MOF upregulates the estrogen receptor α signaling pathway by its acetylase activity in hepatocellular carcinoma.

The histone acetyltransferase MOF (KAT8) is mainly involved in the acetylation of histone H4 at lysine 16 (H4K16) and some non-histone proteins. The MOF expression level is significantly reduced in many cancers, however the biological function of MOF and its underlying mechanism are still elusive in hepatocellular carcinoma (HCC). Estrogen receptor α (ERα) has been considered as a tumor suppressor in HCC. Here, we demonstrated that MOF expression is significantly reduced in HCC samples, and is positively correlated with that of ERα. MOF interacts with ERα, and participates in acetylation of ERα at K266, K268, K299, thereby inhibiting ERα ubiquitination to maintain the stability of ERα. In addition, MOF participates in the upregulation of ERα-mediated transactivation. Depletion of MOF significantly promotes cell growth, migration, and invasion in HCC cell lines. Taken together, our results provide new insights to understand the mechanism underlying the modulation function of MOF on ERα action in HCC, suggesting that MOF might be a potential therapeutic target for HCC.

Mendelian disorders of the epigenetic machinery: postnatal malleability and therapeutic prospects.

The epigenetic machinery in conjunction with the transcriptional machinery is responsible for maintaining genome-wide chromatin states and dynamically regulating gene expression. Mendelian disorders of the epigenetic machinery (MDEMs) are genetic disorders resulting from mutations in components of the epigenetic apparatus. Though individually rare, MDEMs have emerged as a collectively common etiology for intellectual disability (ID) and growth disruption. Studies in model organisms and humans have demonstrated dosage sensitivity of this gene group with haploinsufficiency as a predominant disease mechanism. The epigenetic machinery consists of three enzymatic components (writers, erasers and chromatin remodelers) as well as one non-enzymatic group (readers). A tally of the entire census of such factors revealed that although multiple enzymatic activities never coexist within a single component, individual enzymatic activities often coexist with a reader domain. This group of disorders disrupts both the chromatin and transcription states of target genes downstream of the given component but also DNA methylation on a global scale. Elucidation of these global epigenetic changes may inform our understanding of disease pathogenesis and have diagnostic utility. Moreover, many therapies targeting epigenetic marks already exist, and some have proven successful in treating cancer. This, along with the recent observation that neurological dysfunction in these disorders may in fact be treatable in postnatal life, suggests that the scientific community should prioritize this group as a potentially treatable cause of ID. Here we summarize the recent expansion and major characteristics of MDEMs, as well as the unique therapeutic prospects for this group of disorders.

Early-onset aging and mitochondrial defects associated with loss of histone acetyltransferase 1 (Hat1).

Histone acetyltransferase 1 (Hat1) is responsible for the acetylation of newly synthesized histone H4 on lysines 5 and 12 during the process of chromatin assembly. To understand the broader biological role of Hat1, we have generated a conditional mouse knockout model of this enzyme. We previously reported that Hat1 is required for viability and important for mammalian development and genome stability. In this study, we show that haploinsufficiency of Hat1 results in a significant decrease in lifespan. Defects observed in Hat1+/- mice are consistent with an early-onset aging phenotype. These include lordokyphosis (hunchback), muscle atrophy, minor growth retardation, reduced subcutaneous fat, cancer, and paralysis. In addition, the expression of Hat1 is linked to the normal aging process as Hat1 mRNA and protein becomes undetectable in many tissues in old mice. At the cellular level, fibroblasts from Hat1 haploinsufficient embryos undergo early senescence and accumulate high levels of p21. Hat1+/- mouse embryonic fibroblasts (MEFs) display modest increases in endogenous DNA damage but have significantly higher levels of reactive oxygen species (ROS). Consistently, further studies show that Hat1-/- MEFs exhibit mitochondrial defects suggesting a critical role for Hat1 in mitochondrial function. Taken together, these data show that loss of Hat1 induces multiple hallmarks of early-onset aging.

