P53-dependent Tumor Suppression Research Articles

Ninjurin 2, a Cell Adhesion Molecule and a Target of p53, Modulates Wild-Type p53 in Growth Suppression and Mutant p53 in Growth Promotion.

The nerve injury-induced protein 1 (NINJ1) and NINJ2 constitute a family of homophilic adhesion molecules and are involved in nerve regeneration. Previously, we showed that NINJ1 and p53 are mutually regulated and the NINJ1-p53 loop plays a critical role in p53-dependent tumor suppression. However, the biology of NINJ2 has not been well-explored. By using multiple in vitro cell lines and genetically engineered mouse embryo fibroblasts (MEFs), we showed that NINJ2 is induced by DNA damage in a p53-dependent manner. Moreover, we found that the loss of NINJ2 promotes p53 expression via mRNA translation and leads to growth suppression in wild-type p53-expressing MCF7 and Molt4 cells and premature senescence in MEFs in a wild-type p53-dependent manner. Interestingly, NINJ2 also regulates mutant p53 expression, and the loss of NINJ2 promotes cell growth and migration in mutant p53-expressing MIA-PaCa2 cells. Together, these data indicate that the mutual regulation between NINJ2 and p53 represents a negative feedback loop, and the NINJ2-p53 loop has opposing functions in wild-type p53-dependent growth suppression and mutant p53-dependent growth promotion.

ZFP14 Regulates Cancer Cell Growth and Migration by Modulating p53 Protein Stability as Part of the MDM2 E3 Ubiquitin Ligase Complex.

Multi-zinc finger proteins that contain a KRAB domain are part of the biggest family of transcription factors in mammals. However, the physiological or pathological functions for the majority of them are unknown. Here, we showed that ZFP14 (also known as ZNF531) is a p53 target gene that can be induced upon genotoxic stress in a p53-dependent manner. To determine the function of ZFP14 in mouse and human cancer cell lines, we generated multiple cell lines where ZFP14 was knocked out. We showed that ZFP14-KO inhibits cancer cell growth and migration. We also showed that, as a target of p53, ZFP14, in turn, represses p53 expression and that the knockdown of p53 restores the potential of ZFP14-KO cells to proliferate and migrate. Mechanistically, we found that ZFP14 modulates p53 protein stability by increasing its ubiquitination via associating with and possibly enhancing MDM2/p53 complex integrity through its zinc finger domains. Our findings suggest that the reciprocal regulation of p53 and ZFP14 represents a novel p53-ZFP14 regulatory loop and that ZFP14 plays a role in p53-dependent tumor suppression.

IPLA2\u03b2-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4

Here, we identify iPLA2β as a critical regulator for p53-driven ferroptosis upon reactive oxygen species (ROS)-induced stress. The calcium-independent phospholipase iPLA2β is known to cleave acyl tails from the glycerol backbone of lipids and release oxidized fatty acids from phospholipids. We found that iPLA2β-mediated detoxification of peroxidized lipids is sufficient to suppress p53-driven ferroptosis upon ROS-induced stress, even in GPX4-null cells. Moreover, iPLA2β is overexpressed in human cancers; inhibition of endogenous iPLA2β sensitizes tumor cells to p53-driven ferroptosis and promotes p53-dependent tumor suppression in xenograft mouse models. These results demonstrate that iPLA2β acts as a major ferroptosis repressor in a GPX4-independent manner. Notably, unlike GPX4, loss of iPLA2β has no obvious effect on normal development or cell viability in normal tissues but iPLA2β plays an essential role in regulating ferroptosis upon ROS-induced stress. Thus, our study suggests that iPLA2β is a promising therapeutic target for activating ferroptosis-mediated tumor suppression without serious toxicity concerns.

P53-intact cancers escape tumor suppression through loss of long noncoding RNA Dino.

