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Maf1 is a novel target of PTEN and PI3K signaling that negatively regulates oncogenesis and lipid metabolism.

Palian BM, Rohira AD, Johnson SA, He L, Zheng N, Dubeau L, Stiles BL, Johnson DL - PLoS Genet. (2014)

Bottom Line: PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes.We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation.Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, and the Norris Comprehensive Cancer Center, Los Angeles, California, United States of America.

ABSTRACT
Maf1 was initially identified as a transcriptional repressor of RNA pol III-transcribed genes, yet little is known about its other potential target genes or its biological function. Here, we show that Maf1 is a key downstream target of PTEN that drives both its tumor suppressor and metabolic functions. Maf1 expression is diminished with loss of PTEN in both mouse models and human cancers. Consistent with its role as a tumor suppressor, Maf1 reduces anchorage-independent growth and tumor formation in mice. PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes. This regulatory event is biologically relevant as diet-induced PI3K activation reduces Maf1 expression in mouse liver. We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation. Consistent with these findings, Maf1 inhibits intracellular lipid accumulation and increasing Maf1 expression in mouse liver abrogates diet-mediated induction of lipogenic enzymes and triglycerides. Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

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Maf1 is regulated through PI3K/AKT/FOXO1 signaling.(A) Pharmacologic inhibition of PI3K signaling increases Maf1 expression. MEF and HepG2 cells were treated with LY294002 or DMSO control for 6 hrs. Protein lysates were subjected to immunoblot analysis. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. (B) AKT2 negatively regulates Maf1 expression. Left: Protein lysates from livers of 1 month-old wild-type (n = 4), Pten−/− (PtenloxP/loxP; Alb-Cre+; n = 4), Pten−/−; Akt2−/− (PtenloxP/loxP; Akt2 ; Alb-Cre+; n = 3), and Akt2−/− (PtenloxP/loxP; Akt2 ; Alb-Cre−; n = 2) mice were subjected to immunoblot analysis. A representative example is shown for each genotype. Right: Huh7 cells were transfected with HA-tagged AKT2-Myr or empty vector control. Protein lysates were subjected to immunoblot analysis with antibodies indicted. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means +S.E. (C) Mice fed a high carbohydrate diet display a reduction in Maf1 protein in the liver. Mice were fed control or high carbohydrate diets (HCD), and immunoblot analysis was performed from liver lysates with antibodies against the proteins designated (n = 8 total for each dietary group). The data shown is representative from two independent experiments. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. (D) FoxO1 knockdown decreases Maf1 protein expression and increases Maf1 target gene activity. Left: Protein lysates were isolated from MEF cells stably expressing nonsilencing small hairpin RNA (nsRNA) or two distinct FoxO1-targeting shRNAs and immunoblots were performed. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. Right: RNA was isolated from stable MEF cell lines and qRT-PCR was performed with primers specific for precursor tRNALeu and tRNAiMet. Values shown are the means ±S.E (n = 3). Values are statistically significant: Student t-test, Maf1, p = 0.0429; pre-tRNALeu, p = 0.0001; pre-tRNAiMet, p = 0.011. (E) FoxO1 activation positively regulates Maf1 protein expression and represses Maf1 target gene activity. U87 cells were transfected with a FLAG-tagged constitutively active FoxO1 mutant or empty vector control. Protein lysates and RNA were isolated after 48 hrs and subjected to immunoblot analysis (right) and qRT-PCR (left). The fold-change in Maf1 protein levels was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E (n = 3). qRT-PCR statistics: Maf1, p = 0.0029; pre-tRNALeu, p = 0.0006; pre-tRNAiMet, p = 0.0141.
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pgen-1004789-g003: Maf1 is regulated through PI3K/AKT/FOXO1 signaling.(A) Pharmacologic inhibition of PI3K signaling increases Maf1 expression. MEF and HepG2 cells were treated with LY294002 or DMSO control for 6 hrs. Protein lysates were subjected to immunoblot analysis. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. (B) AKT2 negatively regulates Maf1 expression. Left: Protein lysates from livers of 1 month-old wild-type (n = 4), Pten−/− (PtenloxP/loxP; Alb-Cre+; n = 4), Pten−/−; Akt2−/− (PtenloxP/loxP; Akt2 ; Alb-Cre+; n = 3), and Akt2−/− (PtenloxP/loxP; Akt2 ; Alb-Cre−; n = 2) mice were subjected to immunoblot analysis. A representative example is shown for each genotype. Right: Huh7 cells were transfected with HA-tagged AKT2-Myr or empty vector control. Protein lysates were subjected to immunoblot analysis with antibodies indicted. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means +S.E. (C) Mice fed a high carbohydrate diet display a reduction in Maf1 protein in the liver. Mice were fed control or high carbohydrate diets (HCD), and immunoblot analysis was performed from liver lysates with antibodies against the proteins designated (n = 8 total for each dietary group). The data shown is representative from two independent experiments. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. (D) FoxO1 knockdown decreases Maf1 protein expression and increases Maf1 target gene activity. Left: Protein lysates were isolated from MEF cells stably expressing nonsilencing small hairpin RNA (nsRNA) or two distinct FoxO1-targeting shRNAs and immunoblots were performed. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. Right: RNA was isolated from stable MEF cell lines and qRT-PCR was performed with primers specific for precursor tRNALeu and tRNAiMet. Values shown are the means ±S.E (n = 3). Values are statistically significant: Student t-test, Maf1, p = 0.0429; pre-tRNALeu, p = 0.0001; pre-tRNAiMet, p = 0.011. (E) FoxO1 activation positively regulates Maf1 protein expression and represses Maf1 target gene activity. U87 cells were transfected with a FLAG-tagged constitutively active FoxO1 mutant or empty vector control. Protein lysates and RNA were isolated after 48 hrs and subjected to immunoblot analysis (right) and qRT-PCR (left). The fold-change in Maf1 protein levels was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E (n = 3). qRT-PCR statistics: Maf1, p = 0.0029; pre-tRNALeu, p = 0.0006; pre-tRNAiMet, p = 0.0141.

