P53 Stabilization Research Articles

RNA-interference screen for p53 regulators unveils a role of WDR75 in ribosome biogenesis.

Ribosome biogenesis is an essential, energy demanding process whose deregulation has been implicated in cancer, aging, and neurodegeneration. Ribosome biogenesis is therefore under surveillance of pathways including the p53 tumor suppressor. Here, we first performed a high-content siRNA-based screen of 175 human ribosome biogenesis factors, searching for impact on p53. Knock-down of 4 and 35 of these proteins in U2OS cells reduced and increased p53 abundance, respectively, including p53 accumulation after depletion of BYSL, DDX56, and WDR75, the effects of which were validated in several models. Using complementary approaches including subcellular fractionation, we demonstrate that endogenous human WDR75 is a nucleolar protein and immunofluorescence analysis of ectopic GFP-tagged WDR75 shows relocation to nucleolar caps under chemically induced nucleolar stress, along with several canonical nucleolar proteins. Mechanistically, we show that WDR75 is required for pre-rRNA transcription, through supporting the maintenance of physiological levels of RPA194, a key subunit of the RNA polymerase I. Furthermore, WDR75 depletion activated the RPL5/RPL11-dependent p53 stabilization checkpoint, ultimately leading to impaired proliferation and cellular senescence. These findings reveal a crucial positive role of WDR75 in ribosome biogenesis and provide a resource of human ribosomal factors the malfunction of which affects p53.

Loss of TRIM31 promotes breast cancer progression through regulating K48- and K63-linked ubiquitination of p53

Breast cancer is the most common cancer in the world. Relapse and metastasis are important factors endangering the life of breast cancer patients, but the mechanism is still unclear. The stabilization of p53 is essential for preventing carcinogenesis, and ubiquitination is one of the main ways to regulate the stability of p53. Tripartite motif-containing 31 (TRIM31) is a new member of the TRIM family and functions as an E3 ubiquitin ligase. It acts as a cancer promoter or suppressor in the malignant processes of multiple cancers. However, the function of TRIM31 in breast cancer progression remains unknown. In this study, we showed that TRIM31 is downregulated in breast cancer tissues and negatively correlated with breast cancer progression. Both gain- and loss-of-function assays indicated that TRIM31 inhibits the proliferation, colony formation, migration, and invasion of breast cancer cells. Further investigation demonstrated that TRIM31 directly interacts with p53, and inducing the K63-linked ubiquitination of p53 via its RING domain, Meanwhile, TRIM31 suppresses the MDM2-mediated K48-linked ubiquitination of p53 through competitive inhibiting the interaction of MDM2 and p53, leading to the p53 stabilization and activation. Knockdown of p53 reversed the inhibitory effects of TRIM31 on the growth and metastasis of breast cancer cells. Moreover, we found that the RING and coiled-coil (C–C) domains of TRIM31 were essential for its tumor suppressor function. Taken together, our findings reveal a novel mechanism by which TRIM31 suppresses breast cancer development through the stabilization and activation of p53 and define a promising therapeutic strategy for restoring TRIM31 to treat breast cancer.

UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11.