SUMMARYMany long noncoding RNA (lncRNA) genes exist near cancer-associated loci, yet evidence connecting lncRNA functions to recurrent genetic alterations in cancer are lacking. Here, we report that DINO, the lncRNA transcribed from the cancer-associated DINO/CDKN1A locus, suppresses tumor formation independent of p21, the protein encoded at the locus. Loss of one or two alleles of Dino impairs p53 signaling and apoptosis, resulting in a haplo-insufficient tumor suppressor phenotype in genetically defined mouse models of tumorigenesis. A discrete region of the DINO/CDKN1A locus is recurrently hypermethylated in human cancers, silencing DINO but not CDKN1A, the gene encoding p21. Hypermethylation silences DINO, impairs p53 signaling pathway in trans, and is mutually exclusive with TP53 alterations, indicating that DINO and TP53 comprise a common tumor suppressor module. Therefore, DINO encodes a lncRNA essential for tumor suppression that is recurrently silenced in human cancers as a mechanism to escape p53-dependent tumor suppression.

Ceramide-Rubusoside Nanomicelles, a Potential Therapeutic Approach to Target Cancers Carrying p53 Missense Mutations.

Ceramide (Cer) is an active cellular sphingolipid that can induce apoptosis or proliferation-arrest of cancer cells. Nanoparticle-based delivery offers an effective approach for overcoming bioavailability and biopharmaceutics issues attributable to the pronounced hydrophobicity of Cer. Missense mutations of the protein p53, which have been detected in approximately 42% of cancer cases, not only lose the tumor suppression activity of wild-type p53, but also gain oncogenic functions promoting tumor progression and drug resistance. Our previous works showed that cellular Cer can eradicate cancer cells that carry a p53 deletion-mutation by modulating alternative pre-mRNA splicing, restoring wild-type p53 protein expression. Here, we report that new ceramide-rubusoside (Cer-RUB) nanomicelles considerably enhance Cer in vivo bioavailability and restore p53-dependent tumor suppression in cancer cells carrying a p53 missense mutation. Natural RUB encapsulated short-chain C6-Cer so as to form Cer-RUB nanomicelles (∼32 nm in diameter) that substantially enhanced Cer solubility and its levels in tissues and tumors of mice dosed intraperitoneally. Intriguingly, Cer-RUB nanomicelle treatments restored p53-dependent tumor suppression and sensitivity to cisplatin in OVCAR-3 ovarian cancer cells and xenograft tumors carrying p53 R248Q mutation. Moreover, Cer-RUB nanomicelles showed no signs of significant nonspecific toxicity to noncancerous cells or normal tissues, including bone marrow. Furthermore, Cer-RUB nanomicelles restored p53 phosphorylated protein and downstream function to wild-type levels in p53 R172H/+ transgenic mice. Altogether, this study, for the first time, indicates that natural Cer-RUB nanomicelles offer a feasible approach for efficaciously and safely targeting cancers carrying p53 missense mutations.

P53-Intact Cancers Escape Tumor Suppression Through Loss of Long Noncoding RNA <i>Dino</i>

Many long noncoding RNA (lncRNA) genes are located near cancer-associated loci, suggesting potential roles in cancer development. However, conclusive evidence connecting the function of lncRNAs to the mechanism by which these genetic alterations impact cancer are largely lacking. Here we report that DINO, the long noncoding RNA transcribed from the cancer-associated DINO/CDKN1A locus, suppresses tumor formation in a phenotype that does not require the protein encoded at the locus, p21. In rigorous, genetically-defined mouse models, Dino is a haplo-insufficient tumor suppressor of B cell lymphoma in which p21 does not suppress tumorigenesis. In human cancers, a discrete region of the DINO/CDKN1A locus is recurrently hypermethylated in human cancers, silencing DINO but not CDKN1A, the gene encoding p21. Hypermethylation silences DINO to impair the p53 signaling pathway in trans and is mutually exclusive with TP53 alterations, indicating that DINO and TP53 comprise a common tumor suppressor module. Therefore, DINO encodes a long noncoding RNA essential for tumor suppression that is recurrently silenced in human cancers as a mechanism to escape p53-dependent tumor suppression.

