Depletion Of P53 Research Articles

Haploidy-linked cell proliferation defects limit larval growth in zebrafish.

Haploid larvae in non-mammalian vertebrates are lethal, with characteristic organ growth retardation collectively called 'haploid syndrome'. In contrast to mammals, whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control larvae, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1-3 days post-fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 partially improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are parts of critical cellular causes shared among vertebrates that limit the larval growth in the haploid state, contributing to an evolutionary constraint on allowable ploidy status in the vertebrate life cycle.

Abstract 2065: Activation Of Cgas-sting Axis By DNA Double-strand Breaks In Atherosclerosis: A Possible Link From DNA Damage To Inflammation And Cell Senescence

Aim: Premature atherosclerosis in some progeroid syndromes suggests the involvement of DNA damage in the progression of atherosclerosis. However, causative links between DNA double-strand breaks (DSBs) and atherosclerosis have yet to be determined. The aim of this study was to elucidate the role of DSBs in atherosclerosis using mice and vascular smooth muscle cells (VSMCs) deficient in Ku80, a DSB repair protein. Methods and Results: The plaque size and DSBs were increased in the aorta of Ku80-deficient apolipoprotein E knockout mice (Ku80 +/- ApoE -/- ) compared to those of ApoE -/- control after 4 weeks of a high-fat diet. At the early stages of atherosclerosis (two-week high-fat diet), although the plaque size of Ku80 +/- ApoE -/- was similar to that of ApoE -/- control, the number of DSBs and mRNA levels of inflammatory cytokines such as IL-6 and MCP-1 were significantly increased in the Ku80 +/- ApoE -/- aortas. We further investigated molecular links between DSBs and inflammatory responses using VSMCs isolated from Ku80 +/- mice. In VSMCs from Ku80 +/- mice, IL-6 mRNA level was elevated along with increased cellular senescence markers, such as p16 mRNA and senescence-associated (SA) β-gal activity. Additionally, Ku80 +/- VSMCs showed accumulated cytosolic free DNA, an increased level of cGAMP, and activation of TBK1, interferon regulatory factor 3 (IRF3) and NF-κB, suggesting the activation of cGAS-STING axis, a cytosolic DNA sensing machinery. Activation of cGAS-STING axis was also shown in the aortae from Ku80 +/- ApoE -/- in comparison with those from ApoE -/- control. Silencing IRF3, but not NF-κB attenuated the IL-6 mRNA level in Ku80 +/- VSMCs, so did cGAS-silencing. Of interest, cGAS-silencing also attenuated DNA damage-induced SA β-gal activity in wild-type VSMCs. Furthermore, depletion of either p16 or p53 suppressed IRF3 activation by DNA damage. Conclusions: These results suggest that accumulation of cytosolic DNA induced by DNA damage accelerates atherosclerosis via the activation of cGAS-STING-IRF3 axis. cGAS-STING-IRF3 axis may play a pivotal role as a common signaling pathway that serve as a link from DNA damage to inflammatory and cell senescence response in arterial wall.

Phenotyping adult mice with inducible depletion of sequestosome-1 (p62) in endothelial cells

