Tumor Biol. (2015) 36:747–756 DOI 10.1007/s13277-014-2683-5
RESEARCH ARTICLE
Expression and clinical role of NF45 as a novel cell cycle protein in esophageal squamous cell carcinoma (ESCC) Sujie Ni & Junya Zhu & Jianguo Zhang & Shu Zhang & Mei Li & Runzhou Ni & Jinxia Liu & Huiyuan Qiu & Wenjuan Chen & Huijie Wang & Weijian Guo
Received: 6 August 2014 / Accepted: 25 September 2014 / Published online: 8 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract NF45 (also known as ILF2), as one subunit of NFAT (nuclear factor of activated T cells), repairs DNA breaks, inhibits viral replication, and also functions as a negative regulator in the microRNA processing pathway in combination with NF90. Recently, it was found that implicated in the mitotic control of HeLa cells and deletion of endogenous NF45 decreases growth of HeLa cells. While the role of NF45 in cancer biology remains under debate. In this study, we analyzed the expression and clinical significance of NF45 in esophageal squamous cell carcinoma ESCC. The expression of NF45 was evaluated by Western blot in 8 paired fresh ESCC tissues and immunohistochemistry on 105 paraffinembedded slices. NF45 was highly expressed in ESCC and significantly associated with ESCC cells tumor stage and Ki67. Besides, high NF45 expression was an independent prognostic factor for ESCC patients’ poor survival. To determine whether NF45 could regulate the proliferation of ESCC cells, we increased endogenous NF45 and analyzed the proliferation of TE1 ESCC cells using Western blot, CCK8, flow cytometry assays and colony formation analyses, which together indicated that overexpression of NF45 favors cell cycle progress of TE1 ESCC cells. While knockdown of NF45 resulted in cell cycle arrest at G0/G1-phase and thus abolished the cell growth. These findings suggested that NF45 might play an important role in promoting the tumorigenesis of ESCC, and
Sujie Ni and Junya Zhu have contributed equally to this work. S. Ni : H. Wang : W. Guo (*) Department of Medical Oncology, Shanghai Cancer Center, Fudan University, No. 270 Dong An Road, Shanghai 200032, China e-mail: [email protected] J. Zhu : J. Zhang : S. Zhang : M. Li : R. Ni : J. Liu : H. Qiu : W. Chen Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China
thus be a promising therapeutic target to prevent ESCC progression. Keywords NF45 . Esophageal squamous cell carcinoma (ESCC) . Proliferation . Prognosis
Introduction Esophageal cancer is the eighth most common cancer in the world and the sixth leading cause of cancer mortality [1–3]. Esophageal squamous cell carcinoma (ESCC) accounts for ∼90 % of all esophageal cancer diagnosed in Asian countries [4] and remains one of the most aggressive carcinomas of the gastrointestinal tract. Despite the development of new prognostic and therapeutical strategies, ESCC is still incurable because of its high recurrent rate as well as it’s diagnose at advanced and later stages. A wide range of genetic modifications have been shown to play a pivotal role in the development and tumorigenesis of ESCC. However, the underlying molecular mechanisms contributing to the initiation and development of ESCC remain largely unclear. One subunit of NF-AT (nuclear factor of activated T cells), NF45 (also known as ILF2) appears to function predominantly as a stable heterodimeric complex with another nuclear factor, NF90 (also known as DRBP76, TCP80, ILF3, and NFAR-1). This complex was originally shown to be a transcription factor to regulate gene expression such as the interleukin-2 gene expression and HS4-dependent interleukin-13 expression [5–10]. NF45 and NF90 are proteins that belong to the double-stranded RNA-binding protein family and both are substrates for the dsRNA-activated protein kinase, PKR. NF45 also involves in the replication process of various types of RNA viruses like hepatitis B viral as well as infectious bursal disease virus (IBDV) [11–14]. Recently, NF45 was reported to correlate with malignant grade in
748
gliomas and play a pivotal role in tumor growth [15]. Furthermore, the NF45/NF90 complex was recently shown to repair DNA breaks by nonhomologous end joining and also function as a negative regulator in the microRNA processing pathway [16–18]. Above all, knockdown of endogenous NF90 or NF45 retards cell growth in HeLa cells, possibly owing to the decreased DNA synthesis [17]. All of these studies imply that NF45 may be involved with the pathogenesis of ESCC; however, the expression and significance of NF45 in ESCC are still obscure. In the present study, we investigated the involvement of NF45 in regulation of cell cycle in ESCC, which might help us have a better understanding with the clinical significance of NF45. We examined the expression of NF45 in 8 paired human ESCC tissues and on 105 ESCC slices by Western blot as well as immunohistochemistry (IHC), and determined the correlation between NF45 and Ki-67. The various clinical and pathological features, including prognosis of NF45, were also determined. Furthermore, CCK8, flow cytometry analyses and colony formation analyses were performed to determine the pathogenestic role of NF45 in TE1 ESCC cell lines. Our study firstly reported that NF45 may directly affect the proliferation of human ESCC and targeting this protein might be of great value for experimental adjuvant therapies in ESCC.
