NNAT is a novel mediator of oxidative stress that suppresses ER + breast cancer

Abstract

Background: 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 role(s) of NNAT in ER + breast cancer remains unclear. Based on high protein homology with phospholamban, we hypothesized that NNAT mediates the homeostasis of intracellular calcium [Ca²⁺]_i levels and endoplasmic reticulum (EndoR) function, which is frequently disrupted in ER + breast cancer and other malignancies.

Methods: To evaluate the role of NNAT on [Ca²⁺]_i homeostasis, we used a combination of bioinformatics, gene expression and promoter activity assays, CRISPR gene manipulation, pharmacological tools and confocal imaging to characterize the association between ROS, NNAT and calcium signaling.

Results: 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 maintains Ca²⁺ homeostasis. Pharmacological inhibition of calcium channels revealed that NNAT regulates [Ca²⁺]_i levels in breast cancer cells through the interaction with ORAI but not the TRPC signaling cascade. Furthermore, NNAT is transcriptionally regulated by NRF1, PPARα, and PPARγ and is strongly upregulated by oxidative stress via the ROS and PPAR signaling cascades.

Conclusion: Collectively, these data suggest that NNAT expression is mediated by oxidative stress and acts as a regulator of Ca²⁺ homeostasis to impact ER + breast cancer proliferation, thus providing a molecular link between the longstanding observation that is accumulating ROS and altered Ca²⁺ signaling are key oncogenic drivers of cancer.

Keywords: Breast cancer; Neuronatin; Orai; PPAR; ROS.

Fig. 1

NNAT expression correlated with ROS and PPAR signal-transduction pathway in TCGA-BRCA ER + cohort and ER + breast cancer cells. A Gene expression that correlates with NNAT expression in breast cancer patients. Analysis of NNAT mRNA expression in TCGA-BRCA from ER + tumors (n = 699). Data are presented as log expression values and mean values are statistical different between groups (2.2 × 10^− 16), as tested by ANOVA. B The most significant modules of oxidative stress genes selected from protein–protein interaction network upregulated by NNAT overexpression in both ZR75 and T75D cell lines

Fig. 2

NNAT mRNA expression is regulated by oxidative stress and activation of PPAR signaling cascade. A Luminescence activity of NNAT promoter activity co-transfected with E2F1, E2F4, NRF1, PPARα + RXR, PPARγ + RXR, or pLX304 control plasmid in ER + breast cancer cell lines. Data presented as mean percentage of GLuc/SEAP ratio normalized to pLX304 ± SEM (n = 9 per group; paired t-test, ***p < 0.001 vs. pLX304 control). B Evaluation of mRNA expression of NNAT, tumor suppressors genes CDKN1A and CDKN2B, and the transcription factor NRF1 responsible for cellular growth, during the exposure to H₂O₂. MCF10A, MDA-MB-231, T47D, and ZR75 cell lines untreated or treated with H₂O₂ (n = 3 per group; paired t-test, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control). C NNAT mRNA expression of MCF10A, MDA-MB-231, T47D, and ZR75 cells treated with or without PPAR agonist, Clofibrate (n = 3 per group; paired t-test, *p < 0.05, **p < 0.01 vs. untreated control)

Fig. 3

NNAT regulates intracellular calcium in ER + breast cancer cells through EndoR calcium storage. A Confocal fluorescent imaging revealed NNAT (green) colocalization with EndoR and lysosome (red and yellow merged images). B Basal Ca²⁺ concentration in T47D and ZR75 CRISPR knockout of NNAT cell lines (NNAT CRISPR) (n = 30 per group, paired t-test, p < 0.001 vs. control). C EndoR Ca²⁺ release following knockout of NNAT (n ≥ 16 per group; paired t-test, **p < 0.01 vs. respective CRISPR control). D Basal Ca²⁺ concentration in T47D and ZR75 cell lines overexpressing NNAT (n = 30 per group, paired t-test, p < 0.001 vs. control). E Change in proliferative capacity in T47D and ZR75 breast cancer cell lines overexpressed NNAT deletion construct (wild-type, NNAT, and dER). (n = 9 per group; one-way ANOVA tests (Tukey post hoc test) *p < 0.05, ***p < 0.001 vs. GFP).

Fig. 4

ORAI but not TRPC3 inhibition reduces NNAT-mediated elevation in intracellular Ca²⁺concentration in breast cancer cells. The overexpression of NNAT promotes significantly higher Ca²⁺ release from EndoR of ZR75 breast cancer cells. (n = 30 per group; paired t-test, ***p < 0.001 vs. control GFP) (A). Intracellular Ca²⁺ levels in standard and overexpressing NNAT T47D and ZR75 cell lines in the presence or absence of ORAI (pyr6) (B) or TRPC3 (pyr3) (C) pyrazole compound inhibitors (n = 30 per group; Two-way ANOVA tests (factors: NNAT and inhibitor; Tukey post hoc test) ***p < 0.001)