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

KAT6B Is a Tumor Suppressor Histone H3 Lysine 23 Acetyltransferase Undergoing Genomic Loss in Small Cell Lung Cancer.

Recent efforts to sequence human cancer genomes have highlighted that point mutations in genes involved in the epigenetic setting occur in tumor cells. Small cell lung cancer (SCLC) is an aggressive tumor with poor prognosis, where little is known about the genetic events related to its development. Herein, we have identified the presence of homozygous deletions of the candidate histone acetyltransferase KAT6B, and the loss of the corresponding transcript, in SCLC cell lines and primary tumors. Furthermore, we show, in vitro and in vivo, that the depletion of KAT6B expression enhances cancer growth, while its restoration induces tumor suppressor-like features. Most importantly, we demonstrate that KAT6B exerts its tumor-inhibitory role through a newly defined type of histone H3 Lys23 acetyltransferase activity.

MOZ (MYST3, KAT6A) inhibits senescence via the INK4A-ARF pathway.

Cellular senescence is an important mechanism that restricts tumour growth. The Ink4a-Arf locus (also known as Cdkn2a), which encodes p16(INK4A) and p19(ARF), has a central role in inducing and maintaining senescence. Given the importance of cellular senescence in restraining tumour growth, great emphasis is being placed on the identification of novel factors that can modulate senescence. The MYST-family histone acetyltransferase MOZ (MYST3, KAT6A), first identified in recurrent translocations in acute myeloid leukaemia, has been implicated in both the promotion and inhibition of senescence. In this study, we investigate the role of MOZ in cellular senescence and show that MOZ is a potent inhibitor of senescence via the INK4A-ARF pathway. Primary mouse embryonic fibroblasts (MEFs) isolated from Moz-deficient embryos exhibit premature senescence, which was rescued on the Ink4a-Arf(-/-) background. Importantly, senescence resulting from the absence of MOZ was not accompanied by DNA damage, suggesting that MOZ acts independently of the DNA damage response. Consistent with the importance of senescence in cancer, expression profiling revealed that genes overexpressed in aggressive and highly proliferative cancers are expressed at low levels in Moz-deficient MEFs. We show that MOZ is required to maintain normal levels of histone 3 lysine 9 (H3K9) and H3K27 acetylation at the transcriptional start sites of at least four genes, Cdc6, Ezh2, E2f2 and Melk, and normal mRNA levels of these genes. CDC6, EZH2 and E2F2 are known inhibitors of the INK4A-ARF pathway. Using chromatin immunoprecipitation, we show that MOZ occupies the Cdc6, Ezh2 and Melk loci, thereby providing a direct link between MOZ, H3K9 and H3K27 acetylation, and normal transcriptional levels at these loci. This work establishes that MOZ is an upstream inhibitor of the INK4A-ARF pathway, and suggests that inhibiting MOZ may be one way to induce senescence in proliferative tumour cells.

Histone acetylation mediated by Brd1 is crucial for Cd8 gene activation during early thymocyte development.

During T-cell development, Cd8 expression is controlled via dynamic regulation of its cis-regulatory enhancer elements. Insufficiency of enhancer activity causes variegated Cd8 expression in CD4(+)CD8(+) double-positive (DP) thymocytes. Brd1 is a subunit of the Hbo1 histone acetyltransferase (HAT) complex responsible for acetylation of histone H3 at lysine 14 (H3K14). Here we show that deletion of Brd1 in haematopoietic progenitors causes variegated expression of Cd8, resulting in the appearance of CD4(+)CD8(-)TCRß(-/low) thymocytes indistinguishable from DP thymocytes in their properties. Biochemical analysis confirms that Brd1 forms a HAT complex with Hbo1 in thymocytes. ChIP analysis demonstrates that Brd1 localizes at the known enhancers in the Cd8 genes and is responsible for acetylation at H3K14. These findings indicate that the Brd1-mediated HAT activity is crucial for efficient activation of Cd8 expression via acetylation at H3K14, which serves as an epigenetic mark that promotes the recruitment of transcription machinery to the Cd8 enhancers.