Macroautophagy (autophagy) maintains intracellular homeostasis by targeting damaged / dysregulated organelles for their transport to and degradation by the lysosome. Autophagy regulated gene 3 ( Atg3) enables autophagosome maturation which is required for the process of autophagy to occur. We reported that inducible depletion of Atg3 specifically in endothelial cells (ECs) impairs arterial function in the absence of metabolic disruptions (Cho et al., Cardiovasc Res, 2023). Sequestosome-1 (p62/SQSTM1) tethers proteins into the autophagic pathway for subsequent lysosomal degradation. The contribution from EC p62 to arterial and metabolic function is unknown and we addressed this. Adult male and female C57BL/6 mice with tamoxifen inducible depletion of p62 specifically in ECs ( p62EC−/−) were compared to littermate controls ( p62EC+/+). p62/18S mRNA expression was lower (p<0.05) in ECs but not vascular smooth muscle cells (VSM) from p62EC−/− [n=6] vs. p62EC+/+ [n=9] mice, whereas Beclin-1/18S was similar in ECs and VSM from both groups. Neither body composition, glucose disposal nor insulin tolerance were impacted by EC specific p62 depletion. Vascular contractile responses to: (i) phenylephrine mediated α1-activation and (ii) potassium chloride evoked L-type Ca2+ channel opening; and relaxation responses to (iii) acetylcholine (endothelium-dependent) and (iv) sodium nitroprusside (endothelium-independent); were similar between groups in mesenteric resistance arteries (isometric myography) and middle cerebral arteries (isobaric myography). Further, intrinsic arterial stiffness was not different between p62EC−/− [n=6] and p62EC+/+ [n=9] mice i.e., neither the elastic modulus nor stress vs. strain curves generated using segments of thoracic aorta were altered. These findings indicate EC specific p62 depletion does not impact arterial function or the indexes of systemic metabolism we measured. Because p62 is upregulated during cellular stress, it is possible that a challenge (e.g., hypoxia, ischemia) might be required to unmask a protective role for intact EC p62 and studies are ongoing to test this. Supported by AHA23PRE1025910 (SM); Rural and Underserved Utah Training Experience T32 (CG), Pacific Islander Research Internship Program (LG), NIHR01HL149870-01A1 (SB) and NIHRO1HL141540 (JDS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract PR-002: Uncovering novel therapeutic avenues for glioma by exploring the functional interplay of FGFR1, p53, and ribosome biogenesis

Abstract PR-002: Uncovering novel therapeutic avenues for glioma by exploring the functional interplay of FGFR1, p53, and ribosome biogenesis

Human CST complex restricts excessive PrimPol repriming upon UV induced replication stress by suppressing p21.

DNA replication stress, caused by various endogenous and exogenous agents, halt or stall DNA replication progression. Cells have developed diverse mechanisms to tolerate and overcome replication stress, enabling them to continue replication. One effective strategy to overcome stalled replication involves skipping the DNA lesion using a specialized polymerase known as PrimPol, which reinitiates DNA synthesis downstream of the damage. However, the mechanism regulating PrimPol repriming is largely unclear. In this study, we observe that knockdown of STN1 or CTC1, components of the CTC1/STN1/TEN1 complex, leads to enhanced replication progression following UV exposure. We find that such increased replication is dependent on PrimPol, and PrimPol recruitment to stalled forks increases upon CST depletion. Moreover, we find that p21 is upregulated in STN1-depleted cells in a p53-independent manner, and p21 depletion restores normal replication rates caused by STN1 deficiency. We identify that p21 interacts with PrimPol, and STN1 depletion stimulates p21-PrimPol interaction and facilitates PrimPol recruitment to stalled forks. Our findings reveal a previously undescribed interplay between CST, PrimPol and p21 in promoting repriming in response to stalled replication, and shed light on the regulation of PrimPol repriming at stalled forks.

Targeting Ubiquitin-specific Protease 5 Overcomes Chemoresistance via Negatively Regulating p53 in Gastric Cancer.

Resistance to chemotherapy is a major obstacle in the clinical management of gastric cancer, and the mechanisms underlying chemoresistance remain largely unknown. This study aimed to investigate the involvement of ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, in gastric cancer chemoresistance Methods: USP5 expression was analyzed in fifty paired gastric cancer and adjacent normal tissues, chemo-sensitive and chemo-resistant gastric cancer lines using quantitative ELISA. The role of USP5 was determined using loss-of-function and gainof- function methods. USP5-mediated downstream effectors were analyzed using biochemical methods focusing on p53. USP5 expression was comparable in tumors and normal in the majority of the cohort. Following chemotherapy treatment, USP5 expression significantly increased in gastric cancer cells, while p53 levels remained unaltered. Overexpression of USP5 amplified growth and migration while decreasing apoptosis induced by serum withdrawal across multiple gastric cancer cell lines. Conversely, USP5 knockdown effectively heightened gastric cancer sensitivity to paclitaxel and 5-FU treatments, particularly targeting chemo-resistant gastric cancer cells by inhibiting proliferation and migration and inducing apoptosis. Additionally, USP5 knockdown increased levels of p53 but not MDM2, increased p53 activity and increased transcription of p53 target genes. In contrast, USP5 overexpression decreased the level and activity of p53 and inhibited transcription of p53 target genes. The anti-proliferative, anti-migratory, and pro-apoptotic effects of USP5 were significantly diminished upon p53 depletion, highlighting the interplay between p53 and USP5 in regulating gastric cancer cell activities. Additionally, USP5 inhibition suppressed chemo-resistant gastric cancer cell migration via suppressing epithelial-mesenchymal transition (EMT) and RhoA activity. Targeting USP5 inhibition has emerged as a promising alternative therapeutic approach to overcoming chemoresistance in gastric cancer. Additionally, our study sheds light on the novel role of USP5 as a regulator of p53 in gastric cancer.