Simple SummaryHerein, the oncogenic role of UBE2M as an E2 NEDD8-conjugating enzyme was explored in hepatocellular carcinoma (HCC) cells, since neddylation plays a critical role in tumorigenesis. To address this issue, human tissue array and TCGA analysis were conducted in HCCs to find overexpression of UBE2M in HCCs. In addition, a differential profile was confirmed in UBE2M-depleted HepG2 cells. Furthermore, UBE2M depletion activated p53 expression and stability, while the ectopic expression of UBE2M disturbed p53 activation and enhanced degradation of exogenous p53 mediated by MDM2 in HepG2 cells via binding to MDM2 and ribosomal protein L11 by immunoprecipitation and immunofluorescence. These findings provide evidence that UBE2M is critically involved in liver cancer progression as a p53 negative regulator by binding to MDM2 and ribosomal protein L11.Though UBE2M, an E2 NEDD8-conjugating enzyme, is overexpressed in HepG2, Hep3B, Huh7 and PLC/PRF5 HCCs with poor prognosis by human tissue array and TCGA analysis, its underlying oncogenic mechanism remains unclear. Herein, UBE2M depletion suppressed viability and proliferation and induced cell cycle arrest and apoptosis via cleavages of PARP and caspase 3 and upregulation of p53, Bax and PUMA in HepG2, Huh7 and Hep3B cells. Furthermore, UBE2M depletion activated p53 expression and stability, while the ectopic expression of UBE2M disturbed p53 activation and enhanced degradation of exogenous p53 mediated by MDM2 in HepG2 cells. Interestingly, UBE2M binds to MDM2 or ribosomal protein L11, but not p53 in HepG2 cells, despite crosstalk between p53 and UBE2M. Consistently, the colocalization between UBE2M and MDM2 was observed by immunofluorescence. Notably, L11 was required in p53 activation by UBE2M depletion. Furthermore, UBE2M depletion retarded the growth of HepG2 cells in athymic nude mice along with elevated p53. Overall, these findings suggest that UBE2M promotes cancer progression as a p53 negative regulator by binding to MDM2 and ribosomal protein L11 in HCCs.

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.

Polycyclic aromatic hydrocarbons regulate the pigmentation pathway and induce DNA damage responses in keratinocytes, a process driven by systemic immunity

BackgroundUrban pollution is correlated with an increased prevalence of skin pigmentation disorders, however the physiological processes underlying this association are unclear. ObjectivesTo delineate the relationship between polycyclic aromatic hydrocarbons (PAHs), a key constituent of atmospheric pollution, and immunity/skin pigmentation pathways. MethodsWe exposed peripheral blood mononuclear cells (PBMC) to PAHs and performed cytokines/chemokine profiling. We then examined the effect of immune activation on pigmentation by co-culturing PBMC and Benzo(a)pyrene (BaP) with reconstructed human pigmented epidermis (RHPE). To study the mechanism, we treated keratinocytes with conditioned medium from BaP-exposed PBMC and studied DNA damage responses, aryl hydrocarbon receptor (AhR) activation and pro-pigmentation factor, proopiomelanocortin (POMC) secretion. ResultsPAHs induced up-regulation of inflammatory cytokines/chemokine in PBMC. Co-culturing of RHPE with PBMC+BaP resulted in increased melanin content and localization. BaP-conditioned medium significantly increased DNA damage, p53 stabilization, AhR activation and POMC secretion in keratinocytes. We found that IFNγ induced DNA damage, while TNFα and IL-8 potentiated POMC secretion in keratinocytes. Importantly, BaP-conditioned medium-induced DNA damage and POMC secretion is prevented by antioxidants vitamin E, vitamin C and sulforaphane, as well as the prototypical corticosteroid dexamethasone. Finally, vitamin C and sulforaphane enhanced the genome protective and depigmentation effects of dexamethasone, providing proof-of-concept for a combinatorial approach for the prevention and/or correction of PAH-induced pigment spots formation. ConclusionOur study reveals the importance of systemic immunity in regulating PAH-induced skin pigmentation, and provide a new keratinocyte DNA damage response mechanistic target for the prevention or reversal of pollution-associated skin pigmentation.

Functional characterization of HIC, a P2Y1 agonist, as a p53 stabilizer for prostate cancer cell death induction.