Separation of a functional deubiquitylating module from the SAGA complex by the proteasome regulatory particle.

Gene expression is an intricate process tightly linked from gene activation to the nuclear export of mRNA. Recent studies have indicated that the proteasome is essential for gene expression regulation. The proteasome regulatory particle binds to the SAGA complex and affects transcription in an ATP-dependent manner. Here we report that a specific interaction between the proteasomal ATPase, Rpt2p and Sgf73p of the SAGA complex leads to the dissociation of the H2Bub1-deubiquitylating module (herein designated the Sgf73-DUBm) from SAGA both in vitro and in vivo. We show that the localization of the Sgf73-DUBm on chromatin is perturbed in rpt2-1, a strain of Saccharomyces cerevisiae that is specifically defective in the Rpt2p-Sgf73p interaction. The rpt2-1 mutant also exhibits impaired localization of the TREX-2 and MEX67-MTR2 complexes and is defective in mRNA export. Our findings collectively demonstrate that the proteasome-mediated remodelling of the SAGA complex is a prerequisite for proper mRNA export.

MOZ increases p53 acetylation and premature senescence through its complex formation with PML.

Monocytic leukemia zinc finger (MOZ)/KAT6A is a MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST)-type histone acetyltransferase that functions as a coactivator for acute myeloid leukemia 1 protein (AML1)- and Ets family transcription factor PU.1-dependent transcription. We previously reported that MOZ directly interacts with p53 and is essential for p53-dependent selective regulation of p21 expression. We show here that MOZ is an acetyltransferase of p53 at K120 and K382 and colocalizes with p53 in promyelocytic leukemia (PML) nuclear bodies following cellular stress. The MOZ-PML-p53 interaction enhances MOZ-mediated acetylation of p53, and this ternary complex enhances p53-dependent p21 expression. Moreover, we identified an Akt/protein kinase B recognition sequence in the PML-binding domain of MOZ protein. Akt-mediated phosphorylation of MOZ at T369 has a negative effect on complex formation between PML and MOZ. As a result of PML-mediated suppression of Akt, the increased PML-MOZ interaction enhances p21 expression and induces p53-dependent premature senescence upon forced PML expression. Our research demonstrates that MOZ controls p53 acetylation and transcriptional activity via association with PML.

Long-term memory deficits in Huntington's disease are associated with reduced CBP histone acetylase activity.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by an expanded CAG/polyglutamine repeat in the coding region of the huntingtin (htt) gene. Although HD is classically considered a motor disorder, there is now considerable evidence that early cognitive deficits appear in patients before the onset of motor disturbances. Here we demonstrate early impairment of long-term spatial and recognition memory in heterozygous HD knock-in mutant mice (Hdh(Q7/Q111)), a genetically accurate HD mouse model. Cognitive deficits are associated with reduced hippocampal expression of CREB-binding protein (CBP) and diminished levels of histone H3 acetylation. In agreement with reduced CBP, the expression of CREB/CBP target genes related to memory, such c-fos, Arc and Nr4a2, was significantly reduced in the hippocampus of Hdh(Q7/Q111) mice compared with wild-type mice. Finally, and consistent with a role of CBP in cognitive impairment in Hdh(Q7/Q111) mice, administration of the histone deacetylase inhibitor trichostatin A rescues recognition memory deficits and transcription of selective CREB/CBP target genes in Hdh(Q7/Q111) mice. These findings demonstrate an important role for CBP in cognitive dysfunction in HD and suggest the use of histone deacetylase inhibitors as a novel therapeutic strategy for the treatment of memory deficits in this disease.

Inactivating mutations of acetyltransferase genes in B-cell lymphoma.