Activating TP53 by Dual Inhibition of MDMX and MDM2

TP53 is often inactivated by mutations or other mechanisms in human cancers. Recent work has demonstrated that its endogenous inhibitor MDMX (or MDM4) is frequently overexpressed in patients with hematologic malignancies including AML, lymphoid malignancies, as well as other cancers. Pharmacological disruption of the interactions of TP53 with both its endogenous inhibitors (MDMX and MDM2) has long been sought after as an attractive strategy to restore p53-dependent tumor suppressor activity. However, selective targeting of this pathway has previously been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. More recently, pharmacological dual targeting of MDMX/MDM2 has become feasible through stapled peptides and is currently being tested in clinical trials. This presentation will discuss such MDMX/MDM2 dual-targeting strategies as well as new insights into MDMX-mediated mechanisms of tumor progression at the stem cell level, which have emerged from recent studies. Disclosures Steidl: Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; Celgene: Consultancy; BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding.

Dino Is a DNA Damage-Induced Tumor Suppressing Long Noncoding RNA

While the CKDN1A locus is frequently suppressed in human cancers, the mechanism by which this finding contributes to tumor suppression has not been fully elucidated. We hypothesized that DINO, a novel DNA damage-induced long noncoding RNA encoded in the CDKN1A promoter, is essential to p53-dependent tumor suppression. We used a combination of in vivo mouse models, cell culture experiments and human tumor profiling to examine the contribution of DINO to p53-dependent tumor suppression. We crossed a Dino loss of function mouse to the E-mu-Myc model of B-cell lymphoma to examine p53-dependent, p21-independent tumor suppression. Rescue experiments to restore Dino function in Dino-/- lymphoma cells were performed to confirm its contribution to growth suppression. Finally, we analyzed methylation patterns of the DINO/CDKN1A locus in TCGA datasets to investigate the relationship of DINO inactivation and TP53 mutation in human cancers. We find that loss of Dino significantly accelerates tumorigenesis in the E-mu-Myc mouse model of B-cell lymphoma in a haplo-insufficient manner, while previous studies have shown that loss of Cdkn1a does not impact tumor development. Complementation with Dino suppresses growth of isolated Dino-/-, E-mu-Myc lymphoma cells and delays tumor development when re-engrafted into mice. We show that each of these effects requires an intact p53 signaling pathway. Human DINO and mouse Dino can each functionally restore growth suppression in cell culture, suggesting functional conservation through mammalian evolution. Finally, we find that DINO is hypermethylated in multiple types of human cancer and that DINO hypermethylation is correlated to DINO expression, but not CDNK1A expression. Hypermethylation of DINO is mutually exclusive with TP53 alteration in several specific human cancers. We provide direct evidence that the long noncoding RNA DINO is a tumor suppressor transcribed from the CDKN1A promoter. These findings provide insight into a mechanism by which human tumors inactivate p53-dependent functions and describe a novel mechanism of tumor suppression by a long noncoding RNA. We suggest that inactivation of DINO presents a potential biomarker of tumors with altered p53 function, and provides a future basis for targeted therapy and personalized treatment.

The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.

Primate-specific miR-944 activates p53-dependent tumor suppression in human colorectal cancers

As cancers with a high incidence rate, colorectal cancers are a main cause of cancer-related death. MicroRNAs are often deregulated in cancers. The primate-specific miR-944, located in a p63 intron, is known to be highly expressed in patients exhibiting low colorectal cancer recurrence rates. However, the biological functions of miR-944 in colorectal cancers remain unclear. In this study, we found that miR-944 was downregulated in colorectal cancer tissues, and inhibited cancer cell growth in a xenograft mouse model. The overexpression of miR-944 caused G1 phase arrest and increased p53 expression in cancer cells. p53 stability was enhanced by miR-944s targeting E3 ligases COP1 and MDM2. Overexpression of COP1 and MDM2 restored cell growth inhibition caused by miR-944. Taken together, our results suggest that miR-944 acts as a potential tumor suppressor in colorectal cancers through the ubiquitin-proteasome system.