Targeting PERK-ATF4-P21 axis enhances the sensitivity of osteosarcoma HOS cells to Mppα-PDT.

Osteosarcoma (OS) is the most prevalent type of malignant bone tumor in adolescents. The overall survival of OS patients has reached a plateau recently. Thus, there is an urgent need to develop approaches to improve the sensitivity of OS to therapies. Pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPα-PDT) is a new type of tumor therapy, and elucidating its mechanism is helpful to improve its anti-tumor efficacy. Here, we investigated how PERK signaling promotes the human OS (HOS) cell survival induced by MPPα-PDT, as overcoming this may enhance sensitivity to MPPα-PDT. We found that MPPα-PDT combined with PERK inhibitor GSK2656157 enhanced HOS cell apoptosis by suppressing autophagy and p21. Autophagy inhibition and p21 depletion enhanced cell death, indicating pro-survival effects in MPPα-PDT. Notably, p21 was found to be an effector of the PERK-Atf4 pathway, which could positively regulate autophagy mediated by MPPα-PDT. In conclusion, we found that the combination of MPPα-PDT and GSK2656157 enhanced apoptosis in HOS cells by inhibiting autophagy. Mechanistically, this autophagy is p21-dependent and can be suppressed by GSK2656157, thereby enhancing sensitivity to MPPα-PDT.

Aurora A Kinase Plays a Key Role in Mitosis Skip during Senescence Induced by Ionizing Radiation

ObjectiveTo investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation (IR). MethodsHuman melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora A inhibitor MLN8237 (MLN) and/or p21 depletion by small interfering RNA (siRNA). Cell cycle distribution was determined using flow cytometry and a fluorescent ubiquitin-based cell cycle indicator (FUCCI) system combined with histone H3 phosphorylation at Ser10 (pS10 H3) detection. Senescence was assessed using senescence-associated-β-galactosidase (SA-β-Gal), Ki67, and γH2AX staining. Protein expression levels were determined using western blotting. ResultsTumor cells suffered severe DNA damage and underwent G2 arrest after IR treatment. The damaged cells did not successfully enter M phase nor were they stably blocked at G2 phase but underwent mitotic skipping and entered G1 phase as tetraploid cells, ultimately leading to senescence in G1. During this process, the p53/p21 pathway is hyperactivated. Accompanying p21 accumulation, Aurora A kinase levels declined sharply. MLN treatment confirmed that Aurora A kinase activity is essential for mitosis skipping and senescence induction. ConclusionPersistent p21 activation during IR-induced G2 phase blockade drives Aurora A kinase degradation, leading to senescence via mitotic skipping.

Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments

Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-type p53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.

Impact of diffused versus vasculature targeted DNA damage on the heart of mice depleted of telomeric factor Ft1.

DNA damage is emerging as a driver of heart disease, although the cascade of events, its timing, and the cell types involved are yet to be fully clarified. In this context, the implication of cardiomyocytes has been highlighted, while that of vasculature smooth muscle cells has been implicated but not explored exhaustively. In our previous work we characterized a factor called Ft1 in mice and AKTIP in humans whose depletion generates telomere instability and DNA damage. Herein, we explored the effect of the reduction of Ft1 on the heart with the goal of comparatively defining the impact of DNA damage targeted to vasculature smooth muscle cells to that of diffuse damage. Using two newly generated mouse models, Ft1 constitutively knocked out (Ft1ko) mice, and mice in which we targeted the Ft1 depletion to the smooth muscle cells (Ft1sm22ko), it is shown that both genetic models display cardiac defects but with differences. Both Ft1ko and Ft1sm22ko mice display hypertrophy, fibrosis, and functional heart defects. Interestingly, Ft1sm22ko mice have early milder pathological traits that become manifest with age. Significantly, the defects of Ft1ko mice, including the alteration of the left ventricle and functional heart defects, are rescued by depletion of the DNA damage sensor p53. These results point to Ft1 deficiency as a driver of cardiac disease and show that Ft1 deficiency targeted to vasculature smooth muscle cells generates a pre-pathological profile exacerbated by age.

