(SNX3)'ün EGFR-pozitif meme hücrelerinde erken ve uzun dönem EGF uyarımına duyarlılığı

Sorting nexin 3 (SNX3) is a member of the sorting nexins family, which is involved in the endocytic trafficking network. SNX3 only has a PX domain, which helps endosomes bind to phosphatidyl inositol-3-phosphate and plays a role in the constitution of the retromer complex on early endosomes. Endocytosed receptors are internalized and recycled back to the plasma membrane, the Trans-Golgi Network, or are degraded in the lysosomes. The current understanding of SNX3 function in recycling Epidermal Growth Factor Receptor (EGFR), was demonstrated in an epidermoid carcinoma cell line (A431). Therefore, we hypothesized that SNX3 is an important component of the EGF/EGFR axis in breast cancers and investigated how SNX3 gene is regulated in EGFR positive cells in response to EGF stimulation. To test this hypothesis, we used EGFR (+) MCF10A cells which has been established as an EGF responsive cell line. We performed SNX3 stimulation in a time dependent setup, collected RNA and lysates from treated cells. We observed immediate and sustained upregulation of SNX3 protein upon EGF stimulation. Interestingly, SNX3 upregulation at early time points was independent of transcriptional regulation. EGF stimulation improved protein half life. However, late response to EGF stimulation was due to transcriptional upregulation as was demonstrated by RT-qPCR. We also show that upregulation of SNX3 is also EGFR pathway specific as EGFR inhibitör AG1478 and other inhibitors that blocked EGFR downstream pathways. These observations were further confirmed in another cell line positive for EGFR; HEK293. Overall, these results show SNX3 as an important component of EGF/EGFR axis as an immediate and sustained target of EGF stimulation. Implications for these findings will be important in better understanding EGFR biology.