Maf1 Is a Novel Target of PTEN and PI3K Signaling That Negatively Regulates Oncogenesis and Lipid Metabolism

<div><p>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.</p></div

Maf1 is regulated through PI3K/AKT/FOXO1 signaling.

<p>(<b>A</b>) <u>Pharmacologic inhibition of PI3K signaling increases Maf1 expression.</u> 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>B</b>) <u>AKT2 negatively regulates Maf1 expression</u>. Left: Protein lysates from livers of 1 month-old wild-type (n = 4), <i>Pten</i>−/− (<i>Pten^loxP/loxP; Alb-Cre+</i>; n = 4), <i>Pten</i>−/−; <i>Akt2</i>−/− (<i>Pten^loxP/loxP; Akt2 null; Alb-Cre+</i>; n = 3), and <i>Akt2</i>−<i>/</i>− (<i>Pten^loxP/loxP; Akt2 null; Alb-Cre</i>−; 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 <u>+</u>S.E. (<b>C</b>) <u>Mice fed a high carbohydrate diet display a reduction in Maf1 protein in the liver</u>. 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. (<b>D</b>) <u>FoxO1 knockdown decreases Maf1 protein expression and increases Maf1 target gene activity</u>. 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 tRNA^Leu and tRNA_i^Met. Values shown are the means ±S.E (n = 3). Values are statistically significant: Student t-test, Maf1, <i>p = </i>0.0429; pre-tRNA^Leu, <i>p = </i>0.0001; pre-tRNA_i^Met, <i>p</i> = 0.011. (<b>E</b>) <u>FoxO1 activation positively regulates Maf1 protein expression and represses Maf1 target gene activity</u>. 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, <i>p = </i>0.0029; pre-tRNA^Leu, <i>p = </i>0.0006; pre-tRNA_i^Met, <i>p = </i>0.0141.</p

Maf1 negatively regulates fatty acid synthase (FASN) and acetyl-coA carboxylase (ACC1) expression.

<p>(<b>A</b>) <u>Decreased Maf1 expression increases FASN and ACC1 mRNA</u>. A murine hepatocyte cell line was transiently transfected with non-silencing scrambled mismatch (mm) RNA or siRNA targeting Maf1. RNA was isolated and qRT-PCR was performed. mRNA amounts were normalized to that of GAPDH. (<b>B</b>) <u>Increased Maf1 expression represses FASN and ACC1 expression</u>. HepG2 cells were stably infected with Maf1-HA under the control of a doxycycline-inducible promoter. After treatment with 250 ng/ml doxycycline for 48 hrs, RNA was isolated and qRT-PCR was performed. Fold changes are statistically significant with <i>p</i><0.0001. (<b>C</b>) <u>Maf1 negatively regulates FASN promoter activity</u>. A murine hepatocyte cell line were transfected with Maf1 siRNA or Maf1-HA and a FASN promoter-reporter construct. Luciferase activity was measured from resulting lysates and normalized to protein levels. Values shown are the means <u>+</u>S.E (n = 4). Fold changes are statistically significant with <i>p</i> = 0.0003 (Maf1 siRNA) and <i>p</i><0.0001 (Maf1-HA).</p

Increased Maf1 expression suppresses cellular transformation and tumorigenesis, consistent with its diminished expression in human cancer tissues.

<p><u>Nuclear Maf1 expression is decreased in PTEN negative human prostate and liver cancers.</u> Immunohistochemistry of frozen human liver tissue (<b>A</b>) and paraffin-embedded human prostate tissue (<b>B</b>) with Maf1 or PTEN antibodies. Photomicrographs show representative staining of cancerous tissue (right) and adjacent normal tissue (left). Insets represent enlargements of areas highlighted. Scale bars represent 50 µm. (<b>C</b>) <u>Increased Maf1 expression results in repression of RNA polymerase III-dependent transcription</u>. Immunoblot analysis of protein lysates from Huh7 stable cell lines with vector or human Maf1-HA expression plasmid using HA (ectopic Maf1-HA), Maf1, or β-actin antibodies (left). qRT-PCR was performed using RNA isolated from stable cell lines with primers for pre-tRNA^Leu, pre-tRNA_i^Met, 7SL RNA, using GAPDH as an internal control (right). Values are the mean ±S.E. (n≥3). Fold changes in transcripts were statistically different from vector controls (Student t-test, pre-tRNA^Leu and pre-tRNA_i^Met, <i>p</i> = 0.0001, 7SL RNA, <i>p</i> = 0.0025). (<b>D</b>) <u>Effect of increased Maf1 expression on Huh7 cell doubling time</u>. Stable cell lines described in “C” grown on duplicate plates were trypsinized and counted daily. Values are the mean ±S.E. (n≥3), <i>p</i> = 0.0001. (<b>E</b>) <u>Increased Maf1 expression represses anchorage-independent growth.</u> Stable cell lines were analyzed for growth in soft agar. Colonies>100 uM were counted. Values are the means ±S.E. (n≥3), <i>p</i> = 0.005. (<b>F</b>) <u>Tumor growth rate is repressed and visible tumor formation is delayed in mice with cells expressing increased Maf1</u>. Two independent stable cell lines expressing Maf1 were injected subcutaneously into the groins of nude mice (10 mice per group). Calculated tumor growth rates shown (left). The day of first visible tumors noted are shown in the table (right). Values shown are the means ±S.E., <i>p</i> = 0.02.</p

PTEN regulates Maf1 protein expression.