Background: (1-(2-hydroxy-5-nitrophenyl)(4-hydroxyphenyl)methyl)indoline-4-carbonitrile (HIC), an agonist of theP2Y1 receptor (P2Y1R), induces cell death in prostate cancer cells. However, the molecular mechanism behind the inhibition of HIC in prostate cancerremains elusive. Methods & results: Here, to outline the inhibitory role of HIC on prostate cancer cells, PC-3 and DU145 cell lines were treated with the respective IC50 concentrations, which reduced cell proliferation, adherence propertiesand spheroid formation. HIC was able to arrest the cell cycle at G1/S phaseand also inducedapoptosis and DNA damage, validated by gene expression profiling. HIC inhibited the prostate cancer cells' migration and invasion, revealing its antimetastatic ability. P2Y1R-targeted HIC affects p53, MAPK andNF-κBprotein expression, thereby improvingthe p53 stabilization essential for G1/S arrest and cell death. Conclusion: These findings provide an insight on the potential use of HIC, whichremains the mainstay treatment for prostate cancer.

Increased H2O2 levels and p53 stabilization lead to mitochondrial dysfunction in XPC-deficient cells.

XPC deficiency is associated with mitochondrial dysfunction, increased mitochondrial H2O2 production and sensitivity to the Complex III inhibitor antimycin A (AA), through a yet unclear mechanism. We found an imbalanced expression of several proteins that participate in important mitochondrial function and increased expression and phosphorylation of the tumor suppressor p53 in Xeroderma pigmentosum complementation group C (XP-C) (XPC-null) cells compared with an isogenic line corrected in locus with wild-type XPC (XPC-wt). Interestingly, inhibition of p53 nuclear import reversed the overexpression of mitochondrial proteins, whereas AA treatment increased p53 expression more strongly in the XP-C cells. However, inhibition of p53 substantially increased XP-C cellular sensitivity to AA treatment, suggesting that p53 is a critical factor mediating the cellular response to mitochondrial stress. On the other hand, treatment with the antioxidant N-acetylcysteine increased glutathione concentration and decreased basal H2O2 production, p53 levels and sensitivity to AA treatment in the XPC-null back to the levels found in XPC-wt cells. Thus, the results suggest a critical role for mitochondrially generated H2O2 in the regulation of p53 expression, which in turn modulates XP-C sensitivity to agents that cause mitochondrial stress.

YY1 Promotes Telomerase Activity and Laryngeal Squamous Cell Carcinoma Progression Through Impairment of GAS5-Mediated p53 Stability

Yin Yang 1 (YY1) is a key transcription factor that exerts functional roles in the cell biological process of various cancers. The current study aimed to elucidate the role and mechanism of YY1 in laryngeal squamous cell carcinoma (LSCC). YY1 mRNA and protein expression in human LSCC cell lines was detected by RT-qPCR and Western blot analysis. An interaction of YY1, GAS5, and p53 protein stability was predicted and confirmed by bioinformatics, ChIP, Co-IP, RIP, and FISH assays. Following loss- and gain-function assays, LSCC cell proliferation, colony formation, cell cycle, telomere length and telomerase activity were evaluated by CCK-8 assay, colony formation assay, flow cytometry, and PCR-ELISA, respectively. Nude mice were xenografted with the tumor in vivo. LSCC cell lines presented with upregulated expression of YY1, downregulated GAS5 expression, and decreased p53 stability. YY1 inhibited the expression of GAS5, which in turn recruited p300 and bound to p53, thus stabilizing it. Moreover, YY1 could directly interact with p300 and suppressp53 stability, leading to enhancement of cell proliferation, telomere length and telomerase activity in vitro along with tumor growth in vivo. Collectively, YY1 can stimulate proliferation and telomerase activity of LSCC cells through suppression of GAS5-dependent p53 stabilization or by decreasing p53 stability via a direct interaction with p300, suggesting that YY1 presents a therapeutic target as a potential oncogene in LSCC development and progression.

