Cancer Letters

Cancer Letters

Volume 356, Issue 2, Part B, 28 January 2015, Pages 613-627
Cancer Letters

Original Articles
Mutated K-ras activates CDK8 to stimulate the epithelial-to-mesenchymal transition in pancreatic cancer in part via the Wnt/β-catenin signaling pathway

https://doi.org/10.1016/j.canlet.2014.10.008Get rights and content

Abstract

Cyclin-dependent kinase 8 (CDK8), a gene encoding the cyclin-dependent kinase (CDK) component of the Mediator complex, is known as a colon cancer oncogene. Our recent study showed that CDK8 plays an important role in the formation of pancreatic cancer, but the CDK8 expression levels were not completely identical in different pancreatic cancer samples. The level of CDK8 expression depended on whether the K-ras gene was mutated; its expression was much higher in samples carrying a K-ras mutation than in wild-type K-ras samples. Moreover, CDK8 expression was reduced following mutated K-ras knockdown in K-ras-mutated pancreatic cancer cells, whereas CDK8 expression was increased following expression of mutated K-ras in wild-type K-ras cells. Our study demonstrates that mutated K-ras stimulates CDK8 expression, possibly by regulating HIF-1α, and both CDK8 and mutated K-ras were confirmed to promote cell proliferation and prevent apoptosis in vitro. Additionally, we found that both CDK8 and mutated K-ras promote the invasion and migration of pancreatic cancer cells via the positive regulation of the Wnt/β-catenin signaling pathway, thereby increasing the expression of Snail1 and ZEB1, which act as important stimulating factors of the epithelial-to-mesenchymal transition (EMT). Finally, knockdown of either CDK8 or mutated K-ras contributed to attenuated pancreatic cancer growth in BALB/c nude mice. In conclusion, these findings demonstrate that mutated K-ras promotes CDK8 expression and that the regulatory effects of CDK8 on the EMT are partially attributed to the Wnt/β-catenin signaling pathway.

Introduction

Pancreatic cancer is a common cause of cancer-related death in Western developed countries, with an estimated 37,500 deaths and almost the same number of new cases (approximately 40,000) occurring annually in the USA [1]. Recent research has shown that, in most cases, this disease has spread locally or to distant organs at the time of diagnosis, thus precluding R0 resection, as only approximately 15% of all pancreatic cancer patients have an opportunity to receive surgical R0 resection. Even among the fortunate patients who undergo a potentially curative resection, the probability of long-term survival remains less than 20% due to its early recurrence and metastatic properties [2], [3], [4]. Currently, no effective systemic therapy for the aggressive pathology of this disease is available. Therefore, there is an urgent need for an improved understanding of the molecular mechanisms underlying the aggressive and invasive metastasis of pancreatic cancer [5], [6].

Cyclin-dependent kinase 8 (CDK8) is a serine–threonine protein kinase localized to the nucleus that regulates gene expression by interacting directly with the transcriptional machinery and regulating RNAPII activity [7], [8]. In recent studies of colorectal cancer, breast cancer and malignant melanoma, CDK8 has been confirmed to be an oncogene [9], [10], [11]. However, little is known about the role of this gene in the progression of pancreatic cancer. Therefore, we investigated the underlying mechanisms by which CDK8 acts in pancreatic cancer. Recently, we found the CDK8 expression level to be much higher in pancreatic cancer samples than in non-malignant pancreatic samples. Interestingly, the expression of this gene was not completely identical in different pancreatic cancer samples, and its expression depended on the mutation status of the K-ras gene. Therefore, the relationship between CDK8 expression and K-ras mutation was another basis for this study.

The epithelial-to-mesenchymal transition (EMT) originally acts as a physiological process during embryonic development in which cells lose epithelial characteristics and gain mesenchymal properties. Important EMT characteristics include the loss of cell-to-cell contacts and increased cell motility, which are also implicated in the aggressive invasion and metastasis of late stage tumors [12], [13]. EMT occurs in response to several distinct pathways, including Notch, several receptor tyrosine kinases, and transforming growth factor-β (TGF-β) [14], [15], [16]. Moreover, dysregulation of Wnt/β-catenin signaling has been demonstrated to play a significant role in the development and promotion of both the EMT and cancer metastasis [17], [18], [19], [20]. Additionally, accumulation of stabilized β-catenin, especially nuclear β-catenin, is an important marker of enhanced Wnt signaling [21]. Moreover, numerous studies have revealed that the EMT is regulated by a group of transcription factors downstream of Wnt/β-catenin signaling, such as the Snail family (Snail 1 and Snail 2), ZEB1 and Twist [22], [23], [24]. In summary, in this study, we used vitro and in vivo approaches to examine whether and how mutated K-ras stimulates the expression of CDK8 and whether the regulatory effects of CDK8 on the EMT are partially attributed to the Wnt/β-catenin signaling pathway in pancreatic cancer.

Section snippets

Cell culture

The human pancreatic cancer cell lines BxPC-3, AsPC-1 and CFPAC-1 were purchased from the Chinese Type Culture Collection. The BxPC-3 and AsPC-1 cells were maintained in RPMI 1640 medium (RPMI-1640, Gibco, USA), and the CFPAC-1 cells were maintained in IMDM medium (IMDM, Gibco, USA). These media were supplemented with 10% FBS (Gibco, USA) and 2% penicillin/streptomycin (Hyclone, Shanghai, China). The culture flasks were placed in a humidified atmosphere containing 95% air and 5% CO2 at 37 °C.

Expression of CDK8 in pancreatic cancer samples of different K-ras status, in non-malignant tissues and in human pancreatic cancer cell lines

Codons 12 and 13 of the K-ras gene were amplified from all of the samples and were sequenced. A total of 83.3% (35/42) of the pancreatic cancer samples carried a K-ras mutation, whereas K-ras mutation occurred in none of the non-malignant tissues. Furthermore, the CDK8 protein levels were examined in all tissues via immunohistochemistry. CDK8 expression was predominantly localized to the nucleus of pancreatic cancer cells (Fig. 1A), but its expression was very weak in non-malignant tissues (

Discussion

In several recent studies, CDK8 has been confirmed to be an oncogene in colorectal cancer, breast cancer and malignant melanoma [9], [10], [11]. Although this gene may play a significant role in promoting the formation and development of cancer, some researchers have demonstrated that CDK8 acts as a tumor-suppressive protein in endometrial cancer [31]. These findings prompted us to investigate the role that this gene plays in pancreatic cancer. In this study, we also aimed to carefully

Conflict of interest

The authors declare no financial or other conflict of interest with regard to this work.

Acknowledgement

This work was supported by a grant from the National Natural Science Foundation of China (No. 81272753).

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