Disruption Of P53 Research Articles

Unraveling the mechanisms of glioblastoma's resistance: investigating the influence of tumor suppressor p53 and non-coding RNAs.

Glioblastoma (GB) is one of the most fatal CNS malignancies, and its high resistance to therapy and poor outcomes have made it one of the primary challenges in oncology. Resistance to standard therapy, i.e., radio-chemotherapy with temozolomide, is one of the principal causes of the poor prognostic outcomes of GB. Finding the molecular basis of GB resistance to therapy is key to creating effective solution approaches. The general problem of GB resistance is supervised by cancer suppressive protein, p53, and has become a very special interest in molecular research in recent decades. The principal aim of this manuscript is to perform a comprehensive survey on the complex network of interactions developed by p53 with non-coding RNAs (ncRNA) in the context of GB resistance. The present article details the functional aspects of p53 as a cellular stress response protein, including its roles in apoptosis, cell cycle regulation, and DNA repair in glioblastoma (GB), along with the disruption of p53 and its involvement in chemoresistance (CR). It also highlights several classes of ncRNAs, namely microRNAs, long ncRNAs, and circular RNAs, that manipulate p53 signaling in GB-CR. The article likewise explains how disruption in the expression of these ncRNAs can promote GB-CR and how it interacts with essential cellular functions, such as proliferation, apoptosis, and DNA repair. The manuscript also describes the potential of targeting p53 and ncRNAs with their diagnostic and prognostic potential as novel promising therapeutics for GB. Nevertheless, ncRNA-based biomarkers still present challenges for their suitability in GB resistance. However, modern research continues to discover novel prediction targets, potentially enhancing patient outcomes and therapeutic options. Therefore, the neutralization of this intricate regulatory network of GB resistance might have a primary clinical effect in fighting GB resistance therapy and thus might lead to a substantial increase in patient survival and quality of life.

Activation of mTOR signaling in adult lung microvascular progenitor cells accelerates lung aging.

Reactivation and dysregulation of the mTOR signaling pathway are a hallmark of aging and chronic lung disease; however, the impact on microvascular progenitor cells (MVPCs), capillary angiostasis, and tissue homeostasis is unknown. While the existence of an adult lung vascular progenitor has long been hypothesized, these studies show that Abcg2 enriches for a population of angiogenic tissue-resident MVPCs present in both adult mouse and human lungs using functional, lineage, and transcriptomic analyses. These studies link human and mouse MVPC-specific mTORC1 activation to decreased stemness, angiogenic potential, and disruption of p53 and Wnt pathways, with consequent loss of alveolar-capillary structure and function. Following mTOR activation, these MVPCs adapt a unique transcriptome signature and emerge as a venous subpopulation in the angiodiverse microvascular endothelial subclusters. Thus, our findings support a significant role for mTOR in the maintenance of MVPC function and microvascular niche homeostasis as well as a cell-based mechanism driving loss of tissue structure underlying lung aging and the development of emphysema.

In Vivo Intra-Uterine Delivery of TAT-Fused Cre Recombinase and CRISPR/Cas9 Editing System in Mice Unveil Histopathology of Pten/p53-Deficient Endometrial Cancers.

Phosphatase and TENsin homolog (Pten) and p53 are two of the most frequently mutated tumor suppressor genes in endometrial cancer. However, the functional consequences and histopathological manifestation of concomitant p53 and Pten loss of function alterations in the development of endometrial cancer is still controversial. Here, it is demonstrated that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel intravaginal delivery method using HIV1 trans-activator of transcription cell penetrating peptide fused with a Cre recombinase protein (TAT-Cre), local ablation of both p53 and Pten is achieved specifically in the uterus. These mice developed high-grade endometrial carcinomas and a high percentage of uterine carcinosarcomas resembling those found in humans. To further demonstrate that carcinosarcomas arise from epithelium, double Pten/p53 deficient epithelial cells are mixed with wild type stromal and myometrial cells and subcutaneously transplanted to Scid mice. All xenotransplants resulted in the development of uterine carcinosarcomas displaying high nuclear pleomorphism and metastatic potential. Accordingly, in vivo CRISPR/Cas9 disruption of Pten and p53 also triggered the development of metastatic carcinosarcomas. The results unfadingly demonstrate that simultaneous deletion of p53 and Pten in endometrial epithelial cells is enough to trigger epithelial to mesenchymal transition that is consistently translated to the formation of uterine carcinosarcomas in vivo.

