Clin Oral Invest DOI 10.1007/s00784-015-1482-8
ORIGINAL ARTICLE
Expression of cdk6 in head and neck squamous cell carcinoma Sopee Poomsawat 1 & Sirima Sanguansin 2 & Jirapa Punyasingh 1 & Paisarn Vejchapipat 3 & Phaibul Punyarit 4
Received: 18 December 2014 / Accepted: 23 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Objective Cdk6 is a key regulator during the G1/S cell cycle transition. Aberrant expression of cdk6 protein has been observed in many cancer types. However, little is known about the expression of cdk6 in head and neck squamous cell carcinoma (HNSCC) and its clinical significance. This study evaluated the expression of cdk6 in HNSCC and analyzed the relationship between cdk6 expression and clinicopathological parameters of HNSCC. M a t e r i al s a n d m et ho ds E x pr es s i o n o f c d k6 w a s immunohistochemically investigated in 98 HNSCCs. Nuclear and cytoplasmic positive cells were counted separately. Data were presented as the percentage of positive cells. The correlation between the percentage of positive cells and clinicopathological factors was determined. Results Nuclear and cytoplasmic staining for cdk6 were detected in 91 cases and 97 cases, respectively. A significant correlation was found only between the percentage of nuclear positive cells and T classification (p value=0.0410). Tumors
* Sopee Poomsawat [email protected] 1
Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Mahidol University, Yothi Street, Bangkok 10400, Thailand
2
Department of Oral Biology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand
3
Department of Surgery, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
4
Army Institute of Pathology, Pramongkutklao Hospital, Bangkok, Thailand
with high nuclear cdk6-positive cells showed a linear trend toward advanced tumor status (p value=0.0064). Conclusions Cdk6 was highly expressed in HNSCC. Tumors with high nuclear cdk6 expression tended to have advanced tumor status. These results suggest that cdk6 plays a vital role in HNSCC and is involved in tumor progression of this cancer. Clinical relevance An increased nuclear cdk6 expression is an unfavorable factor for HNSCC. Cdk6 may serve as a therapeutic target in this cancer. Keywords Cdk6 . Head and neck . Immunohistochemistry . Squamous cell carcinoma
Introduction Cell cycle deregulation is a basic process for the development of cancer. Alterations in the genes or proteins involving in the cell cycle have been documented in many cancer types [1–6]. The cyclin-dependent kinases (cdks) are one of essential players in the cell cycle machinery. The association of cdks and cyclin proteins leads to the downstream process of the cell cycle by phosphorylation of the target proteins. Cdk6 is a key regulator during the G1/S cell cycle transition. Cdk6 coupled with cyclin D phosphorylates retinoblastoma protein, leading to the release of E2F transcriptional factors. Subsequently, cells are driving from the G1 phase to the S phase of the cell cycle [7]. The cell proliferation activity mediated by cdk6 can be abolished by the binding of cdk6 with p16, a cyclindependent kinase inhibitor [8]. Therefore, the precise control of the cooperation between cdk6 and p16 is required for normal cell growth. Aberrant expression of cdk6 protein has been observed in esophageal squamous cell carcinoma (ESCC), oral squamous cell carcinoma (OSCC), T cell lymphoma, glioblastoma
Clin Oral Invest