Abstract 4667: The use of fragment-based screening approaches to improve the specificity of inhibitors targeting malignant melanoma

Abstract S100B belongs to the S100 protein family of Ca2+-binding proteins. Prior studies have shown that S100B is a biological marker for malignant melanoma. Increases in S100B levels are predictive of disease progression, recurrence, and overall low survival in the patients. It was shown previously that S100B binds to p53 and down-regulates its tumor suppression activities including growth arrest and apoptosis. Chemical biology and structure-based drug design methods are underway to inhibit S100B and restore p53 function. However, highly specific S100B inhibitors are required, so S100B can be blocked in melanoma without impairing S100A1 function in skeletal and cardiac muscle. With this goal in mind, a fragment-based screening approach using NMR and computational approaches is being applied. Results to date indicate that these methods can be used to identify specific small molecule fragments that target S100B and not S100A1. The fragments that uniquely bind S100B will then be considered with structural data of existing S100B-SBiX complexes to improve their specificity for binding S100B (i.e. versus S100A1). The inhibitor design strategy will also include improving the stability of the compounds their cell permeability, and their overall drug-like properties. The most promising of these new inhibitors will ultimately be used to target high levels of S100B found in malignant and rescue p53-dependent tumor suppression pathways in vivo with the goal of them having therapeutic value for treating malignant melanoma. Citation Format: Sreya Mukherjee, Shardell Spriggs, Paul Wilder, Wenbo Yu, Kristen Varney, Alexander D. MacKerell, David J. Weber. The use of fragment-based screening approaches to improve the specificity of inhibitors targeting malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4667.

DNA repair processes are critical mediators of p53-dependent tumor suppression

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence1,2. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function3-5. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development.

Dual Inhibition of Mdmx and Mdm2 Using an Alpha-Helical P53 Stapled Peptide (ALRN-6924) As a Novel Therapeutic Strategy in Acute Myeloid Leukemia

