An immunohistochemical study of cyclin-dependent kinase 5 (CDK5) expression in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC): a possible prognostic biomarker
- Kanglai Wei†1,
- Zhihua Ye†1,
- Zuyun Li1,
- Yiwu Dang1,
- Xin Chen1,
- Na Huang1,
- Chongxi Bao1,
- Tingqing Gan2,
- Lihua Yang2Email author and
- Gang Chen1Email author
© Wei et al. 2016
Received: 30 July 2015
Accepted: 26 January 2016
Published: 9 February 2016
Cyclin-dependent kinase 5 (CDK5) is an atypical CDK which plays a vital role in several cancers via regulating migration and motility of cancer cells. However, the clinicopathological impact and function of CDK5 in lung cancer remain poorly understood. The present study was aimed at exploring expression and clinicopathological significance of CDK5 in lung cancer.
There were 395 samples of lung tissue including 365 lung tumors (339 non-small cell lung cancers and 26 small cell lung cancers) and 30 samples of normal lung. CDK5 expression was detected by immunohistochemistry on lung tissue microarrays.
Over expression was detected in lung cancer compared with normal lung tissues (P = 0.001). Furthermore, area under curve (AUC) of receiver operating characteristic (ROC) of CDK5 was 0.685 (95 % CI 0.564~0.751, P = 0.004). In lung cancer, we also discovered close correlations between CDK5 and pathological grading (r = 0.310, P < 0.001), TNM stage (r = 0.155, P = 0.003), and lymph node metastasis (r = 0.279, P < 0.001) by using Spearman analysis. In two subgroups of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), the expression of CDK5 was also higher than that of normal lung tissue, respectively (P = 0.001 and P = 0.004). Moreover, in NSCLCs, Spearman analysis revealed that expression of CDK5 was correlated with TNM stages (r = 0.129, P = 0.017), lymph node metastasis (r = 0.365, P < 0.001), and pathological grading (r = 0.307, P < 0.001), respectively. The significant correlation was also found between CDK5 expression and TNM stages (r = 0.415, P = 0.049) and lymphatic metastasis (r = 0.469, P = 0.024) in SCLCs.
The results of this present study suggest that the CDK5 expression is associated with several clinicopathological factors linked with poorer prognosis.
KeywordsLung neoplasms Cyclin-dependent kinase 5 Immunohistochemistry Tissue array analysis Neoplasm metastasis
Lung cancer is the most common type of cancer and the leading cause of cancer-related deaths in the world [1, 2]. In China, the incidence and the mortality of lung cancer increase rapidly, and now, lung cancer is the first dominating cancer . Non-small cell lung cancer (NSCLC) is the most frequent (approximately 85 %) class of lung cancers [4, 5]. As a result of the insufficiency of efficacious biomarkers for early diagnosis, the majority of lung cancer patients are diagnosed in an advanced stage . Although there are increasing proofs of therapeutic targets like EGFR, HER2, ALK, ROS1, BRAF, MET, VEGF, and FGFR1 and perpetual endeavor in clinic, the prognosis for patients with NSCLC still remains poor, with only a 5-year survival rate of 15 % with the normal therapy [7–9]. Therefore, there is an urgent requirement to discover new stable and independent biomarkers for prognosis and molecular therapy for lung cancer.
Cyclin-dependent kinases (CDKs) are serine/threonine kinases activated by cyclins . CDK5 is a member of CDKs and the investigation of CDK5 in cancer is increasing. In addition to western blot analysis, immunohistochemistry has also been performed to detect expression of CDK5 in cancer tissue . CDK5 has been reported to be upregulated in prostate cancer, breast cancer, medullary thyroid carcinoma, pituitary adenoma, and hepatocellular carcinoma, and CDK5 gene amplification was found in lung cancer [11–16]. However, decreased expression of CDK5 was detected in gastric cancer . The results of the studies showed that CDK5 was greatly related to proliferation, migration, and motility of cancer cells [13–17]. Moreover, downregulation of CDK5 indicated higher overall survival in multiple myeloma . With regard to prognostic implications, decreased expression of CDK5 was associated with advanced clinical stage and poor survival in gastric cancer patients and increased CDK5 expression was correlated to high pathological grading in breast cancer [11, 17]. So far, several articles have studied the potential role of CDK5 in lung cancer in vitro [19–22]. However, only one paper mentioned the clinical contribution of CDK5 in lung cancer with only 95 NSCLC patients and without small cell lung cancer (SCLC) cases by Liu et al. . In the current study, we set up a larger sample size of 365 lung cancers, 3.8 times bigger than the previous study performed by Liu et al. . Hence, the objective of this study was to explore the expression and clinicopathological significance of CDK5 in lung cancers and investigate its potential role of CDK5 as a biomarker for diagnosis and prognosis prediction for lung cancer patients.
The classification of lung cancer
Cancer subtype of histology
Squamous cell carcinomas
Large cell carcinoma
Bronchioloalveolar cell carcinomas
Santa Cruz Biotechnology (Heidelberg, Germany) provided the CDK5 antibody (C-8,sc-173,1:50 dilution) for immunostaining, and a ZSGB Kit (PV-6000, ZSGB, Beijing, China) was used for the secondary antibody at room temperature. CDK5 immunostaining score was determined by both positive rate of stained tumor cells and staining intensity. Concretely, the positive rate of stained tumor cells and the corresponding score were assigned as follows: 0 (0 %), 1 (1–25 %), 2 (26–50 %), 3 (51–75 %), and 4 (76–100 %). The intensity of CDK5 staining was scored from 0 to 3, and the detailed standard was as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). Samples were scored by the summation of the percentage of CDK5-positive cells and staining intensity. The total score of immunostaining was more than two which was considered as positive expression of CDK5. Immunostaining was assessed and graded independently by two pathologists (Kanglai Wei and Gang Chen).
The statistical analysis was conducted by SPSS 20.0 completely, and P values less than 0.05 were considered statistically significant. The chi-square test was used in the analysis of contrast of two groups, and when it exceeded two groups, Kruskal-Wallis H test was performed. Further, Spearman analysis was performed to study the relationship between CDK5 expression and clinicopathological characteristics. Moreover, we conducted ROC curve to evaluate the diagnostic significance of CDK5 in lung cancer, and the area under curve (AUC) of CDK5 more than 0.5 was considered significant.
(CDK5) expression in lung cancer
CDK5 expression in lung cancer compared with normal lung tissue
Cancer and normal lung tissue
CDK5 negative (n, %)
CDK5 positive (n, %)
Normal lung tissue
Squamous cell carcinoma
Large cell carcinoma
Bronchioloalveolar cell carcinoma
CDK5 expression associated with the various clinicopathological parameters in lung cancer
CDK5 negative (n, %)
CDK5 positive (n, %)
Tumor diameter (cm)
Cyclin-dependent kinases (CDK5) expression in non-small cell lung cancer (NSCLC)
The correlation of CDK5 with diverse clinical clinicopathological factors in NSCLC
CDK5 negative (n, %)
CDK5 positive (n, %)
Tumor diameter (cm)
Squamous cell carcinoma
Large cell carcinoma
Broncholoalveolar cell carcinoma
Cyclin-dependent kinases (CDK5) expression in small cell lung cancer (SCLC)
The correlation of CDK5 expression with various clinical pathological factors in SCLC
CDK5 negative (n, %)
CDK5 positive (n, %)
Tumor diameter (cm)
Cyclin-dependent kinase 5 (CDK5) is vital in neural cell migration and differentiation and is activated by p35 or p39 , and CDK5 is considered to be essential in neuronal cells [25, 26]. Nevertheless, as a unique member of cyclin-dependent kinases, the function of CDK5 beyond the nervous system has been demonstrated. CDK5 also regulates cell proliferation by alterant expression and its downstream signaling pathways, especially in cancer cells. Up to date, there has been a growing number of evidence that CDK5 has an important effect on cancer progression . The expression of CDK5 was aberrant in several cancers, and CDK5 regulated the proliferation of cancer cell in prostate cancer, medullary thyroid carcinoma, and gastric cancer [14, 15, 17]. In breast cancer, CDK5 was essential for the motility of cancer cell . Moreover, CDK5 can be used to predict the prognosis of multiple myeloma . In a word, the role of CDK5 in cancer is attracting increasing attention. To date, the expression of CDK5 was investigated in several cancers [13, 15, 17]. Higher expression of CDK5 was observed in hepatocellular carcinoma, ampullary adenocarcinoma, breast cancer, and medullary thyroid carcinoma, and Zachary et al. confirmed the expression of CDK5 was upregulated in colorectal, head/neck, breast, lung, ovarian, lymphoma, prostatic, sarcoma, myeloma, and bladder cancers via the Oncomine microarray online data mining software [11, 12, 18, 28]. However, downregulated expression of CDK5 was observed in gastric cancer. Thus, CDK5 might be heterogeneously expressed in different cancers.
In the present study, immunohistochemistry on lung tissue microarrays was performed to explore the expression of CDK5 in lung cancer and normal lung tissues. There was prominently higher expression of CDK5 in lung cancer, independent of various pathological subtypes, than in normal lung tissue. In the study of Liu et al., CDK5 was upregulated in cancer tissue as compared to benign pulmonary disease with a sample size of 95 non-small cell lung cancers (NSCLCs) . Our study, with a bigger sample size, approximately four times, confirmed that increased expression of CDK5 could be detected in lung cancer tissue compared with normal lung tissue and further supported that CDK5 was considered as an oncogene in lung cancer. No significant difference of CDK5 expression was found between NSCLC and SCLC in this current study. Although NSCLC and SCLC are commonly regarded as different diseases owing to their distinct biology and genomic abnormalities, the role and function of CDK5 may be consistent, as CDK5 level was both upregulated in NSCLC and SCLC tissues than the non-cancerous lung. However, the exact role of CDK5 in SCLC needs further investigation, since only a limited sample size (n = 26) was included in the current study. Taken together, CDK5 might be a potential biomarker of lung cancer despite its histology types.
