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p63 promotes cell survival through fatty acid synthase.

Sabbisetti V, Di Napoli A, Seeley A, Amato AM, O'Regan E, Ghebremichael M, Loda M, Signoretti S - PLoS ONE (2009)

Bottom Line: Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN.FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival.Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

ABSTRACT
There is increasing evidence that p63, and specifically DeltaNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN), a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT) was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity, suggesting that the rescue from apoptosis documented in the p63-silenced cells expressing myr-AKT cells may be partially mediated by FASN. Finally, we demonstrated that p63 and FASN expression are positively associated in clinical squamous cell carcinoma samples as well as in the developing prostate. Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

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p63 silencing induces a decrease in FASN expression and enzymatic activity.A. Quantitation of FASN mRNA by real time RT-PCR in SCC9 cells transfected with Tp63 or Dp63 siRNA relative to cells transfected with Scr siRNA (left panel). Quantitation of FASN mRNA in SCC9-Tp63 and iPrEC-Tp63 cells treated with tetracycline relative to the correspondent untreated cells (middle and right panels, respectively). Data are shown as means +/− standard error and are representative of three independent experiments. B. Immunoblot analysis of FASN in SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (left panel), in SCC9-Tp63 cells with or without treatment with tetracycline (middle panel) and in iPrEC-Tp63 cells with or without treatment with tetracycline (right panel). C. Quantitation of C14 incorporation within cellular lipids in SCC9 cells transfected with Tp63 or Dp63 siRNA relative to cells transfected with Scr siRNA (left panel) and in SCC9-Tp63 and iPrEC-Tp63 cells treated with tetracycline relative to the correspondent untreated cells (middle and right panels, respectively). Data are shown as means +/− standard error and are representative of three independent experiments.
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pone-0005877-g003: p63 silencing induces a decrease in FASN expression and enzymatic activity.A. Quantitation of FASN mRNA by real time RT-PCR in SCC9 cells transfected with Tp63 or Dp63 siRNA relative to cells transfected with Scr siRNA (left panel). Quantitation of FASN mRNA in SCC9-Tp63 and iPrEC-Tp63 cells treated with tetracycline relative to the correspondent untreated cells (middle and right panels, respectively). Data are shown as means +/− standard error and are representative of three independent experiments. B. Immunoblot analysis of FASN in SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (left panel), in SCC9-Tp63 cells with or without treatment with tetracycline (middle panel) and in iPrEC-Tp63 cells with or without treatment with tetracycline (right panel). C. Quantitation of C14 incorporation within cellular lipids in SCC9 cells transfected with Tp63 or Dp63 siRNA relative to cells transfected with Scr siRNA (left panel) and in SCC9-Tp63 and iPrEC-Tp63 cells treated with tetracycline relative to the correspondent untreated cells (middle and right panels, respectively). Data are shown as means +/− standard error and are representative of three independent experiments.

Mentions: As first step, we assessed whether p63 regulates FASN expression by evaluating the effects p63 downregulation on FASN mRNA and protein levels. Knockdown of p63, with either Tp63 or Dp63 siRNAs decreased the mRNA expression of FASN by approximately 50% in the SCC9 cell line (Figure 3A, left and middle panels). In addition, we observed a similar decrease in FASN transcript levels after p63 knockdown in iPrEC cells (Figure 3A, right panel). Consistent with these results, we detected a significant decrease in FASN protein expression upon knockdown of p63 in both SCC9 and iPrEC cells (Figure 3B). These data confirm that p63 modulates FASN expression at both mRNA and protein levels in both transformed and immortalized epithelial cells.


p63 promotes cell survival through fatty acid synthase.

Sabbisetti V, Di Napoli A, Seeley A, Amato AM, O'Regan E, Ghebremichael M, Loda M, Signoretti S - PLoS ONE (2009)

p63 silencing induces a decrease in FASN expression and enzymatic activity.A. Quantitation of FASN mRNA by real time RT-PCR in SCC9 cells transfected with Tp63 or Dp63 siRNA relative to cells transfected with Scr siRNA (left panel). Quantitation of FASN mRNA in SCC9-Tp63 and iPrEC-Tp63 cells treated with tetracycline relative to the correspondent untreated cells (middle and right panels, respectively). Data are shown as means +/− standard error and are representative of three independent experiments. B. Immunoblot analysis of FASN in SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (left panel), in SCC9-Tp63 cells with or without treatment with tetracycline (middle panel) and in iPrEC-Tp63 cells with or without treatment with tetracycline (right panel). C. Quantitation of C14 incorporation within cellular lipids in SCC9 cells transfected with Tp63 or Dp63 siRNA relative to cells transfected with Scr siRNA (left panel) and in SCC9-Tp63 and iPrEC-Tp63 cells treated with tetracycline relative to the correspondent untreated cells (middle and right panels, respectively). Data are shown as means +/− standard error and are representative of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691576&req=5

pone-0005877-g003: p63 silencing induces a decrease in FASN expression and enzymatic activity.A. Quantitation of FASN mRNA by real time RT-PCR in SCC9 cells transfected with Tp63 or Dp63 siRNA relative to cells transfected with Scr siRNA (left panel). Quantitation of FASN mRNA in SCC9-Tp63 and iPrEC-Tp63 cells treated with tetracycline relative to the correspondent untreated cells (middle and right panels, respectively). Data are shown as means +/− standard error and are representative of three independent experiments. B. Immunoblot analysis of FASN in SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (left panel), in SCC9-Tp63 cells with or without treatment with tetracycline (middle panel) and in iPrEC-Tp63 cells with or without treatment with tetracycline (right panel). C. Quantitation of C14 incorporation within cellular lipids in SCC9 cells transfected with Tp63 or Dp63 siRNA relative to cells transfected with Scr siRNA (left panel) and in SCC9-Tp63 and iPrEC-Tp63 cells treated with tetracycline relative to the correspondent untreated cells (middle and right panels, respectively). Data are shown as means +/− standard error and are representative of three independent experiments.
Mentions: As first step, we assessed whether p63 regulates FASN expression by evaluating the effects p63 downregulation on FASN mRNA and protein levels. Knockdown of p63, with either Tp63 or Dp63 siRNAs decreased the mRNA expression of FASN by approximately 50% in the SCC9 cell line (Figure 3A, left and middle panels). In addition, we observed a similar decrease in FASN transcript levels after p63 knockdown in iPrEC cells (Figure 3A, right panel). Consistent with these results, we detected a significant decrease in FASN protein expression upon knockdown of p63 in both SCC9 and iPrEC cells (Figure 3B). These data confirm that p63 modulates FASN expression at both mRNA and protein levels in both transformed and immortalized epithelial cells.

Bottom Line: Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN.FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival.Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

ABSTRACT
There is increasing evidence that p63, and specifically DeltaNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN), a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT) was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity, suggesting that the rescue from apoptosis documented in the p63-silenced cells expressing myr-AKT cells may be partially mediated by FASN. Finally, we demonstrated that p63 and FASN expression are positively associated in clinical squamous cell carcinoma samples as well as in the developing prostate. Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

Show MeSH
Related in: MedlinePlus