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PPARβ/δ promotes HRAS-induced senescence and tumor suppression by potentiating p-ERK and repressing p-AKT signaling.

Zhu B, Ferry CH, Blazanin N, Bility MT, Khozoie C, Kang BH, Glick AB, Gonzalez FJ, Peters JM - Oncogene (2013)

Bottom Line: PPARβ/δ expression increased p-ERK and decreased p-AKT activity.Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARβ/δ.Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Biomedical Sciences, The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA, USA.

ABSTRACT
Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits skin tumorigenesis through mechanisms that may be dependent on HRAS signaling. The present study examined the hypothesis that PPARβ/δ promotes HRAS-induced senescence resulting in suppression of tumorigenesis. PPARβ/δ expression increased p-ERK and decreased p-AKT activity. Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARβ/δ. Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression. Decreased p-AKT activity in turn promotes cellular senescence through upregulation of p53 and p27 expression. Both over-expression of RASGRP1 and shRNA-mediated knockdown of ILK partially restore cellular senescence in Pparβ/δ- cells. Higher PPARβ/δ expression is also correlated with increased senescence observed in human benign neurofibromas and colon adenoma lesions in vivo. These results demonstrate that PPARβ/δ promotes senescence to inhibit tumorigenesis and provide new mechanistic insights into HRAS-induced cellular senescence.

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PPARβ/δ promotes cellular senescence in human benign lesions. (a) p16 mRNA expression in different neurofibroma tumors (malignant peripheral nerve sheet tumor (MPNST) or benign dermal neurofibroma) and tumor-derived cell lines (MPNST or NF1−/− benign dermal neurofibroma Schwann cell lines). (b) Scatter plots of the log2 value of PPARβ/δ mRNA compared to p16, RASGRP1 and PDPK1 in NF1-derived primary benign neurofibroma Schwann cells with NF1−/− mutation. (c) Western blot analysis of paired human normal colon and human colon adenomas and scatter plots of PPARβ/δ protein level with protein level of DcR2, p-AKT (S473), ILK and mRNA level of ILK. Relative expression level of proteins was normalized to the intensity of Ponceau staining and is shown as the relative fold change as compared to the normal colon for the first sample in lane 1. The mRNA level of ILK was normalized to that of ACTIN. Close and open circles represent normal tissue and colon adenomas respectively.
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Figure 7: PPARβ/δ promotes cellular senescence in human benign lesions. (a) p16 mRNA expression in different neurofibroma tumors (malignant peripheral nerve sheet tumor (MPNST) or benign dermal neurofibroma) and tumor-derived cell lines (MPNST or NF1−/− benign dermal neurofibroma Schwann cell lines). (b) Scatter plots of the log2 value of PPARβ/δ mRNA compared to p16, RASGRP1 and PDPK1 in NF1-derived primary benign neurofibroma Schwann cells with NF1−/− mutation. (c) Western blot analysis of paired human normal colon and human colon adenomas and scatter plots of PPARβ/δ protein level with protein level of DcR2, p-AKT (S473), ILK and mRNA level of ILK. Relative expression level of proteins was normalized to the intensity of Ponceau staining and is shown as the relative fold change as compared to the normal colon for the first sample in lane 1. The mRNA level of ILK was normalized to that of ACTIN. Close and open circles represent normal tissue and colon adenomas respectively.

Mentions: To determine if the changes observed in mouse models were also found in human tumors, the correlation between PPARβ/δ expression and the senescence marker p16 in human benign dermal neurofibroma lesions was examined. These lesions were chosen because: 1) benign dermal neurofibromas harbor an NF1 loss-of-function mutation, resulting in activation of RAS signaling pathway 27 similar to the activated HRAS model used in the present studies and 2) β-gal positive staining of senescent cells are found in these lesions 27. Examination of a publicly available database 35 revealed that expression of mRNA encoding the senescence marker p16 is significantly higher in benign dermal neurofibromas and NF1-derived primary benign neurofibroma Schwann cells compared to malignant peripheral nerve sheath tumors and cell lines, respectively (Figure 7a). This suggests that p16 mRNA is a good senescence marker in these types of lesions. Neurofibromas are heterogeneous tumors that consist of Schwann cells with initiating homozygous NF1 mutations, but also recruited fibroblasts, peripheral cells, neurons and mast cells that are only heterozygous for NF1 mutations. Since β-gal positive staining was only found in cells with homozygous loss-of-function NF1 mutations 27, eight samples of NF1-derived primary benign neurofibroma Schwann cells with a homozygous NF1 mutation were examined. A positive correlation between PPARβ/δ and p16 mRNA was found in these cells (Figure 7b). In addition, a positive correlation was also found between PPARβ/δ and RASGPR1 (Figure 7b), similar to effects observed in the mouse models. While no correlation between PPARβ/δ and ILK mRNA was found, a negative correlation between PPARβ/δ and PDPK1 mRNA was found in these cells (Figure 7b). Human colon adenomas were also examined because 1) they contain cells exhibiting both strong p16 immunoreactivity and the absence of Ki-67 staining as previously reported 36, 37, and 2) the KRAS oncogene is mutated in approximately 35%–45% of colorectal cancers 38. In the five human colon adenomas that contained a KRAS mutation at codon 13 (data not shown), the average PPARβ/δ protein level was higher compared to untransformed colon (Figure 7c). In addition, PPARβ/δ protein in both normal colon and colon adenomas negatively correlated with both mRNA and protein of ILK and p-AKT (Figure 7c). The expression of PPARβ/δ protein in colon adenomas also positively correlates with expression of senescence markers (Figure 7c). These data suggest that PPARβ/δ promotes cellular senescence in benign human tumors.