<p>(<b>A</b>) <u>Maf1 expression is decreased in mouse cells and tissues lacking PTEN</u>. Protein lysates were isolated from wild-type and <i>Pten</i>−/− MEFs; livers of 1 month old <i>Pten^loxP/loxP; Alb-Cre</i>− (+/+; n = 4) and <i>Pten^loxP/loxP</i>; <i>Alb-Cre+</i> (−/−; n = 3) mice; and prostates of 5.2 week old <i>Pten^loxP/loxP; PB-Cre+</i> mice (+/+; n = 3) and <i>Pten^loxP/loxP</i>; <i>PB-Cre</i>− littermate controls (−/−; n = 3). Lysates were subjected to immunoblot analysis with antibodies against the proteins indicated. Densitometry analysis revealed statistically significant changes in Maf1 expression (Student t-test, MEF <i>p</i> = 0.0426; liver <i>p</i> = 0.0097; prostate <i>p</i> = 0.0046) (<b>B</b>) <u>Induction of wild type PTEN, but not a phosphatase defective mutant form induces Maf1 expression in cells lacking endogenous PTEN</u>. U87 cells engineered to stably express inducible PTEN or phosphatase defective PTEN-C124S were used. Protein lysates were isolated from cells treated with 1 µg/ml doxycycline at times indicated. Immunoblot analysis was performed using antibodies as indicated. Densitometry analysis revealed significant differences between no doxycycline control and 24 and 48 hr doxycycline treatment (ANOVA, <i>p</i><0.0001). Maf1 amounts were normalized to β-actin. The graphs represent quantification of 3 independent experiments. Values shown are the means ±S.E.</p

Maf1 occupies the FASN promoter to repress lipogenic gene expression.

<p>(<b>A</b>) <u>Maf1 negatively regulates cellular FASN and ACC1 protein expression.</u> Protein lysates isolated from Huh7 cells stably infected with nsRNA or Maf1 shRNAs (left) or HepG2 cells engineered to express doxycycline-inducible Maf1-HA as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004789#pgen-1004789-g004" target="_blank">Fig. 4</a> (right) were subjected to immunoblot analysis using antibodies against ACC1, FASN, SREBP, Maf1, HA (right, ectopic Maf1-HA), or β-actin. (<b>B</b>) <u>Altering Maf1 levels changes occupancy of Maf1 but not SREBP on the FASN promoter</u>. Chromatin immunoprecipitations were performed with Huh7 cells stably expressing Maf1 shRNA or HepG2-doxycycline-inducible Maf1-HA cells. ChIP analysis was performed with antibodies against Maf1, SREBP1c, and IgG. qPCR was performed with an upstream primers set (gray bars) and a set encompassing the transcription start site TSS (black and white bars). Bars represent Maf1 (top) or SREBP1c (bottom) occupancy relative to input and IgG. Maf1 occupancy at the TSS displayed statistically significant differences, Maf1 siRNA compared to non-silencing control, <i>p</i> = 0.001, and doxycycline versus no doxycycline treatment, <i>p</i> = 0.0001. (<b>C</b>) <u>Altering Maf1 levels changes Maf1 occupancy at the tRNA^Leu gene promoter. </u>ChIP analysis was performed as in (B) with primers specific for the tRNA^Leu promoter. Values shown for all graphs are the means ±S.E. (n = 4); Student t-test, Maf1 shRNA <i>p</i> = 0.0026, Maf1-HA <i>p</i> = 0.0013.</p

Maf1 controls intracellular lipid accumulation and de novo lipogenesis.

<p>(<b>A</b>) <u>Maf1 represses intracellular lipid accumulation</u>. Huh-7 cells infected with nsRNA (left) or Maf1 shRNA (right) were stained with Oil Red O and Mayer's Hematoxylin. Intracellular lipid droplets were detected as red spheres and nuclei are shown in purple. Magnification 40x. (<b>B</b>) <u>Maf1 overexpression inhibits diet-induced lipogenic gene expression</u>. Male C57BL mice injected with adenovirus overexpressing either Maf1-HA or LacZ were fed a high carbohydrate diet for 2 or 4 days. RNA was isolated from livers and qRT-PCR was performed with primers specific for FASN and ACC1. Fold changes are statistically significant: Student t-test, FASN; 2 day <i>p</i> = 0.0067, 4 day <i>p</i> = 0.0028; ACC1; 2 day <i>p</i> = 0.006, 4 day <i>p</i> = 0.021. (<b>C</b>) Protein lysates were isolated from the mouse livers fed a high carbohydrate diet for 4 days and were subjected to immunoblot analysis using antibodies against FASN, LacZ, HA (ectopic Maf1-HA), or vinculin. Representative samples shown. (<b>D</b>) <u>Maf1 overexpression reduces triglyceride accumulation</u>. Triglycerides were isolated from the livers of mice that were fed a high carbohydrate diet for 4 days and quantified. Values represent means ±S.E. (n = 4). Fold changes are statistically significant with <i>p</i><0.018.</p

Maf1 is a central coordinator of metabolic signals.

<p>Shown is a model depicting the proposed mechanism for the regulation of Maf1 expression by PTEN and the consequences of altered Maf1 expression on gene expression pathways that regulate the oncogenic potential of cells. New findings are highlighted in grey.</p