B-cell non-Hodgkin's lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types--follicular lymphoma and diffuse large B-cell lymphoma--harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300, two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin's lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms.

Loss of steroid receptor co-activator-3 attenuates carbon tetrachloride-induced murine hepatic injury and fibrosis.

Hepatic fibrosis, a disease characterized by altered accumulation of extracellular matrix, can cause cirrhosis and liver failure. There is growing interest in the impact of co-activators on hepatic fibrogenesis. Here, we provided genetic evidence that mice lacking steroid receptor co-activator-3 (SRC-3) were protected against carbon tetrachloride (CCl4)-induced acute liver necrosis and chronic hepatic fibrosis. After acute CCl4 treatment, SRC-3(-/-) mice showed attenuated profibrotic response and hepatocyte apoptosis, whereas hepatocyte proliferation was elevated in SRC-3(-/-) mice versus SRC-3+/+ mice. Similarly, chronically CCl4-treated SRC-3(-/-) mice showed significant weakening of inflammatory infiltrates, hepatic stellate cell activation and collagen accumulation in the liver compared with SRC-3+/+ mice. Further investigation revealed that TGFbeta1/Smad signaling pathway was impaired in the absence of SRC-3. Moreover, the expression levels of SRC-3, as assessed in human tissue microarray of liver diseases, correlated positively with degrees of fibrosis. These data revealed that SRC-3(-/-) mice were resistant to CCl4-induced acute and chronic hepatic damage and TGFbeta1/Smad signaling was suppressed in the lack of SRC-3. Our results established an essential involvement of SRC-3 in liver fibrogenesis, which might provide new clues to the future treatment of hepatic fibrosis.

Mig-6 modulates uterine steroid hormone responsiveness and exhibits altered expression in endometrial disease.

Normal endometrial function requires a balance of progesterone (P4) and estrogen (E2) effects. An imbalance caused by increased E2 action and/or decreased P4 action can result in abnormal endometrial proliferation and, ultimately, endometrial adenocarcinoma, the fourth most common cancer in women. We have identified mitogen-inducible gene 6 (Mig-6) as a downstream target of progesterone receptor (PR) and steroid receptor coactivator (SRC-1) action in the uterus. Here, we demonstrate that absence of Mig-6 in mice results in the inability of P4 to inhibit E2-induced uterine weight gain and E2-responsive target genes expression. At 5 months of age, the absence of Mig-6 results in endometrial hyperplasia. Ovariectomized Mig-6(d/d) mice exhibit this hyperplastic phenotype in the presence of E2 and P4 but not without ovarian hormone. Ovariectomized Mig-6(d/d) mice treated with E2 developed invasive endometrioid-type endometrial adenocarcinoma. Importantly, the observation that endometrial carcinomas from women have a significant reduction in MIG-6 expression provides compelling support for an important growth regulatory role for Mig-6 in the uterus of both humans and mice. This demonstrates the Mig-6 is a critical regulator of the response of the endometrium to E2 in regulating tissue homeostasis. Since Mig-6 is regulated by both PR and SRC-1, this identifies a PR, SRC-1, Mig-6 regulatory pathway that is critical in the suppression of endometrial cancer.

Steroid receptor coactivator 3 maintains circulating insulin-like growth factor I (IGF-I) by controlling IGF-binding protein 3 expression.

