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Acidosis decreases c-Myc oncogene expression in human lymphoma cells: a role for the proton-sensing G protein-coupled receptor TDAG8.

Li Z, Dong L, Dean E, Yang LV - Int J Mol Sci (2013)

Bottom Line: The pH-sensing receptor TDAG8 (GPR65) is involved in acidosis-induced c-Myc downregulation.Acidic pH alone is insufficient to reduce c-Myc expression, as it does not decrease c-Myc in H1299 lung cancer cells expressing very low levels of pH-sensing G protein-coupled receptors (GPCRs).Collectively, our results identify a novel mechanism of c-Myc regulation by acidosis in the tumor microenvironment and indicate that modulation of TDAG8 and related pH-sensing receptor pathways may be exploited as a new approach to inhibit Myc expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA. yangl@ecu.edu.

ABSTRACT
Acidosis is a biochemical hallmark of the tumor microenvironment. Here, we report that acute acidosis decreases c-Myc oncogene expression in U937 human lymphoma cells. The level of c-Myc transcripts, but not mRNA or protein stability, contributes to c-Myc protein reduction under acidosis. The pH-sensing receptor TDAG8 (GPR65) is involved in acidosis-induced c-Myc downregulation. TDAG8 is expressed in U937 lymphoma cells, and the overexpression or knockdown of TDAG8 further decreases or partially rescues c-Myc expression, respectively. Acidic pH alone is insufficient to reduce c-Myc expression, as it does not decrease c-Myc in H1299 lung cancer cells expressing very low levels of pH-sensing G protein-coupled receptors (GPCRs). Instead, c-Myc is slightly increased by acidosis in H1299 cells, but this increase is completely inhibited by ectopic overexpression of TDAG8. Interestingly, TDAG8 expression is decreased by more than 50% in human lymphoma samples in comparison to non-tumorous lymph nodes and spleens, suggesting a potential tumor suppressor function of TDAG8 in lymphoma. Collectively, our results identify a novel mechanism of c-Myc regulation by acidosis in the tumor microenvironment and indicate that modulation of TDAG8 and related pH-sensing receptor pathways may be exploited as a new approach to inhibit Myc expression.

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Ectopic overexpression of TDAG8 inhibits the upregulation of c-Myc protein by acidosis in H1299 cells. (A) c-Myc protein is moderately increased by acidosis in H1299 cells. Cells treated with HEM-buffered RPMI medium at pH 7.4 or pH 6.4 for three and 6 h were subject to Western blot assay using anti-c-Myc antibody. β-Actin was used as a loading control; (B) Low expression of proton-sensing GPCRs in H1299 cells. Total RNAs were isolated from H1299 parental, H1299/vector and H1299/hTDAG8 cells and subject to RT-PCR using gene-specific primers of GPR4, G2A, TDAG8 and OGR1. β-Actin was used as a positive control; (C) c-Myc protein is reduced by ectopic overexpression of TDAG8. Cells were treated with HEM-buffered RPMI medium at pH 7.4 or pH 6.4 for 3 h and, then, subject to Western blot assay using anti-c-Myc antibody. β-Actin was used as a loading control; (D–F) Quantification of Western blot results from (C). The expression of the total (D), big isoform (E) and small isoform (F) of c-Myc were quantified based on six Western blot repeats. The expression of c-Myc under the 3-h pH 7.4 treatment was set as one. Error bars indicate ±SEM. *p < 0.05; **p < 0.01; ns, not significant (p > 0.05).
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f5-ijms-14-20236: Ectopic overexpression of TDAG8 inhibits the upregulation of c-Myc protein by acidosis in H1299 cells. (A) c-Myc protein is moderately increased by acidosis in H1299 cells. Cells treated with HEM-buffered RPMI medium at pH 7.4 or pH 6.4 for three and 6 h were subject to Western blot assay using anti-c-Myc antibody. β-Actin was used as a loading control; (B) Low expression of proton-sensing GPCRs in H1299 cells. Total RNAs were isolated from H1299 parental, H1299/vector and H1299/hTDAG8 cells and subject to RT-PCR using gene-specific primers of GPR4, G2A, TDAG8 and OGR1. β-Actin was used as a positive control; (C) c-Myc protein is reduced by ectopic overexpression of TDAG8. Cells were treated with HEM-buffered RPMI medium at pH 7.4 or pH 6.4 for 3 h and, then, subject to Western blot assay using anti-c-Myc antibody. β-Actin was used as a loading control; (D–F) Quantification of Western blot results from (C). The expression of the total (D), big isoform (E) and small isoform (F) of c-Myc were quantified based on six Western blot repeats. The expression of c-Myc under the 3-h pH 7.4 treatment was set as one. Error bars indicate ±SEM. *p < 0.05; **p < 0.01; ns, not significant (p > 0.05).

