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Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation

View Article: PubMed Central - PubMed

ABSTRACT

Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer. The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells. To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST. MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1). Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells. Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells. Both NF-κB and MYC have been shown to regulate MCT1 expression. While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels. Several MCC lines had high levels of MYCL and MYCN but not MYC. Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells. Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis.

No MeSH data available.


MCPyV ST increases aerobic glycolysis and MCT1 sensitivity.A) Media glucose (Glc) and lactate (Lac) levels (mM) from cultures of IMR90 cells expressing ST or GFP were measured at the indicated day following dox addition. **P < 0.005 calculated using unpaired student’s T test between the marked GFP and ST points. B) ECAR (mpH/min) of IMR90 cells inducibly expressing ST with and without dox addition for 48 hours. ***P < 0.0005 calculated using unpaired student’s T test. C) ECAR of IMR90 cells expressing ST or GFP with CHC (5 mM) or DMSO (minutes) following 48 hours of dox treatment. Cells were treated with oligomycin (1 μM) at the indicated time point. ***P < 0.0005 calculated using unpaired student’s T test between GFP-DMSO and ST-DMSO samples. D) OCR (pmoles/min) of cells (minutes) as in C. E) Growth of IMR90 cells expressing ST or GFP treated with dox and CHC or DMSO was assessed by crystal violet every day for 5 days. ***P < 0.0005 calculated using unpaired, two-tailed student’s T test between ST-DMSO and ST-CHC treatments. Key same as in C.
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ppat.1006020.g003: MCPyV ST increases aerobic glycolysis and MCT1 sensitivity.A) Media glucose (Glc) and lactate (Lac) levels (mM) from cultures of IMR90 cells expressing ST or GFP were measured at the indicated day following dox addition. **P < 0.005 calculated using unpaired student’s T test between the marked GFP and ST points. B) ECAR (mpH/min) of IMR90 cells inducibly expressing ST with and without dox addition for 48 hours. ***P < 0.0005 calculated using unpaired student’s T test. C) ECAR of IMR90 cells expressing ST or GFP with CHC (5 mM) or DMSO (minutes) following 48 hours of dox treatment. Cells were treated with oligomycin (1 μM) at the indicated time point. ***P < 0.0005 calculated using unpaired student’s T test between GFP-DMSO and ST-DMSO samples. D) OCR (pmoles/min) of cells (minutes) as in C. E) Growth of IMR90 cells expressing ST or GFP treated with dox and CHC or DMSO was assessed by crystal violet every day for 5 days. ***P < 0.0005 calculated using unpaired, two-tailed student’s T test between ST-DMSO and ST-CHC treatments. Key same as in C.

Mentions: Glycolysis is a multistep biochemical process. HK2 serves as an upstream regulator that irreversibly commits glucose to enter the pathway. A byproduct of aerobic glycolysis is lactate. Production of lactate from pyruvate is mediated by lactate dehydrogenase (LDH), comprised of homo- or heterotetramers of two subunits, LDHA and LDHB. A major function of MCTs, including MCT1, is to prevent the toxic buildup of lactate in the intracellular milieu by co-exporting lactate together with protons out of the cell [27]. We observed that levels of glucose were depleted from the media and lactate levels increased at a significantly higher rate following ST expression compared to GFP cells (Fig 3A).


Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation
MCPyV ST increases aerobic glycolysis and MCT1 sensitivity.A) Media glucose (Glc) and lactate (Lac) levels (mM) from cultures of IMR90 cells expressing ST or GFP were measured at the indicated day following dox addition. **P < 0.005 calculated using unpaired student’s T test between the marked GFP and ST points. B) ECAR (mpH/min) of IMR90 cells inducibly expressing ST with and without dox addition for 48 hours. ***P < 0.0005 calculated using unpaired student’s T test. C) ECAR of IMR90 cells expressing ST or GFP with CHC (5 mM) or DMSO (minutes) following 48 hours of dox treatment. Cells were treated with oligomycin (1 μM) at the indicated time point. ***P < 0.0005 calculated using unpaired student’s T test between GFP-DMSO and ST-DMSO samples. D) OCR (pmoles/min) of cells (minutes) as in C. E) Growth of IMR90 cells expressing ST or GFP treated with dox and CHC or DMSO was assessed by crystal violet every day for 5 days. ***P < 0.0005 calculated using unpaired, two-tailed student’s T test between ST-DMSO and ST-CHC treatments. Key same as in C.
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ppat.1006020.g003: MCPyV ST increases aerobic glycolysis and MCT1 sensitivity.A) Media glucose (Glc) and lactate (Lac) levels (mM) from cultures of IMR90 cells expressing ST or GFP were measured at the indicated day following dox addition. **P < 0.005 calculated using unpaired student’s T test between the marked GFP and ST points. B) ECAR (mpH/min) of IMR90 cells inducibly expressing ST with and without dox addition for 48 hours. ***P < 0.0005 calculated using unpaired student’s T test. C) ECAR of IMR90 cells expressing ST or GFP with CHC (5 mM) or DMSO (minutes) following 48 hours of dox treatment. Cells were treated with oligomycin (1 μM) at the indicated time point. ***P < 0.0005 calculated using unpaired student’s T test between GFP-DMSO and ST-DMSO samples. D) OCR (pmoles/min) of cells (minutes) as in C. E) Growth of IMR90 cells expressing ST or GFP treated with dox and CHC or DMSO was assessed by crystal violet every day for 5 days. ***P < 0.0005 calculated using unpaired, two-tailed student’s T test between ST-DMSO and ST-CHC treatments. Key same as in C.
Mentions: Glycolysis is a multistep biochemical process. HK2 serves as an upstream regulator that irreversibly commits glucose to enter the pathway. A byproduct of aerobic glycolysis is lactate. Production of lactate from pyruvate is mediated by lactate dehydrogenase (LDH), comprised of homo- or heterotetramers of two subunits, LDHA and LDHB. A major function of MCTs, including MCT1, is to prevent the toxic buildup of lactate in the intracellular milieu by co-exporting lactate together with protons out of the cell [27]. We observed that levels of glucose were depleted from the media and lactate levels increased at a significantly higher rate following ST expression compared to GFP cells (Fig 3A).

View Article: PubMed Central - PubMed

ABSTRACT

Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer. The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells. To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST. MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1). Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells. Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells. Both NF-&kappa;B and MYC have been shown to regulate MCT1 expression. While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-&kappa;B subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels. Several MCC lines had high levels of MYCL and MYCN but not MYC. Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells. Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis.

No MeSH data available.