Mentions: A major function of PTEN is to repress the activation of PI3K signaling. We therefore assessed the potential role of PI3K in regulating Maf1 expression. MEFs and HepG2 human hepatoma cell lines were treated with the PI3K inhibitor, LY294002 (Fig. 3A). Increased Maf1 expression was observed with a corresponding decrease in the activation of AKT. As AKT2 is the predominant form of AKT in liver, we further analyzed its role in regulating Maf1 expression. Maf1 protein expression was measured in lysates derived from wild type livers, and those conditionally deleted for Pten, those for Akt, or both (Fig. 3B). Loss of Akt2 resulted in an increase in Maf1 expression compared with liver lysates derived from wild type mice. Compared with Pten-deficient livers, additional loss of Akt2 restored Maf1 amounts to that observed in wild type mice, supporting a role for AKT2 in negatively regulating cellular Maf1 concentrations. Consistent with these results, expression of a constitutively activated form of AKT2 resulted in a reduction in Maf1 expression in Huh 7 cells (Fig. 3B). Together these results demonstrate that the ability of PTEN to negatively regulate PI3K/AKT signaling is responsible for PTEN-mediated regulation of Maf1 expression.


Maf1 is a novel target of PTEN and PI3K signaling that negatively regulates oncogenesis and lipid metabolism.

Palian BM, Rohira AD, Johnson SA, He L, Zheng N, Dubeau L, Stiles BL, Johnson DL - PLoS Genet. (2014)

Maf1 is regulated through PI3K/AKT/FOXO1 signaling.(A) Pharmacologic inhibition of PI3K signaling increases Maf1 expression. MEF and HepG2 cells were treated with LY294002 or DMSO control for 6 hrs. Protein lysates were subjected to immunoblot analysis. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. (B) AKT2 negatively regulates Maf1 expression. Left: Protein lysates from livers of 1 month-old wild-type (n = 4), Pten−/− (PtenloxP/loxP; Alb-Cre+; n = 4), Pten−/−; Akt2−/− (PtenloxP/loxP; Akt2 ; Alb-Cre+; n = 3), and Akt2−/− (PtenloxP/loxP; Akt2 ; Alb-Cre−; n = 2) mice were subjected to immunoblot analysis. A representative example is shown for each genotype. Right: Huh7 cells were transfected with HA-tagged AKT2-Myr or empty vector control. Protein lysates were subjected to immunoblot analysis with antibodies indicted. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means +S.E. (C) Mice fed a high carbohydrate diet display a reduction in Maf1 protein in the liver. Mice were fed control or high carbohydrate diets (HCD), and immunoblot analysis was performed from liver lysates with antibodies against the proteins designated (n = 8 total for each dietary group). The data shown is representative from two independent experiments. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. (D) FoxO1 knockdown decreases Maf1 protein expression and increases Maf1 target gene activity. Left: Protein lysates were isolated from MEF cells stably expressing nonsilencing small hairpin RNA (nsRNA) or two distinct FoxO1-targeting shRNAs and immunoblots were performed. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. Right: RNA was isolated from stable MEF cell lines and qRT-PCR was performed with primers specific for precursor tRNALeu and tRNAiMet. Values shown are the means ±S.E (n = 3). Values are statistically significant: Student t-test, Maf1, p = 0.0429; pre-tRNALeu, p = 0.0001; pre-tRNAiMet, p = 0.011. (E) FoxO1 activation positively regulates Maf1 protein expression and represses Maf1 target gene activity. U87 cells were transfected with a FLAG-tagged constitutively active FoxO1 mutant or empty vector control. Protein lysates and RNA were isolated after 48 hrs and subjected to immunoblot analysis (right) and qRT-PCR (left). The fold-change in Maf1 protein levels was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E (n = 3). qRT-PCR statistics: Maf1, p = 0.0029; pre-tRNALeu, p = 0.0006; pre-tRNAiMet, p = 0.0141.
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Related In: Results  -  Collection