Mentions: To further elucidate the connection between TDAG8 and acidosis-induced c-Myc downregulation, we identified a human lung cancer cell line, H1299, in which c-Myc expression was not decreased by acidic pH (Figure 5A). Western blotting revealed two isoforms of c-Myc protein around 59 and 62 kDa in H1299 cells (Figure 5A). The treatment with pH 6.4, in comparison to pH 7.4, did not result in any reduction of c-Myc protein. Instead, c-Myc protein, particularly the big isoform, was moderately increased by acidosis (Figure 5A). In addition to H1299 cells, we also found that acidic pH did not affect c-Myc protein level in A375 human melanoma cells (data not shown). RT-PCR revealed that the expression of the proton-sensing GPCRs, TDAG8 in particular, was very low in H1299 lung cancer cells (Figure 5B). To test whether ectopic overexpression of TDAG8 can inhibit c-Myc expression, H1299 cells were stably transduced with MSCV-TDAG8-IRES-GFP (H1299/TDAG8 cells) or MSCV-IRES-GFP (H1299/vector cells). The overexpression of TDAG8 in H1299/TDAG8 cells was confirmed by RT-PCR (Figure 5B). As observed in parental H1299 cells (Figure 5A), treatment with pH 6.4 slightly increased the expression of c-Myc protein, especially the big isoform, in H1299/vector cells, whereas ectopic overexpression of TDAG8 completely suppressed the increase of c-Myc by pH 6.4 in H1299/TDAG8 cells (Figure 5C–F).


Acidosis decreases c-Myc oncogene expression in human lymphoma cells: a role for the proton-sensing G protein-coupled receptor TDAG8.

Li Z, Dong L, Dean E, Yang LV - Int J Mol Sci (2013)