Andrographolide causes p53-independent HCC cell death through p62 accumulation and impaired DNA damage repair

BackgroundHepatocellular carcinoma (HCC) is a highly lethal cancer characterized by dominant driver mutations, including p53. Consequently, there is an urgent need to search for novel therapeutic agents to treat HCC. Andrographolide (Andro), a clinically available anti-inflammatory phytochemical agent, has shown inhibitory effects against various types of cancer, including HCC. However, the underlying molecular mechanisms of its action remain poorly understood. PurposeThis study aims to investigate the molecular mechanisms by which p53 and p62 collectively affect Andro-induced HCC cell death, using both in vitro and in vivo models. MethodsIn vitro cellular experiments were conducted to examine the effects of Andro on cell viability and elucidate its mechanisms of action. In vivo xenograft experiments further validated the anti-cancer effects of Andro. ResultsAndro induced dose- and time-dependent HCC cell death while sparing normal HL-7702 hepatocytes. Furthermore, Andro caused DNA damage through the generation of reactive oxygen species (ROS), a critical event leading to cell death. Notably, HCC cells expressing p53 exhibited greater resistance to Andro-induced cell death compared to p53-deficient cells, likely due to the ability of p53 to induce G2/M cell cycle arrest. Additionally, Andro-induced p62 aggregation led to the proteasomal degradation of RAD51 and 53BP1, two key proteins involved in DNA damage repair. Consequently, silencing or knocking out p62 facilitated DNA damage repair and protected HCC cells. Importantly, disruption of either p53 or p62 did not affect the expression of the other protein. These findings were further supported by the observation that xenograft tumors formed by p62-knockout HCC cells displayed increased resistance to Andro treatment. ConclusionThis study elucidates the mechanistic basis of Andro-induced HCC cell death. It provides valuable insights for repurposing Andro for the treatment of HCC, regardless of the presence of functional p53.

Synergistic effects of combined immunotherapy strategies in a model of multifocal hepatocellular carcinoma

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4+ anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive Tcell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance Tcell infiltration and the intratumoral performance of T lymphocytes.

Congenital aflatoxicosis, mal-detoxification genomics & ontogeny trigger immune-mediated Kotb disease biliary atresia variant: SANRA compliant review.

Biliary atresia (BA) is the most common indication for pediatric liver transplantation. We describe The BA variant: Kotb disease. Liver tissue in the Kotb disease BA is massively damaged by congenital aflatoxicosis resulting in inflammation, adhesions, fibrosis, bile duct proliferation, scarring, cholestasis, focal syncytial giant cell transformation, and typical immune response involving infiltration by CD4+, CD8+, CD68+, CD14+, neutrophil infiltration, neutrophil elastase spill, heavy loads of aflatoxin B1, accelerated cirrhosis, disruption of p53 and GSTPi, and have null glutathione S transferase M1 (GSTM1). All their mothers are heterozygous for GSTM1. This inability to detoxify aflatoxicosis results in progressive inflammatory adhesions and obliterative cholangiopathy early in life. The typical disruption of both p53 and GSTPi causes loss of fidelity of hepatic regeneration. Hence, regeneration in Kotb disease BA typically promotes accelerated cirrhosis. The immune response in Kotb disease BA is for damage control and initiation of regeneration, yet, this friendly fire incurs massive structural collateral damage. The Kotb disease BA is about actual ongoing hepatic entrapment of aflatoxins with lack of ability of safe disposal due to child detoxification-genomics disarray.The Kotb disease BA is a product of the interaction of persistent congenital aflatoxicosis, genetic lack of GSTM1 detoxification, ontogenically impaired activity of other hepatic detoxification, massive neutrophil-elastase, immune-induced damage, and disturbed regeneration. Ante-natal and neonatal screening for aflatoxicosis, avoiding cord milking, and stringent control of aflatoxicosis content of human, poultry and live-stock feeds might prove effective for prevention, prompt diagnosis and management based on our recent understanding of its patho-genomics.

