Estrogen receptors influence the epithelial-to-mesenchymal transition of pancreatic cancer cell lines

Pancreatic ductal adenocarcinoma (PDAC) represents the majority of diagnosed pancreatic cancers and its mortality rates are well above 90%. This bleak scenario is mostly due to its very difficult early diagnosis, scarce surgical treatability, high relapse rate and poor response to systemic and targeted therapies. Apparently, an imbalance between male and female incidence of PDAC could suggest some sort of “protective mechanism” in women against the onset of this neoplasia, possibly played by estrogen receptors. Estrogen receptor Beta (ER Beta) and the G-protein coupled estrogen receptor (GPER, formerly known as GPR30) are often expressed in pancreatic cancer but their role is still unknown. These two receptors work in agonistic and antagonistic ways in different cancer cells and are used as prognostic markers. To this end, we aim to correlate the expression of ER Beta and GPR30 with PDAC cell lines behavior and to investigate their role in biological processes involved in cancer, hoping to identify novel biomarkers or therapeutic targets for patients’ management and treatment. Using various approaches in 4 different commercially available pancreatic cell lines (KP-2, SUIT-2, PANC-1 and Mia-Paca2) we characterized how the two receptors ER Beta and GPR30, alone or together, can control or interfere with the EGFR regulation of different cellular processes such as proliferation, invasion and, particularly, EMT. By using different approaches, we evaluated the expression (Western blot analysis) and the localization (immunofluorescence microscopy) of these two receptors in pancreatic cancer cell lines. Subsequently, by in vitro experiments, we evaluated the contribution each of the two receptors gives to the analyzed cellular processes alone and with the EGFR. Data obtained in 4 different pancreatic cancer cell lines demonstrates that estrogen stimulation is responsible for the increased E-cadherin level and for the inhibition of EMT. On the other hand, triggering pancreatic cancer cells with EGF increases Vimentin synthesis, while the combination of the two stimuli does not have the same effects in the 4 cell lines, underlying that ER Beta and GPR30 differentially participate to EMT of pancreatic cancer cells. In the cell lines expressing both receptors, the combined use of E2 and EGF increases the expression of vimentin, a result similar to that observed after EGF stimulation. In the cell line expressing only GPR30 we observe a slight increase of E-cadherin after estradiol stimulation but, surprisingly, the combination of E2 and EGF is not able to increase vimentin synthesis as observed in the other cell lines expressing both receptors. These results indicate that while ER Beta could play a role in controlling E-cadherin transcription, GPR30 is primarily involved in EGFR transactivation and vimentin synthesis. The knowledge of the differential roles of ER beta and GPR30 in EMT machinery can allow to identify two new prognostic and diagnostic markers and to support the design of new personalized and effective therapies to cure aggressive and incurable pancreatic cancer.