Steroid receptor coactivator 3 (SRC-3/AIB1/ACTR/NCoA-3) is a transcriptional coactivator for nuclear receptors including vitamin D receptor (VDR). Growth hormone (GH) regulates insulin-like growth factor I (IGF-I) expression, and IGF-I forms complexes with acid-labile subunit (ALS) and IGF-binding protein 3 (IGFBP-3) to maintain its circulating concentration and endocrine function. This study demonstrated that the circulating IGF-I was significantly reduced in SRC-3(-/-) mice with the C57BL/6J background. However, SRC-3 deficiency affected neither GH nor ALS expression. The low IGF-I level in SRC-3(-/-) mice was not due to the failure of IGF-I mRNA and protein synthesis but was a consequence of rapid degradation. The rapid IGF-I degradation was associated with drastically reduced IGFBP-3 levels. Because IGF-I and IGFBP-3 stabilize each other, SRC-3(-/-) mice were crossbred with the liver-specific transthyretin (TTR)-IGF-I transgenic mice to assess the relationship between reduced IGF-I and IGFBP-3. In SRC-3(-/-)/TTR-IGF-I mice, the IGF-I level was significantly increased over that in SRC-3(-/-) mice, but the IGFBP-3 level failed to increase proportionally, indicating that the low IGFBP-3 level is a responsible factor that limits the IGF-I level in SRC-3(-/-) mice. Furthermore, IGFBP-3 mRNA was reduced in SRC-3(-/-) mice. The IGFBP-3 promoter activity induced by vitamin D, through VDR, was diminished in SRC-3(-/-) cells, suggesting an important role of SRC-3 in VDR-mediated transactivation of the IGFBP-3 gene. In agreement with the role of SRC-3 in VDR function, the expression of several VDR target genes was also reduced in SRC-3(-/-) mice. Therefore, SRC-3 maintains IGF-I in the circulation through enhancing VDR-regulated IGFBP-3 expression.

The skeletal response to estrogen is impaired in female but not in male steroid receptor coactivator (SRC)-1 knock out mice.

Estrogen (E) is critical for the maintenance of bone mass in both female and male mice and steroid receptor coactivator (SRC)-1 has been shown to be important for mediating E effects on bone, at least in female mice. In the present study, we defined the skeletal phenotype of male SRC-1 knock out (KO) mice and compared it with their female littermates. Further, to determine the role of SRC-1 in mediating effects of E on bone in male mice, we examined the skeletal effects of gonadectomy (gnx) with or without E replacement in male mice and placed these findings in the context of our previous studies in female SRC-1 KO mice. Analysis of a large group of male (WT, n=67; SRC-1 KO, n=56) and female (WT, n=66; SRC-1 KO, n=70) mice showed a significant decrease in trabecular volumetric bone mineral density (vBMD) in SRC-1 KO mice compared to their WT littermates in both genders (male SRC-1 KO, 275+/-3 vs. WT, 295+/-3 mg/cm(3), P<0.001; female SRC-1 KO, 210+/-2 vs. WT, 221+/-2 mg/cm(3), P<0.001). Following gnx and E replacement (10 microg/kg/day), we previously demonstrated that SRC-1 KO female mice have a defect in E action in trabecular, but not in cortical bone. In contrast, we now demonstrate that the same dose of E administered to gnx'd male SRC-1 KO mice was sufficient to prevent trabecular bone loss in these mice. For example, in WT female mice, gnx followed by E replacement maintained spine BMD (1.2+/-3.4% vs. baseline) as compared to gnx without E replacement (-12.7+/-2.6%, P<0.001 vs. sham); this effect of E was absent in SRC-1 KO female mice. By contrast, the identical dose of E was equally effective in maintaining spine BMD in E-treated gnx'd male WT (-5.2+/-5.1% vs. baseline) and male SRC-1 KO (-5.4+/-5.3%) mice, respectively, as compared to gnx'd mice without E treatment (WT, -17.6+/-2.5%, P=0.02; SRC-1 KO, -28.6+/-2.6%, P<0.001 vs. sham). E treatment was effective in suppressing cancellous bone turnover in both gnx'd WT and SRC-1 KO male mice as determined by significant reductions in osteoblast and osteoclast numbers; however, in female mice, E treatment only suppressed bone turnover in WT but not in SRC-1 KO mice. Collectively, these findings demonstrate that loss of SRC-1 results in trabecular osteopenia in male and female mice, but in contrast to female mice, this is not due to any detectable resistance to E action in trabecular bone in male SRC-1 KO mice.

p300 protein acetyltransferase activity suppresses systemic lupus erythematosus-like autoimmune disease in mice.