Cinobufagin-induced DNA damage response activates G2/M checkpoint and apoptosis to cause selective cytotoxicity in cancer cells

BackgroundProcessed extracts from toad skin and parotoid gland have long been used to treat various illnesses including cancer in many Asian countries. Recent studies have uncovered a family of bufadienolides as the responsible pharmacological compounds, and the two major molecules, cinobufagin and bufalin, have been shown to possess robust antitumor activity; however, the underlying mechanisms remain poorly understood.MethodsIntracellular reactive oxygen species (ROS) were measured by DCFH-DA staining and flow cytometry, and DNA damage was analyzed by immunofluorescent staining and the alkaline comet assay. Cytotoxicity was measured by MTT as well as colony formation assays, and cell cycle and apoptosis were analyzed by flow cytometry. In addition, apoptosis was further characterized by TUNEL and mitochondrial membrane potential assays.ResultsHere we showed that sublethal doses of cinobufagin suppressed the viability of many cancer but not noncancerous cell lines. This tumor-selective cytotoxicity was preceded by a rapid, cancer-specific increase in cellular ROS and was significantly reduced by the ROS inhibitor N-acetyl cysteine (NAC), indicating oxidative stress as the primary source of cinobufagin-induced cancer cell toxicity. Sublethal cinobufagin-induced ROS overload resulted in oxidative DNA damage and intense replication stress in cancer cells, leading to strong DNA damage response (DDR) signaling. Subsequent phosphorylation of CDC25C and stabilization of p53 downstream of DDR resulted in activation of the G2/M checkpoint followed by induction of apoptosis. These data indicate that cinobufagin suppresses cancer cell viability via DDR-mediated G2 arrest and apoptosis.ConclusionAs elevated oxidative pressure is shared by most cancer cells that renders them sensitive to further oxidative insult, these studies suggest that nontoxic doses of cinobufagin can be used to exploit a cancer vulnerability for induction of cancer-specific cytotoxicity.

UBCH5 Family Members Differentially Impact Stabilization of Mutant p53 via RNF128 Iso1 During Barrett's Progression to Esophageal Adenocarcinoma.

Background & AimsTP53 mutations underlie Barrett’s esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown.MethodsSingle-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability.ResultsThe UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2–E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate β-TrCP1–mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding–deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells.ConclusionsLoss of RNF128 Iso2–UBCH5C and persistence of the Iso1–UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression.

LNX1 Contributes to Cell Cycle Progression and Cisplatin Resistance.

Simple SummaryThe ligand of numb-protein X1 (LNX1) is reported to be upregulated in various cancers, however the cellular function of LNX1 is not clearly characterized. The aim of the present study was to elucidate the regulation of LNX1 expression and clarify the role of LNX1 in cell-cycle progression and resistance to the cancer therapeutic agent, cisplatin. We found that LNX1 expression is decreased by DNA damage including cisplatin treatment and the levels of S and G2/M populations were correlated with LNX1 expression. We also showed that the upregulation of LNX1 contributes to cell-cycle progression and cisplatin resistance. Our data suggest that LNX1 is the important regulator of the cell cycle, and contributes to tumor progression.The ligand of numb-protein X1 (LNX1) acts as a proto-oncogene by inhibiting p53 stability; however, the regulation of LNX1 expression has not been investigated. In this study, we screened chemicals to identify factors that potentially regulate LNX1 expression. We found that LNX1 expression levels were decreased by DNA damage, including that by cisplatin. Upon treatment with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), LNX1 expression levels increased. In addition, cell-cycle progression increased upon LNX1 expression; the levels of S and G2/M populations were correlated with LNX1 expression. Moreover, in CRISPR-Cas9-mediated LNX1 knockout cells, we observed a delay in cell-cycle progression and a downregulation of genes encoding the cell-cycle markers cyclin D1 and cyclin E1. Finally, the upregulation of LNX1-activated cell-cycle progression and increased resistance to cisplatin-mediated cell death. Taken together, these results suggest that LNX1 contributes to cell-cycle progression and cisplatin resistance.

Deacetylation of BmAda3 is required for cell apoptosis caused by Bombyx mori nucleopolyhedrovirus infection.