Ectopic overexpression of TDAG8 inhibits the upregulation of c-Myc protein by acidosis in H1299 cells. (A) c-Myc protein is moderately increased by acidosis in H1299 cells. Cells treated with HEM-buffered RPMI medium at pH 7.4 or pH 6.4 for three and 6 h were subject to Western blot assay using anti-c-Myc antibody. β-Actin was used as a loading control; (B) Low expression of proton-sensing GPCRs in H1299 cells. Total RNAs were isolated from H1299 parental, H1299/vector and H1299/hTDAG8 cells and subject to RT-PCR using gene-specific primers of GPR4, G2A, TDAG8 and OGR1. β-Actin was used as a positive control; (C) c-Myc protein is reduced by ectopic overexpression of TDAG8. Cells were treated with HEM-buffered RPMI medium at pH 7.4 or pH 6.4 for 3 h and, then, subject to Western blot assay using anti-c-Myc antibody. β-Actin was used as a loading control; (D–F) Quantification of Western blot results from (C). The expression of the total (D), big isoform (E) and small isoform (F) of c-Myc were quantified based on six Western blot repeats. The expression of c-Myc under the 3-h pH 7.4 treatment was set as one. Error bars indicate ±SEM. *p < 0.05; **p < 0.01; ns, not significant (p > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5-ijms-14-20236: Ectopic overexpression of TDAG8 inhibits the upregulation of c-Myc protein by acidosis in H1299 cells. (A) c-Myc protein is moderately increased by acidosis in H1299 cells. Cells treated with HEM-buffered RPMI medium at pH 7.4 or pH 6.4 for three and 6 h were subject to Western blot assay using anti-c-Myc antibody. β-Actin was used as a loading control; (B) Low expression of proton-sensing GPCRs in H1299 cells. Total RNAs were isolated from H1299 parental, H1299/vector and H1299/hTDAG8 cells and subject to RT-PCR using gene-specific primers of GPR4, G2A, TDAG8 and OGR1. β-Actin was used as a positive control; (C) c-Myc protein is reduced by ectopic overexpression of TDAG8. Cells were treated with HEM-buffered RPMI medium at pH 7.4 or pH 6.4 for 3 h and, then, subject to Western blot assay using anti-c-Myc antibody. β-Actin was used as a loading control; (D–F) Quantification of Western blot results from (C). The expression of the total (D), big isoform (E) and small isoform (F) of c-Myc were quantified based on six Western blot repeats. The expression of c-Myc under the 3-h pH 7.4 treatment was set as one. Error bars indicate ±SEM. *p < 0.05; **p < 0.01; ns, not significant (p > 0.05).
Mentions: To further elucidate the connection between TDAG8 and acidosis-induced c-Myc downregulation, we identified a human lung cancer cell line, H1299, in which c-Myc expression was not decreased by acidic pH (Figure 5A). Western blotting revealed two isoforms of c-Myc protein around 59 and 62 kDa in H1299 cells (Figure 5A). The treatment with pH 6.4, in comparison to pH 7.4, did not result in any reduction of c-Myc protein. Instead, c-Myc protein, particularly the big isoform, was moderately increased by acidosis (Figure 5A). In addition to H1299 cells, we also found that acidic pH did not affect c-Myc protein level in A375 human melanoma cells (data not shown). RT-PCR revealed that the expression of the proton-sensing GPCRs, TDAG8 in particular, was very low in H1299 lung cancer cells (Figure 5B). To test whether ectopic overexpression of TDAG8 can inhibit c-Myc expression, H1299 cells were stably transduced with MSCV-TDAG8-IRES-GFP (H1299/TDAG8 cells) or MSCV-IRES-GFP (H1299/vector cells). The overexpression of TDAG8 in H1299/TDAG8 cells was confirmed by RT-PCR (Figure 5B). As observed in parental H1299 cells (Figure 5A), treatment with pH 6.4 slightly increased the expression of c-Myc protein, especially the big isoform, in H1299/vector cells, whereas ectopic overexpression of TDAG8 completely suppressed the increase of c-Myc by pH 6.4 in H1299/TDAG8 cells (Figure 5C–F).

Bottom Line: The pH-sensing receptor TDAG8 (GPR65) is involved in acidosis-induced c-Myc downregulation.Acidic pH alone is insufficient to reduce c-Myc expression, as it does not decrease c-Myc in H1299 lung cancer cells expressing very low levels of pH-sensing G protein-coupled receptors (GPCRs).Collectively, our results identify a novel mechanism of c-Myc regulation by acidosis in the tumor microenvironment and indicate that modulation of TDAG8 and related pH-sensing receptor pathways may be exploited as a new approach to inhibit Myc expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA. yangl@ecu.edu.

ABSTRACT
Acidosis is a biochemical hallmark of the tumor microenvironment. Here, we report that acute acidosis decreases c-Myc oncogene expression in U937 human lymphoma cells. The level of c-Myc transcripts, but not mRNA or protein stability, contributes to c-Myc protein reduction under acidosis. The pH-sensing receptor TDAG8 (GPR65) is involved in acidosis-induced c-Myc downregulation. TDAG8 is expressed in U937 lymphoma cells, and the overexpression or knockdown of TDAG8 further decreases or partially rescues c-Myc expression, respectively. Acidic pH alone is insufficient to reduce c-Myc expression, as it does not decrease c-Myc in H1299 lung cancer cells expressing very low levels of pH-sensing G protein-coupled receptors (GPCRs). Instead, c-Myc is slightly increased by acidosis in H1299 cells, but this increase is completely inhibited by ectopic overexpression of TDAG8. Interestingly, TDAG8 expression is decreased by more than 50% in human lymphoma samples in comparison to non-tumorous lymph nodes and spleens, suggesting a potential tumor suppressor function of TDAG8 in lymphoma. Collectively, our results identify a novel mechanism of c-Myc regulation by acidosis in the tumor microenvironment and indicate that modulation of TDAG8 and related pH-sensing receptor pathways may be exploited as a new approach to inhibit Myc expression.

Show MeSH
Related in: MedlinePlus