PPARβ/δ promotes HRAS-induced senescence and tumor suppression by potentiating p-ERK and repressing p-AKT signaling.

Zhu B, Ferry CH, Blazanin N, Bility MT, Khozoie C, Kang BH, Glick AB, Gonzalez FJ, Peters JM - Oncogene (2013)

PPARβ/δ promotes cellular senescence in human benign lesions. (a) p16 mRNA expression in different neurofibroma tumors (malignant peripheral nerve sheet tumor (MPNST) or benign dermal neurofibroma) and tumor-derived cell lines (MPNST or NF1−/− benign dermal neurofibroma Schwann cell lines). (b) Scatter plots of the log2 value of PPARβ/δ mRNA compared to p16, RASGRP1 and PDPK1 in NF1-derived primary benign neurofibroma Schwann cells with NF1−/− mutation. (c) Western blot analysis of paired human normal colon and human colon adenomas and scatter plots of PPARβ/δ protein level with protein level of DcR2, p-AKT (S473), ILK and mRNA level of ILK. Relative expression level of proteins was normalized to the intensity of Ponceau staining and is shown as the relative fold change as compared to the normal colon for the first sample in lane 1. The mRNA level of ILK was normalized to that of ACTIN. Close and open circles represent normal tissue and colon adenomas respectively.
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Related In: Results  -  Collection

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Figure 7: PPARβ/δ promotes cellular senescence in human benign lesions. (a) p16 mRNA expression in different neurofibroma tumors (malignant peripheral nerve sheet tumor (MPNST) or benign dermal neurofibroma) and tumor-derived cell lines (MPNST or NF1−/− benign dermal neurofibroma Schwann cell lines). (b) Scatter plots of the log2 value of PPARβ/δ mRNA compared to p16, RASGRP1 and PDPK1 in NF1-derived primary benign neurofibroma Schwann cells with NF1−/− mutation. (c) Western blot analysis of paired human normal colon and human colon adenomas and scatter plots of PPARβ/δ protein level with protein level of DcR2, p-AKT (S473), ILK and mRNA level of ILK. Relative expression level of proteins was normalized to the intensity of Ponceau staining and is shown as the relative fold change as compared to the normal colon for the first sample in lane 1. The mRNA level of ILK was normalized to that of ACTIN. Close and open circles represent normal tissue and colon adenomas respectively.
Mentions: To determine if the changes observed in mouse models were also found in human tumors, the correlation between PPARβ/δ expression and the senescence marker p16 in human benign dermal neurofibroma lesions was examined. These lesions were chosen because: 1) benign dermal neurofibromas harbor an NF1 loss-of-function mutation, resulting in activation of RAS signaling pathway 27 similar to the activated HRAS model used in the present studies and 2) β-gal positive staining of senescent cells are found in these lesions 27. Examination of a publicly available database 35 revealed that expression of mRNA encoding the senescence marker p16 is significantly higher in benign dermal neurofibromas and NF1-derived primary benign neurofibroma Schwann cells compared to malignant peripheral nerve sheath tumors and cell lines, respectively (Figure 7a). This suggests that p16 mRNA is a good senescence marker in these types of lesions. Neurofibromas are heterogeneous tumors that consist of Schwann cells with initiating homozygous NF1 mutations, but also recruited fibroblasts, peripheral cells, neurons and mast cells that are only heterozygous for NF1 mutations. Since β-gal positive staining was only found in cells with homozygous loss-of-function NF1 mutations 27, eight samples of NF1-derived primary benign neurofibroma Schwann cells with a homozygous NF1 mutation were examined. A positive correlation between PPARβ/δ and p16 mRNA was found in these cells (Figure 7b). In addition, a positive correlation was also found between PPARβ/δ and RASGPR1 (Figure 7b), similar to effects observed in the mouse models. While no correlation between PPARβ/δ and ILK mRNA was found, a negative correlation between PPARβ/δ and PDPK1 mRNA was found in these cells (Figure 7b). Human colon adenomas were also examined because 1) they contain cells exhibiting both strong p16 immunoreactivity and the absence of Ki-67 staining as previously reported 36, 37, and 2) the KRAS oncogene is mutated in approximately 35%–45% of colorectal cancers 38. In the five human colon adenomas that contained a KRAS mutation at codon 13 (data not shown), the average PPARβ/δ protein level was higher compared to untransformed colon (Figure 7c). In addition, PPARβ/δ protein in both normal colon and colon adenomas negatively correlated with both mRNA and protein of ILK and p-AKT (Figure 7c). The expression of PPARβ/δ protein in colon adenomas also positively correlates with expression of senescence markers (Figure 7c). These data suggest that PPARβ/δ promotes cellular senescence in benign human tumors.

Bottom Line: PPARβ/δ expression increased p-ERK and decreased p-AKT activity.Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARβ/δ.Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Biomedical Sciences, The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA, USA.

ABSTRACT
Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits skin tumorigenesis through mechanisms that may be dependent on HRAS signaling. The present study examined the hypothesis that PPARβ/δ promotes HRAS-induced senescence resulting in suppression of tumorigenesis. PPARβ/δ expression increased p-ERK and decreased p-AKT activity. Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARβ/δ. Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression. Decreased p-AKT activity in turn promotes cellular senescence through upregulation of p53 and p27 expression. Both over-expression of RASGRP1 and shRNA-mediated knockdown of ILK partially restore cellular senescence in Pparβ/δ- cells. Higher PPARβ/δ expression is also correlated with increased senescence observed in human benign neurofibromas and colon adenoma lesions in vivo. These results demonstrate that PPARβ/δ promotes senescence to inhibit tumorigenesis and provide new mechanistic insights into HRAS-induced cellular senescence.

Show MeSH
Related in: MedlinePlus