Materials and methods Tissue specimens The ESCC tumors and matched adjacent normal esophageal epithelia were retrospectively selected from 105 ESCC patients who underwent surgery as their first and only treatment between 2005 and 2011 at a single institution, the Department of Pathology, affiliated Hospital of Nantong University. All tissue samples were snapped frozen in liquid nitrogen immediately after surgery and stored at−80 °C for specific analysis. For histological examination, all tumorous and surrounding nontumorous tissue portions were fixed in 10 % buffered formalin and embedded in paraffin for sectioning. Resected specimens were classified according to the International Union Against Cancer TNM classification system [19]. The study population consisted of 76 males and 29 females, and the age ranged from 31 to 85 years. The main clinical and pathologic variables were summarized in Table 1. Approval for this study was obtained from the Ethics Committee of Affiliated Hospital of Nantong University. Signed informed consent was also obtained. Western blot analysis Western blot experiments were used to measure certain proteins. Briefly, tissues and cell samples were promptly
Tumor Biol. (2015) 36:747–756
homogenized in a homogenization buffer containing 1 M Tris-HCl pH 7.5, 1 % Triton X-100, 1 % NP-40 (Nonidet P-40), 10 % sodium dodecyl sulfate (SDS), 0.5 % sodium deoxycholate, 0.5 M EDTA, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 1 mM PMSF; then centrifuged at 10,000 g for 30 min to collect the supernatant. The supernatant was diluted in 2× SDS loading buffer and boiled. An equivalent amount of protein from each sample was electrophoresed by 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and then transferred to a PVDF membrane (Millipore, Bedford, MA). After blocking with phosphate-buffered saline (PBS) containing 5 % nonfat milk and 0.1 % Tween 20 overnight, the membrane was incubated overnight at 4 °C with primary polyclonal antibody which were anti-GAPDH (1:1000, Sigma), anti-NF45 (1:500), anti-Cyclin A (1:500), and anti-PCNA (1:1000, all the above antibodies from Santa Cruz Biotechnology). After washing with PBS containing 0.1 % Tween 20 three times, each for 5 min, the membrane was then incubated with horseradish peroxidase-linked IgG as the secondary antibody for another 2 h at room temperature. The membrane was then developed using the ECL detection systems. Immunoreactive bands were visualized by chemiluminescence detection system (Pierce). After the chemiluminescence was exposed to X-ray films, the films were scanned with a Molecular Dynamics densitometer (Imaging Technology, Ontario, Canada). The band density was measured with a computer-assisted image analysis system (Adobe Systems, San Jose, CA, USA) and normalized against GAPDH level. The experiments were carried out on three separate occasions. Immunohistochemical analyses Serial sections measuring 5 μm thick were mounted on glass slides coated with 10 % polylysine. Tissue sections were dewaxed in xylene, rehydrated through graded alcohol, and quenched in 3 % hydrogen peroxide to block endogenous peroxidase activity, and thereafter, immunoreactivity was enhanced by high temperature and pressure in an autoclave and incubating the tissue sections for 3 min in 10 mM citrate buffer (pH 6.0). The following panel of antibodies was used: (1) anti-NF45 (1:100, Santa Cruz Biotechnology) and (2) antiKi-67 (1:400, Santa Cruz Biotechnology). Immunostaining was performed using the avidin-biotin-peroxidase complex method, and antigen-antibody reactions were visualized with chromogen diaminobenzidine. Negative control slides were processed in parallel using a nonspecific immunoglobulin IgG (Sigma Chemical Co, St. Louis, MO, USA) at the same concentration as the primary antibody. All the immunostained sections were evaluated in a blinded manner without knowledge of the clinical and pathological parameters of the ESCC patients. Five high-power fields were chosen randomly, and at least 300 cells were counted per field. Expression score was determined by staining intensity and immunoreactive cell
Tumor Biol. (2015) 36:747–756 Table 1 Clinicopathological features of ESCC in relation to the NF45 expression pattern
749
Clinicopathological features
NF45 Low (0–4.5) n=43
Age (years) Gender Tumor grade
Tumor metastasis Tumor size (cm) Tumor invasion (T)
Statistical analyses were carried out using Pearson’s χ2 test
Total
Ki-67 expression
*P < 0.05 was considered significant
75 %, score of 3), middle-expression group (50–75 %, score of 2), and low-expression group (
RESEARCH ARTICLE
Expression and clinical role of NF45 as a novel cell cycle protein in esophageal squamous cell carcinoma (ESCC) Sujie Ni & Junya Zhu & Jianguo Zhang & Shu Zhang & Mei Li & Runzhou Ni & Jinxia Liu & Huiyuan Qiu & Wenjuan Chen & Huijie Wang & Weijian Guo