Phenotype screens of murine pancreatic cancer identify a Tgf-α-Ccl2-paxillin axis driving human-like neural invasion.

Solid cancers like pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer, frequently exploit nerves for rapid dissemination. This neural invasion (NI) is an independent prognostic factor in PDAC, but insufficiently modeled in genetically engineered mouse models (GEMM) of PDAC. Here, we systematically screened for human-like NI in Europe's largest repository of GEMM of PDAC, comprising 295 different genotypes. This phenotype screen uncovered 2 GEMMs of PDAC with human-like NI, which are both characterized by pancreas-specific overexpression of transforming growth factor α (TGF-α) and conditional depletion of p53. Mechanistically, cancer-cell-derived TGF-α upregulated CCL2 secretion from sensory neurons, which induced hyperphosphorylation of the cytoskeletal protein paxillin via CCR4 on cancer cells. This activated the cancer migration machinery and filopodia formation toward neurons. Disrupting CCR4 or paxillin activity limited NI and dampened tumor size and tumor innervation. In human PDAC, phospho-paxillin and TGF-α-expression constituted strong prognostic factors. Therefore, we believe that the TGF-α-CCL2-CCR4-p-paxillin axis is a clinically actionable target for constraining NI and tumor progression in PDAC.

Effectiveness of Nonfunctionalized Graphene Oxide Nanolayers as Nanomedicine against Colon, Cervical, and Breast Cancer Cells.

Recent studies in nanomedicine have intensively explored the prospective applications of surface-tailored graphene oxide (GO) as anticancer entity. However, the efficacy of nonfunctionalized graphene oxide nanolayers (GRO-NLs) as an anticancer agent is less explored. In this study, we report the synthesis of GRO-NLs and their in vitro anticancer potential in breast (MCF-7), colon (HT-29), and cervical (HeLa) cancer cells. GRO-NLs-treated HT-29, HeLa, and MCF-7 cells showed cytotoxicity in the MTT and NRU assays via defects in mitochondrial functions and lysosomal activity. HT-29, HeLa, and MCF-7 cells treated with GRO-NLs exhibited substantial elevations in ROS, disturbances of the mitochondrial membrane potential, an influx of Ca2+, and apoptosis. The qPCR quantification showed the upregulation of caspase 3, caspase 9, bax, and SOD1 genes in GRO-NLs-treated cells. Western blotting showed the depletion of P21, P53, and CDC25C proteins in the above cancer cell lines after GRO-NLs treatment, indicating its function as a mutagen to induce mutation in the P53 gene, thereby affecting P53 protein and downstream effectors P21 and CDC25C. In addition, there may be a mechanism other than P53 mutation that controls P53 dysfunction. We conclude that nonfunctionalized GRO-NLs exhibit prospective biomedical application as a putative anticancer entity against colon, cervical, and breast cancers.

Diclofenac exhibits cytotoxic activity associated with metabolic alterations and p53 induction in ESCC cell lines and decreases ESCC tumor burden in vivo.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human malignancy, often displaying limited therapeutic response. Here, we examine the non-steroidal anti-inflammatory drug diclofenac (DCF) as a novel therapeutic agent in ESCC using complementary in vitro and in vivo models. DCF selectively reduced viability of human ESCC cell lines TE11, KYSE150, and KYSE410 as compared with normal primary or immortalized esophageal keratinocytes. Apoptosis and altered cell cycle profiles were documented in DCF-treated TE11 and KYSE 150. In DCF-treated TE11, RNA-Sequencing identified differentially expressed genes and Ingenuity Pathway Analysis predicted alterations in pathways associated with cellular metabolism and p53 signaling. Downregulation of proteins associated with glycolysis was documented in DCF-treated TE11 and KYSE150. In response to DCF, TE11 cells further displayed reduced levels of ATP, pyruvate, and lactate. Evidence of mitochondrial depolarization and superoxide production was induced by DCF in TE11 and KYSE150. In DCF-treated TE11, the superoxide scavenger MitoTempo improved viability, supporting a role for mitochondrial reactive oxygen species in DCF-mediated toxicity. DCF treatment resulted in increased expression of p53 in TE11 and KYSE150. p53 was further identified as a mediator of DCF-mediated toxicity in TE11 as genetic depletion of p53 partially limited apoptosis in response to DCF. Consistent with the anticancer activity of DCF in vitro, the drug significantly decreased tumor burdene in syngeneic ESCC xenograft tumors and 4-nitroquinoline 1-oxide-mediated ESCC lesions in vivo. These preclinical findings identify DCF as an experimental therapeutic that should be explored further in ESCC.

