MECHANISTIC INSIGHT INTO THE PROLACTIN-/ANDROGEN-INDUCIBLE CARBOXYPEPTIDASE-D AND EDD E3 UBIQUITIN LIGASE GENES IN TRIPLE-NEGATIVE BREAST CANCER

Abstract

Membrane-bound Carboxypeptidase-D (CPD) hydrolyzes C-terminal arginine residues from extracellular substrates. Released arginine, taken into cells, is converted into nitric oxide (NO). Our laboratory has shown that CPD mRNA/protein levels are upregulated by prolactin (PRL), 17β-estradiol, and androgen testosterone in breast cancer (BCa) cells, in turn, increasing NO production to promote BCa cell survival. EDD E3 ubiquitin ligase identified by differential display (EDD) is an E3 ubiquitin ligase protein normally involved in protein turnover, however it is frequently overexpressed in cancer cells. EDD has been implicated by our laboratory in the mammalian-target-of-rapamycin (mTOR) signalling pathway that regulates the initiation of protein translation. Like CPD, EDD protein levels are upregulated by hormones, specifically PRL, progesterone, and synthetic androgen R1881. This study investigated the role of PRL/androgen-inducible EDD and CPD in BCa, particularly in the aggressive triple-negative breast cancer (TNBC) subtype. This was accomplished in vivo by immunohistochemical analysis of EDD in archival human breast specimens as well as examining the effect of high EDD or CPD mRNA expression on the survival probability of BCa patients. A multitude of in vitro experiments were conducted to demonstrate hormonal upregulation of EDD or CPD and the biological effects of altered EDD or CPD gene expression in TNBC cell lines. The present study showed that high CPD mRNA expression in BCa patients correlated with a poorer probability of relapse-free survival. CPD protein expression was upregulated by PRL and R1881 in a time-/dose-dependent manner and was accompanied by increased intracellular NO production, which enhanced TNBC cell survival in vitro. Depletion of CPD from TNBC cell lines using small-interfering ribonucleic acid (siRNA) resulted in decreased TNBC cell viability. EDD immunostaining was shown to be low in benign human breast tissue but increased with breast cancer progression in vivo. High EDD mRNA expression correlated with a poorer probability of overall and relapse-free survival in BCa and TNBC patients, respectively. EDD protein expression was upregulated by PRL and R1881 in a time-/dose-dependent manner in vitro and was speculated to occur at the transcriptional level due to the presence of putative hormone response elements in the EDD gene promoter. SiRNA-mediated knockdown of EDD gene expression induced a variety of effects including decreased TNBC cell viability, increased expression of pro-apoptotic-associated proteins MOAP-1 and Bax, cleavage of Caspase-7 and PARP-1, decreased PRL-/R1881-induced phosphorylation of initiation factor 4E binding protein-1 (4E-BP1), and subsequent 4E release, as well as decreased resistance to anti-cancer drugs. In summary, PRL/R1881-inducible CPD increased TNBC cell survival through the production of NO. EDD levels increased with BCa progression and loss of PRL/R1881-inducible EDD decreased TORC1 signalling, promoted pro-apoptotic protein expression, and decreased anti-cancer drug resistance in BCa cells. Collectively, this work supports EDD and CPD as therapeutic targets for BCa, including TNBC, and suggest that EDD and CPD expression may predict BCa responsiveness to various anti-cancer drug treatments.