Background: IL15RA forms part of a heterotrimeric receptor with IL2RB and IL2RG and plays a role in proliferation and differentiation of cytotoxic T-cells. IL15RA gene copy number is amplified in triple negative breast cancer leading to higher expression of an isolated IL15RA receptor component and trans-presentation of IL15 driving both an autocrine proliferative signalling cascade and activation of immune cells (Marra et al., Cancer Res. 2014 Sep 1;74(17):4908-21). GPR89/GPHR is a Golgi-complex-associated proton channel that controls Golgi complex intraluminal pH and post-translational modification of plasma membrane and secretory proteins (Maeda et al., Nat Cell Biol 10(10): 1135-1145). We have found GPR89/GPHR upregulated and overexpressed in breast cancer.
Aim: To investigate the dynamics of IL15RA/IL15 in TNBC-derived cells in response to 1) inflammatory cytokines typically abundant in cancer tissue such as interferon gamma (IFN-G) and 2) upon perturbation of the putative Golgi complex associated trafficking function caused by TN-specific upregulation of GPR89/GPHR
Methods: We measured the expression and plasma membrane localization of Il15RA in different TNBC-derived cell lines by flow cytometry on intact cells and by immunoblot analysis on whole cell lysates, with or without stimulation by 20 ng/ml IFN-G for 36h. The effect of GPR89/GPHR on the above mentioned phenomena was assessed by siRNA mediated GPR89/GPHR silencing or lentiviral mediated TET-ON mediated conditional expression.
Results: Flow-cytometry analysis of multiple breast cancer cell lines showed a dramatic increase in the plasma membrane-associated IL15RA in response to treatment with IFN-G. The overall amount of IL15RA didn't appear to have changed by immunoblot analysis on total cell lysates, thus suggesting that IFN-G mobilises IL15RA from intracellular stores which we could identify by IF in resting cells largely colocalising with endosomal structures. The upregulation of GPR89/GPHR which we observed occurs in ca 40% of TNBC, as well as in other breast cancer subtypes, dramatically alters the postranslational modification of plasma membrane proteins in breast cancer cell lines and concomitantly reduces the proportion of IL15RA transported to the PM in response to IFN-G stimulation.
These data shed light on the intracellular dynamics of IL15RA in breast cancer cell lines, suggesting an unprecedented IFN-G regulated mobilization and transpresentation of the IL15RA/IL15 complex which is sensitive to specific and yet unknown, post-translational modifications controlled by GPR89/GPHR a novel breast cancer associated Golgi-complex proton channel.
Citation Format: Pierfrancesco Marra, Sumi Mathew, Steven Catchpole, Alessandra Facchetti, Andrew Tutt. Interferon gamma and post-translational modifications control the dynamics and plasma membrane association of IL15RA expressed by Triple Negative Breast Cancer (TNBC). [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A093.
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