Retinoic acid inhibits pancreatic cancer cell migration and EMT through the downregulation of IL-6 in cancer associated fibroblast cells
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is histologically characterized by an abundant desmoplastic stroma, which is even significantly present in premalignant conditions [1]. Fibrogenesis involves a specific cell type in the stroma of pancreatic cancer, namely cancer associated fibroblast cells (CAFs). An increasing number of studies have reported that CAFs support the proliferation, migration, metastasis and chemotherapy resistance of tumor cells by producing extracellular matrix, secreting cytokines, and activating signaling pathways in tumor cells [2], [3], [4], [5], [6]. As important components of tumor microenvironment, CAFs have become a promising target for biological therapy. For example, Oliver et al. found that the combination therapy of gemcitabine and IPI-926, a drug that inhibits cancer-associated stromal tissue through the Hedgehog signaling pathway, was more effective than gemcitabine treatment alone; this finding was due to the increased gemcitabine accumulation in the pancreatic cancer stroma [7].
Evidence has shown that a loss of vitamin A is related to the activation of fibroblast cells and fibrogenesis [8]. To attenuate the function of these activated fibroblast cells, researchers tried to re-induce vitamin A in activated fibroblast cells to restore the cells’ quiescent status [9]. Retinoic acid (RA) is a small, lipophilic molecule derived from vitamin A. Studies in hepatic cells showed that RA could prevent the activation of hepatic stellate cells by decreasing cell proliferation, α-SMA expression and collagen production [10]. A similar phenomenon was observed in rat pancreas when treating with RA; inhibition of all three classes of MAPKs [9] and suppression of AP-1 were also observed in these cells [11]. Additionally, RA has an inhibitory effect on tumor epithelial cells. RA can inhibit the proliferation of the pancreatic cancer lines Capan-2 and Hs766T through TGF-β2 [12]. It was also observed that combined treatment with retinoids and gemcitabine chemotherapy could effectively treat pancreatic cancer [13]. However, whether RA could inhibit the tumor behavior by influencing CAFs, the activated fibroblast cells in the tumor microenvironment, remains unclear.
Based on the above considerations, we hypothesized that, through inhibition of CAFs, RA can decrease the support that the CAFs provide to the tumor cells. In our study, we induced CAFs into a relatively quiescent state by treating the cells with two forms of RA: ATRA and 9-cis-RA, then utilized the conditioned media from CAFs to culture the pancreatic cancer cell line that was not sensitive to RA. We found that the conditioned media from these CAFs resulted in a decrease in the migration distance and EMT of tumor cells. The reduction of IL-6 in the conditioned media with RA treatment was responsible for the decreased support of CAFs to EMT of tumor cells.
Section snippets
Cell culture and immunohistochemistry
Panc-1 and Aspc-1 pancreatic tumor cells were obtained from the American Type Culture Collection, and these cells were cultured in DMEM and RPMI, respectively, containing 10% FBS, 2 mmol/L glutamine and penicillin/streptomycin at 37 °C in a humidified atmosphere with 5% CO2.
Human fibroblast cells were cultured using the outgrowth method [8]. Fresh tissue was obtained from residual pancreatic adenocarcinoma specimens from patients who underwent primary surgical resection at Peking Union Medical
The effect of RA on the pancreatic cancer cell lines
Parenchyma and mesenchyma are two components involved in the study of tumor microenvironment. To focus our study on the cancer associated fibroblast cells, we first had to find a tumor cell line that was not sensitive to RA. According to previous study [9], Aspc-1 and Panc-1 are two pancreatic cancer cell lines in which RA had relatively low cytotoxicity. To find the cancer cell line that was less sensitive to RA, we tested the apoptotic rate, cell cycle status and proliferation rate of the two
Discussion
CAFs in the tumor microenvironment play many roles in supporting tumor cell progression. In the current study, we demonstrated that CAFs promoted pancreatic cancer cell migration by secreting cytokines, such as IL-6, and treatment with RA could decrease the migration of tumor cells by inhibiting the IL-6 secretion of CAFs.
Conclusions
Our study suggested that IL-6 secreted by CAFs supports EMT progression of tumor cells, resulting in increased migration potential. Additionally, our data add new evidence that supports the uses of RA as a treatment for pancreatic adenocarcinoma, as the mesenchymal transition was inhibited with RA treatment due to the reduced IL-6 secretion of CAFs, demonstrating the indirect influence that RA had on tumor cells. We also verified that tumor associated stroma supports the migration and EMT of
Conflict of interest
None of the authors have any conflict of interest to disclose regarding this study.
Acknowledgements
This work was supported by the National Natural Science Foundation, China (Project No. 81172334).
We would like to thank Dr. Diane Simeone and her staff at the University of Michigan, Department of Surgery for her instruction and the study experience in UM.
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