Cellular localization of the estrogen receptor GPER1 in colorectal cancer cell lines

Abstract: Colorectal cancer is the third most common cancer type in the world with a mortality rate of around 2000 people in Sweden, year 2018. Women tend to have a lower risk of developing and progressing colorectal cancer, specifically premenopausal women. This indicates that Estrogen plays an important role in protecting from colorectal cancer. Estrogen has been shown to both inhibit colon cancer cell proliferation and stimulate cell apoptosis through several pathways. Estrogen signaling mediates through the estrogen receptors (ER) ERα, ERβ and GPER1. ERβ has been linked to suppression of colorectal cancer tumors but is downregulated in colon cancer tissue. The trans-membrane receptor GPER1 has also been found present in colorectal cells, however its effect on the CRC progression is still debated. It has been shown to both contribute to cell proliferation and the downregulation of cell proliferation, depending on the cell conditions. The location of the GPER1 receptor plays an important role to understand the signaling pathways and how the receptor could be a potential therapeutic target for cancer treatment. The localization of the receptor affects all types of molecular interactions with the protein and decides the cellular environment of the signaling protein. The location of GPER1 is still debatable and the localization has not been confirmed with a validated primary antibody. Anti-GPR30 703480 and three anti-FLAG (F7425, F1804, 14793) primary antibodies were used for detection on HT29, HCT116 and SiHa cells. Immunofluorescence (IF) followed by visualization with a confocal fluorescence microscope was used to localize the receptor tagged with a FLAG (Flag-GPER1). Western Blot (WB) and qPCR were used to validate the primary antibodies. The results obtained with the antibody anti-GPR30 show a signal mainly limited to the cytoplasm of the cells. However, the negative control SiHa presented a similar fluorescent signal while a low expression of GPER1 in WB and qPCR were measured, which indicates that the signal detected with the anti-GPR30 is not specific in IF. The anti-FLAG antibodies did not give sufficient signal detection for the FLAG protein on the different cellular populations. Further tests with different antibody dilutions for anti-GPR30 are required and new anti-Flag antibodies should be tested for IF detection.