multiforme, medulloblastoma, bladder cancer, and gastric cancer [3–5, 9–12]. Cdk6 contributed to tumorigenesis in breast cancer as mice lacking cdk6 were resistant to ErbB2induced tumor [13]. Based on cell lines derived from medulloblastoma, bladder cancer, gastric cancer, and OSCC, cdk6mediated cell proliferation as using a cdk6 inhibitor or microRNAs suppressed cell proliferation in these cells [3, 6, 14, 15]. By contrast, overexpression of cdk6 inhibited cell proliferation of BCR-ABL-transformed B cell leukemia/ lymphoma cells [16]. Injection of leukemic cells overexpressed cdk6 into recipient mice delayed tumor formation. In these lymphoid models, it has been suggested that the overexpression of cdk6 may induce high level of p16, leading to the cell cycle arrest. Besides the role in cell cycle, cdk6 also induced angiogenesis in lymphoid malignancies [16]. Therefore, it is possible that the function of cdk6 is a cancertype specific. Little is known about the role of cdk6 in head and neck SCC (HNSCC). One study demonstrated that cdk6 was overexpressed in OSCC tissue as compared with tissues from normal oral mucosa and oral leukoplakia with or without dysplasia [5]. The expression level of cdk6 was upregulated in OSCC cell lines [17, 18]. It has been shown that cdk6 is involved in the aggressive behavior of many cancers. The expression of cdk6 was significantly increased in the invasive bladder cancer cases compared with the noninvasive superficial cases [12]. Furthermore, overexpression of cdk6 protein correlated significantly with poor prognosis in ESCC, medulloblastoma, myxofibrosarcoma, and gastric cancer [3, 4, 9, 19]. OSCC cell growth was suppressed by decreasing cdk6 expression [14]. In cell lines derived from Ewing’s sarcoma, bladder cancer, and pancreatic cancer, the knockdown of cdk6 could inhibit important cellular activities, including proliferation, invasion, and migration [15, 20, 21]. Recently, it has been shown that the expression of CDK6 mRNA and cdk6 protein in ESCC was associated with the levels of long interspersed nucleotide element-1 (LINE-1) [9]. LINE-1 methylation is considered to be a surrogate marker of global DNA methylation, and LINE-1 hypomethylation was related to poor prognosis or aggressive behaviors in many cancers including ESCC, lung cancer, breast cancer, and gastric cancer [9, 22–24]. Taken together, it is possible that cdk6 also plays an important role in HNSCC, and its expression may be correlated with the behavior of this cancer. Based on the English literature, the expression of cdk6 in HNSCC is very limited [5] and the correlation between cdk6 expression and clinicopathological factors of this cancer has never been reported. The purposes of this study were to evaluate the expression of cdk6 in HNSCC by immunohistochemistry and to analyze the relationship between cdk6 expression and clinicopathological parameters of HNSCC.
Materials and methods Specimens Cases diagnosed as squamous cell carcinoma from the Department of Oral and Maxillofacial Pathology, Faculty of Dentistry and Army Institute of Pathology, Phramongkutklao Hospital were reviewed. Only cases from the head and neck area were selected and retrieved as formalin-fixed paraffinembedded blocks. Hematoxylin and eosin-stained sections from all tissue blocks were reviewed by a board certified oral pathologist (SP). Clinical data including age of occurrence, sex, site of involvement, histologic grade, and tumor size, node, metastasis (TNM) status were recorded. Histologic grade and TNM classification of these tumors were defined based on the WHO classification [25]. The patients’ oral habits including smoking, consumption of alcohol, or betel quid were also recorded. This study was approved by the Institutional Review Board (MU-DT/PY-IRB 2013/015.1203 and IRB/ RTA 1510/2555).
