Original ArticlesMutated K-ras activates CDK8 to stimulate the epithelial-to-mesenchymal transition in pancreatic cancer in part via 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).
References (56)
- et al.
Randomized clinical trials in pancreatic cancer
Surg. Oncol. Clin. N. Am
(2002) - et al.
Pancreatic cancer: current and future treatment strategies
Cancer Treat. Rev
(2009) - et al.
Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways
Cell
(2009) - et al.
Epithelial-mesenchymal transitions in development and disease
Cell
(2009) - et al.
Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element
Blood
(1997) - et al.
MAPK and Akt act cooperatively but independently on hypoxia inducible factor-1a in rasV12 upregulation of VEGF
Biochem. Biophys. Res. Commun
(2001) - et al.
HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia
Cell
(2013) - et al.
The molecular genetics of pancreatic cancer
Surg. Oncol
(1997) - et al.
Epithelial-mesenchymal transitions: twist in development and metastasis
Cell
(2004) - et al.
Inhibition of cytoplasmic GSK-3β increases cisplatin resistance through activation of Wnt/β-catenin signaling in A549/DDP cells
Cancer Lett
(2013)
Wnt/beta-catenin signaling: components, mechanisms, and diseases
Dev. Cell
Wnt signaling from membrane to nucleus: β-catenin caught in a loop
Int. J. Biochem. Cell Biol
Peroxiredoxin 2 knockdown by RNA interference inhibits the growth of colorectal cancer cells by downregulating Wnt/β-catenin signaling
Cancer Lett
Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis
Cell
Cancer statistics, 2012
CA Cancer J. Clin
Metastatic pancreatic cancer 2008: is the glass less empty
Oncologist
1423 pancreaticoduodenectomies for pancreatic cancer: a single-institution experience
J. Gastrointest. Surg
Advanced pancreatic carcinoma: current treatment and future challenges
Nat. Rev. Clin. Oncol
Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C
PNAS
CDK8 is a colorectal cancer oncogene that regulates beta-catenin activity
Nature
siRNA-mediated silencing of CDK8 inhibits proliferation and growth in breast cancer cells
Int. J. Clin. Exp. Pathol
The histone variant macroH2A suppresses melanoma progression through regulation of CDK8
Nature
Cancer metastasis facilitated by developmental pathways: sonic hedgehog, Notch, and bone morphogenic proteins
J. Cell. Biochem
Epithelial-mesenchymal transitions in tumour progression
Nat. Rev. Cancer
Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits
Nat. Rev. Cancer
Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer
Genes Dev
Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway
Cancer Res
β-catenin as a potential key target for tumor suppression
Int. J. Cancer
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2021, European Journal of Medicinal ChemistryCitation Excerpt :The CDK8-coding gene was conferred an oncogene status first in colorectal cancer in 2008 [11]. Since then, the kinase activity of CDK8 has been found oncogenic in melanoma [12], breast cancer [13], acute myeloid leukaemia (AML) [14], pancreatic cancer [15], prostate cancer [16], and so forth [17]. In addition, a previous study has shown that CDK8 kinase activity attenuates the defence of natural killer cells against malignant cells and restrains the tumour surveillance of the former cells [18]; this concept has been further validated both genetically (via deletion of the CDK8-coding gene in natural killer cells) [19] and pharmacologically (by use of several exogenous small-molecule inhibitors of CDK8) [20].