Pathophysiological role of NNAT in ER+ breast cancer

Abstract

Neuronatin (NNAT) was recently identified as a novel mediator of estrogen receptor‐positive (ER+) breast cancer cell proliferation and migration, which correlated with decreased tumorigenic potential and prolonged patient survival. However, despite these observations, the molecular and pathophysiological mechanism(s) of NNAT in ER+ breast cancer remain unclear. Based on high protein homology with phospholamban, we hypothesized that NNAT mediates homeostasis of intracellular calcium [Ca2+]i) levels and endoplasmic reticulum (EndoR) function, which is frequently disrupted in ER+ breast cancer and other malignancies.To evaluate the role of NNAT on [Ca2+]i homeostasis, we used intracellular labeling and subcellular confocal imaging of multiple breast cancer models (ER+ breast cancer cell lines T47D and ZR75, MCF10A normal mammary epithelial cells, and MDA‐MB‐231 triple‐negative breast cancer cell lines originally obtained from ATCC (American Type Culture Collection) to better understand the role of NNAT in Ca2+‐mediated cellular functions.Our data indicate that NNAT localizes predominantly to EndoR and lysosome, and genetic manipulation of NNAT levels demonstrated that NNAT modulates [Ca2+]i influx and maintaining Ca2+homeostasis. The application of thapsigargin and consecutive inhibition of SERCA in ER+ breast cancer cells overexpressed NNAT show a significant elevation in the amount of Ca2+ in EndoR (184 ± 5 %, p<0.001 comparing to GFP). At the same time, overexpression of NNAT increased basal cytosolic Ca2+ concentration up to 50% (p<0.001 comparing to GFP). Pharmacological inhibition of calcium channels revealed that NNAT regulates [Ca2+]i levels in breast cancer cells through the interaction with ORAI1 but not the TRPC3 signaling cascade. Furthermore, NNAT transcriptionally regulated by NRF1, PPARα, and PPARγ and is strongly upregulated by oxidative stress via the ROS and PPAR signaling cascades.Collectively, these data suggest that NNAT regulates Ca2+homeostasis in response to oxidative stress, thus providing a molecular link between the longstanding observation that accumulating ROS and altered Ca2+signaling are key oncogenic drivers of cancer.