Silkworm is not only an ideal insect model with a biological significance, but it is also crucially important in sericulture and bioreactors. Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of silkworm. However, the molecular mechanism underlying BmNPV invasion is still unclear. Based on our previous acetylome research findings of B. mori after BmNPV infection, here, we focused on silkworm alteration/deficiency in activation-3 (BmAda3). The acetylation of K124 and K128 were significantly reduced (0.66-fold) upon the virus challenge. Due to the interaction between Ada3 and P53, acetylation-mimic K124Q/K128Q and deacetylation-mimic K124R/K128R mutants of BmAda3 were constructed to explore the roles exerted by the acetylation modification of BmAda3 on P53. Yeast two-hybrid and IP results revealed that both BmAda3 and its deacetylation mutants (K124R/K128R) interacted with P53. Interestingly, we found that the deacetylation mutants (K124R/K128R) of BmAda3 significantly promoted the stability of P53. Since P53 is a proapoptotic factor, cell apoptosis was detected. We established that the deacetylation of BmAda3 at K124/K128 facilitated cellular apoptosis during BmNPV infection. Finally, viral proliferation was analyzed, and the results indicated that virus generation was reduced by K124/K128 deacetylation. Our report, based on the deacetylation of two lysine sites 124/128 of BmAda3, shows possible regulatory pathways of BmNPV proliferation and provides novel insights into the development of antiviral agents.

TRAIL-receptor 2\u2014a novel negative regulator of p53

TNF-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2) can induce apoptosis in cancer cells upon crosslinking by TRAIL. However, TRAIL-R2 is highly expressed by many cancers suggesting pro-tumor functions. Indeed, TRAIL/TRAIL-R2 also activate pro-inflammatory pathways enhancing tumor cell invasion, migration, and proliferation. In addition, nuclear TRAIL-R2 (nTRAIL-R2) promotes malignancy by inhibiting miRNA let-7-maturation. Here, we show that TRAIL-R2 interacts with the tumor suppressor protein p53 in the nucleus, assigning a novel pro-tumor function to TRAIL-R2. Knockdown of TRAIL-R2 in p53 wild-type cells increases the half-life of p53 and the expression of its target genes, whereas its re-expression decreases p53 protein levels. Interestingly, TRAIL-R2 also interacts with promyelocytic leukemia protein (PML), a major regulator of p53 stability. PML-nuclear bodies are also the main sites of TRAIL-R2/p53 co-localization. Notably, knockdown or destruction of PML abolishes the TRAIL-R2-mediated regulation of p53 levels. In summary, our finding that nTRAIL-R2 facilitates p53 degradation and thereby negatively regulates p53 target gene expression provides insight into an oncogenic role of TRAIL-R2 in tumorigenesis that particularly manifests in p53 wild-type tumors.

MHY1485 enhances X-irradiation-induced apoptosis and senescence in tumor cells.

The mammalian target of rapamycin (mTOR) is a sensor of nutrient status and plays an important role in cell growth and metabolism. Although inhibition of mTOR signaling promotes tumor cell death and several mTOR inhibitors have been used clinically, recent reports have shown that co-treatment with MHY1485, an mTOR activator, enhances the anti-cancer effects of anti-PD-1 antibody and 5-fluorouracil. However, it remains unclear whether MHY1485 treatment alters the effects of radiation on tumor cells. In this study, the radiosensitizing effects of MHY1485 were investigated using murine CT26 and LLC cell lines. We examined mTOR signaling, tumor cell growth, colony formation, apoptosis, senescence, oxidative stress, p21 accumulation and endoplasmic reticulum (ER) stress levels in cells treated with MHY1485 and radiation, either alone or together. We found that MHY1485 treatment inhibited growth and colony formation in both cell lines under irradiation and no-irradiation conditions, results that were not fully consistent with MHY1485’s known role in activating mTOR signaling. Furthermore, we found that combined treatment with MHY1485 and radiation significantly increased apoptosis and senescence in tumor cells in association with oxidative stress, ER stress and p21 stabilization, compared to radiation treatment alone. Our results suggested that MHY1485 enhances the radiosensitivity of tumor cells by a mechanism that may differ from MHY1485’s role in mTOR activation.

