Original ArticlesREG3A accelerates pancreatic cancer cell growth under IL-6-associated inflammatory condition: Involvement of a REG3A–JAK2/STAT3 positive feedback loop
Introduction
Pancreatic cancer (PaC) is one of the most aggressive and lethal types of cancer with poor prognosis, and has become the fourth leading cause of cancer deaths in the USA. Various factors including pancreatic inflammation and injury are reported to contribute to the development of PaC [1]. A large amount of genetic alterations in cellular signaling pathways and processes form the basis for progression from pancreatic inflammation to PaC [2]. Thereinto, signaling cascades regulated by interleukin-6 (IL-6) are well accepted to play an important role in the tumor-promoting effects of inflammation during the development of various malignancies including pancreatic carcinogenesis [3]. IL-6 is a potent inflammatory cytokine that mediates a plethora of biologic processes, including cell proliferation, survival, migration, invasion and anti-apoptosis [4], [5]. The binding of IL-6 to a specific receptor complex activates the downstream kinases such as Janus kinase 2 (JAK2), which subsequently triggers the phosphorylation and nuclear localization of signal transducers and activators of transcription-1 and -3 (STAT1 and STAT3) [3]. In particular, STAT3 is widely considered as an oncogene upregulating a number of proliferative or anti-apoptotic genes (for example, CyclinD1 and Bcl2 respectively) [6], [7], [8], and its leading role in promoting pro-oncogenic inflammatory pathways has been recognized [9]. Further understanding the regulation of IL-6/JAK2/STAT3 is of great importance and could provide a new basis for the rationalization of therapeutic strategy.
Our previous study [10] found that regenerating gene protein (REG) 3A also functions as a tumor promoter in inflammation-associated pancreatic carcinogenesis. REG3A, also named as pancreatic associated protein (PAP) or the encoded protein of genes expressed in heptocarcinoma-intestine-pancreas (HIP) [11], [12], is one of important members of the REG family (REG1A, REG1B, REG3A, REG4) in humans [13]. It is well-established that REG3A expression is significantly increased after pancreatic inflammation damage. And REG3A overexpression is correlated with over-proliferation, invasion, migration, distant metastases and tumor aggressiveness [14], [15]. Nevertheless, the underlying mechanism by which REG3A regulates pancreatic cell growth and contributes to the pathogenesis of inflammation-associated PaC had been poorly explored.
Intriguingly, there might be a potential interaction between REG3A and classic IL-6 signaling. It has been demonstrated that gene expression of REG1A, another REG protein similar to REG3A, in gastric cancer cells is strongly induced by IL-6 [16], while IL-6 regulates the multifaceted biological function through activating the JAK2/STAT3 pathway [3]. And REG1A has been elucidated to promote cell proliferation and has the function of anti-apoptosis by inducing c-myc, CyclinD1, Bcl-XL and so on, and thus has been involved in the pathophysiology of gastrointestinal inflammation and its associated cancer [17]. The high conservatism of REG family in structure and functional properties [13] led us to hypothesize that there is also the potential relationship between IL-6-mediated pathway and REG3A in promoting cell malignant proliferation and carcinogenesis. Most importantly, we have previously demonstrated that the JAK2/STAT3 pathway appears involved in the effect of REG3A on pancreatic cell growth [10]. In the present study, we aimed to define the function of REG3A in PaC cell growth under inflammatory environments (especially associated with IL-6), and further explored how the JAK2/STAT3 pathway contributes to the tumor promoting function of REG3A in the pathogenesis of the inflammation-related pancreatic malignancy.
Section snippets
Cell culture and treatment
Human PaC cell lines BxPC-3, AsPC-1 and SW1990 were purchased from American Type Culture Collection (ATCC, USA). Cell line HPDE6c7 (immortalized but not transformed pancreatic epithelial cell line), used as normal pancreas cell line in this research, was purchased from GuangZhou Jennio Biotech Co., Ltd. The cell lines were maintained at 37 °C under a 5% CO2 environment in RPMI 1640 (Hyclone, USA) enriched with 10% fetal calf serum (Invitrogen, USA) and antibiotics of penicillin/streptomycin
IL-6 stimulation induces REG3A expression via a STAT3-dependent mechanism
To clarify the direct effect of pro-inflammatory cytokines on REG3A signaling in the progress of inflammation-linked pancreatic carcinogenesis, endogenous levels of REG3A in human ductal epithelial HPDE6c7 cell were detected by qRT-PCR and Western blot after incubation with classic pro-inflammatory cytokines (including IL-6, TNF-α, and IL-1β) at different doses respectively for 24 h. In HPDE6c7 cells, the mRNA and protein expression levels of endogenous REG3A were enhanced by IL-6 stimulation
Discussion
Proinflammatory cytokines play important roles in promoting PaC progression by modulating the tumor microenvironment as well as directly acting on cancer cells for growth, invasion and metastasis [25], [26]. In particular, the level of circulating IL-6 in PaC patients is directly correlated with cachexia, advanced tumor stage and poor survival. IL-6 and its downstream pathways, especially the JAK2/STAT3 pathway, have emerged as the most important factors in the modulation of
Conclusions
For the first time, our study provides the proof of the synergism of REG3A with IL-6 to initiate tumorigenesis and a REG3A–JAK2/STAT3 feedback loop-associated mechanism. We suppose that, under pancreatic inflammatory conditions, proinflammatory cytokine IL-6 combines with IL-6R, activates downstream JAK2, which further triggers the dimerization and nuclear localization of STATs by tyrosine phosphorylation, then directly accelerates cell cycle progression from the G1 into the S phase by
Conflict of interest
The contents of this manuscript have not been submitted or published in any form in any other journals. All authors have read the final version of the manuscript and agreed upon the content. Xiulan Liu, Jun Wang, Guoxiao Yin, Yang Liu and Xiang Lei can be contacted at Ming Xiang’s address. Ming Xiang affirms that there is no financial interest with regard to this manuscript that might be construed as a conflict of interest on my part or on the part of any of the co-authors.
Acknowledgement
This work is supported by the National Natural Science Foundation of China (nos. 91229114).
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These authors contributed equally to this work.