Loss of the methylarginine reader function of SND1 confers resistance to hepatocellular carcinoma.

Staphylococcal nuclease Tudor domain containing 1 (SND1) protein is an oncogene that 'reads' methylarginine marks through its Tudor domain. Specifically, it recognizes methylation marks deposited by protein arginine methyltransferase 5 (PRMT5), which is also known to promote tumorigenesis. Although SND1 can drive hepatocellular carcinoma (HCC), it is unclear whether the SND1 Tudor domain is needed to promote HCC. We sought to identify the biological role of the SND1 Tudor domain in normal and tumorigenic settings by developing two genetically engineered SND1 mouse models, an Snd1 knockout (Snd1 KO) and an Snd1 Tudor domain-mutated (Snd1 KI) mouse, whose mutant SND1 can no longer recognize PRMT5-catalyzed methylarginine marks. Quantitative PCR analysis of normal, KO, and KI liver samples revealed a role for the SND1 Tudor domain in regulating the expression of genes encoding major acute phase proteins, which could provide mechanistic insight into SND1 function in a tumor setting. Prior studies indicated that ectopic overexpression of SND1 in the mouse liver dramatically accelerates the development of diethylnitrosamine (DEN)-induced HCC. Thus, we tested the combined effects of DEN and SND1 loss or mutation on the development of HCC. We found that both Snd1 KO and Snd1 KI mice were partially protected against malignant tumor development following exposure to DEN. These results support the development of small molecule inhibitors that target the SND1 Tudor domain or the use of upstream PRMT5 inhibitors, as novel treatments for HCC.

Genome-wide CRISPR-Cas9 screen analyzed by SLIDER identifies network of repressor complexes that regulate TRIM24.

TRIM24 is an oncogenic chromatin reader that is frequently overexpressed in human tumors and associated with poor prognosis. However, TRIM24 is rarely mutated, duplicated, or rearranged in cancer. This raises questions about how TRIM24 is regulated and what changes in its regulation are responsible for its overexpression. Here, we perform a genome-wide CRISPR-Cas9 screen by fluorescence-activated cell sorting (FACS) that nominated 220 negative regulators and elucidated a regulatory network that includes the KAP1 corepressor, CNOT deadenylase, and GID/CTLH E3 ligase. Knocking out required components of these three complexes caused TRIM24 overexpression, confirming their negative regulation of TRIM24. Our findings identify regulators of TRIM24 that nominate previously unexplored contexts for this oncoprotein in biology and disease. These findings were enabled by SLIDER, a new scoring system designed and vetted in our study as a broadly applicable tool for analysis of CRISPR screens performed by FACS.

Mammary-specific expression of Trim24 establishes a mouse model of human metaplastic breast cancer.

Conditional overexpression of histone reader Tripartite motif containing protein 24 (TRIM24) in mouse mammary epithelia (Trim24COE) drives spontaneous development of mammary carcinosarcoma tumors, lacking ER, PR and HER2. Human carcinosarcomas or metaplastic breast cancers (MpBC) are a rare, chemorefractory subclass of triple-negative breast cancers (TNBC). Comparison of Trim24COE metaplastic carcinosarcoma morphology, TRIM24 protein levels and a derived Trim24COE gene signature reveals strong correlation with human MpBC tumors and MpBC patient-derived xenograft (PDX) models. Global and single-cell tumor profiling reveal Met as a direct oncogenic target of TRIM24, leading to aberrant PI3K/mTOR activation. Here, we find that pharmacological inhibition of these pathways in primary Trim24COE tumor cells and TRIM24-PROTAC treatment of MpBC TNBC PDX tumorspheres decreased cellular viability, suggesting potential in therapeutically targeting TRIM24 and its regulated pathways in TRIM24-expressing TNBC.

TMEM9-v-ATPase Activates Wnt/ß-Catenin Signaling Via APC Lysosomal Degradation for Liver Regeneration and Tumorigenesis.