Structural and Drug Targeting Insights on Mutant p53.

Simple SummaryThe tumor suppressor protein p53 is frequently mutated in human cancers, and its reactivation represents an encouraging hope for precision anticancer therapy. Despite this, the clinical use of compounds capable of restoring the wild-type-like function to mutant p53 (mutp53) is not yet a reality. A more detailed understanding of the structural features of p53 mutations and the molecular mechanisms of mutp53 reactivators is critical to drive an efficient translation of these compounds into the clinic. In addition, this knowledge will provide insights into wild-type and mutp53 pharmacology at molecular levels, fostering the design and development of improved mutp53-targeted therapies to halt cancer.p53 is a transcription factor with a pivotal role in cell homeostasis and fate. Its impairment is a major event in tumor onset and development. In fact, about half of human cancers bear TP53 mutations that not only halt the normal function of p53, but also may acquire oncogenic gain of functions that favor tumorigenesis. Although considered undruggable for a long time, evidence has proven the capability of many compounds to restore a wild-type (wt)-like function to mutant p53 (mutp53). However, they have not reached the clinic to date. Structural studies have strongly contributed to the knowledge about p53 structure, stability, dynamics, function, and regulation. Importantly, they have afforded relevant insights into wt and mutp53 pharmacology at molecular levels, fostering the design and development of p53-targeted anticancer therapies. Herein, we provide an integrated view of mutp53 regulation, particularly focusing on mutp53 structural traits and on targeting agents capable of its reactivation, including their biological, biochemical and biophysical features. With this, we expect to pave the way for the development of improved small molecules that may advance precision cancer therapy by targeting p53.

Abstract 1398: Impact of proteasome inhibitor specificity and efficacy on apoptosis induction by combination with ABT-199

Abstract Metastatic Soft-Tissue Sarcomas (STS) are a rare and highly heterogeneous group of mesenchymal malignancies that carry a poor prognosis. Therapy is hampered by a limited number of effective treatments, and the almost non-existing long-term survival rate illustrates the need of effective targeted treatment. As a potential approach, we investigated combination of the clinically approved BH3-mimetic drug ABT-199 with different proteasome inhibitors (PIs): Bortezomib, Carfilzomib and Ixazomib, each with proven efficacy, e.g., in multiple myeloma. ABT-199 selectively inhibits the anti-apoptotic protein Bcl-2 while Bortezomib (BOZ) and Ixazomib (IXZ) bind reversible to the β1 and β5 subunits of the 20S proteasome and Carfilzomib (CFZ) irreversibly blocks the subunits β2 and β5. We investigated whether the combination of ABT-199 with CFZ or IXZ shows synergistic activity as recently published for BOZ. SW982 sarcoma cells were cultured with PIs alone or in combination with ABT-199. Cell death was detected by flow cytometric analysis of mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin V). To elucidate potential differences due to specificity of PIs, we analyzed expression of BCL-2 family proteins by Western Blot and performed analogue experiments in knock-out (BAXKO, BAKKO, BOKKO) cell lines. In combination, ABT-199&BOZ or ABT-199&CFZ showed comparable synergistic cell death induction in SW982 while ABT-199&IXZ less efficiently induced cell death. Engineered SW982 knock-out cell lines suggests specific relevance of BOK in IXZ induced apoptosis, whereas all tested PIs crucially depend on BAX for apoptosis induction in combination with ABT-199. Also, as shown for ABT-199&BOZ, both CFZ and IXZ, alone and in combination with ABT-199, efficiently induced expression of NOXA. Strikingly, and in line with augmented cell death induction, ABT-199&CFZ resulted in highest expression of NOXA as compared to BOZ and IXZ. ABT-199&PIs synergistically induce apoptotic cell death in SW982 and corresponding knock-out cell lines with CFZ showing exacerbated expression of NOXA. ABT-199&CFZ also induced strongest stabilization of p53, a transcriptional key regulator of NOXA. We suggest that simultaneous inhibition of anti-apoptotic BCL-2 by ABT-199 and the enhanced expression of NOXA double-hit on the BCL-2 and the MCL-1 signaling axis force the cells over the threshold of apoptosis. Enhanced efficacy of CFZ is dually caused by reduced degradation of NOXA and enhanced stabilization of p53, which in turn transactivates expression of NOXA. In conclusion, PI specificity and efficacy modulate apoptosis induction by combination with ABT-199. Future efforts, e.g. knock-out of relevant BH3-only proteins, will elucidate the observed variable efficacy of these and other PIs for prospective translation in clinical applications. Citation Format: Sandra Weller, Benjamin Schaefer, Tobias Beigl, Kathrin Böpple, Walter E. Aulitzky, Hans-Georg Kopp, Frank Essmann. Impact of proteasome inhibitor specificity and efficacy on apoptosis induction by combination with ABT-199 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1398.