While the TP53 tumor suppressor gene is mutated in more than 50% of human tumors, in Acute Myeloid Leukemia (AML) TP53 mutations are rare, occurring in less than 10% of cases. Yet, functional inactivation of wild-type p53 due to non-mutational abnormalities occurs frequently in AML and other hematological malignancies. A major mechanism of p53 inactivation results from the overexpression of its endogenous inhibitors MDMX (also known as MDM4, HDMX, and HDM4) and MDM2 which are frequently overexpressed in various p53 wild-type human cancers, including AML. Strikingly, MDMX has been reported to be overexpressed in over 92% of AML cases, while MDM2 overexpression is less frequent. Pharmacological disruption of both these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in AML and other cancers with wild-type p53. Nonetheless, selective targeting of this pathway has thus far been limited to MDM2-only small molecule inhibitors which lack affinity for MDMX. ALRN-6924 is an optimized alpha helical p53 stapled peptide and first-in-class dual MDMX/MDM2 inhibitor which has recently entered phase I/II clinical testing (NCT02264613, NCT02909972) in solid tumors and lymphomas with, thus far, excellent tolerability and objective responses as single agent1. The goal of our study was to evaluate the molecular, cellular, and biochemical mechanisms of action of ALRN-6924 in AML.We used biochemical affinity studies as well as single molecule FISH and live single cell imaging to assess MDMX/MDM2 binding as well as p21 transactivation by p53 in response to ALRN-6924. Effects on cellular proliferation, apoptosis, DNA repair, cell cycle, clonogenic capacity, and serial replating were determined using AML cell lines and primary human AML patients' cells, including in leukemic stem (CD34+ CD38-) and progenitor (CD34+CD38+) cells. Genome-wide molecular effects were determined by RNA seq. P53 activity in a patient undergoing treatment with ALRN-6924 was measured by intracellular staining of p53 and its target gene p21 in CD34+ cells by flow cytometry. Furthermore, we evaluated ALRN-6924 activity in a xeno-transplantation model of human AML in NSG mice.We found that MDMX is significantly overexpressed in highly fractionated leukemic stem (Lin-CD34+CD38-CD90-) and progenitor (Lin-CD34+CD38+CD123+CD45+) cells in AML patients compared to identically sorted, age-matched healthy controls (p<0.0001). Dual MDMX/MDM2 inhibition using ALRN-6924 led to striking anti-leukemic effects in suspension culture of AML cell lines and primary cells, with negligible effects on healthy controls. ALRN-6924 robustly activated p53-dependent transcription at the single cell and single molecule level, and exhibited strong biochemical and molecular biological on-target activity in AML cell lines and primary cells in vitro, as well as in a patient who received ALRN-6924 treatment. Co-immunoprecipitation experiments demonstrated that ALRN-6924 disrupts both MDMX/P53 as well as MDM2/P53 interactions at relevant therapeutic dosages in AML cells. RNA seq data showed that dual MDMX/MDM2 inhibition led to global transcriptional activation of p53-dependent pathways in AML cells. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibited cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patients' cells, including in leukemic stem cell-enriched populations. Furthermore, ALRN-6924 disrupted functional clonogenic and serial replating capacity of AML cells, and showed superiority over MDM2-only inhibition. Most strikingly, ALRN-6924 led to highly significant improved survival in an AML xenograft model in vivo (p<0.0001).Our study provides insight into the molecular, cell biological, and in vivo effects of pharmacological dual MDMX/MDM2 inhibition in AML, which may have important implications for other MDMX/MDM2-related cancers. Our findings provide proof-of-concept for ALRN-6924 as a novel therapeutic in p53-wildtype AML, and provide a rationale for its further preclinical and clinical development in AML and other cancers.Furthermore, the success of targeting p53 raises the intriguing prospect that the same development path is possible for other helix-in-groove targets, and may thus pave the way for a new class of targeted therapeutics.1 ASCO 2017 annual meeting: J Clin Oncol 35, 2017 (suppl; abstr 2505) DisclosuresGuerlavais:Aileron: Employment. Annis:Aileron Therapeutics: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding. Aivado:Aileron: Employment. Steidl:Novartis: Research Funding; Celgene: Consultancy; Bayer Healthcare: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding.

Reprimo, a Potential p53-Dependent Tumor Suppressor Gene, Is Frequently Hypermethylated in Estrogen Receptor α-Positive Breast Cancer.

Aberrant DNA methylation is a hallmark of many cancers. Currently, there are four intrinsic molecular subtypes in breast cancer (BC): Luminal A, B, Her2-positive, and triple negative (TNBC). Recently, The Cancer Genome Atlas (TCGA) project has revealed that Luminal subtypes have higher levels of genome-wide methylation that may be a result of Estrogen/Estrogen receptor α (E2/ERα) signaling pathway activation. In this study, we analyze promoter CpG-island (CGIs) of the Reprimo (RPRM) gene in breast cancers (n = 77), cell lines (n = 38), and normal breast tissue (n = 10) using a MBDCap-seq database. Then, a validation cohort (n = 26) was used to confirm the results found in the MBDCap-seq platform. A differential methylation pattern was found between BC and cell lines compared to normal breast tissue. In BC, a higher DNA methylation was observed in tissues that were ERα-positive than in ERα-negative ones; more precisely, subtypes Luminal A compared to TNBC. Also, significant reverse correlation was observed between DNA methylation and RPRM mRNA expression in BC. Our data suggest that ERα expression in BC may affect the DNA methylation of CGIs in the RPRM gene. This approach suggests that DNA methylation status in CGIs of some tumor suppressor genes could be driven by E2 availability, subsequently inducing the activation of the ERα pathway.

Ferredoxin reductase is critical for p53-dependent tumor suppression via iron regulatory protein 2.