BACKGROUND AND AIMS: How Wnt signaling is orchestrated in liver regeneration and tumorigenesis remains elusive. Recently, we identified transmembrane protein 9 (TMEM9) as a Wnt signaling amplifier. APPROACH AND RESULTS: TMEM9 facilitates v-ATPase assembly for vesicular acidification and lysosomal protein degradation. TMEM9 is highly expressed in regenerating liver and hepatocellular carcinoma (HCC) cells. TMEM9 expression is enriched in the hepatocytes around the central vein and acutely induced by injury. In mice, Tmem9 knockout impairs hepatic regeneration with aberrantly increased adenomatosis polyposis coli (Apc) and reduced Wnt signaling. Mechanistically, TMEM9 down-regulates APC through lysosomal protein degradation through v-ATPase. In HCC, TMEM9 is overexpressed and necessary to maintain ß-catenin hyperactivation. TMEM9-up-regulated APC binds to and inhibits nuclear translocation of ß-catenin, independent of HCC-associated ß-catenin mutations. Pharmacological blockade of TMEM9-v-ATPase or lysosomal degradation suppresses Wnt/ß-catenin through APC stabilization and ß-catenin cytosolic retention. CONCLUSIONS: Our results reveal that TMEM9 hyperactivates Wnt signaling for liver regeneration and tumorigenesis through lysosomal degradation of APC.

CARM1 inhibition reduces histone acetyltransferase activity causing synthetic lethality in CREBBP/EP300-mutated lymphomas.

Somatic mutations affecting CREBBP and EP300 are a hallmark of diffuse large B-cell lymphoma (DLBCL). These mutations are frequently monoallelic, within the histone acetyltransferase (HAT) domain and usually mutually exclusive, suggesting that they might affect a common pathway, and their residual WT expression is required for cell survival. Using in vitro and in vivo models, we found that inhibition of CARM1 activity (CARM1i) slows DLBCL growth, and that the levels of sensitivity are positively correlated with the CREBBP/EP300 mutation load. Conversely, treatment of DLBCLs that do not have CREBBP/EP300 mutations with CARM1i and a CBP/p300 inhibitor revealed a strong synergistic effect. Our mechanistic data show that CARM1i further reduces the HAT activity of CBP genome wide and downregulates CBP-target genes in DLBCL cells, resulting in a synthetic lethality that leverages the mutational status of CREBBP/EP300 as a biomarker for the use of small-molecule inhibitors of CARM1 in DLBCL and other cancers.

ZEB1/NuRD complex suppresses TBC1D2b to stimulate E-cadherin internalization and promote metastasis in lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, due in part to the propensity of lung cancer to metastasize. Aberrant epithelial-to-mesenchymal transition (EMT) is a proposed model for the initiation of metastasis. During EMT cell-cell adhesion is reduced allowing cells to dissociate and invade. Of the EMT-associated transcription factors, ZEB1 uniquely promotes NSCLC disease progression. Here we apply two independent screens, BioID and an Epigenome shRNA dropout screen, to define ZEB1 interactors that are critical to metastatic NSCLC. We identify the NuRD complex as a ZEB1 co-repressor and the Rab22 GTPase-activating protein TBC1D2b as a ZEB1/NuRD complex target. We find that TBC1D2b suppresses E-cadherin internalization, thus hindering cancer cell invasion and metastasis.

TRIM24 suppresses development of spontaneous hepatic lipid accumulation and hepatocellular carcinoma in mice.

BACKGROUND & AIMS: Aberrantly high expression of TRIM24 occurs in human cancers, including hepatocellular carcinoma. In contrast, TRIM24 in the mouse is reportedly a liver-specific tumour suppressor. To address this dichotomy and to uncover direct regulatory functions of TRIM24 in vivo, we developed a new mouse model that lacks expression of all Trim24 isoforms, as the previous model expressed normal levels of Trim24 lacking only exon 4. METHODS: To produce germline-deleted Trim24(dlE1) mice, deletion of the promoter and exon 1 of Trim24 was induced in Trim24(LoxP) mice by crossing with a zona pellucida 3-Cre line for global deletion. Liver-specific deletion (Trim24(hep)) was achieved by crossing with an albumin-Cre line. Phenotypic analyses were complemented by protein, gene-specific and global RNA expression analyses and quantitative chromatin immunoprecipitation. RESULTS: Global loss of Trim24 disrupted hepatic homeostasis in 100% of mice with highly significant, decreased expression of oxidation/reduction, steroid, fatty acid, and lipid metabolism genes, as well as increased expression of genes involved in unfolded protein response, endoplasmic reticulum stress and cell cycle pathways. Trim24(dlE1/dlE1) mice have markedly depleted visceral fat and, like Trim24(hep/hep) mice, spontaneously develop hepatic lipid-filled lesions, steatosis, hepatic injury, fibrosis and hepatocellular carcinoma. CONCLUSIONS: TRIM24, an epigenetic co-regulator of transcription, directly and indirectly represses hepatic lipid accumulation, inflammation, fibrosis and damage in the murine liver. Complete loss of Trim24 offers a model of human non-alcoholic fatty liver disease, steatosis, fibrosis and development of hepatocellular carcinoma in the absence of high-fat diet or obesity.

p53 regulates a mitotic transcription program and determines ploidy in normal mouse liver.

