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MYC regulates the unfolded protein response and glucose and glutamine uptake in endocrine resistant breast cancer.

Shajahan-Haq AN, Cook KL, Schwartz-Roberts JL, Eltayeb AE, Demas DM, Warri AM, Facey CO, Hilakivi-Clarke LA, Clarke R - Mol. Cancer (2014)

Bottom Line: Inhibition of MYC reduced cell growth and uptake of both glucose and glutamine in resistant cells.These disparate effects are regulated, at different signaling junctions, by MYC more robustly in resistant cells.Endocrine resistant cells overexpress MYC and are better adapted to withstand periods of glucose deprivation and can use glutamine in the short term to maintain adequate metabolism to support cell survival.

View Article: PubMed Central - PubMed

Affiliation: Lombardi Comprehensive Cancer Center and Department of Oncology, Georgetown University School of Medicine, 3970 Reservoir Road NW, Washington, DC 20057, USA. ans33@georgetown.edu.

ABSTRACT

Background: About 70% of all breast cancers are estrogen receptor alpha positive (ER+) and are treated with antiestrogens. However, 50% of ER + tumors develop resistance to these drugs (endocrine resistance). In endocrine resistant cells, an adaptive pathway called the unfolded protein response (UPR) is elevated that allows cells to tolerate stress more efficiently than in sensitive cells. While the precise mechanism remains unclear, the UPR can trigger both pro-survival and pro-death outcomes that depend on the nature and magnitude of the stress. In this study, we identified MYC, an oncoprotein that is upregulated in endocrine resistant breast cancer, as a regulator of the UPR in glucose-deprived conditions.

Methods: ER+ human breast cancer cell lines (LCC1, LCC1, LY2 and LCC9) and rat mammary tumors were used to confirm upregulation of MYC in endocrine resistance. To evaluate functional relevance of proteins, siRNA-mediated inhibition or small molecule inhibitors were used. Cell density/number was evaluated with crystal violet assay; cell cycle and apoptosis were measured by flow cytometry. Relative quantification of glutamine metabolites were determined by mass spectrometry. Signaling molecules of the UPR, apoptosis or autophagy pathways were investigated by western blotting.

Results: Increased MYC function in resistant cells correlated with increased dependency on glutamine and glucose for survival. Inhibition of MYC reduced cell growth and uptake of both glucose and glutamine in resistant cells. Interestingly, in glucose-deprived conditions, glutamine induced apoptosis and necrosis, arrested autophagy, and triggered the unfolded protein response (UPR) though GRP78-IRE1α with two possible outcomes: (i) inhibition of cell growth by JNK activation in most cells and, (ii) promotion of cell growth by spliced XBP1 in the minority of cells. These disparate effects are regulated, at different signaling junctions, by MYC more robustly in resistant cells.

Conclusions: Endocrine resistant cells overexpress MYC and are better adapted to withstand periods of glucose deprivation and can use glutamine in the short term to maintain adequate metabolism to support cell survival. Our findings reveal a unique role for MYC in regulating cell fate through the UPR, and suggest that targeting glutamine metabolism may be a novel strategy in endocrine resistant breast cancer.

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Glutamine induces apoptosis and arrests autophagy via the UPR in glucose-deprived conditions.A, Significantly higher levels of apoptosis were seen in LCC9 compared with LCC1 cells following treatment with 2 or 4 mM glutamine at 48 h. ANOVA, p < 0.05; *p < 0.05 for LCC9 versus LCC1 for indicated treatment. B, Time-course, 0, 24 and 48 h, analysis of the autophagosome-associated proteins LC3II (marker for autophagosome formation or enlargement) and p62/SQSTM1 (marker for autophagosome activity, degradation of cargo). Increased formation of autophagosomes but arrested cargo degradation was seen within 24 h in both LCC1 and LCC9 cells in glutamine only media (and in no-glucose + no glutamine) conditions at 24 and 48 h but not in glucose-only (or in glucose + glutamine) media. C, In presence of 2 or 4 mM glutamine at 48 h, LCC9 cells showed increased levels of MYC and MAX and LC3II but no change in SQSTM1/p62. D, Cellular levels of total reactive species (RS) was significantly elevated in LCC9 compared to LCC1 cells in incomplete media (ANOVA, p < 0.001; *p < 0.05 for LCC9 versus LCC1 with no glucose + no glutamine).
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Fig7: Glutamine induces apoptosis and arrests autophagy via the UPR in glucose-deprived conditions.A, Significantly higher levels of apoptosis were seen in LCC9 compared with LCC1 cells following treatment with 2 or 4 mM glutamine at 48 h. ANOVA, p < 0.05; *p < 0.05 for LCC9 versus LCC1 for indicated treatment. B, Time-course, 0, 24 and 48 h, analysis of the autophagosome-associated proteins LC3II (marker for autophagosome formation or enlargement) and p62/SQSTM1 (marker for autophagosome activity, degradation of cargo). Increased formation of autophagosomes but arrested cargo degradation was seen within 24 h in both LCC1 and LCC9 cells in glutamine only media (and in no-glucose + no glutamine) conditions at 24 and 48 h but not in glucose-only (or in glucose + glutamine) media. C, In presence of 2 or 4 mM glutamine at 48 h, LCC9 cells showed increased levels of MYC and MAX and LC3II but no change in SQSTM1/p62. D, Cellular levels of total reactive species (RS) was significantly elevated in LCC9 compared to LCC1 cells in incomplete media (ANOVA, p < 0.001; *p < 0.05 for LCC9 versus LCC1 with no glucose + no glutamine).

Mentions: We next examined how the presence of glutamine in glucose-deprived conditions triggered a rapid decrease in cell number in antiestrogen resistant cells. To determine whether the decrease in cell survival in the presence of glutamine in glucose-deprived conditions was caused by induction of apoptosis, we measured apoptosis following 48 h of glutamine-only treatment in LCC1 and LCC9 cells. Apoptosis was significantly increased in LCC9 compared with LCC1 cells in the absence of both glutamine and glucose (Figure 7A). Moreover, in the presence of glutamine-only conditions, cells underwent significantly higher levels of apoptosis in LCC9 cells than in LCC1 cells. To determine autophagic flux, total protein from both LCC1 and LCC9 cells in the differ conditions (glucose + glutamine, glucose-only, glutamine-only, no glucose + no glutamine) were analyzed at 0, 24 and 48 h for p62/SQSTM1, LC3II and actin (Figure 7B). p62/SQSTM1 are adapter proteins that are autophagosome cargo markers used to determine activity within autolysosomes [31, 32]; however, each protein is selectively degraded by autophagy depending on the signaling cues and nature of stress [31]. An increase in LC3II expression is a marker of increased autophagosome formation and enlargement [33]. Increase in number of autophagosomes in the absence cargo degradation indicates interrupted autophagy that can promote apoptosis [34]. Moreover, Western blot analysis of total proteins from LCC9 cells treated with increasing concentrations of glutamine had higher levels of MYC, MAX and LC3II expression when compared with LCC1 cells; p62/SQSTM1 levels did not change (Figure 7C). Thus, while formation of autophagosomes may be triggered by the glutamine-only condition, autophagy-mediated degradation of cellular substrates is halted. Moreover, the induction of MYC suggests a possible role for this protein in regulating autophagy (see next section and Figure 8B). Disruption in cellular metabolic processes can lead to accumulation of reactive oxygen species (ROS) [35] and reactive nitrogen species (RNS) [36]. Figure 7D shows that deprivation of both glucose and glutamine significantly increased total reactive species (RS) levels in LCC9 cells. However, in both LCC1 and LCC9 cells, the presence of either glucose alone or glutamine alone did not change cellular RS levels compared with conditions where both metabolites are present. Thus, the decrease in cell number in glutamine-only conditions is independent of RS.Figure 7


MYC regulates the unfolded protein response and glucose and glutamine uptake in endocrine resistant breast cancer.

Shajahan-Haq AN, Cook KL, Schwartz-Roberts JL, Eltayeb AE, Demas DM, Warri AM, Facey CO, Hilakivi-Clarke LA, Clarke R - Mol. Cancer (2014)

Glutamine induces apoptosis and arrests autophagy via the UPR in glucose-deprived conditions.A, Significantly higher levels of apoptosis were seen in LCC9 compared with LCC1 cells following treatment with 2 or 4 mM glutamine at 48 h. ANOVA, p < 0.05; *p < 0.05 for LCC9 versus LCC1 for indicated treatment. B, Time-course, 0, 24 and 48 h, analysis of the autophagosome-associated proteins LC3II (marker for autophagosome formation or enlargement) and p62/SQSTM1 (marker for autophagosome activity, degradation of cargo). Increased formation of autophagosomes but arrested cargo degradation was seen within 24 h in both LCC1 and LCC9 cells in glutamine only media (and in no-glucose + no glutamine) conditions at 24 and 48 h but not in glucose-only (or in glucose + glutamine) media. C, In presence of 2 or 4 mM glutamine at 48 h, LCC9 cells showed increased levels of MYC and MAX and LC3II but no change in SQSTM1/p62. D, Cellular levels of total reactive species (RS) was significantly elevated in LCC9 compared to LCC1 cells in incomplete media (ANOVA, p < 0.001; *p < 0.05 for LCC9 versus LCC1 with no glucose + no glutamine).
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Related In: Results  -  Collection

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Fig7: Glutamine induces apoptosis and arrests autophagy via the UPR in glucose-deprived conditions.A, Significantly higher levels of apoptosis were seen in LCC9 compared with LCC1 cells following treatment with 2 or 4 mM glutamine at 48 h. ANOVA, p < 0.05; *p < 0.05 for LCC9 versus LCC1 for indicated treatment. B, Time-course, 0, 24 and 48 h, analysis of the autophagosome-associated proteins LC3II (marker for autophagosome formation or enlargement) and p62/SQSTM1 (marker for autophagosome activity, degradation of cargo). Increased formation of autophagosomes but arrested cargo degradation was seen within 24 h in both LCC1 and LCC9 cells in glutamine only media (and in no-glucose + no glutamine) conditions at 24 and 48 h but not in glucose-only (or in glucose + glutamine) media. C, In presence of 2 or 4 mM glutamine at 48 h, LCC9 cells showed increased levels of MYC and MAX and LC3II but no change in SQSTM1/p62. D, Cellular levels of total reactive species (RS) was significantly elevated in LCC9 compared to LCC1 cells in incomplete media (ANOVA, p < 0.001; *p < 0.05 for LCC9 versus LCC1 with no glucose + no glutamine).
Mentions: We next examined how the presence of glutamine in glucose-deprived conditions triggered a rapid decrease in cell number in antiestrogen resistant cells. To determine whether the decrease in cell survival in the presence of glutamine in glucose-deprived conditions was caused by induction of apoptosis, we measured apoptosis following 48 h of glutamine-only treatment in LCC1 and LCC9 cells. Apoptosis was significantly increased in LCC9 compared with LCC1 cells in the absence of both glutamine and glucose (Figure 7A). Moreover, in the presence of glutamine-only conditions, cells underwent significantly higher levels of apoptosis in LCC9 cells than in LCC1 cells. To determine autophagic flux, total protein from both LCC1 and LCC9 cells in the differ conditions (glucose + glutamine, glucose-only, glutamine-only, no glucose + no glutamine) were analyzed at 0, 24 and 48 h for p62/SQSTM1, LC3II and actin (Figure 7B). p62/SQSTM1 are adapter proteins that are autophagosome cargo markers used to determine activity within autolysosomes [31, 32]; however, each protein is selectively degraded by autophagy depending on the signaling cues and nature of stress [31]. An increase in LC3II expression is a marker of increased autophagosome formation and enlargement [33]. Increase in number of autophagosomes in the absence cargo degradation indicates interrupted autophagy that can promote apoptosis [34]. Moreover, Western blot analysis of total proteins from LCC9 cells treated with increasing concentrations of glutamine had higher levels of MYC, MAX and LC3II expression when compared with LCC1 cells; p62/SQSTM1 levels did not change (Figure 7C). Thus, while formation of autophagosomes may be triggered by the glutamine-only condition, autophagy-mediated degradation of cellular substrates is halted. Moreover, the induction of MYC suggests a possible role for this protein in regulating autophagy (see next section and Figure 8B). Disruption in cellular metabolic processes can lead to accumulation of reactive oxygen species (ROS) [35] and reactive nitrogen species (RNS) [36]. Figure 7D shows that deprivation of both glucose and glutamine significantly increased total reactive species (RS) levels in LCC9 cells. However, in both LCC1 and LCC9 cells, the presence of either glucose alone or glutamine alone did not change cellular RS levels compared with conditions where both metabolites are present. Thus, the decrease in cell number in glutamine-only conditions is independent of RS.Figure 7

Bottom Line: Inhibition of MYC reduced cell growth and uptake of both glucose and glutamine in resistant cells.These disparate effects are regulated, at different signaling junctions, by MYC more robustly in resistant cells.Endocrine resistant cells overexpress MYC and are better adapted to withstand periods of glucose deprivation and can use glutamine in the short term to maintain adequate metabolism to support cell survival.

View Article: PubMed Central - PubMed

Affiliation: Lombardi Comprehensive Cancer Center and Department of Oncology, Georgetown University School of Medicine, 3970 Reservoir Road NW, Washington, DC 20057, USA. ans33@georgetown.edu.

ABSTRACT

Background: About 70% of all breast cancers are estrogen receptor alpha positive (ER+) and are treated with antiestrogens. However, 50% of ER + tumors develop resistance to these drugs (endocrine resistance). In endocrine resistant cells, an adaptive pathway called the unfolded protein response (UPR) is elevated that allows cells to tolerate stress more efficiently than in sensitive cells. While the precise mechanism remains unclear, the UPR can trigger both pro-survival and pro-death outcomes that depend on the nature and magnitude of the stress. In this study, we identified MYC, an oncoprotein that is upregulated in endocrine resistant breast cancer, as a regulator of the UPR in glucose-deprived conditions.

Methods: ER+ human breast cancer cell lines (LCC1, LCC1, LY2 and LCC9) and rat mammary tumors were used to confirm upregulation of MYC in endocrine resistance. To evaluate functional relevance of proteins, siRNA-mediated inhibition or small molecule inhibitors were used. Cell density/number was evaluated with crystal violet assay; cell cycle and apoptosis were measured by flow cytometry. Relative quantification of glutamine metabolites were determined by mass spectrometry. Signaling molecules of the UPR, apoptosis or autophagy pathways were investigated by western blotting.

Results: Increased MYC function in resistant cells correlated with increased dependency on glutamine and glucose for survival. Inhibition of MYC reduced cell growth and uptake of both glucose and glutamine in resistant cells. Interestingly, in glucose-deprived conditions, glutamine induced apoptosis and necrosis, arrested autophagy, and triggered the unfolded protein response (UPR) though GRP78-IRE1α with two possible outcomes: (i) inhibition of cell growth by JNK activation in most cells and, (ii) promotion of cell growth by spliced XBP1 in the minority of cells. These disparate effects are regulated, at different signaling junctions, by MYC more robustly in resistant cells.

Conclusions: Endocrine resistant cells overexpress MYC and are better adapted to withstand periods of glucose deprivation and can use glutamine in the short term to maintain adequate metabolism to support cell survival. Our findings reveal a unique role for MYC in regulating cell fate through the UPR, and suggest that targeting glutamine metabolism may be a novel strategy in endocrine resistant breast cancer.

Show MeSH
Related in: MedlinePlus