The regulative mechanism of CDK5 in several cancers was investigated. CDK5 regulates DNA damage response via phosphorylating Ataxia telangiectasia mutated (ATM) kinase and thereby affecting its downstream signal pathways which was crucial to progression of hepatocellular carcinoma . In ampullary adenocarcinoma, over expression of nestin/CDK5 was involved in several oncogenic pathways (the activation of NOTCH, TGF-β1, or PDGFR pathways) that facilitated invasiveness of cancer . In breast cancer, CDK5 takes part in epithelial-mesenchymal transition induced by TGF-β1which is vital for tumor metastasis . In medullary thyroid carcinoma, CDK5 is essential to tumorigenesis and progression by retinoblastoma protein (Rb) and inhibition of Rb reduced proliferation of medullary thyroid carcinoma . The mechanisms of tumorigenesis and progression in lung cancer might be similar to the mechanisms aforementioned in other cancers in consideration of a consistent trend of CDK5 expression. However, this hypothesis needs to be verified with in vitro and in vivo studies.
Though expression of CDK5 was detected in lung cancer tissue and regulative mechanism of CDK5 was investigated in other cancers, the mechanism and exact role of CDK5 in the carcinogenesis and development of lung cancer remain unclear. A study of Korean population shows that CDK5 promoter polymorphisms contribute to the genetic susceptibility to lung cancer . As one of the downstream components of the EGFR-family-signaling pathway, the gene of CDK5 was amplified in lung cancer and it might be the common mechanism of oncogene activation in carcinogenesis . Tripathi et al. demonstrated that the CDK5 phosphorylates four serines located N-terminal to the Rho-GTPase activating protein(Rho-GAP) domain in DLC1(deleted in lung cancer 1), a tumor suppressor protein, and thereby activates DLC1 . Through the reconstruction of an integrated genome-scale co-expression network, Bidkhori et al. exhibited that CDK5 played a vital role in cell cycle progression in lung adenocarcinoma . The studies above of CDK5 in lung cancer suggested CDK5 may play an oncogenic role in lung cancer.
In addition to the expression of CDK5 in cancer tissues, the relationship between CDK5 and pathological parameters has been paid more and more attention to. There were a few researches of the relationship between CDK5 and clinical factors in the patients with cancers. In breast cancer, upregulated expression of CDK5 was related to higher grading (grading III) . In multiple myeloma, downregulated expression of cdk5 predicted favorable overall survival after bortezomib treatment . Liu et al. demonstrated that higher expression of CDK5 was correlated with low/undifferentiated, high pathological stage, lymph node metastasis, shorter median survival, and lower 5-year overall survival in the patients with NSCLC . Similarly, with a larger sample size, the consistent trend in this current study was confirmed that higher positive rate of CDK5 expression was greatly correlated with unfavorable clinicopathological parameters, including advanced TNM stage, lymphatic metastasis, and high pathological grading, which commonly indicate poorer prognosis. Thus, CDK5 might be used for the prediction to prognosis of lung cancer.
Further, Demelash et al.  demonstrated that CDK5 played a vital role in the regulation of lung cancer cell migration and invasion through Wound closure and Boyden chamber assay and certified that achaete-scute homologue-1 (ASH1), a basic transcription factor which was expressed in lung cancer cells with neuroendocrine features , could stimulate migration of lung cancer cells through CDK5/p35 pathway. The mechanism may support that CDK5 was closed related to lymphatic metastasis in lung cancer.
In summary, in this current study, the expression of CDK5 was investigated by lung tissue microarrays and immunohistochemistry. We demonstrated that CDK5 was highly expressed in lung cancer, including non-small cell lung cancer and small cell lung cancer, compared to normal lung tissue. Higher positive rate of CDK5 was associated with several clinicopathological parameters, which are representative of the progression and deterioration of lung cancer. These results suggest that the CDK5 expression associated with several unfavorable clinicopathological factors linked with poorer prognosis. Nevertheless, further plans are needed to explore the potential function of CDK5 in vitro and in vivo in the carcinogenesis of and progression in lung cancer.
The study was supported by the Fund of Guangxi Zhuang Autonomous Region University Student Innovative Plan (no. WLXSZX1555), China, the Fund of National Natural Science Foundation of China (NSFC 81360327), and the fund of Guangxi Provincial Health Bureau Scientific Research Project (Z2013201, Z2014055). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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