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and invivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how invivo tissue-specific phenotypes emerge in ribosomopathies.

Abstract 962: Small-molecule NSC58874 releases and activates p73 via induction of mutant p53 degradation in cancer cells

Abstract The tumor suppressor p53 is the most frequently mutated gene in cancer. Mutant p53 is a challenge to target in cancer therapy due to its many mutations and the fact that it is an inactivated transcription factor. p73 is a member of the p53 family but very rarely mutated in cancer cells. Therefore, activation of p73 is one of the promising strategies to restore the p53 pathway signaling in cancer therapy. Our laboratory has reported that p73 can be activated by small molecules to restore p53 pathway signaling via induction of p73 expression or interruption of p73 interaction with mutant p53. In the present study, we explored a strategy for releasing p73 from a mutant p53 inhibitory complex by induction of mutant p53 degradation using small molecule NSC59984. We previously reported that NSC59984 induces mutant p53 degradation and restores p53 pathway signaling through p73 in p53-mutant cancer cells. To address the mechanism of whether p73 is released from a mutant p53 inhibitory complex to transcriptionally regulate p53 targets, we performed p53-RE-luc reporter assays and Chromatin Immunoprecipitation (ChIP)-PCR in cancer cells with overexpression of p73. We found NSC59984 enhances p73-mediated signaling based on p53 target (such as p21 and noxa) induction at the protein level and p53-RE-luc reporter assay. Further ChIP-PCR analysis showed that NSC59984 treatment increases p73-binding to the p21 and Noxa promoters. These results taken together suggest that NSC59984 enhances p73 transcriptional activity to restore the p53 pathway signaling. It is also well known that p73 activity is regulated through a complex mechanism such as post-translational modifications and protein-protein interactions. We found that NSC59984 can stimulate the ERK2 signaling pathway. The MEK1 inhibitor U0126 partially blocked p73 binding to the p21 and Noxa promoters and their gene expression in cells treated with NSC59984, and this correlated with the rescue of mutant p53 stabilization. These results suggest that p73 is released from mutant p53 inhibitory complex and further stimulated through ERK2 signaling, as a mechanism of tumor suppression in tumors with mutant p53. Citation Format: Shengliang Zhang, Wafik S. El-Deiry. Small-molecule NSC58874 releases and activates p73 via induction of mutant p53 degradation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 962.

Natural Merosesquiterpenes Activate the DNA Damage Response via DNA Strand Break Formation and Trigger Apoptotic Cell Death in p53-Wild-type and Mutant Colorectal Cancer.