Immunohistochemistry New sections of 4 μm thickness were cut from the formalinfixed paraffin-embedded blocks and mounted on glass slides coated by aminopropyltriethoxysilane (APES; Sigma Chemical Co., St Louis, MO, USA). Sections were deparaffinized and rehydrated with serial dilutions of alcohol. Endogenous peroxidase activity was blocked with 10-min incubation in 3 % H2O2. Antigen retrieval was done by heating the sections in a microwave for 15 min in 10 mM citrate buffer pH 6.0. After washing with 0.1 % Tween 20 (MERCKSchuchardt, Hohanbrunn, Germany) in phosphate-buffered saline (PBS), the sections were treated with 5 % bovine serum albumin (Sigma Chemical Co.) in PBS for 30 min and then treated with a primary antibody for 2 h at room temperature. The primary antibody used in this study was against cdk6 (Santa Cruz Biotechnology; SC177) diluted at 1:100 in PBS. After thorough washing in 0.1 % Tween 20 in PBS, labeled polymer (Dako Envision System, Dako Corporation, Carpinteria, CA, USA) was applied to the sections for 30 min and followed by three washes of 0.1 % Tween 20 in PBS. Color was developed in freshly made diaminobenzidine (Sigma Chemical Co.). Sections were washed briefly in running tap water and lightly stained with Mayer’s hematoxylin. Negative controls were done by replacing the primary antibody with nonimmune rabbit serum with the same dilution to the primary antibody. Sections of an oral squamous cell carcinoma known to have nuclear staining for cdk6 were stained at the same run as positive controls [5]. All sections were processed under identical parameters.
Clin Oral Invest
Evaluation of cdk6 expression Epithelial cells, which demonstrate golden-brown staining in the nucleus and/or cytoplasm, regarding the staining intensity, were considered to be positive. Five to eight fields under light microscope (×200 magnification) were randomly chosen and photographed. Then, the numbers of both positive cells and all cells in each photo were counted with a 10×10 grid to avoid repetition of counted cells. The nuclear positive cells and the cytoplasmic positive cells were counted separately. The counted cells were calculated and presented as the percentage of positive cells. At least 1000 cells were counted in each case. Positive cases were determined according to a previous study [5]. In order to minimize the inter-observer variation, each slide was independently examined by two of the authors (SP and JP). The specimens with more than 5 % deviation in the percentage of positive cells were reevaluated together. Statistical analysis The association between nuclear positive cells or cytoplasmic positive cells and clinicopathological parameters were analyzed using one-way ANOVA or unpaired Student’s t tests. A linear trend was used as a post-test analysis. It is a choice on the multiple comparisons of the parameters dialog for oneway ANOVA. This test determined whether the means of the positive cells increased or decreased systematically as the clinicopathological parameters advanced. A value of p
ORIGINAL ARTICLE
Expression of cdk6 in head and neck squamous cell carcinoma Sopee Poomsawat 1 & Sirima Sanguansin 2 & Jirapa Punyasingh 1 & Paisarn Vejchapipat 3 & Phaibul Punyarit 4
Received: 18 December 2014 / Accepted: 23 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Objective Cdk6 is a key regulator during the G1/S cell cycle transition. Aberrant expression of cdk6 protein has been observed in many cancer types. However, little is known about the expression of cdk6 in head and neck squamous cell carcinoma (HNSCC) and its clinical significance. This study evaluated the expression of cdk6 in HNSCC and analyzed the relationship between cdk6 expression and clinicopathological parameters of HNSCC. M a t e r i al s a n d m et ho ds E x pr es s i o n o f c d k6 w a s immunohistochemically investigated in 98 HNSCCs. Nuclear and cytoplasmic positive cells were counted separately. Data were presented as the percentage of positive cells. The correlation between the percentage of positive cells and clinicopathological factors was determined. Results Nuclear and cytoplasmic staining for cdk6 were detected in 91 cases and 97 cases, respectively. A significant correlation was found only between the percentage of nuclear positive cells and T classification (p value=0.0410). Tumors
* Sopee Poomsawat [email protected] 1
Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Mahidol University, Yothi Street, Bangkok 10400, Thailand
2
Department of Oral Biology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand
3
Department of Surgery, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
4