UNLABELLED: Functions of p53 during mitosis reportedly include prevention of polyploidy and transmission of aberrant chromosomes. However, whether p53 plays these roles during genomic surveillance in vivo and, if so, whether this is done via direct or indirect means remain unknown. The ability of normal, mature hepatocytes to respond to stimuli, reenter the cell cycle, and regenerate liver mass offers an ideal setting to assess mitosis in vivo. In quiescent liver, normally high ploidy levels in adult mice increased with loss of p53. Following partial hepatectomy, p53(-/-) hepatocytes exhibited early entry into the cell cycle and prolonged proliferation with an increased number of polyploid mitoses. Ploidy levels increased during regeneration of both wild-type (WT) and p53(-/-) hepatocytes, but only WT hepatocytes were able to dynamically resolve ploidy levels and return to normal by the end of regeneration. We identified multiple cell cycle and mitotic regulators, including Foxm1, Aurka, Lats2, Plk2, and Plk4, as directly regulated by chromatin interactions of p53 in vivo. Over a time course of regeneration, direct and indirect regulation of expression by p53 is mediated in a gene-specific manner. CONCLUSION: Our results show that p53 plays a role in mitotic fidelity and ploidy resolution in hepatocytes of normal and regenerative liver.

Genome-wide location analysis reveals distinct transcriptional circuitry by paralogous regulators Foxa1 and Foxa2.

Gene duplication is a powerful driver of evolution. Newly duplicated genes acquire new roles that are relevant to fitness, or they will be lost over time. A potential path to functional relevance is mutation of the coding sequence leading to the acquisition of novel biochemical properties, as analyzed here for the highly homologous paralogs Foxa1 and Foxa2 transcriptional regulators. We determine by genome-wide location analysis (ChIP-Seq) that, although Foxa1 and Foxa2 share a large fraction of binding sites in the liver, each protein also occupies distinct regulatory elements in vivo. Foxa1-only sites are enriched for p53 binding sites and are frequently found near genes important to cell cycle regulation, while Foxa2-restricted sites show only a limited match to the forkhead consensus and are found in genes involved in steroid and lipid metabolism. Thus, Foxa1 and Foxa2, while redundant during development, have evolved divergent roles in the adult liver, ensuring the maintenance of both genes during evolution.

Direct activation of forkhead box O3 by tumor suppressors p53 and p73 is disrupted during liver regeneration in mice.

UNLABELLED: The p53 family of proteins regulates the expression of target genes that promote cell cycle arrest and apoptosis, which may be linked to cellular growth control as well as tumor suppression. Within the p53 family, p53 and the transactivating p73 isoform (TA-p73) have hepatic-specific functions in development and tumor suppression. Here, we determined TA-p73 interactions with chromatin in the adult mouse liver and found forkhead box O3 (Foxo3) to be one of 158 gene targets. Global profiling of hepatic gene expression in the regenerating liver versus the quiescent liver revealed specific, functional categories of genes regulated over the time of regeneration. Foxo3 is the most responsive gene among transcription factors with altered expression during regenerative cellular proliferation. p53 and TA-p73 bind a Foxo3 p53 response element (p53RE) and maintain active expression in the quiescent liver. During regeneration of the liver, the binding of p53 and TA-p73, the recruitment of acetyltransferase p300, and the active chromatin structure of Foxo3 are disrupted along with a loss of Foxo3 expression. In agreement with the loss of Foxo3 transcriptional activation, a decrease in histone activation marks (dimethylated histone H3 at lysine 4, acetylated histone H3 at lysine 14, and acetylated H4) at the Foxo3 p53RE was detected after partial hepatectomy in mice. These parameters of Foxo3 regulation are reestablished with the completion of liver growth and regeneration and support a temporary suspension of p53 and TA-p73 regulatory functions in normal cells during tissue regeneration. p53-dependent and TA-p73-dependent activation of Foxo3 was also observed in mouse embryonic fibroblasts and in mouse hepatoma cells overexpressing p53, TA-p73alpha, and TA-p73beta isoforms. CONCLUSION: p53 and p73 directly bind and activate the expression of the Foxo3 gene in the adult mouse liver and murine cell lines. p53, TA-p73, and p300 binding and Foxo3 expression decrease during liver regeneration, and this suggests a critical growth control mechanism mediated by these transcription factors in vivo.