Simple SummaryBowel cancer is a serious disease, which affects many people worldwide. Unfortunately, the disease is often diagnosed in an advanced stage, which impairs the chance of survival. Furthermore, resistance to therapy occurs frequently. Thus, novel therapeutic approaches are required to improve cancer therapy. Here, we studied whether merosesquiterpenes might be useful for cancer treatment. These compounds occur in marine sponges and were isolated by our group. We were able to identify three compounds with potent cytotoxic activity in different cell lines established from human large bowel cancer. Our experiments provided evidence that the compounds cause DNA damage and trigger cell death, so-called mitochondrial apoptosis, which was attested in cancer cells with expression of wild-type and mutated p53 tumor suppressor. Finally, we show that merosesquiterpenes also kill intestinal tumor organoids, an ex vivo model of large bowel cancer.Colorectal cancer (CRC) is a frequently occurring malignant disease with still low survival rates, highlighting the need for novel therapeutics. Merosesquiterpenes are secondary metabolites from marine sponges, which might be useful as antitumor agents. To address this issue, we made use of a compound library comprising 11 isolated merosesquiterpenes. The most cytotoxic compounds were smenospongine > ilimaquinone ≈ dactylospontriol, as shown in different human CRC cell lines. Alkaline Comet assays and γH2AX immunofluorescence microscopy demonstrated DNA strand break formation in CRC cells. Western blot analysis revealed an activation of the DNA damage response with CHK1 phosphorylation, stabilization of p53 and p21, which occurred both in CRC cells with p53 knockout and in p53-mutated CRC cells. This resulted in cell cycle arrest followed by a strong increase in the subG1 population, indicative of apoptosis, and typical morphological alterations. In consistency, cell death measurements showed apoptosis following exposure to merosesquiterpenes. Gene expression studies and analysis of caspase cleavage revealed mitochondrial apoptosis via BAX, BIM, and caspase-9 as the main cell death pathway. Interestingly, the compounds were equally effective in p53-wild-type and p53-mutant CRC cells. Finally, the cytotoxic activity of the merosesquiterpenes was corroborated in intestinal tumor organoids, emphasizing their potential for CRC chemotherapy.

Novel CDK9 inhibitor oroxylin A promotes wild-type P53 stability and prevents hepatocellular carcinoma progression by disrupting both MDM2 and SIRT1 signaling.

Hepatocellular carcinoma (HCC) is one of the most lethal tumours worldwide. However, the effects of first-line sorafenib treatment in advanced HCC fail to prolong patients' survival due to the highly heterogeneous characteristics of HCC etiology. Cyclin-dependent kinase 9 (CDK9) is an important target in the continuous development of cancer therapy. Here, we demonstrate that CDK9 is closely associated with the progression of HCC and can serve as an HCC therapeutic target by modulating the recovery of wild-type p53 (wt-p53) function. We prove that mouse double minute 2 homologue (MDM2) and Sirtuin 1 (SIRT1) are phosphorylated by CDK9 at Ser166 and Ser47, respectively. Inhibition of CDK9 not only reduces the MDM2-mediated ubiquitination and degradation of wt-p53 but also increases wt-p53 stability by suppressing deacetylase activity of SIRT1. Thus, inhibition of CDK9 promotes the wt-p53 stabilization and prevents HCC progression. However, excessive inhibition by high concentrations of specific CDK9 inhibitors counteracts the promotion of p53 stability and reduces their anti-HCC activity because of extreme general transcription repression. The effects of a novel CDK9 inhibitor named oroxylin A (OA) from Scutellaria baicalensis are explored, with the results indicating that OA shows moderate and controlled inhibition of CDK9 activity and expression, and stabilizes wt-p53 by inhibiting CDK9-regulated MDM2 and SIRT1 signaling. These outcomes indicate the high therapeutic potential of OA against HCC and its low toxicity in normal tissue. This study demonstrates a novel mechanism for the regulation of wt-p53 by CDK9 and indicates that OA is a potential candidate for HCC therapy.