Date Published: May 14, 2012
Publisher: Public Library of Science
Author(s): Eran Schmukler, Ben Shai, Marcelo Ehrlich, Ronit Pinkas-Kramarski, Antimo Migliaccio. http://doi.org/10.1371/journal.pone.0036828
Growth factors activating the ErbB receptors have been described in prostate tumors. The androgen dependent prostate cancer cell line, LNCaP, expresses the ErbB-1, ErbB-2 and ErbB-3 receptor tyrosine kinases. Previously, it was demonstrated that NRG activates ErbB-2/ErbB-3 heterodimers to induce LNCaP cell death, whereas, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers to induce cell growth and survival. It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death.
In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited.
Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.
Prostatic carcinoma is one of the most common male cancers. Prostate cells growth is regulated by hormones, growth factors and their respective receptors. Among the most frequent group of receptors implicated in human cancers is the ErbB subfamily of receptor tyrosine kinases , , . This family includes four receptors ErbB-1-ErbB-4. Whereas ErbB-1 receptor (known as epidermal growth factor receptor, EGFR), is activated by EGF and EGF-like ligands, ErbB-3 and ErbB-4 receptors are activated by NRG/neuregulin isoforms and ErbB-2 receptor has no known ligand . These receptors are expressed in the prostate epithelium, whereas, ErbB-1 ligands are expressed in the stroma and NRGs are expressed in the stroma and in the basal and secretory epithelium .
Prostate cancers typically start as androgen-sensitive lesions but frequently develop into androgen-insensitive lesions with the progression to advanced stages. The LNCaP androgen-dependent cell line expresses high levels of ErbB-2 and ErbB-3 compared to other human prostate cancer cells . In addition, these cells do not express NRG but they do express TGF-α and EGF, which can function as autocrine activators of the EGFR . Previously, it was demonstrated that in LNCaP cells grown without androgen mimetic, NRG but not EGF induces cell death. This effect of NRG on cell death is mediated by ErbB-2/ErbB-3 heterodimers . It was also demonstrated that the cell death induced by NRG is inhibited by PI3K inhibitors, indicating that NRG may induce autophagic cell death . In the present study, we demonstrate for the first time that NRG indeed induces autophagy in LNCaP cells, using two assays: immunoblot analysis with anti-LC3 antibodies and microscopic analysis of LC3-GFP staining in LNCaP cells. This effect of NRG was also inhibited by 3-MA. However, the autophagy induced by NRG is incomplete since no degradation of p62 protein following NRG treatment was detected. Thus, NRG activating ErbB2/ErbB3 heterodimers induces incomplete autophagy and cell death in LNCaP cells.