Conditional knock-in mice expressing a histone acetyltransferase-deficient version of the transcriptional coregulator p300 exclusively in B lymphocytes die prematurely with full penetrance. The mice develop an autoimmune disease similar to systemic lupus erythematosus in its pathological manifestations, such as splenomegaly, glomerulonephritis, vasculitis, deposition of immune complexes, and production of autoantibodies against dsDNA. Aged mice show a severe reduction of transitional and marginal zone B cells and generate aberrant mature B cells. These B cells show diminished proliferation in response to stimulation of the BCR, but respond normally to other stimuli. Yet, the mice mount a normal primary immune response against a T-dependent Ag. In contrast, the memory response is impaired. In addition, serum Ig levels, in particular IgG2b, are increased. We conclude that p300 acetyltransferase activity is essential for maintaining self-tolerance of B lymphocytes. These findings support the concept of treating lupus with inhibitors of protein deacetylases and point to B cells as a critical target of these drugs.

Loss of Gcn5 acetyltransferase activity leads to neural tube closure defects and exencephaly in mouse embryos.

Gcn5 was the first transcription-related histone acetyltransferase (HAT) to be identified. However, the functions of this enzyme in mammalian cells remain poorly defined. Deletion of Gcn5 in mice leads to early embryonic lethality with increased apoptosis in mesodermal lineages. Here we show that deletion of p53 allows Gcn5(-/-) embryos to survive longer, but Gcn5(-/-) p53(-/-) embryos still die in midgestation. Interestingly, embryos homozygous for point mutations in the Gcn5 catalytic domain survive significantly longer than Gcn5(-/-) or Gcn5(-/-) p53(-/-) mice. In contrast to Gcn5(-/-) embryos, Gcn5(hat/hat) embryos do not exhibit increased apoptosis but do exhibit severe cranial neural tube closure defects and exencephaly. Together, our results indicate that Gcn5 has important, HAT-independent functions in early development and that Gcn5 acetyltransferase activity is required for cranial neural tube closure in the mouse.

Steroid receptor coactivator-3 and activator protein-1 coordinately regulate the transcription of components of the insulin-like growth factor/AKT signaling pathway.

Steroid receptor coactivator (SRC)-3, also called amplified in breast cancer 1, is a member of the p160 nuclear receptor coactivator family involved in transcriptional regulation of target genes. SRC-3 is frequently amplified and/or overexpressed in hormone-sensitive and hormone-insensitive tumors. We reported previously that SRC-3 stimulated prostate cell growth in a hormone-independent manner through activation of AKT signaling pathway. However, the underlying mechanism remains undefined. Here, we exploited the mifepristone-induced SRC-3 LNCaP prostate cancer cell line generated in our laboratory to identify SRC-3-regulated genes by oligonucleotide microarray analysis. We found that SRC-3 up-regulates the expression of multiple genes in the insulin-like growth factor (IGF)/AKT signaling pathway that are involved in cell proliferation and survival. In contrast, knockdown of SRC-3 in PC3 (androgen receptor negative) prostate cancer cells and MCF-7 breast cancer cells reduces their expression. Similarly, in prostate glands of SRC-3 null mice, expressions of these components in the IGF/AKT signal pathway are also reduced. Chromatin immunoprecipitation assay revealed that SRC-3 was directly recruited to the promoters of these genes, indicating that they are direct targets of SRC-3. Interestingly, we showed that recruitment of SRC-3 to two target promoters, IRS-2 and IGF-I, requires transcription factor activator protein-1 (AP-1). Taken together, our results clearly show that SRC-3 and AP-1 can coordinately regulate the transcription of multiple components in the IGF/AKT pathway to ensure ligand-independent cell proliferation and survival of cancer cells.