Introduction: Molecular apocrine is a subtype of estrogen receptor (ER)-negative breast cancer that is characterized by a steroid-response gene signature. We have recently identified a positive feedback loop between androgen receptor (AR) and extracellular signal-regulated kinase (ERK) signaling in this subtype. In this study, we investigated the transcriptional regulation of molecular apocrine genes by the AR-ERK feedback loop.
Methods: The transcriptional effects of AR and ERK inhibition on molecular apocrine genes were assessed in cell lines. The most regulated gene in this process, prolactin-induced protein (PIP), was further studied using immunohistochemistry of breast tumors and xenograft models. The transcriptional regulation of PIP was assessed by luciferase reporter assay and chromatin immunoprecipitation. The functional significance of PIP in cell invasion and viability was assessed using siRNA knockdown experiments and the mechanism of PIP effect on integrin-β1 signaling was studied using immunoblotting and immunoprecipitation.
Results: We found that PIP is the most regulated molecular apocrine gene by the AR-ERK feedback loop and is overexpressed in ER-/AR+ breast tumors. In addition, PIP expression is regulated by AR-ERK signaling in xenograft models. These observations are explained by the fact that PIP is a target gene of the ERK-CREB1 pathway and is also induced by AR activation. Furthermore, we demonstrated that PIP has a significant functional role in maintaining cell invasion and viability of molecular apocrine cells because of a positive regulatory effect on the Integrin-ERK and Integrin-Akt signaling pathways. In fact, PIP-knockdown markedly decreases the phosphorylation of ERK, Akt, and CREB1. Importantly, PIP knockdown leads to a marked reduction of integrin-β1 binding to ILK1 and ErbB2 that can be reversed by the addition of fibronectin fragments.
Conclusions: We have identified a novel feedback loop between PIP and CREB1 mediated through the Integrin signaling pathway. In this process, PIP cleaves fibronectin to release fragments that activate integrin signaling, which in turn increases PIP expression through the ERK-CREB1 pathway. In addition, we demonstrated that PIP expression has a profound effect on cell invasion and the viability of molecular apocrine cells. Therefore, PIP signaling may be a potential therapeutic target in molecular apocrine breast cancer.