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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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Co-localization of p63 and FASN in vivo.A. Immunohistochemical staining for FASN and p63 in TMAs containing cores from HNSCC tissues. Two representative cases are shown: one case displays both low p63 and low FASN expression (upper panel) and one case displays both high p63 and high FASN expression (lower panel). B. Co-localization of p63 and FASN in the UGS epithelium and prostatic buds. (a) A coronal section of the UGS from a wild-type E18.5 embryo was double immunostained for p63 (Fast Red) and FASN (DAB) and counterstained with hematoxylin. Deconvolved images of the DAB (b) and Fast Red (c) color channels as well a pseudo-colorized image (c) are also shown. In the pseudo-colorized image, the DAB channel (FASN) is displayed in green, the Fast Red channel (p63) is displayed in red and the hematoxylin channel is displayed in blue. Expression of both FASN and p63 proteins is observed in the vast majority of the cells. The cells lining the UGS lumen are consistently negative for both FASN and p63.
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pone-0005877-g006: Co-localization of p63 and FASN in vivo.A. Immunohistochemical staining for FASN and p63 in TMAs containing cores from HNSCC tissues. Two representative cases are shown: one case displays both low p63 and low FASN expression (upper panel) and one case displays both high p63 and high FASN expression (lower panel). B. Co-localization of p63 and FASN in the UGS epithelium and prostatic buds. (a) A coronal section of the UGS from a wild-type E18.5 embryo was double immunostained for p63 (Fast Red) and FASN (DAB) and counterstained with hematoxylin. Deconvolved images of the DAB (b) and Fast Red (c) color channels as well a pseudo-colorized image (c) are also shown. In the pseudo-colorized image, the DAB channel (FASN) is displayed in green, the Fast Red channel (p63) is displayed in red and the hematoxylin channel is displayed in blue. Expression of both FASN and p63 proteins is observed in the vast majority of the cells. The cells lining the UGS lumen are consistently negative for both FASN and p63.

Mentions: Our cell-based assays indicate that FASN is a functionally relevant target of p63 in SCC cells. If FASN were a main mediator of p63 function also in human tumors, the levels of the two proteins would be positively associated in SCC tissues. In order to test this hypothesis, we examined FASN and p63 protein expression by immunohistochemistry in a tissue microarray (TMA) containing cores from 83 cases of head and neck SCC (HNSCC). The characteristics of the tumors are described in Table 1. In the morphologically normal epithelium, FASN expression was weak and restricted to the lower layers. In line with previous reports, p63 expression was observed in both basal and parabasal epithelial cell layers (data not shown). Among the 83 tumors, FASN was detected in 95.2% of the cases and p63 in 98.8% of the cases. p63 protein expression was scored by estimating the percentage of positive nuclei in tumor cells. Cytoplasmic expression of FASN was scored as negative (no expression), weak expression (1+), moderate expression (2+), and strong (3+). Statistical analysis revealed a significant positive association (p<0.007, Fisher's exact test) between FASN and p63 expression levels (Table 2). Representative cases are shown in Figure 6A and Figure S3. Of note, FASN expression was more intense in the non-keratinizing poorly differentiated tumors with basaloid morphology, where p63 was found to be positive in almost 100% of tumor cells (Figure 6A, lower panel). No significant association was found between tumor grade and FASN or p63 expression levels (data not shown). These findings are in line with our in vitro data and further support a role for FASN in mediating pro-oncogenic p63 functions in vivo.


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)

Co-localization of p63 and FASN in vivo.A. Immunohistochemical staining for FASN and p63 in TMAs containing cores from HNSCC tissues. Two representative cases are shown: one case displays both low p63 and low FASN expression (upper panel) and one case displays both high p63 and high FASN expression (lower panel). B. Co-localization of p63 and FASN in the UGS epithelium and prostatic buds. (a) A coronal section of the UGS from a wild-type E18.5 embryo was double immunostained for p63 (Fast Red) and FASN (DAB) and counterstained with hematoxylin. Deconvolved images of the DAB (b) and Fast Red (c) color channels as well a pseudo-colorized image (c) are also shown. In the pseudo-colorized image, the DAB channel (FASN) is displayed in green, the Fast Red channel (p63) is displayed in red and the hematoxylin channel is displayed in blue. Expression of both FASN and p63 proteins is observed in the vast majority of the cells. The cells lining the UGS lumen are consistently negative for both FASN and p63.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691576&req=5

pone-0005877-g006: Co-localization of p63 and FASN in vivo.A. Immunohistochemical staining for FASN and p63 in TMAs containing cores from HNSCC tissues. Two representative cases are shown: one case displays both low p63 and low FASN expression (upper panel) and one case displays both high p63 and high FASN expression (lower panel). B. Co-localization of p63 and FASN in the UGS epithelium and prostatic buds. (a) A coronal section of the UGS from a wild-type E18.5 embryo was double immunostained for p63 (Fast Red) and FASN (DAB) and counterstained with hematoxylin. Deconvolved images of the DAB (b) and Fast Red (c) color channels as well a pseudo-colorized image (c) are also shown. In the pseudo-colorized image, the DAB channel (FASN) is displayed in green, the Fast Red channel (p63) is displayed in red and the hematoxylin channel is displayed in blue. Expression of both FASN and p63 proteins is observed in the vast majority of the cells. The cells lining the UGS lumen are consistently negative for both FASN and p63.
Mentions: Our cell-based assays indicate that FASN is a functionally relevant target of p63 in SCC cells. If FASN were a main mediator of p63 function also in human tumors, the levels of the two proteins would be positively associated in SCC tissues. In order to test this hypothesis, we examined FASN and p63 protein expression by immunohistochemistry in a tissue microarray (TMA) containing cores from 83 cases of head and neck SCC (HNSCC). The characteristics of the tumors are described in Table 1. In the morphologically normal epithelium, FASN expression was weak and restricted to the lower layers. In line with previous reports, p63 expression was observed in both basal and parabasal epithelial cell layers (data not shown). Among the 83 tumors, FASN was detected in 95.2% of the cases and p63 in 98.8% of the cases. p63 protein expression was scored by estimating the percentage of positive nuclei in tumor cells. Cytoplasmic expression of FASN was scored as negative (no expression), weak expression (1+), moderate expression (2+), and strong (3+). Statistical analysis revealed a significant positive association (p<0.007, Fisher's exact test) between FASN and p63 expression levels (Table 2). Representative cases are shown in Figure 6A and Figure S3. Of note, FASN expression was more intense in the non-keratinizing poorly differentiated tumors with basaloid morphology, where p63 was found to be positive in almost 100% of tumor cells (Figure 6A, lower panel). No significant association was found between tumor grade and FASN or p63 expression levels (data not shown). These findings are in line with our in vitro data and further support a role for FASN in mediating pro-oncogenic p63 functions in vivo.

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