Army Institute of Pathology, Pramongkutklao Hospital, Bangkok, Thailand
with high nuclear cdk6-positive cells showed a linear trend toward advanced tumor status (p value=0.0064). Conclusions Cdk6 was highly expressed in HNSCC. Tumors with high nuclear cdk6 expression tended to have advanced tumor status. These results suggest that cdk6 plays a vital role in HNSCC and is involved in tumor progression of this cancer. Clinical relevance An increased nuclear cdk6 expression is an unfavorable factor for HNSCC. Cdk6 may serve as a therapeutic target in this cancer. Keywords Cdk6 . Head and neck . Immunohistochemistry . Squamous cell carcinoma
Introduction Cell cycle deregulation is a basic process for the development of cancer. Alterations in the genes or proteins involving in the cell cycle have been documented in many cancer types [1–6]. The cyclin-dependent kinases (cdks) are one of essential players in the cell cycle machinery. The association of cdks and cyclin proteins leads to the downstream process of the cell cycle by phosphorylation of the target proteins. Cdk6 is a key regulator during the G1/S cell cycle transition. Cdk6 coupled with cyclin D phosphorylates retinoblastoma protein, leading to the release of E2F transcriptional factors. Subsequently, cells are driving from the G1 phase to the S phase of the cell cycle [7]. The cell proliferation activity mediated by cdk6 can be abolished by the binding of cdk6 with p16, a cyclindependent kinase inhibitor [8]. Therefore, the precise control of the cooperation between cdk6 and p16 is required for normal cell growth. Aberrant expression of cdk6 protein has been observed in esophageal squamous cell carcinoma (ESCC), oral squamous cell carcinoma (OSCC), T cell lymphoma, glioblastoma
Clin Oral Invest
multiforme, medulloblastoma, bladder cancer, and gastric cancer [3–5, 9–12]. Cdk6 contributed to tumorigenesis in breast cancer as mice lacking cdk6 were resistant to ErbB2induced tumor [13]. Based on cell lines derived from medulloblastoma, bladder cancer, gastric cancer, and OSCC, cdk6mediated cell proliferation as using a cdk6 inhibitor or microRNAs suppressed cell proliferation in these cells [3, 6, 14, 15]. By contrast, overexpression of cdk6 inhibited cell proliferation of BCR-ABL-transformed B cell leukemia/ lymphoma cells [16]. Injection of leukemic cells overexpressed cdk6 into recipient mice delayed tumor formation. In these lymphoid models, it has been suggested that the overexpression of cdk6 may induce high level of p16, leading to the cell cycle arrest. Besides the role in cell cycle, cdk6 also induced angiogenesis in lymphoid malignancies [16]. Therefore, it is possible that the function of cdk6 is a cancertype specific. Little is known about the role of cdk6 in head and neck SCC (HNSCC). One study demonstrated that cdk6 was overexpressed in OSCC tissue as compared with tissues from normal oral mucosa and oral leukoplakia with or without dysplasia [5]. The expression level of cdk6 was upregulated in OSCC cell lines [17, 18]. It has been shown that cdk6 is involved in the aggressive behavior of many cancers. The expression of cdk6 was significantly increased in the invasive bladder cancer cases compared with the noninvasive superficial cases [12]. Furthermore, overexpression of cdk6 protein correlated significantly with poor prognosis in ESCC, medulloblastoma, myxofibrosarcoma, and gastric cancer [3, 4, 9, 19]. OSCC cell growth was suppressed by decreasing cdk6 expression [14]. In cell lines derived from Ewing’s sarcoma, bladder cancer, and pancreatic cancer, the knockdown of cdk6 could inhibit important cellular activities, including proliferation, invasion, and migration [15, 20, 21]. Recently, it has been shown that the expression of CDK6 mRNA and cdk6 protein in ESCC was associated with the levels of long interspersed nucleotide element-1 (LINE-1) [9]. LINE-1 methylation is considered to be a surrogate marker of global DNA methylation, and LINE-1 hypomethylation was related to poor prognosis or aggressive behaviors in many cancers including ESCC, lung cancer, breast cancer, and gastric cancer [9, 22–24]. Taken together, it is possible that cdk6 also plays an important role in HNSCC, and its expression may be correlated with the behavior of this cancer. Based on the English literature, the expression of cdk6 in HNSCC is very limited [5] and the correlation between cdk6 expression and clinicopathological factors of this cancer has never been reported. The purposes of this study were to evaluate the expression of cdk6 in HNSCC by immunohistochemistry and to analyze the relationship between cdk6 expression and clinicopathological parameters of HNSCC.
