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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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Analysis of NRG-induced autophagy compared to Rapamycin-mediated autophagy.(A) LNCaP cells were treated with either 100 ng/ml NRG or 50 nM rapamycin (Rapa) for 24 h, in the presence or in the absence of 10 mM NAC. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3, anti-phospho-S6K and anti-S6K antibodies. (B) Densitometric analysis of the results described in A is represented as fold induction of the control untreated cells (n = 3, means ± S.D). (C) Representative images of cell morphology following treatments with NRG and rapamycin are shown (Olympus, 20×magnification). (D) LNCaP cells were treated with either 100 ng/ml NRG, 50 nM rapamycin or both for 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 antibodies. This experiment was repeated three times with similar results.
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pone-0036828-g005: Analysis of NRG-induced autophagy compared to Rapamycin-mediated autophagy.(A) LNCaP cells were treated with either 100 ng/ml NRG or 50 nM rapamycin (Rapa) for 24 h, in the presence or in the absence of 10 mM NAC. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3, anti-phospho-S6K and anti-S6K antibodies. (B) Densitometric analysis of the results described in A is represented as fold induction of the control untreated cells (n = 3, means ± S.D). (C) Representative images of cell morphology following treatments with NRG and rapamycin are shown (Olympus, 20×magnification). (D) LNCaP cells were treated with either 100 ng/ml NRG, 50 nM rapamycin or both for 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 antibodies. This experiment was repeated three times with similar results.

Mentions: Since NRG induced S6K phosphorylation but also induced autophagy, we next compared autophagy induced by mTOR inhibition to the autophagy induced by NRG. The mTOR inhibitor, rapamycin, was previously shown to induce autophagy by downregulating mTOR activity [37], [40]. We found that rapamycin treatment as well as NRG treatment induced autophagy, as judged by the increased LC3-II/LC3-I ratio (Figure 5A and B) and by enhanced LC3 puncta formation (Figure 1B). However, as expected, S6K phosphorylation was increased following NRG treatment but reduced following rapamycin treatment. In addition, NAC treatment inhibited autophagy induced by rapamycin and NRG, however it had no effect on NRG-induced S6K phosphorylation. Interestingly, rapamycin treatment although inhibited cell growth [29], had no effect on cells morphology, while NRG caused a dramatic morphological change as previously described [28], [29] and as demonstrated in Figure 5C and Figure S1 (round and detached cells). These results demonstrate that NRG-induced autophagy differs from autophagy triggered by rapamycin. Next, we examined the effect of NRG and rapamycin co-treatment on autophagy-inducion in LNCaP cells (Figure 5D). As shown, combined treatment induced higher levels of LC3II compared to each treatment alone, suggesting that rapamycin and NRG might act through different signaling pathways to induce 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)

Analysis of NRG-induced autophagy compared to Rapamycin-mediated autophagy.(A) LNCaP cells were treated with either 100 ng/ml NRG or 50 nM rapamycin (Rapa) for 24 h, in the presence or in the absence of 10 mM NAC. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3, anti-phospho-S6K and anti-S6K antibodies. (B) Densitometric analysis of the results described in A is represented as fold induction of the control untreated cells (n = 3, means ± S.D). (C) Representative images of cell morphology following treatments with NRG and rapamycin are shown (Olympus, 20×magnification). (D) LNCaP cells were treated with either 100 ng/ml NRG, 50 nM rapamycin or both for 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 antibodies. This experiment was repeated three times with similar results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351469&req=5

pone-0036828-g005: Analysis of NRG-induced autophagy compared to Rapamycin-mediated autophagy.(A) LNCaP cells were treated with either 100 ng/ml NRG or 50 nM rapamycin (Rapa) for 24 h, in the presence or in the absence of 10 mM NAC. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3, anti-phospho-S6K and anti-S6K antibodies. (B) Densitometric analysis of the results described in A is represented as fold induction of the control untreated cells (n = 3, means ± S.D). (C) Representative images of cell morphology following treatments with NRG and rapamycin are shown (Olympus, 20×magnification). (D) LNCaP cells were treated with either 100 ng/ml NRG, 50 nM rapamycin or both for 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 antibodies. This experiment was repeated three times with similar results.
Mentions: Since NRG induced S6K phosphorylation but also induced autophagy, we next compared autophagy induced by mTOR inhibition to the autophagy induced by NRG. The mTOR inhibitor, rapamycin, was previously shown to induce autophagy by downregulating mTOR activity [37], [40]. We found that rapamycin treatment as well as NRG treatment induced autophagy, as judged by the increased LC3-II/LC3-I ratio (Figure 5A and B) and by enhanced LC3 puncta formation (Figure 1B). However, as expected, S6K phosphorylation was increased following NRG treatment but reduced following rapamycin treatment. In addition, NAC treatment inhibited autophagy induced by rapamycin and NRG, however it had no effect on NRG-induced S6K phosphorylation. Interestingly, rapamycin treatment although inhibited cell growth [29], had no effect on cells morphology, while NRG caused a dramatic morphological change as previously described [28], [29] and as demonstrated in Figure 5C and Figure S1 (round and detached cells). These results demonstrate that NRG-induced autophagy differs from autophagy triggered by rapamycin. Next, we examined the effect of NRG and rapamycin co-treatment on autophagy-inducion in LNCaP cells (Figure 5D). As shown, combined treatment induced higher levels of LC3II compared to each treatment alone, suggesting that rapamycin and NRG might act through different signaling pathways to induce 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