Received: 6 August 2014 / Accepted: 25 September 2014 / Published online: 8 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract NF45 (also known as ILF2), as one subunit of NFAT (nuclear factor of activated T cells), repairs DNA breaks, inhibits viral replication, and also functions as a negative regulator in the microRNA processing pathway in combination with NF90. Recently, it was found that implicated in the mitotic control of HeLa cells and deletion of endogenous NF45 decreases growth of HeLa cells. While the role of NF45 in cancer biology remains under debate. In this study, we analyzed the expression and clinical significance of NF45 in esophageal squamous cell carcinoma ESCC. The expression of NF45 was evaluated by Western blot in 8 paired fresh ESCC tissues and immunohistochemistry on 105 paraffinembedded slices. NF45 was highly expressed in ESCC and significantly associated with ESCC cells tumor stage and Ki67. Besides, high NF45 expression was an independent prognostic factor for ESCC patients’ poor survival. To determine whether NF45 could regulate the proliferation of ESCC cells, we increased endogenous NF45 and analyzed the proliferation of TE1 ESCC cells using Western blot, CCK8, flow cytometry assays and colony formation analyses, which together indicated that overexpression of NF45 favors cell cycle progress of TE1 ESCC cells. While knockdown of NF45 resulted in cell cycle arrest at G0/G1-phase and thus abolished the cell growth. These findings suggested that NF45 might play an important role in promoting the tumorigenesis of ESCC, and
Sujie Ni and Junya Zhu have contributed equally to this work. S. Ni : H. Wang : W. Guo (*) Department of Medical Oncology, Shanghai Cancer Center, Fudan University, No. 270 Dong An Road, Shanghai 200032, China e-mail: [email protected] J. Zhu : J. Zhang : S. Zhang : M. Li : R. Ni : J. Liu : H. Qiu : W. Chen Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China
thus be a promising therapeutic target to prevent ESCC progression. Keywords NF45 . Esophageal squamous cell carcinoma (ESCC) . Proliferation . Prognosis
Introduction Esophageal cancer is the eighth most common cancer in the world and the sixth leading cause of cancer mortality [1–3]. Esophageal squamous cell carcinoma (ESCC) accounts for ∼90 % of all esophageal cancer diagnosed in Asian countries [4] and remains one of the most aggressive carcinomas of the gastrointestinal tract. Despite the development of new prognostic and therapeutical strategies, ESCC is still incurable because of its high recurrent rate as well as it’s diagnose at advanced and later stages. A wide range of genetic modifications have been shown to play a pivotal role in the development and tumorigenesis of ESCC. However, the underlying molecular mechanisms contributing to the initiation and development of ESCC remain largely unclear. One subunit of NF-AT (nuclear factor of activated T cells), NF45 (also known as ILF2) appears to function predominantly as a stable heterodimeric complex with another nuclear factor, NF90 (also known as DRBP76, TCP80, ILF3, and NFAR-1). This complex was originally shown to be a transcription factor to regulate gene expression such as the interleukin-2 gene expression and HS4-dependent interleukin-13 expression [5–10]. NF45 and NF90 are proteins that belong to the double-stranded RNA-binding protein family and both are substrates for the dsRNA-activated protein kinase, PKR. NF45 also involves in the replication process of various types of RNA viruses like hepatitis B viral as well as infectious bursal disease virus (IBDV) [11–14]. Recently, NF45 was reported to correlate with malignant grade in
748
gliomas and play a pivotal role in tumor growth [15]. Furthermore, the NF45/NF90 complex was recently shown to repair DNA breaks by nonhomologous end joining and also function as a negative regulator in the microRNA processing pathway [16–18]. Above all, knockdown of endogenous NF90 or NF45 retards cell growth in HeLa cells, possibly owing to the decreased DNA synthesis [17]. All of these studies imply that NF45 may be involved with the pathogenesis of ESCC; however, the expression and significance of NF45 in ESCC are still obscure. In the present study, we investigated the involvement of NF45 in regulation of cell cycle in ESCC, which might help us have a better understanding with the clinical significance of NF45. We examined the expression of NF45 in 8 paired human ESCC tissues and on 105 ESCC slices by Western blot as well as immunohistochemistry (IHC), and determined the correlation between NF45 and Ki-67. The various clinical and pathological features, including prognosis of NF45, were also determined. Furthermore, CCK8, flow cytometry analyses and colony formation analyses were performed to determine the pathogenestic role of NF45 in TE1 ESCC cell lines. Our study firstly reported that NF45 may directly affect the proliferation of human ESCC and targeting this protein might be of great value for experimental adjuvant therapies in ESCC.
Materials and methods Tissue specimens The ESCC tumors and matched adjacent normal esophageal epithelia were retrospectively selected from 105 ESCC patients who underwent surgery as their first and only treatment between 2005 and 2011 at a single institution, the Department of Pathology, affiliated Hospital of Nantong University. All tissue samples were snapped frozen in liquid nitrogen immediately after surgery and stored at−80 °C for specific analysis. For histological examination, all tumorous and surrounding nontumorous tissue portions were fixed in 10 % buffered formalin and embedded in paraffin for sectioning. Resected specimens were classified according to the International Union Against Cancer TNM classification system [19]. The study population consisted of 76 males and 29 females, and the age ranged from 31 to 85 years. The main clinical and pathologic variables were summarized in Table 1. Approval for this study was obtained from the Ethics Committee of Affiliated Hospital of Nantong University. Signed informed consent was also obtained. Western blot analysis Western blot experiments were used to measure certain proteins. Briefly, tissues and cell samples were promptly
Tumor Biol. (2015) 36:747–756
homogenized in a homogenization buffer containing 1 M Tris-HCl pH 7.5, 1 % Triton X-100, 1 % NP-40 (Nonidet P-40), 10 % sodium dodecyl sulfate (SDS), 0.5 % sodium deoxycholate, 0.5 M EDTA, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 1 mM PMSF; then centrifuged at 10,000 g for 30 min to collect the supernatant. The supernatant was diluted in 2× SDS loading buffer and boiled. An equivalent amount of protein from each sample was electrophoresed by 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and then transferred to a PVDF membrane (Millipore, Bedford, MA). After blocking with phosphate-buffered saline (PBS) containing 5 % nonfat milk and 0.1 % Tween 20 overnight, the membrane was incubated overnight at 4 °C with primary polyclonal antibody which were anti-GAPDH (1:1000, Sigma), anti-NF45 (1:500), anti-Cyclin A (1:500), and anti-PCNA (1:1000, all the above antibodies from Santa Cruz Biotechnology). After washing with PBS containing 0.1 % Tween 20 three times, each for 5 min, the membrane was then incubated with horseradish peroxidase-linked IgG as the secondary antibody for another 2 h at room temperature. The membrane was then developed using the ECL detection systems. Immunoreactive bands were visualized by chemiluminescence detection system (Pierce). After the chemiluminescence was exposed to X-ray films, the films were scanned with a Molecular Dynamics densitometer (Imaging Technology, Ontario, Canada). The band density was measured with a computer-assisted image analysis system (Adobe Systems, San Jose, CA, USA) and normalized against GAPDH level. The experiments were carried out on three separate occasions. Immunohistochemical analyses Serial sections measuring 5 μm thick were mounted on glass slides coated with 10 % polylysine. Tissue sections were dewaxed in xylene, rehydrated through graded alcohol, and quenched in 3 % hydrogen peroxide to block endogenous peroxidase activity, and thereafter, immunoreactivity was enhanced by high temperature and pressure in an autoclave and incubating the tissue sections for 3 min in 10 mM citrate buffer (pH 6.0). The following panel of antibodies was used: (1) anti-NF45 (1:100, Santa Cruz Biotechnology) and (2) antiKi-67 (1:400, Santa Cruz Biotechnology). Immunostaining was performed using the avidin-biotin-peroxidase complex method, and antigen-antibody reactions were visualized with chromogen diaminobenzidine. Negative control slides were processed in parallel using a nonspecific immunoglobulin IgG (Sigma Chemical Co, St. Louis, MO, USA) at the same concentration as the primary antibody. All the immunostained sections were evaluated in a blinded manner without knowledge of the clinical and pathological parameters of the ESCC patients. Five high-power fields were chosen randomly, and at least 300 cells were counted per field. Expression score was determined by staining intensity and immunoreactive cell
Tumor Biol. (2015) 36:747–756 Table 1 Clinicopathological features of ESCC in relation to the NF45 expression pattern
749
Clinicopathological features
NF45 Low (0–4.5) n=43
Age (years) Gender Tumor grade
Tumor metastasis Tumor size (cm) Tumor invasion (T)
Statistical analyses were carried out using Pearson’s χ2 test
Total
Ki-67 expression
*P < 0.05 was considered significant
75 %, score of 3), middle-expression group (50–75 %, score of 2), and low-expression group (
