Increased expression of Cks1 protein is associated with lymph node metastasis and poor prognosis in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a common head and neck malignancy with a concentrated incidence rate in Southeast Asia compared with global distribution [1, 2]. Due to its sensibility to radiotherapy, early diagnosis and radiotherapy alone or in combination with chemotherapy have remarkably increased NPC survival rate. However, local recurrence and metastasis frequently lead to failure of clinical therapy in advanced stage patients [3]. The initiation and progression of NPC involves various factors, including Epstein-Barr virus (EBV) infection, genetic susceptibility, exposure to chemical carcinogens and mutant expression of tumor-suppressor genes, etc [4, 5]. Over the past decade, essential biomarkers involved in critical genetic events that contribute to the carcinogenesis of NPC have been increasingly found. Further investigation of novel factors associated with prognosis would provide new clue for exploring new effective therapeutic methods in NPC.

The cyclin–dependent protein kinase regulatory subunit 1 (Cks1) gene which encodes a 9KD protein Cks1 takes important roles in cell growth, proliferation and apoptosis. The protein Cks1 is an essential adaptor of the SCF-SKP2 E3 ligase ubiquitin ligase complex which appends ubiquitin to targets for degradation through the ubiquitin proteasomal system [6]. One of crucial functional roles of Cks1 is to mediate the ubiquitination of CDK inhibitor p27^Kip1 and lead to promote cell cycle progression from G1 to S phase [7, 8]. Aberrant expression of Cks1 and p27^KIP1 has been found in multiple human cancers and is significantly associated with tumor invasion and metastasis [9‐13]. Although Cks1 expression is generally inversely related to p27^Kip1 expression, but some studies reported different expression pattern of Cks1 and p27^Kip1 proteins which emphasize potentially p27^Kip1 independent mechanisms of Cks1 in cancer progression [14, 15]. The relevance between Cks1 and p27^Kip1 protein and the clinicopathological characteristics in NPC remains unclear. In this present study, we aimed to identify the expression level of Cks1 and p27^KIP1 in NPC and their potential relationship with clinicopathological features.

The alterations of expression or activity of proteins which is related to cell cycle regulation are of extensive interest, because uncontrolled proliferation is a critical character in tumor progression. Cyclin-dependent kinase (CDK) inhibitor p27^Kip1 inhibits the activity of G1-cyclin–CDK complexes and arrests cell-cycle progression in G1 phase. P27^Kip1 is degraded in the late G1 phase via the ubiquitin–proteasome pathway [17]. The Cks1 protein is a member of the highly conserved family of Cks/Suc1 proteins which interact with Cdks and participates in numerous cellular processes including cell proliferation, growth and survival [18, 19]. One of well established mechanisms of Cks1 modulating cell cycle is to bind with the C-terminal of Skp2 to degrade p27^Kip1 and to promote cell cycle progression from G1 to S phase [7, 8, 18]. Recent evidence has revealed that Cks1 is over-expressed in a majority of tumors, solid tumors such as gastric carcinoma [20], oral squamous cell carcinoma [21], colorectal carcinoma [22], salivary gland tumors [9], esophagus carcinomas [10], hepatocellular carcinoma [11], breast cancer [12], non-small cell lung carcinoma [23], and hematologic tumors, like lymphoma [14], multiple myeloma [13]. In those studies, high expression of Cks1 is associated with tumor formation and aggressiveness. It is frequently observed that high Cks1 expression is correlated with high SKP2 and low p27^Kip1 and is associated with tumor progression in some cases. Those studies coincide with our results and further indicate the important roles of Cks1 in tumor progression.

In this study, we found that the expression of Cks1 increased in NPC tissue, and high expression of Cks1 protein was correlated with LNM status and survival status in NPC patients. But, p27^Kip1 expression was not correlated with the clinicopathological characteristics in NPC, although IHC analysis result showed that p27^Kip1 expression was attenuated in NPC patients and statistical analysis data confirmed that the expression of p27^kip1 was inversely related to Cks1 in NPC. The non-significance of p27^Kip1 in prognostic evaluation of tumor has been observed in esophagus carcinomas [10], hepatocellular carcinoma [11], and non-small cell lung carcinoma [23] too. Those evidences support our results and suggest that Cks1 might influence progression of NPC through p27^KIP1-independent ways.

In fact, some studies also indicate Cks1 is involved in regulating cell cycle transitions by other targets, not only p27^KIP1. For instance, Cks1 promotes cell to enter from G0 to G1 by mediating the ubiquitination of CDK1 inhibitor p130 in breast cancer [15]. Besides the mechanisms of regulating cell cycle, a few studies have improved that Cks1 is required in cell transcriptional events. Cdc20 has been reported to be a transcriptional target of Cks1 [24]. Cks1 protein is primarily involved in modulating the transcriptional activation of the APC/C protein-ubiquitin ligase activator Cdc20 to promote mitosis [24, 25]. Cks1 has also been reported to transcriptionally regulate the expression of cdc2, cyclin B and cyclin A in mammalian cells [26]. Moreover, the involvement of Cks1 in MAPK [27], JAK-STAT [28] and NF- κB [29] cell signaling pathways has been reported recently. Cks1 influences cell proliferation and apoptosis through activating the phosphorylation of MEK1/2 and ERK1/2 in breast cancer, and the phosphorylation of MEK1/2, ERK1/2 and STAT3 in multiple myeloma [28]. Our research results suggested that Cks1 might promote NPC invasion and progression through multiple ways, not only by p27^Kip1-dependent mechanism.

In recent years, due to radiotherapy and chemotherapy resistance, the effect of treatment in NPC is at a standstill. Increased expression of Cks1 promotes the radiation resistance ability of ESCC cells [30]. Cks1 over-expression leads to multidrug resistance in multiple myeloma cells in vitro by activating MAPK and STAT3 pathways [28]. But, enforced expression of Cks1 enhanced chemotherapeutic sensitivity by overriding DNA damage checkpoints in breast cancer cell in vitro and in vivo [31]. These inconsistent research results suggest that Cks1 protein might play complex roles in mechanism of tumor cell tolerance to radiotherapy and chemotherapy. Our research results uncovered that high expression of Cks1 was correlated with lymph node metastasis and survival rate in NPC, which may offer potential target for effective treatment in NPC.

In summary, we first reported that the expression of Cks1 protein increased and p27^KIP1 decreased in NPC. Over-expression of Cks1 was associated with the poor overall survival rate of NPC. Furthermore, multivariate analysis suggested that high expression of Cks1 protein might be regarded as the independent prognostic factor for poor prognosis in NPC patients. These results may provide novel clue for NPC therapy methods.