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Neuregulin promotes incomplete autophagy of prostate cancer cells that is independent of mTOR pathway inhibition.

Schmukler E, Shai B, Ehrlich M, Pinkas-Kramarski R - PLoS ONE (2012)

Bottom Line: 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.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.These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Tel-Aviv University, Ramat-Aviv, Israel.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

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NRG-mediated signaling in LNCaP cells.(A) LNCaP cells were treated with 100 ng/ml NRG with or without 10 mM NAC for 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with the indicated antibodies. (B) Densitometric analysis of several repeats is presented as fold induction of the control untreated cells. The means of bands intensity were standardized compared to the total unphosphorylated protein signals (Data are the mean fold induction ± S.D; *p<0.05).
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pone-0036828-g004: NRG-mediated signaling in LNCaP cells.(A) LNCaP cells were treated with 100 ng/ml NRG with or without 10 mM NAC for 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with the indicated antibodies. (B) Densitometric analysis of several repeats is presented as fold induction of the control untreated cells. The means of bands intensity were standardized compared to the total unphosphorylated protein signals (Data are the mean fold induction ± S.D; *p<0.05).

Mentions: In order to determine the signaling pathway leading to NRG-induced autophagy and cell death, we first examined the Akt/mTOR signaling pathway. LNCaP cells express PTEN mutation that leads to Akt activation [35], [36]. Akt activates mTOR, which is a known negative regulator of autophagy [37], [38]. Thus, it was reasonable to examine the phosphorylation and activation of these proteins. LNCaP cells were treated with NRG for 24 h and the activation of Akt and mTOR were examined using anti-phospho Akt and anti-phospho S6K antibodies. As shown in Figure 4A, basal level of phosphorylated Akt and S6K was observed in the control untreated cells, however, NRG treatment increased the level of phosphorylated Akt and S6K. NAC, which inhibits NRG-induced autophagy, had no effect on the phosphorylation levels of neither Akt nor S6K. These results may suggest that NRG-induced autophagy is independent of mTOR inhibition. To further explore the signaling pathway involved in NRG-induced autophagy in LNCaP cells, we next examined the activation of Erk and JNK, two known mitogen activated protein kinases (MAPKs) which are downstream signaling components of ErbB receptors [39]. Specific anti phospho-protein antibodies to Erk1/2 and JNK were used. As shown in Figure 4, NRG induced an increase in the phosphorylation of Erk1/2 and JNK. NAC, which inhibits NRG-induced autophagy, had no effect on Erk1/2 phosphorylation, indicating that Erk activation is not involved in NRG-induced autophagy or that NAC acts downstream to Erk activation. On the other hand, JNK phosphorylation was strongly reduced in the presence of NAC, indicating that JNK may be a potential mediator of NRG-induced autophagy.


Neuregulin promotes incomplete autophagy of prostate cancer cells that is independent of mTOR pathway inhibition.

Schmukler E, Shai B, Ehrlich M, Pinkas-Kramarski R - PLoS ONE (2012)

NRG-mediated signaling in LNCaP cells.(A) LNCaP cells were treated with 100 ng/ml NRG with or without 10 mM NAC for 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with the indicated antibodies. (B) Densitometric analysis of several repeats is presented as fold induction of the control untreated cells. The means of bands intensity were standardized compared to the total unphosphorylated protein signals (Data are the mean fold induction ± S.D; *p<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351469&req=5

pone-0036828-g004: NRG-mediated signaling in LNCaP cells.(A) LNCaP cells were treated with 100 ng/ml NRG with or without 10 mM NAC for 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with the indicated antibodies. (B) Densitometric analysis of several repeats is presented as fold induction of the control untreated cells. The means of bands intensity were standardized compared to the total unphosphorylated protein signals (Data are the mean fold induction ± S.D; *p<0.05).
Mentions: In order to determine the signaling pathway leading to NRG-induced autophagy and cell death, we first examined the Akt/mTOR signaling pathway. LNCaP cells express PTEN mutation that leads to Akt activation [35], [36]. Akt activates mTOR, which is a known negative regulator of autophagy [37], [38]. Thus, it was reasonable to examine the phosphorylation and activation of these proteins. LNCaP cells were treated with NRG for 24 h and the activation of Akt and mTOR were examined using anti-phospho Akt and anti-phospho S6K antibodies. As shown in Figure 4A, basal level of phosphorylated Akt and S6K was observed in the control untreated cells, however, NRG treatment increased the level of phosphorylated Akt and S6K. NAC, which inhibits NRG-induced autophagy, had no effect on the phosphorylation levels of neither Akt nor S6K. These results may suggest that NRG-induced autophagy is independent of mTOR inhibition. To further explore the signaling pathway involved in NRG-induced autophagy in LNCaP cells, we next examined the activation of Erk and JNK, two known mitogen activated protein kinases (MAPKs) which are downstream signaling components of ErbB receptors [39]. Specific anti phospho-protein antibodies to Erk1/2 and JNK were used. As shown in Figure 4, NRG induced an increase in the phosphorylation of Erk1/2 and JNK. NAC, which inhibits NRG-induced autophagy, had no effect on Erk1/2 phosphorylation, indicating that Erk activation is not involved in NRG-induced autophagy or that NAC acts downstream to Erk activation. On the other hand, JNK phosphorylation was strongly reduced in the presence of NAC, indicating that JNK may be a potential mediator of NRG-induced autophagy.

Bottom Line: 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.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.These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Tel-Aviv University, Ramat-Aviv, Israel.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus