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Mitochondrial p32 is upregulated in Myc expressing brain cancers and mediates glutamine addiction.

Fogal V, Babic I, Chao Y, Pastorino S, Mukthavaram R, Jiang P, Cho YJ, Pingle SC, Crawford JR, Piccioni DE, Kesari S - Oncotarget (2015)

Bottom Line: Loss of p32 in glutamine addicted glioma cells induced resistance to glutamine deprivation and imparted sensitivity to glucose withdrawal.Finally, we provide evidence that p32 expression contributes to Myc-induced glutamine addiction of cancer cells.Our findings suggest that Myc promotes the expression of p32, which is required to maintain sufficient respiratory capacity to sustain glutamine metabolism in Myc transformed cells.

View Article: PubMed Central - PubMed

Affiliation: Translational Neuro-Oncology Laboratories, Moores Cancer Center, University of California San Diego, La Jolla, CA.

ABSTRACT
Metabolic reprogramming is a key feature of tumorigenesis that is controlled by oncogenes. Enhanced utilization of glucose and glutamine are the best-established hallmarks of tumor metabolism. The oncogene c-Myc is one of the major players responsible for this metabolic alteration. However, the molecular mechanisms involved in Myc-induced metabolic reprogramming are not well defined. Here we identify p32, a mitochondrial protein known to play a role in the expression of mitochondrial respiratory chain complexes, as a critical player in Myc-induced glutamine addiction. We show that p32 is a direct transcriptional target of Myc and that high level of Myc in malignant brain cancers correlates with high expression of p32. Attenuation of p32 expression reduced growth rate of glioma cells expressing Myc and impaired tumor formation in vivo. Loss of p32 in glutamine addicted glioma cells induced resistance to glutamine deprivation and imparted sensitivity to glucose withdrawal. Finally, we provide evidence that p32 expression contributes to Myc-induced glutamine addiction of cancer cells. Our findings suggest that Myc promotes the expression of p32, which is required to maintain sufficient respiratory capacity to sustain glutamine metabolism in Myc transformed cells.

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Myc binds to the p32 promoter(A) Schematic of the human p32 promoter sequence starting from 3 kb upstream of exon 1 to exon 6 of the c1qbp gene. Exons (ex) are represented by black boxes and the E box is indicated with a vertical bar. Horizontal bars indicate the regions amplified for scanning ChIP analysis. E2 is the p32 promoter region containing the E-box and amplified by conventional PCR (Figure 3B). (B and C) A ChIP assay was performed on SF188 cells with anti Myc antibody and IgG as a control. Precipitated chromatin was PCR-amplified using E2 primers (B). Quantitative PCRs were performed to amplify and quantify E1, ex1, in1, and in3 promoter regions. Shown are averages with standard deviations of triplicate independent experiments. Binding to amplicons is shown as a percentage of total input DNA plotted relative to the signal obtained from IgG precipitation. (D) Quantification of MycER binding to the p32 promoter after addition of OHT. MRC5 MycER cells were serum starved for 24 hrs and either treated with vehicle (EtOH) or 250nM OHT for 4 hours. Subsequently ChIP assays were conducted as described in (B). The bar graph presented is indicative of three independent experiments.
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Figure 4: Myc binds to the p32 promoter(A) Schematic of the human p32 promoter sequence starting from 3 kb upstream of exon 1 to exon 6 of the c1qbp gene. Exons (ex) are represented by black boxes and the E box is indicated with a vertical bar. Horizontal bars indicate the regions amplified for scanning ChIP analysis. E2 is the p32 promoter region containing the E-box and amplified by conventional PCR (Figure 3B). (B and C) A ChIP assay was performed on SF188 cells with anti Myc antibody and IgG as a control. Precipitated chromatin was PCR-amplified using E2 primers (B). Quantitative PCRs were performed to amplify and quantify E1, ex1, in1, and in3 promoter regions. Shown are averages with standard deviations of triplicate independent experiments. Binding to amplicons is shown as a percentage of total input DNA plotted relative to the signal obtained from IgG precipitation. (D) Quantification of MycER binding to the p32 promoter after addition of OHT. MRC5 MycER cells were serum starved for 24 hrs and either treated with vehicle (EtOH) or 250nM OHT for 4 hours. Subsequently ChIP assays were conducted as described in (B). The bar graph presented is indicative of three independent experiments.

Mentions: Myc is known to bind to a canonical consensus DNA sequence CACGTG, termed the E-box, but can also bind several other non-canonical DNA motifs [48]. Analysis of the p32 promoter sequence identified several described consensus sequences for Myc binding (not shown), with an E-box among them, just upstream (−24 to −19 bp) of the p32 transcriptional start codon (Fig. 4A). We used ChIP to test whether p32/C1QBP may be a direct downstream target of Myc transactivation. Using SF188 cells and primers flanking the E-box, we found that the p32 promoter was significantly enriched in the Myc ChIP sample compared to IgG control (Fig. 4B). Primers amplifying other p32 promoter regions (ex1, in1 and in3 Fig. 4B and 4C) identified specific Myc binding in regions proximal but not distal to the E-box. To further confirm these data, a ChIP analysis was performed in MRC5 MycER cells. Compared with vehicle-treated cells, activation of MycER by OHT led to a 3.5-fold enrichment in p32 promoter binding at the E-box (Fig. 4D). There was no binding enrichment in an area of p32 gene distal to the E-box. Taken together, these data indicate that Myc protein can directly bind to a regulatory region of p32 promoter suggesting that this gene is a direct target of Myc.


Mitochondrial p32 is upregulated in Myc expressing brain cancers and mediates glutamine addiction.

Fogal V, Babic I, Chao Y, Pastorino S, Mukthavaram R, Jiang P, Cho YJ, Pingle SC, Crawford JR, Piccioni DE, Kesari S - Oncotarget (2015)

Myc binds to the p32 promoter(A) Schematic of the human p32 promoter sequence starting from 3 kb upstream of exon 1 to exon 6 of the c1qbp gene. Exons (ex) are represented by black boxes and the E box is indicated with a vertical bar. Horizontal bars indicate the regions amplified for scanning ChIP analysis. E2 is the p32 promoter region containing the E-box and amplified by conventional PCR (Figure 3B). (B and C) A ChIP assay was performed on SF188 cells with anti Myc antibody and IgG as a control. Precipitated chromatin was PCR-amplified using E2 primers (B). Quantitative PCRs were performed to amplify and quantify E1, ex1, in1, and in3 promoter regions. Shown are averages with standard deviations of triplicate independent experiments. Binding to amplicons is shown as a percentage of total input DNA plotted relative to the signal obtained from IgG precipitation. (D) Quantification of MycER binding to the p32 promoter after addition of OHT. MRC5 MycER cells were serum starved for 24 hrs and either treated with vehicle (EtOH) or 250nM OHT for 4 hours. Subsequently ChIP assays were conducted as described in (B). The bar graph presented is indicative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Myc binds to the p32 promoter(A) Schematic of the human p32 promoter sequence starting from 3 kb upstream of exon 1 to exon 6 of the c1qbp gene. Exons (ex) are represented by black boxes and the E box is indicated with a vertical bar. Horizontal bars indicate the regions amplified for scanning ChIP analysis. E2 is the p32 promoter region containing the E-box and amplified by conventional PCR (Figure 3B). (B and C) A ChIP assay was performed on SF188 cells with anti Myc antibody and IgG as a control. Precipitated chromatin was PCR-amplified using E2 primers (B). Quantitative PCRs were performed to amplify and quantify E1, ex1, in1, and in3 promoter regions. Shown are averages with standard deviations of triplicate independent experiments. Binding to amplicons is shown as a percentage of total input DNA plotted relative to the signal obtained from IgG precipitation. (D) Quantification of MycER binding to the p32 promoter after addition of OHT. MRC5 MycER cells were serum starved for 24 hrs and either treated with vehicle (EtOH) or 250nM OHT for 4 hours. Subsequently ChIP assays were conducted as described in (B). The bar graph presented is indicative of three independent experiments.
Mentions: Myc is known to bind to a canonical consensus DNA sequence CACGTG, termed the E-box, but can also bind several other non-canonical DNA motifs [48]. Analysis of the p32 promoter sequence identified several described consensus sequences for Myc binding (not shown), with an E-box among them, just upstream (−24 to −19 bp) of the p32 transcriptional start codon (Fig. 4A). We used ChIP to test whether p32/C1QBP may be a direct downstream target of Myc transactivation. Using SF188 cells and primers flanking the E-box, we found that the p32 promoter was significantly enriched in the Myc ChIP sample compared to IgG control (Fig. 4B). Primers amplifying other p32 promoter regions (ex1, in1 and in3 Fig. 4B and 4C) identified specific Myc binding in regions proximal but not distal to the E-box. To further confirm these data, a ChIP analysis was performed in MRC5 MycER cells. Compared with vehicle-treated cells, activation of MycER by OHT led to a 3.5-fold enrichment in p32 promoter binding at the E-box (Fig. 4D). There was no binding enrichment in an area of p32 gene distal to the E-box. Taken together, these data indicate that Myc protein can directly bind to a regulatory region of p32 promoter suggesting that this gene is a direct target of Myc.

Bottom Line: Loss of p32 in glutamine addicted glioma cells induced resistance to glutamine deprivation and imparted sensitivity to glucose withdrawal.Finally, we provide evidence that p32 expression contributes to Myc-induced glutamine addiction of cancer cells.Our findings suggest that Myc promotes the expression of p32, which is required to maintain sufficient respiratory capacity to sustain glutamine metabolism in Myc transformed cells.

View Article: PubMed Central - PubMed

Affiliation: Translational Neuro-Oncology Laboratories, Moores Cancer Center, University of California San Diego, La Jolla, CA.

ABSTRACT
Metabolic reprogramming is a key feature of tumorigenesis that is controlled by oncogenes. Enhanced utilization of glucose and glutamine are the best-established hallmarks of tumor metabolism. The oncogene c-Myc is one of the major players responsible for this metabolic alteration. However, the molecular mechanisms involved in Myc-induced metabolic reprogramming are not well defined. Here we identify p32, a mitochondrial protein known to play a role in the expression of mitochondrial respiratory chain complexes, as a critical player in Myc-induced glutamine addiction. We show that p32 is a direct transcriptional target of Myc and that high level of Myc in malignant brain cancers correlates with high expression of p32. Attenuation of p32 expression reduced growth rate of glioma cells expressing Myc and impaired tumor formation in vivo. Loss of p32 in glutamine addicted glioma cells induced resistance to glutamine deprivation and imparted sensitivity to glucose withdrawal. Finally, we provide evidence that p32 expression contributes to Myc-induced glutamine addiction of cancer cells. Our findings suggest that Myc promotes the expression of p32, which is required to maintain sufficient respiratory capacity to sustain glutamine metabolism in Myc transformed cells.

Show MeSH
Related in: MedlinePlus