Asparagine and Glutamine Deprivation Alters Ionizing Radiation Response, Migration and Adhesion of a p53null Colorectal Cancer Cell Line.

Colorectal cancer (CRC) is the most prominent form of colon cancer for both incidence (38.7 per 100,000 people) and mortality (13.9 per 100,000 people). CRC's poor response to standard therapies is linked to its high heterogeneity and complex genetic background. Dysregulation or depletion of the tumor suppressor p53 is involved in CRC transformation and its capability to escape therapy, with p53null cancer subtypes known, in fact, to have a poor prognosis. In such a context, new therapeutic approaches aimed at reducing CRC proliferation must be investigated. In clinical practice, CRC chemotherapy is often combined with radiation therapy with the aim of blocking the expansion of the tumor mass or removing residual cancer cells, though contemporary targeting of amino acid metabolism has not yet been explored. In the present study, we used the p53null Caco-2 model cell line to evaluate the effect of a possible combination of radiation and L-Asparaginase (L-ASNase), a protein drug that blocks cancer proliferation by impairing asparagine and glutamine extracellular supply. When L-ASNase was administered immediately after IR, we observed a reduced proliferative capability, a delay in DNA-damage response and a reduced capability to adhere and migrate. Our data suggest that a correctly timed combination of X-rays and L-ASNase treatment could represent an advantage in CRC therapy.

Drugs Targeting p53 Mutations with FDA Approval and in Clinical Trials.

Mutations in the tumor suppressor p53 (p53) promote cancer progression. This is mainly due to loss of function (LOS) as a tumor suppressor, dominant-negative (DN) activities of missense mutant p53 (mutp53) over wild-type p53 (wtp53), and wtp53-independent oncogenic activities of missense mutp53 by interacting with other tumor suppressors or oncogenes (gain of function: GOF). Since p53 mutations occur in ~50% of human cancers and rarely occur in normal tissues, p53 mutations are cancer-specific and ideal therapeutic targets. Approaches to target p53 mutations include (1) restoration or stabilization of wtp53 conformation from missense mutp53, (2) rescue of p53 nonsense mutations, (3) depletion or degradation of mutp53 proteins, and (4) induction of p53 synthetic lethality or targeting of vulnerabilities imposed by p53 mutations (enhanced YAP/TAZ activities) or deletions (hyperactivated retrotransposons). This review article focuses on clinically available FDA-approved drugs and drugs in clinical trials that target p53 mutations and summarizes their mechanisms of action and activities to suppress cancer progression.

BNIP3-dependent mitophagy safeguards ESC genomic integrity via preventing oxidative stress-induced DNA damage and protecting homologous recombination

Embryonic stem cells (ESCs) have a significantly lower mutation load compared to somatic cells, but the mechanisms that guard genomic integrity in ESCs remain largely unknown. Here we show that BNIP3-dependent mitophagy protects genomic integrity in mouse ESCs. Deletion of Bnip3 increases cellular reactive oxygen species (ROS) and decreases ATP generation. Increased ROS in Bnip3−/− ESCs compromised self-renewal and were partially rescued by either NAC treatment or p53 depletion. The decreased cellular ATP in Bnip3−/− ESCs induced AMPK activation and deteriorated homologous recombination, leading to elevated mutation load during long-term propagation. Whereas activation of AMPK in X-ray-treated Bnip3+/+ ESCs dramatically ascended mutation rates, inactivation of AMPK in Bnip3−/− ESCs under X-ray stress remarkably decreased the mutation load. In addition, enhancement of BNIP3-dependent mitophagy during reprogramming markedly decreased mutation accumulation in established iPSCs. In conclusion, we demonstrated a novel pathway in which BNIP3-dependent mitophagy safeguards ESC genomic stability, and that could potentially be targeted to improve pluripotent stem cell genomic integrity for regenerative medicine.