Materials and methods Specimens Cases diagnosed as squamous cell carcinoma from the Department of Oral and Maxillofacial Pathology, Faculty of Dentistry and Army Institute of Pathology, Phramongkutklao Hospital were reviewed. Only cases from the head and neck area were selected and retrieved as formalin-fixed paraffinembedded blocks. Hematoxylin and eosin-stained sections from all tissue blocks were reviewed by a board certified oral pathologist (SP). Clinical data including age of occurrence, sex, site of involvement, histologic grade, and tumor size, node, metastasis (TNM) status were recorded. Histologic grade and TNM classification of these tumors were defined based on the WHO classification [25]. The patients’ oral habits including smoking, consumption of alcohol, or betel quid were also recorded. This study was approved by the Institutional Review Board (MU-DT/PY-IRB 2013/015.1203 and IRB/ RTA 1510/2555).
Immunohistochemistry New sections of 4 μm thickness were cut from the formalinfixed paraffin-embedded blocks and mounted on glass slides coated by aminopropyltriethoxysilane (APES; Sigma Chemical Co., St Louis, MO, USA). Sections were deparaffinized and rehydrated with serial dilutions of alcohol. Endogenous peroxidase activity was blocked with 10-min incubation in 3 % H2O2. Antigen retrieval was done by heating the sections in a microwave for 15 min in 10 mM citrate buffer pH 6.0. After washing with 0.1 % Tween 20 (MERCKSchuchardt, Hohanbrunn, Germany) in phosphate-buffered saline (PBS), the sections were treated with 5 % bovine serum albumin (Sigma Chemical Co.) in PBS for 30 min and then treated with a primary antibody for 2 h at room temperature. The primary antibody used in this study was against cdk6 (Santa Cruz Biotechnology; SC177) diluted at 1:100 in PBS. After thorough washing in 0.1 % Tween 20 in PBS, labeled polymer (Dako Envision System, Dako Corporation, Carpinteria, CA, USA) was applied to the sections for 30 min and followed by three washes of 0.1 % Tween 20 in PBS. Color was developed in freshly made diaminobenzidine (Sigma Chemical Co.). Sections were washed briefly in running tap water and lightly stained with Mayer’s hematoxylin. Negative controls were done by replacing the primary antibody with nonimmune rabbit serum with the same dilution to the primary antibody. Sections of an oral squamous cell carcinoma known to have nuclear staining for cdk6 were stained at the same run as positive controls [5]. All sections were processed under identical parameters.
Clin Oral Invest
Evaluation of cdk6 expression Epithelial cells, which demonstrate golden-brown staining in the nucleus and/or cytoplasm, regarding the staining intensity, were considered to be positive. Five to eight fields under light microscope (×200 magnification) were randomly chosen and photographed. Then, the numbers of both positive cells and all cells in each photo were counted with a 10×10 grid to avoid repetition of counted cells. The nuclear positive cells and the cytoplasmic positive cells were counted separately. The counted cells were calculated and presented as the percentage of positive cells. At least 1000 cells were counted in each case. Positive cases were determined according to a previous study [5]. In order to minimize the inter-observer variation, each slide was independently examined by two of the authors (SP and JP). The specimens with more than 5 % deviation in the percentage of positive cells were reevaluated together. Statistical analysis The association between nuclear positive cells or cytoplasmic positive cells and clinicopathological parameters were analyzed using one-way ANOVA or unpaired Student’s t tests. A linear trend was used as a post-test analysis. It is a choice on the multiple comparisons of the parameters dialog for oneway ANOVA. This test determined whether the means of the positive cells increased or decreased systematically as the clinicopathological parameters advanced. A value of p