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pgen-1004789-g003: Maf1 is regulated through PI3K/AKT/FOXO1 signaling.(A) Pharmacologic inhibition of PI3K signaling increases Maf1 expression. MEF and HepG2 cells were treated with LY294002 or DMSO control for 6 hrs. Protein lysates were subjected to immunoblot analysis. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. (B) AKT2 negatively regulates Maf1 expression. Left: Protein lysates from livers of 1 month-old wild-type (n = 4), Pten−/− (PtenloxP/loxP; Alb-Cre+; n = 4), Pten−/−; Akt2−/− (PtenloxP/loxP; Akt2 ; Alb-Cre+; n = 3), and Akt2−/− (PtenloxP/loxP; Akt2 ; Alb-Cre−; n = 2) mice were subjected to immunoblot analysis. A representative example is shown for each genotype. Right: Huh7 cells were transfected with HA-tagged AKT2-Myr or empty vector control. Protein lysates were subjected to immunoblot analysis with antibodies indicted. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means +S.E. (C) Mice fed a high carbohydrate diet display a reduction in Maf1 protein in the liver. Mice were fed control or high carbohydrate diets (HCD), and immunoblot analysis was performed from liver lysates with antibodies against the proteins designated (n = 8 total for each dietary group). The data shown is representative from two independent experiments. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. (D) FoxO1 knockdown decreases Maf1 protein expression and increases Maf1 target gene activity. Left: Protein lysates were isolated from MEF cells stably expressing nonsilencing small hairpin RNA (nsRNA) or two distinct FoxO1-targeting shRNAs and immunoblots were performed. The fold-change in Maf1 was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E. Right: RNA was isolated from stable MEF cell lines and qRT-PCR was performed with primers specific for precursor tRNALeu and tRNAiMet. Values shown are the means ±S.E (n = 3). Values are statistically significant: Student t-test, Maf1, p = 0.0429; pre-tRNALeu, p = 0.0001; pre-tRNAiMet, p = 0.011. (E) FoxO1 activation positively regulates Maf1 protein expression and represses Maf1 target gene activity. U87 cells were transfected with a FLAG-tagged constitutively active FoxO1 mutant or empty vector control. Protein lysates and RNA were isolated after 48 hrs and subjected to immunoblot analysis (right) and qRT-PCR (left). The fold-change in Maf1 protein levels was calculated by normalizing to β-actin where the control is set to 1. Values shown are the means ±S.E (n = 3). qRT-PCR statistics: Maf1, p = 0.0029; pre-tRNALeu, p = 0.0006; pre-tRNAiMet, p = 0.0141.
Mentions: A major function of PTEN is to repress the activation of PI3K signaling. We therefore assessed the potential role of PI3K in regulating Maf1 expression. MEFs and HepG2 human hepatoma cell lines were treated with the PI3K inhibitor, LY294002 (Fig. 3A). Increased Maf1 expression was observed with a corresponding decrease in the activation of AKT. As AKT2 is the predominant form of AKT in liver, we further analyzed its role in regulating Maf1 expression. Maf1 protein expression was measured in lysates derived from wild type livers, and those conditionally deleted for Pten, those for Akt, or both (Fig. 3B). Loss of Akt2 resulted in an increase in Maf1 expression compared with liver lysates derived from wild type mice. Compared with Pten-deficient livers, additional loss of Akt2 restored Maf1 amounts to that observed in wild type mice, supporting a role for AKT2 in negatively regulating cellular Maf1 concentrations. Consistent with these results, expression of a constitutively activated form of AKT2 resulted in a reduction in Maf1 expression in Huh 7 cells (Fig. 3B). Together these results demonstrate that the ability of PTEN to negatively regulate PI3K/AKT signaling is responsible for PTEN-mediated regulation of Maf1 expression.

Bottom Line: PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes.We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation.Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, and the Norris Comprehensive Cancer Center, Los Angeles, California, United States of America.

ABSTRACT
Maf1 was initially identified as a transcriptional repressor of RNA pol III-transcribed genes, yet little is known about its other potential target genes or its biological function. Here, we show that Maf1 is a key downstream target of PTEN that drives both its tumor suppressor and metabolic functions. Maf1 expression is diminished with loss of PTEN in both mouse models and human cancers. Consistent with its role as a tumor suppressor, Maf1 reduces anchorage-independent growth and tumor formation in mice. PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes. This regulatory event is biologically relevant as diet-induced PI3K activation reduces Maf1 expression in mouse liver. We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation. Consistent with these findings, Maf1 inhibits intracellular lipid accumulation and increasing Maf1 expression in mouse liver abrogates diet-mediated induction of lipogenic enzymes and triglycerides. Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

Show MeSH
Related in: MedlinePlus