Depletion of R270C Mutant p53 in Osteosarcoma Attenuates Cell Growth but Does Not Prevent Invasion and Metastasis In Vivo.

Novel therapeutic targets are needed to better treat osteosarcoma, which is the most common bone malignancy. We previously developed mouse osteosarcoma cells, designated AX (accelerated bone formation) cells from bone marrow stromal cells. AX cells harbor both wild-type and mutant forms of p53 (R270C in the DNA-binding domain, which is equivalent to human R273C). In this study, we showed that mutant p53 did not suppress the transcriptional activation function of wild-type p53 in AX cells. Notably, AXT cells, which are cells derived from tumors originating from AX cells, lost wild-type p53 expression, were devoid of the intact transcription activation function, and were resistant to doxorubicin. ChIP-seq analyses revealed that this mutant form of p53 bound to chromatin in the vicinity of the transcription start sites of various genes but exhibited a different binding profile from wild-type p53. The knockout of mutant p53 in AX and AXT cells by CRISPR-Cas9 attenuated tumor growth but did not affect the invasion of these cells. In addition, depletion of mutant p53 did not prevent metastasis in vivo. Therefore, the therapeutic potency targeting R270C (equivalent to human R273C) mutant p53 is limited in osteosarcoma. However, considering the heterogeneous nature of osteosarcoma, it is important to further evaluate the biological and clinical significance of mutant p53 in various cases.

Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin.

Simple SummaryThe tumor suppressor p53 is frequently mutated in human cancer. Accumulation of missense mutant p53 (mutp53) in tumors is crucial for malignant progression, and cancers are often addicted to oncogenic mutp53. However, strategies to deplete mutp53 have not yet been established. Recent studies have shown that misfolded or conformational mutp53 is stabilized by DNAJA1, a member of HSP40, also known as J-domain proteins (JDPs). However, no selective DNAJA1 inhibitor is clinically available. Through a molecular docking study, we identified a potential DNAJA1 inhibitor, called PLTFBH, derived from the natural compound plumbagin, as a compound that bound to and reduced protein levels of DNAJA1 and several other HSP40/JDPs. PLTFBH reduced the levels of conformational mutp53 and inhibited cancer cell migration in a manner dependent on DNAJA1 and mutp53.Accumulation of missense mutant p53 (mutp53) in cancers promotes malignant progression. DNAJA1, a member of HSP40 (also known as J-domain proteins: JDPs), is shown to prevent misfolded or conformational mutp53 from proteasomal degradation. Given frequent addiction of cancers to oncogenic mutp53, depleting mutp53 by DNAJA1 inhibition is a promising approach for cancer therapy. However, there is no clinically available inhibitor for DNAJA1. Our in silico molecular docking study with a natural compound-derived small molecule library identified a plumbagin derivative, PLIHZ (plumbagin–isoniazid analog), as a potential compound binding to the J domain of DNAJA1. PLIHZ efficiently reduced the levels of DNAJA1 and several conformational mutp53 with minimal impact on DNA contact mutp53 and wild-type p53 (wtp53). An analog, called PLTFBH, which showed a similar activity to PLIHZ in reducing DNAJA1 and mutp53 levels, inhibited migration of cancer cells specifically carrying conformational mutp53, but not DNA contact mutp53, p53 null, and wtp53, which was attenuated by depletion of DNAJA1 or mutp53. Moreover, PLTFBH reduced levels of multiple other HSP40/JDPs with tyrosine 7 (Y7) and/or tyrosine 8 (Y8) but failed to deplete DNAJA1 mutants with alanine substitution of these amino acids. Our study suggests PLTFBH as a potential inhibitor for multiple HSP40/JDPs.

Bioinformatics-Driven Identification of p62 as A Crucial Oncogene in Liver Cancer.