P53 Pathway Inactivation Drives SMARCB1-deficient p53-wildtype Epithelioid Sarcoma Onset Indicating Therapeutic Vulnerability Through MDM2 Inhibition.

Loss of the gene SMARCB1 drives the development of malignant rhabdoid tumors, epithelioid sarcomas, and other malignancies. The SMARCB1 protein is a core component of the SWI/SNF (SWItch/Sucrose Non-Fermentable) family of chromatin remodeling complexes, which are important regulators of gene expression and cell differentiation. Here, we use CRISPR-Cas9 to create germline smarcb1 loss of function in zebrafish. We demonstrate that the combination of smarcb1 deficiency with mutant p53 results in the development of epithelioid sarcomas, angiosarcomas, and carcinomas of the thyroid and colon. Although human epithelioid sarcomas do not frequently harbor p53 mutations, smarcb1-deficient tumors in zebrafish were only observed following disruption of p53, indicating that p53 signaling in human tumors might be attenuated through alternative mechanisms, such as MDM2-mediated proteasomal degradation of p53. To leverage this possibility for the treatment of human epithelioid sarcoma, we tested small molecule-mediated disruption of the p53-MDM2 interaction, which stabilized p53 protein leading to p53-pathway reactivation, cell-cycle arrest, and increased apoptosis. Moreover, we found that MDM2 inhibition and the topoisomerase II inhibitor doxorubicin synergize in targeting epithelioid sarcoma cell viability. This could be especially relevant for patients with epithelioid sarcoma because doxorubicin represents the current gold standard for their clinical treatment. Our results therefore warrant reactivating p53 protein in SMARCB1-deficient, p53-wildtype epithelioid sarcomas using combined doxorubicin and MDM2 inhibitor therapy.

Disruption of tp53 leads to cutaneous nevus and melanoma formation in Xenopus tropicalis.

In humans, germline TP53 mutations predispose carriers to a wide spectrum of cancers, which is known as Li–Fraumeni syndrome (LFS). To date, the association of melanomas with LFS remains unestablished. No melanomas have been reported in any P53‐modified mouse models either. In this study, we show that targeted disruption of P53 at the DNA‐binding domain in Xenopus tropicalis recapitulates LFS, with the formation of soft‐tissue sarcomas and pancreatic ductal adenocarcinoma. Interestingly, 19% of the 14‐month‐old tp53 Δ7/Δ7 homozygotes and 18% of tp53 +/Δ7 heterozygotes spontaneously developed small nevi and non‐invasive melanomas. Large invasive melanomas were also observed in other older homozygous mutants, with about 7.9% penetrance. Our data suggest that more dermatologic investigation of LFS patients should be able to settle the association of melanoma with LFS in epidemiology. Our model is also valuable for further investigation of the molecular mechanism underlying melanoma progression upon germline alteration of the tp53 locus.

E2f2 Attenuates Apoptosis of Activated T Lymphocytes and Protects from Immune-Mediated Injury through Repression of Fas and FasL.

Targeted disruption of E2f2 in mice causes T-cell hyperactivation and a disproportionate cell cycle entry upon stimulation. However, E2f2−/− mice do not develop a lymphoproliferative condition. We report that E2f2 plays a Fas-dependent anti-apoptotic function in vitro and in vivo. TCR-stimulated murine E2f2−/− T cells overexpress the proapoptotic genes Fas and FasL and exhibit enhanced apoptosis, which is prevented by treatment with neutralizing anti-FasL antibodies. p53 pathway is activated in TCR-stimulated E2f2−/− lymphocytes, but targeted disruption of p53 in E2f2−/− mice does not abrogate Fas/FasL expression or apoptosis, implying a p53-independent apoptotic mechanism. We show that E2f2 is recruited to Fas and FasL gene promoters to repress their expression. in vivo, E2f2−/− mice are prone to develop immune-mediated liver injury owing to an aberrant lymphoid Fas/FasL activation. Taken together, our results suggest that E2f2-dependent inhibition of Fas/FasL pathway may play a direct role in limiting the development of immune-mediated pathologies.

P53 coordinates glucose and choline metabolism during the mesendoderm differentiation of human embryonic stem cells.

Metabolism plays crucial roles in the fate decision of human embryonic stem cells (hESCs). Here, we show that the depletion of p53 in hESCs enhances glycolysis and reduces oxidative phosphorylation, and delays mesendoderm differentiation of hESCs. More intriguingly, the disruption of p53 in hESCs leads to dramatic upregulation of phosphatidylcholine and decrease of total choline in both pluripotent and differentiated state of hESCs, suggesting abnormal choline metabolism in the absence of p53. Collectively, our study reveals the indispensable role of p53 in orchestrating both glucose and lipid metabolism to maintain proper hESC identity.

Cell Cycle Checkpoints Cooperate to Suppress DNA- and RNA-Associated Molecular Pattern Recognition and Anti-Tumor Immune Responses

SUMMARYThe DNA-dependent pattern recognition receptor, cGAS (cyclic GMP-AMP synthase), mediates communication between the DNA damage and the immune responses. Mitotic chromosome missegregation stimulates cGAS activity; however, it is unclear whether progression through mitosis is required for cancercell-intrinsic activation of anti-tumor immune responses. Moreover, it is unknown whether cell cycle checkpoint disruption can restore responses in cancer cells that are recalcitrant to DNAdamage-induced inflammation. Here, we demonstrate that prolonged cell cycle arrest at the G2-mitosis boundary from either excessive DNA damage or CDK1 inhibition prevents inflammatory-stimulated gene expression and immune-mediated destruction of distal tumors. Remarkably, DNAdamage-induced inflammatory signaling is restored in a RIG-I-dependent manner upon concomitant disruption of p53 and the G2 checkpoint. These findings link aberrant cell progression and p53 loss to an expanded spectrum of damage-associated molecular pattern recognition and have implications for the design of rational approaches to augment anti-tumor immune responses.

P53 aggregation, interactions with tau, and impaired DNA damage response in Alzheimer\u2019s disease

The transcription factor, p53, is critical for many important cellular functions involved in genome integrity, including cell cycle control, DNA damage response, and apoptosis. Disruption of p53 results in a wide range of disorders including cancer, metabolic diseases, and neurodegenerative diseases. Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by protein aggregates that contribute to disease pathology. Although p53 is known to aggregate, its propensity to aggregate in AD has never been assessed. Moreover, AD neuropathology includes lethal cell cycle re-entry, excessive DNA damage, and abnormal cell death which are all controlled by p53. Here, we show p53 forms oligomers and fibrils in human AD brain, but not control brain. p53 oligomers can also be detected in htau and P301L mouse models. Additionally, we demonstrate that p53 interacts with tau, specifically tau oligomers, in AD brain and can be recapitulated by in vitro exogenous tau oligomer treatment in C57BL/6 primary neurons. p53 oligomers also colocalize, potentially seeding, endogenous p53 in primary neurons. Lastly, we demonstrate that in the presence of DNA damage, phosphorylated p53 is mislocalized outside the nucleus and p53-mediated DNA damage responders are significantly decreased in AD brain. Control brain shows a healthy DNA damage response, indicating a loss of nuclear p53 function in AD may be due to p53 aggregation and/or interactions with tau oligomers. Given the critical role of p53 in cellular physiology, the disruption of this crucial transcription factor may set an irreversible course towards neurodegeneration in AD and potentially other tauopathies, warranting further investigation.

Metabolic Regulation of Hippocampal Neuroprogenitor Apoptosis After Irradiation.

The tumor suppressor p53 is an important regulator of cell fate response after DNA damage. Cell fate response following metabolic stresses has also been linked to p53-dependent pathways. In this study, we asked if 5'-adenosine monophosphate-activated protein kinase (AMPK), the master sensor of cellular energy balance, played a role in p53-dependent apoptosis of neural progenitor cells (NPCs) in the hippocampus after irradiation. Adult mice with targeted disruption of p53 or prkaa2 (gene that encodes AMPKα) in the brain were used to determine the role of p53 and AMPK, respectively, in radiation-induced apoptosis of NPCs in the hippocampus. The p53-dependent apoptosis of NPCs was associated with an increase in phospho-AMPK expression in the dentate gyrus at 8 hours after irradiation. Activation of AMPK was seen in granule neurons and subgranular NPCs. Compared with wildtype mice, apoptosis of NPCs was significantly attenuated in AMPK deficient (nestinCre: prkaa2fl/fl) mice after irradiation. AMPK deficiency did not however alter p53 activation in NPCs after irradiation. We conclude that AMPK may regulate apoptosis of hippocampal NPCs after irradiation. These findings suggest that cellular metabolism may play a role in determining cell fate response such as apoptosis after DNA damage in NPCs.

Monocyte-lineage dendritic cell (DC) precursors are pre-positioned in normal lymph nodes and rapidly differentiate into Batf3+ cross-presenting APCs during inflammation

Abstract We have shown (Immunity 48:91–106, 2018) that a subset of murine monocytic precursor cells are plastic, and can up-regulate a Batf3-driven differentiation program resembling classical cDC1 cells. The resulting inflammatory DCs are important in tumor immunity, but it has been unclear whether they played a role in normal immune responses. We now identify the precursor cells for these inflammatory DCs as a population of Ly6c+ ckit+ c− fms+ CD11b-neg cells. These are constitutively present in all resting LNs (comprising up to half of the Ly6c+ cells). In response to a standard vaccination model of cross-presented antigen (whole OVA protein plus CpG adjuvant in IFA), the precursor cells rapidly up-regulated Batf3 and differentiated into CD103+ IRF8+ DCs. Just as in tumors, maturation was dependent on intracellular p53 signaling in the precursor cells. Targeted disruption of p53 (LysMcre/p53-KO) had no effect on the number of precursors, but abrogated their ability to mature into Batf3+ DCs. In KO mice, the loss of this single monocytic DC population entirely abrogated the ability to create an inflammatory milieu in the vaccine-draining LN, and abolished cross-presentation of protein antigen to T cells. In contrast, direct presentation (soluble siinfekl peptide) remained intact. We hypothesize that the ability of monocyte-lineage cells to differentiate into Batf3+ APCs during inflammation is an important component of the normal cross-presentation process.

82 Disruption of p53 in bovine somatic cells affected cloned embryonic development

The p53 gene is a tumour suppressor gene with many roles in cellular reprogramming, senescence, or cancers. Knockdown of p53 in somatic cells improves generation of induced pluripotent stem cells and developmental competence of cloned embryos. In this study, we hypothesised that disruption of p53 in bovine somatic cells using CRISPR-Cas9 may improve reprogramming and result in increased blastocyst formation. For the study, ovary-derived immature bovine cumulus-oocyte complexes were matured in vitro. To prepare the donor cells with disruption of p53, primary cells from the skin tissue were transfected with spCas9 and single guide RNA for bovine p53. Two days later, genomic DNA was extracted and the mutation was evaluated on the target region using the T7E1 enzyme. Several single cell colonies were isolated to investigate the mutation and sequencing for mutation positive colonies was performed. One colony with high proliferative activity and heterozygotic mutation (−7bp deletion) was selected and used as the donor cell for somatic cell nuclear transfer. The donor cell was microinjected into an enucleated oocyte. Reconstructed oocytes were fused by electrical pulse, activated by chemical stimulation, and cultured in chemically defined media for 7 days at 5% CO2 and 5% O2. Data were subjected to ANOVA and least significant different test to determine differences between experimental groups by using SAS software (SAS Institute Inc., Cary, NC, USA). Statistical significance was determined when the P-value was less than 0.05. Blastocyst formation rate (13.5±7.6%) from the mutated cell line was significantly lower than wild type cells (43.9±12.1%). Moreover, total cell number (115.4±13.3) of the blastocysts from knockout cells was significantly decreased compared with the control group (143.6±22.5). Some randomly selected blastocysts derived from mutated cells were subjected to mutation assay to confirm deletion of p53. As expected, all the samples were positive to mutation. These data did not support our hypothesis that p53 disruption could improve blastocyst formation competence of bovine cloned embryos. In the future, the reason for impaired embryonic development of clones of p53 deleted somatic cells will be further investigated. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.

A molecular mechanism of nickel (II): reduction of nucleotide excision repair activity by structural and functional disruption of p53.

A molecular mechanism of nickel (II): reduction of nucleotide excision repair activity by structural and functional disruption of p53.

A molecular mechanism of nickel (II): reduction of nucleotide excision repair activity by structural and functional disruption of p53.

Nickel is a major carcinogen that is implicated in tumor development through occupational and environmental exposure. Although the exact molecular mechanisms of carcinogenesis by low-level nickel remain unclear, inhibition of DNA repair is frequently considered to be a critical mechanism of carcinogenesis. Here, we investigated whether low concentrations of nickel would affect p53-mediated DNA repair, especially nucleotide excision repair. Our results showed that nickel inhibited the promoter binding activity of p53 on the downstream gene GADD45A, as a result of the disturbance of p53 oligomerization by nickel. In addition, we demonstrated that nickel exposure trigger the reduction of GADD45A-mediated DNA repair by impairing the physical interactions between GADD45A and proliferating cell nuclear antigen or xeroderma pigmentosum G. Notably, in the GADD45A-knockdown system, the levels of unrepaired DNA photoproducts were higher than wild-type cells, elucidating the importance of GADD45A in the nickel-associated inhibition of DNA repair. These results imply that inhibition of p53-mediated DNA repair can be considered a potential carcinogenic mechanism of nickel at low concentrations.

Murine models of osteosarcoma: A piece of the translational puzzle

Osteosarcoma (OS) is the most common cancer of bone in children and young adults. Despite extensive research efforts, there has been no significant improvement in patient outcome for many years. An improved understanding of the biology of this cancer and how genes frequently mutated contribute to OS may help improve outcomes for patients. While our knowledge of the mutational burden of OS is approaching saturation, our understanding of how these mutations contribute to OS initiation and maintenance is less clear. Murine models of OS have now been demonstrated to be highly valid recapitulations of human OS. These models were originally based on the frequent disruption of p53 and Rb in familial OS syndromes, which are also common mutations in sporadic OS. They have been applied to significantly improve our understanding about the functions of recurrently mutated genes in disease. The murine models can be used as a platform for preclinical testing and identifying new therapeutic targets, in addition to testing the role of additional mutations in vivo. Most recently these models have begun to be used for discovery based approaches and screens, which hold significant promise in furthering our understanding of the genetic and therapeutic sensitivities of OS. In this review, we discuss the mouse models of OS that have been reported in the last 3-5 years and newly identified pathways from these studies. Finally, we discuss the preclinical utilization of the mouse models of OS for identifying and validating actionable targets to improve patient outcome.

Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice

It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing. We performed a genome-wide CRISPR/Cas9-based knockout screen of P53-null mouse embryonic liver progenitor cells that overexpressed MYC. We infected p53-/-;Myc;Cas9 hepatocytes with the mGeCKOa lentiviral library of 67,000 single-guide RNAs (sgRNAs), targeting 20,611 mouse genes, and transplanted the transduced cells subcutaneously into nude mice. Within 1 month, all the mice that received the sgRNA library developed subcutaneous tumors. We performed high-throughput sequencing of tumor DNA and identified sgRNAs increased at least 8-fold compared to the initial cell pool. To validate the top 10 candidate tumor suppressors from this screen, we collected data from patients with hepatocellular carcinoma (HCC) using the Cancer Genome Atlas and COSMIC databases. We used CRISPR to inactivate candidate tumor suppressor genes in p53-/-;Myc;Cas9 cells and transplanted them subcutaneously into nude mice; tumor formation was monitored and tumors were analyzed by histology and immunohistochemistry. Mice with liver-specific disruption of p53 were given hydrodynamic tail-vein injections of plasmids encoding Myc and sgRNA/Cas9 designed to disrupt candidate tumor suppressors; growth of tumors and metastases was monitored. We compared gene expression profiles of liver cells with vs without tumor suppressor gene disrupted by sgRNA/Cas9. Genes found to be up-regulated after tumor suppressor loss were examined in liver cancer cell lines; their expression was knocked down using small hairpin RNAs, and tumor growth was examined in nude mice. Effects of the MEK inhibitors AZD6244, U0126, and trametinib, or the multi-kinase inhibitor sorafenib, were examined in human and mouse HCC cell lines. We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS up-regulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 messenger RNA or high levels of HMGA2 messenger RNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of mitogen-activated protein kinase phosphorylation. Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via mitogen-activated protein kinase, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.

NF-Y loss triggers p53 stabilization and apoptosis in HPV18-positive cells by affecting E6 transcription

The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NF-YA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response.