Ferredoxin reductase (FDXR), a target of p53, modulates p53-dependent apoptosis and is necessary for steroidogenesis and biogenesis of iron-sulfur clusters. To determine the biological function of FDXR, we generated a Fdxr-deficient mouse model and found that loss of Fdxr led to embryonic lethality potentially due to iron overload in developing embryos. Interestingly, mice heterozygous in Fdxr had a short life span and were prone to spontaneous tumors and liver abnormalities, including steatosis, hepatitis, and hepatocellular carcinoma. We also found that FDXR was necessary for mitochondrial iron homeostasis and proper expression of several master regulators of iron metabolism, including iron regulatory protein 2 (IRP2). Surprisingly, we found that p53 mRNA translation was suppressed by FDXR deficiency via IRP2. Moreover, we found that the signal from FDXR to iron homeostasis and the p53 pathway was transduced by ferredoxin 2, a substrate of FDXR. Finally, we found that p53 played a role in iron homeostasis and was required for FDXR-mediated iron metabolism. Together, we conclude that FDXR and p53 are mutually regulated and that the FDXR-p53 loop is critical for tumor suppression via iron homeostasis.

P53 coordinates DNA repair with nucleotide synthesis by suppressing PFKFB3 expression and promoting the pentose phosphate pathway.

Activation of p53 in response to DNA damage is essential for tumor suppression. Although previous studies have emphasized the importance of p53-dependent cell cycle arrest and apoptosis for tumor suppression, recent studies have suggested that other areas of p53 regulation, such as metabolism and DNA damage repair (DDR), are also essential for p53-dependent tumor suppression. However, the intrinsic connections between p53-mediated DDR and metabolic regulation remain incompletely understood. Here, we present data suggesting that p53 promotes nucleotide biosynthesis in response to DNA damage by repressing the expression of the phosphofructokinase-2 (PFK2) isoform 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a rate-limiting enzyme that promotes glycolysis. PFKFB3 suppression increases the flux of glucose through the pentose phosphate pathway (PPP) to increase nucleotide production, which results in more efficient DNA damage repair and increased cell survival. Interestingly, although p53-mediated suppression of PFKFB3 could increase the two major PPP products, NADPH and nucleotides, only nucleotide production was essential to promote DDR. By identifying the novel p53 target PFKFB3, we report an important mechanistic connection between p53-regulated metabolism and DDR, both of which play crucial roles in tumor suppression.

The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression.

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is a stress-response gene that has been associated with cancer, but no studies have differentiated among or defined the regulation or function of any of its several recently described expression variants. We found that TNFAIP8 variant 2 (v2) is overexpressed in multiple human cancers, whereas other variants are commonly downregulated in cancer (v1) or minimally expressed in cancer or normal tissue (v3-v6). Silencing v2 in cancer cells induces p53-independent inhibition of DNA synthesis, widespread binding of p53, and induction of target genes and p53-dependent cell cycle arrest and DNA damage sensitization. Cell cycle arrest induced by v2 silencing requires p53-dependent induction of p21. In response to the chemotherapeutic agent doxorubicin, p53 regulates v2 through binding to an intragenic enhancer, together indicating that p53 and v2 engage in complex reciprocal regulation. We propose that TNFAIP8 v2 promotes human cancer by broadly repressing p53 function, in essence offsetting p53-dependent tumor suppression.

Suppression of iASPP-dependent aggressiveness in cervical cancer through reversal of methylation silencing of microRNA-124

Derepression of wild-type p53 by suppressing its negative inhibitor iASPP (Inhibitor of apoptosis-stimulating protein of p53) represents a potential therapeutic option for cervical cancer (CC). Here, we reported a novel functional significance of iASPP upregulation in cervical tumorigenesis: iASPP acts as a key promoter of CC cell proliferation, epithelial-mesenchymal transition, invasion and cancer stemness, by interacting with p53 to suppress p53-mediated transcription of target genes and reducing p53-responsive microRNA-34a levels. Moreover, we demonstrate that miR-124, directly targeting iASPP, reduces expression of iASPP and attenuates CC cell growth and invasiveness. Low miR-124 expression is inversely correlated with increased expression of iASPP mRNA in CC tissues. In a cohort of 40 patients with CC, the low miR-124 expression was correlated with poor 5-year overall survival (P = 0.0002) and shorter disease-free survival 5-year (P = 0006). Treatment with the DNA methyltransferase inhibitor Zebularine increases miR-124 expression and retards CC cell growth and invasion with minimal toxicity to normal cells. Even at a non-toxic concentration, Zebularine was effective in suppressing CC cell invasion and migration. Altogether, the restoration of miR-124 reduces iASPP expression and leads to p53-dependent tumor suppression, suggesting a therapeutic strategy to treat iASPP-associated CC.

Abstract 675: Ninjurin1, a target of p53, modulates p53-dependent tumor suppression in vivo

Abstract The nerve injury-induced protein 1 (Ninjurin1, Ninj1), a homophilic adhesion molecule, is involved in nerve regeneration, inflammation and vascular regression. Ninj1 is found to be overexpressed in human cancers including hepatocellular carcinoma and acute lymphoblastic leukemia. However, the role of Ninj1 in tumorigenesis has not been elucidated. Recently, we found that Ninj1 is transcriptionally regulated by p53, which in turn regulates p53 expression through mRNA translation. Thus, the mutual regulation between p53 and Ninj1 represents a novel loop in the p53 pathway. To examine the biological significance of the p53-Ninj1 loop, we generated and monitored 4 cohorts of mice: (i) WT, (ii) Ninj1-het, (iii) p53-null, (iv) Ninj1-het; p53-null. We found that Ninj1-het mice were highly prone to skin lesions. We also found that Ninj1 deficiency shortened the life-span but decreased the incidence of sarcoma in mice without p53. Additionally, we found that Ninj1-het; p53-null mice were prone to hydrocephalus and kidney abnormality. To examine the association of Ninj1 with mutant p53, we generated and characterized Ninj1+/-; p53 R270H/- and p53 R270H/- mice. We found that Ninj1 deficiency shortened the life-span and promoted the frequency and aggressiveness of tumors in p53 R270H/- mice. Together, we conclude that Ninj1 differently regulates the tumor susceptibility in a p53-dependent manner in vivo and that the Ninj1-p53 loop plays a pivotal role in tumor suppression and longevity. Citation Format: Hee Jung Yang, Seong Jun Cho, Jin Zhang, Wensheng Yan, Xinbin Chen. Ninjurin1, a target of p53, modulates p53-dependent tumor suppression in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 675.

MicroRNA-34a Negatively Regulates Efferocytosis by Tissue Macrophages in Part via SIRT1.

Apoptotic cell (AC) clearance (efferocytosis) is an evolutionarily conserved process essential for immune health, particularly to maintain self-tolerance. Despite identification of many recognition receptors and intracellular signaling components of efferocytosis, its negative regulation remains incompletely understood and has not previously been known to involve microRNAs (miRs). In this article, we show that miR-34a (gene ID 407040), well recognized as a p53-dependent tumor suppressor, mediates coordinated negative regulation of efferocytosis by resident murine and human tissue macrophages (Mø). The miR-34a expression varied greatly between Mø from different tissues, correlating inversely with their capacity for AC uptake. Transient or genetic knockdown of miR-34a increased efferocytosis, whereas miR-34a overexpression decreased efferocytosis, without altering recognition of live, necrotic, or Ig-opsonized cells. The inhibitory effect of miR-34a was mediated both by reduced expression of Axl, a receptor tyrosine kinase known to recognize AC, and of the deacetylase silent information regulator T1, which had not previously been linked to efferocytosis by tissue Mø. Exposure to AC downregulated Mø miR-34a expression, resulting in a positive feedback loop that increased subsequent capacity to engulf AC. These findings demonstrate that miR-34a both specifically regulates and is regulated by efferocytosis. Given the ability of efferocytosis to polarize ingesting Mø uniquely and to reduce their host-defense functions, dynamic negative regulation by miR-34a provides one means of fine-tuning Mø behavior toward AC in specific tissue environments with differing potentials for microbial exposure.