Liver hepatocellular carcinoma (LIHC) is the major form of liver cancer that is the fourth most common cause of cancer death worldwide. It has been reported that the multifunctional protein p62 (also known as SQSTM1) plays a cancer-promoting role in LIHC, but the detailed mechanisms underlying p62 interaction with LIHC remains unclear. To gain a comprehensive understanding of p62 interaction with LIHC in clinical settings, we performed bioinformatic analyses using various online algorithms derived from high throughput profiling. Our results indicate that p62 expression is significantly upregulated, partially due to its promoter demethylation, rather than p62 gene mutation, in LIHC. Mutation of TP53, CTNNB1, or ALB significantly correlates with, and mutation of AXIN1 reversely correlates with, the p62 expression level. Its upregulation occurs as early as liver cirrhosis, and go through all stages of the carcinogenesis. HCV infection makes a significant contribution to p62 upregulation in LIHC. We further identified p62-associated molecular signatures in LIHC, including many genes that are involved in antioxidant stress and metabolism, such as SRX1 and TXNRD1. Regarding to the clinical outcome, p62 expression level reversely correlates with the survival of LIHC patients (p<0.01). Importantly, we experimentally validated that p62 depletion in liver cancer cell lines downregulates the expression of SRX1 and TXNRD1 at both transcriptional and translational levels, and reduces cell proliferation. As the potential mechanisms underlying the tumor-promoting role of p62, we show that p62 upregulation is remarkably associated with reprogramming of pathways mediated by p53, Wnt/β-catenin, and Keap1-NRF2, which are crucial for oncogenesis in many contexts. Our findings provide a comprehensive insight into the interaction between p62 and LIHC, offering valuable information for understanding of LIHC pathogenesis.

Abstract 422: Novel approach to destabilization of oncogenic mutant p53 and therapeutic implications

Abstract Introduction: Mutant p53 drives several hallmarks of cancer through a gain-of-function oncogenic program. The oncogenicity of mutant p53 includes sustained proliferation, increased survival and resistance to therapy, increased invasion and metastasis, and genomic instability. The oncogenicity of mutant p53 is related to its stability and accumulation in cells by evading degradation by the proteasome. Therefore, destabilization of mutant p53 has been sought as a therapeutic strategy, but so far without clinical success. We and others observe that proteasome inhibitors cause paradoxical depletion of mutant p53 in lung cancer cell lines. Objectives: In this project, we sought to determine the mechanism(s) of mutant p53 depletion by proteasome inhibition and its therapeutic implications in lung cancer. Methods: Human lung cancer cell lines bearing mutant p53 and wildtype p53 (WTp53) were used in this study. An isogenic panel of cell lines with mutant p53, WTp53 and a vector control was also used to determine mutant p53 dependency of the observed effects. Western blotting, immunoprecipitation, siRNA technology, DNA transfection were used for protein detection, protein-protein interactions, gene knock-down and overexpression respectively. Cycloheximide chase assay was used to determine protein degradation. The cancer genome atlas (TCGA) analysis was performed using the cBioPortal platform. Results: Proteasome inhibitors resulted in depletion of mutant p53 in the lung cancer cell line H1975. This effect was also confirmed using the isogenic model described above. Depletion of mutant p53 occurred through a degradation mechanism and was time dependent. While WTp53 accumulated at initial timepoints of proteasome inhibition, mutant p53 did not accumulate but instead depleted with time. This suggested that the proteasome is not the primary mechanism for mutant p53, in contrast to WTp53. Since autophagy is an alternative mechanism of protein degradation in eukaryotes, we evaluated for, and found evidence of autophagy upon proteasome inhibition. We further showed that autophagic degradation of mutant p53 could involve the heat shock protein 70. Our TCGA data analysis showed proteasome genes amplification in mutant p53 lung cancers, suggesting a possible functional dependency. Furthermore, RNA sequencing analysis showed upregulation of most proteasome genes upon stable expression of the R273H mutant in p53-null lung cancer cell line. Finally, in vitro cell viability assays using two different proteasome inhibitors showed increased sensitivity of the lung cancer cell line with mutant p53 compared to WTp53. Conclusion: The cumulative evidence of proteasome hyperactivity, degradation of mutant p53 and in vitro evidence of increased susceptibility of mutant p53 lung cancer cell lines suggests that proteasome inhibitors warrant a closer attention for therapeutic targeting of mutant p53 in lung cancer. Citation Format: Eziafa I. Oduah, Larisa Litovchick, Steven R. Grossman. Novel approach to destabilization of oncogenic mutant p53 and therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 422.