Family members p53 and p73 act together in chromatin modification and direct repression of alpha-fetoprotein transcription.

Aberrant expression of the alpha-fetoprotein (AFP) gene is a diagnostic tumor marker of hepatocellular carcinoma. We find that AFP gene expression is repressed by the TP53 family member p73 during normal hepatic development and when p73alpha or p73beta is introduced into cultured hepatoma cells that express AFP. Transient co-transfection of p53 family members showed that p53 and transactivating (TA)-p73, but not TA-p63, repress endogenous AFP transcription additively or independently. p53-independent functions of p73 are further supported by delayed, p73-associated compensation of AFP repression during development of the p53-null mouse. Chromatin immunoprecipitation assays of normal and p53-null mouse liver tissue showed that TA-p73 binds at a previously identified p53 repressor site (-860/-830) within the distal promoter of AFP at a level equivalent to p53 in wild type liver, with increased binding of TA-p73 to chromatin in the absence of p53. Sequential chromatin immunoprecipitation analyses revealed that TA-p73 and p53 bind simultaneously to their shared regulatory site in wild type liver. Like the founding family member p53, TA-p73 represses AFP expression by chromatin structure alteration, targeting reduction of acetylated histone H3 lysine 9 and increased dimethylated histone H3 lysine 9 levels. However, chromatin-bound TA-p73 is associated with elevated di- and tri-methylated histone H3 lysine 4 levels in p53-null liver and hepatoma cells, concomitant with a reduced ability to repress transcription compared with p53.

Transcription factor interactions and chromatin modifications associated with p53-mediated, developmental repression of the alpha-fetoprotein gene.

We performed chromatin immunoprecipitation (ChIP) analyses of developmentally staged solid tissues isolated from wild-type and p53-null mice to determine specific histone N-terminal modifications, histone-modifying proteins, and transcription factor interactions at the developmental repressor region (-850) and core promoter of the hepatic tumor marker alpha-fetoprotein (AFP) gene. Both repression of AFP during liver development and silencing in the brain, where AFP is never expressed, are associated with dimethylation of histone H3 lysine 9 (DiMetH3K9) and the presence of heterochromatin protein 1 (HP1). These heterochromatic markers remain localized to AFP during developmental repression but spread to the upstream albumin gene during silencing. Developmentally regulated decreases in levels of acetylated H3 (AcH3K9) and H4 (AcH4) and of di- and trimethylated H3K4 (DiMetH3K4 and TriMetH3K4) occur at both the core promoter and distal repressor regions of AFP. Hepatic expression of AFP correlates with FoxA interaction at the repressor region and the binding of RNA polymerase II and TATA-binding protein to the core promoter. p53 acts as a developmental repressor of AFP in the liver by binding to chromatin, excluding FoxA interaction and targeting mSin3A/HDAC1 to the distal repressor region. p53-null mice exhibit developmentally delayed AFP repression, concomitant with acetylation of H3K9, methylation of H3K4, and loss of DiMetH3K9, mSin3A/HDAC1, and HP1 interactions.

A direct intersection between p53 and transforming growth factor beta pathways targets chromatin modification and transcription repression of the alpha-fetoprotein gene.

We purified the oncoprotein SnoN and found that it functions as a corepressor of the tumor suppressor p53 in the regulation of the hepatic alpha-fetoprotein (AFP) tumor marker gene. p53 promotes SnoN and histone deacetylase interaction at an overlapping Smad binding, p53 regulatory element (SBE/p53RE) in AFP. Comparison of wild-type and p53-null mouse liver tissue by using chromatin immunoprecipitation (ChIP) reveals that the absence of p53 protein correlates with the disappearance of SnoN at the SBE/p53RE and loss of AFP developmental repression. Treatment of AFP-expressing hepatoma cells with transforming growth factor-beta1 (TGF-beta1) induced SnoN transcription and Smad2 activation, concomitant with AFP repression. ChIP assays show that TGF-beta1 stimulates p53, Smad4, P-Smad2 binding, and histone H3K9 deacetylation and methylation, at the SBE/p53RE. Depletion, by small interfering RNA, of SnoN and/or p53 in hepatoma cells disrupted repression of AFP transcription. These findings support a model of cooperativity between p53 and TGF-beta effectors in chromatin modification and transcription repression of an oncodevelopmental tumor marker gene.