastasis and prognosis have been frequently reported [1,15,16]. However, the factors that affect ß 2014 WILEY PERIODICALS, INC.
these molecules are very complicated and not fully discovered. Zbed3 (Zinc-finger BED Domaincontaining 3), a zinc finger protein, was recently found to combine Axin, a negative regulatory factor in the Wnt signaling pathway, inhibit b-catenin degradation, and eventually promote proliferation of NIH3T3 and HEK293T cells [17]. Zbed3 contains a zinc finger domain, and has a total of 234 amino acids in which the first 107–113 amino acid sequence, namely “PPPPSPT” was defined as the key site of the combination of Zbed3 with Axin [17]. However the function of Zbed3 in malignant tumors is largely unknown. The purpose of this study is to reveal Zbed3 expression, clinical significance, biological function and possible roles in regulating b-catenin in nonsmall cell lung cancer (NSCLC).
Conflict of interest: The authors declare that they have no conflict of interest. Grant sponsor: National Natural Science Foundation of China; Grant number: 81071905 *Correspondence to: Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences of China Medical University, 110001 Shenyang, China. Received 5 February 2014; Revised 2 July 2014; Accepted 30 July 2014 DOI 10.1002/mc.22216 Published online 27 September 2014 in Wiley Online Library (wileyonlinelibrary.com).
Zbed3 EXPRESSION IN LUNG CANCER
MATERIALS AND METHODS Tissue Samples Tumor specimens including NSCLC tissues and paired non-tumor portion (with >5 cm distance from the primary tumor’s edge) from 105 patients with NSCLC were obtained between 2003 and 2009 following surgical resection at the First Affiliated Hospital of China Medical University. Of the 105 lung cancer cases, 70 contained complete follow-up data. None of the patients had received radiotherapy, chemotherapy, or immunotherapy prior to tumor excision. The tumors were classified according to the TNM stage revised by the International Union Against Cancer (UICC) [18]. All specimens were re-evaluated for diagnosis following the criteria for classification of lung cancer by the World Health Organization (WHO) [19] and the revised edition for lung carcinoma in 2010 [20], and 49 squamous cell carcinomas (SCCs) and 56 adenocarcinomas were confirmed. This study was conducted under the regulations of the Institutional Review Board of China Medical University. Informed consent was obtained from all enrolled patients prior to surgery. Immunohistochemistry Formalin-fixed, paraffin-embedded specimens were cut into 4-mm-thick sequential sections. The sections were dewaxed in xylene and rehydrated stepwise in descending ethanol series. Endogenous peroxidase activity and non-specific binding were blocked with 3% H2O2 and non-immune sera, respectively. The sections were then incubated with primary rabbit anti-human polyclonal antibody Zbed3 (ab106383, Abcam, Hongkong; dilution 1:50), Ki67 (Ab-4, Lab Vision, Rockford; dilution, 1:100), mouse anti-human monoclonal antibody p120 (6H11) (sc-23873, Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:50) and P53 (Ab-2, Lab Vision; dilution, 1:00) overnight at 48C. Thereafter, the catalyzed signal amplification system (Maixin Biotechnology, Fuzhou, Fujian, China) was used for staining of these proteins according to the manufacturer’s instructions. The antibodies were detected by a standard avidin-biotin complex method with biotinylated secondary antibodies (Maixin) and an avidin-biotin complex (Maixin), and developed with diaminobenzidine. Counterstaining was done lightly with hematoxylin, and the sections were dehydrated in alcohol before mounting. The sections were assessed by at least three observers independently from each other. Scoring of IHC was based on two parameters. The proportion of immunopositive cells was categorized as follows: 0:
