MDM2-mediated Ubiquitination Research Articles

P-3F, a microtubule polymerization inhibitor enhances P53 stability through the change in localization of RPS27a.

Previously, we demonstrated that P-3F, a podophyllum derivative, exhibits a 297-fold enhancement in antitumor activity than VP-16, used as anticancer agent in clinical. The purpose of our present study was to investigate the precise antitumor mechanism action of P-3F. It showed that P-3F inhibited microtubule polymerization in a concentration-dependent manner. The results were in overall agreement with modeling and docking studies performed on P-3F and tubulin. In addition, P-3F increased the levels of P53, this in turn prolonged P53 half-life. Note as well that levels of P21 protein were increased along with P53 in a concentration dependent change. It suggested that enhancement in stabilization of P53 induced by P-3F may be critical for P53/P21 signaling pathway, resulting in cell cycle arrest at G2/M. Furthermore, release of RPS27a from the nucleolus into the nucleoplasm led to decrease phosphorylation of Mdm2 at serine residue 166 and inhibit Mdm2-mediated ubiquitination of P53 in (P-3F)-treated HeLa cells. Together, these data suggest that P-3F, a microtubule polymerization inhibitor, causes P53 accumulation via P53 stability enhancement, due to blockage of the P53-Mdm2 network through the change in localization of RPS27a.

Accumulation of Smooth Muscle 22α Protein Accelerates Senescence of Vascular Smooth Muscle Cells via Stabilization of p53 In Vitro and In Vivo.

Smooth muscle (SM) 22α, an actin-binding protein, displays an upregulated expression as a marker during cellular senescence. However, the causal relationship between SM22α and senescence is poorly understood. This study aimed to investigate the role of SM22α in angiotensin II (Ang II)-induced senescence of vascular smooth muscle cells (VSMCs). We prepared a model of VSMC senescence induced by Ang II and found that the expression of SM22α in VSMCs was increased in response to chronic Ang II treatment. Overexpression of SM22α promoted Ang II-induced VSMC senescence, whereas knockdown of SM22α suppressed this process. Moreover, this effect of SM22α was p53 dependent. Increased SM22α protein obstructed ubiquitination and degradation of p53 and subsequently improved its stability. Furthermore, SM22α inhibited phosphorylation of Mdm2 (mouse double minute 2 homolog), an E3 ubiquitin-protein ligase, accompanied by a decreased interaction between Mdm2 and p53. Using LY294002, a PI3K/Akt inhibitor, we found that PI3K/Akt-mediated Mdm2 phosphorylation and activation was inhibited in senescent or SM22α-overexpressed VSMCs, in parallel with decreased p53 ubiquitination. We further found that SM22α inhibited activation of PI3K/Akt/Mdm2 pathway via strengthening actin cytoskeleton. In the in vivo study, we showed that the disruption of SM22α reduced the increase of blood pressure induced by Ang II, associated with decreased VSMC senescence through a mechanism similar to that in VSMCs in vitro. In conclusion, these findings suggest that the accumulation of SM22α promotes Ang II-induced senescence via the suppression of Mdm2-mediated ubiquitination and degradation of p53 in VSMCs in vitro and in vivo.

Abstract 2156: NSC59984 induces mutant p53 degradation via activating ERK2 pathway-MDM2 axis

Abstract Mutant p53 protein is highly expressed in most cancer cells due to its protein stabilization. Mutant p53 loses wild-type p53 function but gains new oncogenic functions in driving tumor growth and drug resistance. Depletion of mutant p53 is an attractive strategy for cancer therapy. We reported a small-molecule NSC59984 destabilizes mutant p53 protein via protein degradation (Zhang et al., Cancer Research, 2015). In this study, we demonstrate that the ERK2 pathway plays a specific role in NSC59984-induced mutant p53 degradation via MDM2-mediated ubiquitination. We observe a sustained-phosphorylation of ERK1/2 in cancer cells treated with NSC59984. ERK1/2 pathway is negatively regulated by MKP-1. We find that MKP-1 activity is partially inhibited by NSC59984 in a dose dependent manner in vitro. These results suggest that NSC5998 sustains ERK1/2 phosphorylation partially via disruption of the feedback-loop between MKP-1 and ERK2 pathway. Knockdown of ERK2 rescues mutant p53 from NSC59984-mediated degradation, and inhibits NSC59984-restoration of p53 signaling in mutant p53-expressing cells. On the contrary, the effect of NSC59984 on the mutant p53 is not inhibited by the blockage of JNK and P38, two pathways negatively regulated by MKP-1. These results suggest that the ERK2 phosphorylation is specifically required for NSC59984-mediated mutant p53 degradation. We further find that NSC59984 induces ERK2-dependent MDM2 phosphorylation. The effect of NSC59984 on MDM2 phosphoryation is partially inhibited by the blockage of ERK2 signaling (via U0126 treatment or Knockdown of ERK2), not by the attenuation of the JNK pathway. Furthermore, NSC59984-mediated mutant p53 protein degradation is inhibited by MDM2 knockdown, and enhanced by MDM2 overexpression in cancer cells. NSC59984-increased ubiquitination of mutant p53 is attenuated by U0126. Our results suggest that the ERK2 signaling pathway-MDM2 axis is a major determinant of NSC59984-mediated mutant p53 degradation. We further demonstrate that NSC59984 induces cell death via apoptosis, and NSC59984-induced cell death is mainly rescued by inhibition of the ERK2 pathway in p53 mutant cancer cells. Taken together, our results suggest that phosphorylated-ERK2 is an important factor required for NSC59984-mediated mutant p53 degradation which may further contribute to NSC59984-induced cell death in cancer cells. Citation Format: Shengliang Zhang, Lanlan Zhou, David Dicker, Wafik S. El-Deiry. NSC59984 induces mutant p53 degradation via activating ERK2 pathway-MDM2 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2156. doi:10.1158/1538-7445.AM2017-2156

PIG3 Regulates p53 Stability by Suppressing Its MDM2-Mediated Ubiquitination.

Under normal, non-stressed conditions, intracellular p53 is continually ubiquitinated by MDM2 and targeted for degradation. However, in response to severe genotoxic stress, p53 protein levels are markedly increased and apoptotic cell death is triggered. Inhibiting the ubiquitination of p53 under conditions where DNA damage has occurred is therefore crucial for preventing the development of cancer, because if cells with severely damaged genomes are not removed from the population, uncontrolled growth can result. However, questions remain about the cellular mechanisms underlying the regulation of p53 stability. In this study, we show that p53-inducible gene 3 (PIG3), which is a transcriptional target of p53, regulates p53 stability. Overexpression of PIG3 stabilized both endogenous and transfected wild-type p53, whereas a knockdown of PIG3 lead to a reduction in both endogenous and UV-induced p53 levels in p53-proficient human cancer cells. Using both in vivo and in vitro ubiquitination assays, we found that PIG3 suppressed both ubiquitination- and MDM2-dependent proteasomal degradation of p53. Notably, we demonstrate that PIG3 interacts directly with MDM2 and promoted MDM2 ubiquitination. Moreover, elimination of endogenous PIG3 in p53-proficient HCT116 cells decreased p53 phosphorylation in response to UV irradiation. These results suggest an important role for PIG3 in regulating intracellular p53 levels through the inhibition of p53 ubiquitination.

PAK4 regulates G6PD activity by p53 degradation involving colon cancer cell growth

The p21-activated kinase 4 (PAK4) is overexpressed in different cancers and promotes proliferation of cancer cells. Reprogramming of glucose metabolism is found in most cancer cells which in turn supports rapid proliferation. However, the relationship between PAK4 and glucose metabolism in cancer cells has not been explored. In this study, we reported that PAK4 promoted glucose intake, NADPH production and lipid biosynthesis, leading to an increased proliferation of colon cancer cells. Mechanistically, PAK4 interacted with glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway and increased G6PD activity via enhancing Mdm2-mediated p53 ubiquitination degradation. In addition, we demonstrated a close positive correlation between PAK4 and G6PD expression in colon cancer specimens. Furthermore, expression of PAK4 or G6PD was positively correlated with an aggressive phenotype of clinical colon cancer. These findings revealed a novel glucose metabolism-related mechanism of PAK4 in promoting colon cancer cell growth, suggesting that PAK4 and/or G6PD blockage might be a potential therapeutic strategy for colon cancer.

Bre Enhances Osteoblastic Differentiation by Promoting the Mdm2-Mediated Degradation of p53.

Bre is a conserved cellular protein expressed in various tissues. Its major function includes DNA damage repair and anti-apoptosis. Recent studies indicate that Bre is potentially involved in stem cell differentiation although pathophysiological significance along with the molecular mechanisms is still unclear. Here, we report that Bre protein was substantially expressed in the bone tissue and its expression was highly upregulated during the osteogenic differentiation. To test a hypothesis that Bre plays functional roles in the process of osteogenic differentiation, we examined the expression of Bre in an osteoporosis mouse model. Compared with the normal bone tissue, Bre expression in osteoporotic bone was also significantly reduced. Moreover, knockdown of Bre in the mouse bone marrow mesenchymal cells significantly reduced the expression of osteogenic marker genes, the alkaline phosphatase activity, and the mineralization capacity, while overexpression of Bre greatly promoted the osteogenesis both in vitro and in vivo. Interestingly, we founded that knockdown of Bre led to activation of the p53 signaling pathways exhibited by increased p53, p21, and Mdm2. However, when we inhibited the p53 by siRNA silencing or pifithrin-α, the impaired osteogenesis caused by Bre knockdown was greatly restored. Finally, we found that Bre promoted the Mdm2-mediated p53 ubiquitination and degradation by physically interacting with p53. Taken together, our results revealed a novel function of Bre in osteoblast differentiation through modulating the stability of p53. Stem Cells 2017;35:1760-1772.

Selective targeting p53WT lung cancer cells harboring homozygous p53 Arg72 by an inhibitor of CypA

TP53 plays essential roles in tumor initiation and progression, and is frequently mutated in cancer. However, pharmacological stabilization and reactivation of p53 have not been actively explored for targeted cancer therapies. Herein, we identify a novel Cyclophilin A (CypA) small molecule inhibitor (HL001) that induces non-small cell lung cancer (NSCLC) cell cycle arrest and apoptosis via restoring p53 expression. We find that HL001 stabilizes p53 through inhibiting the MDM2-mediated p53 ubiquitination. Further mechanistic studies reveal that the downregulation of G3BP1 and the induction of reactive oxygen species and DNA damage by HL001 contribute to p53 stabilization. Surprisingly, HL001 selectively suppresses tumor growth in p53 wild-type NSCLC harboring Arg72 homozygous alleles (p53-72R) through disrupting interaction between MDM2 and p53-72R in a CypA-dependent manner. Moreover, combining HL001 with cisplatin synergistically enhance tumor regression in orthotopic NSCLC mouse model. Collectively, this study demonstrates that pharmacologic inhibition of CypA offers a potential therapeutic strategy via specific activation of p53-72R in NSCLC.

Ubiquitin-specific peptidase 48 regulates Mdm2 protein levels independent of its deubiquitinase activity

The overexpression of Mdm2 has been linked to the loss of p53 tumour suppressor activity in several human cancers. Here, we present results suggesting that ubiquitin-specific peptidase 48 (USP48), a deubiquitinase that has been linked in previous reports to the NF-κB signaling pathway, is a novel Mdm2 binding partner that promotes Mdm2 stability and enhances Mdm2-mediated p53 ubiquitination and degradation. In contrast to other deubiquitinating enzymes (DUBs) that have been previously implicated in the regulation of Mdm2 protein stability, USP48 did not induce Mdm2 stabilization by significantly reducing Mdm2 ubiquitination levels. Moreover, two previously characterized USP48 mutants lacking deubiquitinase activity were also capable of efficiently stabilizing Mdm2, indicating that USP48 utilizes a non-canonical, deubiquitination-independent mechanism to promote Mdm2 oncoprotein stability. This study represents, to the best of our knowledge, the first report suggesting DUB-mediated target protein stabilization that is independent of its deubiquitinase activity. In addition, our results suggest that USP48 might represent a new mechanism of crosstalk between the NF-κB and p53 stress response pathways.

Regulation of NUB1 Activity through Non-Proteolytic Mdm2-Mediated Ubiquitination.

NUB1 (Nedd8 ultimate buster 1) is an adaptor protein which negatively regulates the ubiquitin-like protein Nedd8 as well as neddylated proteins levels through proteasomal degradation. However, molecular mechanisms underlying this function are not completely understood. Here, we report that the oncogenic E3 ubiquitin ligase Mdm2 is a new NUB1 interacting protein which induces its ubiquitination. Interestingly, we found that Mdm2-mediated ubiquitination of NUB1 is not a proteolytic signal. Instead of promoting the conjugation of polyubiquitin chains and the subsequent proteasomal degradation of NUB1, Mdm2 rather induces its di-ubiquitination on lysine 159. Importantly, mutation of lysine 159 into arginine inhibits NUB1 activity by impairing its negative regulation of Nedd8 and of neddylated proteins. We conclude that Mdm2 acts as a positive regulator of NUB1 function, by modulating NUB1 ubiquitination on lysine 159.

Otub1 stabilizes MDMX and promotes its proapoptotic function at the mitochondria.

Otub1 regulates p53 stability and activity via non-canonical inhibition of UbcH5, the MDM2 cognate ubiquitin-conjugating enzyme (E2). However, whether Otub1 regulates MDMX stability and activity is not clear. Here we report that Otub1 also suppresses MDM2-mediated MDMX ubiquitination in cells and in vitro, independently of its deubiquitinating enzyme activity. Consequently, overexpression of Otub1 markedly stabilized MDMX and increased its levels, whereas knockdown of Otub1 reduced the levels of MDMX. Interestingly, MDMX induced by Otub1 can localize to mitochondria in addition to the cytosol, enhance p53 phosphorylation at S46 (p53S46P) and promote mitochondria-mediated apoptotic pathway. Knockdown of MDMX reduced Otub1-induced p53S46P, which was shown to be critical for p53's mitochondrial function and apoptotic activity. Furthermore, Otub1 promotes UV-irradiation-induced p53S46P and apoptosis, which can be significantly inhibited by MDMX depletion. Together, these results suggest that Otub1 stabilizes MDMX and promotes p53S46P and mitochondria-mediated apoptosis, providing an alternative mechanism of Otub1's role in apoptosis.

Pleckstrin homology domain-containing protein PHLDB3 supports cancer growth via a negative feedback loop involving p53.

The tumour suppressor p53 transactivates the expression of its target genes to exert its functions. Here, we identify a pleckstrin homology domain-containing protein (PHLDB3)-encoding gene as a p53 target. PHLDB3 overexpression increases proliferation and restrains apoptosis of wild-type p53-harboring cancer cells by reducing p53 protein levels. PHLDB3 binds to MDM2 (mouse double minute 2 homolog) and facilitates MDM2-mediated ubiquitination and degradation of p53. Knockdown of PHLDB3 more efficiently inhibits the growth of mouse xenograft tumours derived from human colon cancer HCT116 cells that contain wild type p53 compared with p53-deficient HCT116 cells, and also sensitizes tumour cells to doxorubicin and 5-Fluorouracil. Analysis of cancer genomic databases reveals that PHLDB3 is amplified and/or highly expressed in numerous human cancers. Altogether, these results demonstrate that PHLDB3 promotes tumour growth by inactivating p53 in a negative feedback fashion and suggest PHLDB3 as a potential therapeutic target in various human cancers.

Regulation of the MDM2-p53 pathway by the nucleolar protein CSIG in response to nucleolar stress.

Nucleolar proteins play an important role in the regulation of the MDM2–p53 pathway, which coordinates cellular response to stress. However, the mechanism underlying this regulation remains poorly understood. Here, we report that the nucleolar protein CSIG is a novel and crucial regulator of the MDM2–p53 pathway. We demonstrate that CSIG translocates from the nucleolus to the nucleoplasm in response to nucleolar stress. Moreover, knockdown of CSIG attenuates the induction of p53 and abrogates G1 phase arrest in response to nucleolar stress. CSIG interacts directly with the MDM2 RING finger domain and inhibits MDM2 E3 ubiquitin ligase activity, thus resulting in a decrease in MDM2-mediated p53 ubiquitination and degradation. Our results suggest that the CSIG–MDM2–p53 regulatory pathway plays an important role in the cellular response to nucleolar stress.

Overexpression of PKM2 promotes mitochondrial fusion through attenuated p53 stability.

M2-type pyruvate kinase (PKM2) contributes to the Warburg effect. However, it remains unknown as to whether PKM2 has an inhibitory effect on mitochondrial function. We report in this work that PKM2 overexpression inhibits the expression of Drp1 and results in the mitochondrial fusion. The ATP production was found to be decreased, the mtDNA copy number elevated and the expression level of electron transport chain (ETC) complex I, III, V depressed in PKM2 overexpressed cells. PKM2 overexpression showed a decreased p53 protein level and a shorter p53 half-life. In contrast, PKM2 knockdown resulted in increased p53 expression and prolonged half-life of p53. PKM2 could directly bind with both p53 and MDM2 and promote MDM2-mediated p53 ubiquitination. The dimeric PKM2 significantly suppressed p53 expression compared with the other PKM2 mutants. The reverse relationship between PKM2 and Drp1 was further confirmed in a large number of clinical samples. Taken together, the present results highlight a new mechanism that link PKM2 to mitochondrial function, based on p53-Drp1 axis down regulation, revealing a novel therapeutic target in patients with abnormal mitochondria.

Abstract 241: MDM2 E3 Ligase-mediated Ubiquitination and Degradation of HDAC1 in Vascular Calcification

Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC.

Abstract 3683: The stress response mediator ATF3 stabilizes p53 by competing with it for MDM2-mediated ubiquitination

Abstract While the tumor suppressor p53 is crucial for the maintenance of genetic integrity upon DNA damage and oncogenic stresses, its level and activity are tightly controlled by a complex network largely mediated by the E3 ubiquitin ligase MDM2. MDM2 catalyzes the addition of ubiqutin chains to the C-terminus of p53 and is the major ubiquitin ligase to promote proteosomal degradation of p53. We previously demonstrated that ATF3, a common stress response mediator, can bind p53 and prevent it from MDM2-mediated ubiquitination. As a consequence, ATF3 can stabilize p53 upon DNA damage and prevent cells from oncogene-induced transformation by promoting p53-mediated senescence, in line with accumulating evidence supporting that ATF3 can act as a tumor suppressor. Interestingly, ATF3 is also a bona fide substrate of MDM2, and thus, the ATF3-MDM2 interaction can fine tune p53 activity in the DNA damage response. However, the mechanism by which ATF3 blocks p53 ubiquitination to activate the latter protein remains largely undefined. Efficient ubiquitination of p53 requires the binding of MDM2 to the N-terminus of p53, and the MDM2 binding to MDMX. Here we show that neither the p53-MDM2 interaction nor the MDM2-MDMX interaction is disrupted by the binding of ATF3 to p53 or MDM2. As both ATF3 and p53 are substrates of MDM2, we explored a possibility that ATF3 competes with p53 for MDM2-mediated ubiquitination. By in-vitro ubiquitination assays, we identified that the 5 lysine residues clustering in aa 100-115 of ATF3 are required for MDM2-mediated ubiquitination. Site-specific mutagenesis indicates that MDM2 mainly ubiquitinates ATF3 at lysine 107 (K107) and lysine 108 (K108) as MDM2 fails to add ubiquitin chains to ATF3 when these two residues were mutated to arginine. Importantly, while mutations at these two residues do not affect the ATF3 binding to MDM2 or p53, the ATF3 mutant devoid of ubiquitination fails to stabilize p53 and prevent p53 from MDM2-mediated ubiquitination. These results thus demonstrate that ATF3 can activate p53 by competing with the tumor suppressor for MDM2-mediated ubiquitination. Citation Format: Xingyao Li, Chunhong Yan. The stress response mediator ATF3 stabilizes p53 by competing with it for MDM2-mediated ubiquitination. [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 3683.

Abstract 3509: Knockdown of RNF2 induces apoptosis by regulating MDM2 and p53 stability

Abstract RNF2, also known as Ring1B/Ring2, is a component of the polycomb repression complex 1 (PRC1). RNF2 is highly expressed in many tumors, suggesting that it might have an oncogenic function, but the mechanism is unknown. Here we show that knockdown of RNF2 significantly inhibits both cell proliferation and colony formation in soft agar, and induces apoptosis of cancer cells. Knockdown of RNF2 in HCT116 p53+/+ cells resulted in significantly more apoptosis than was observed in RNF2 knockdown HCT116 p53−/− cells, indicating that RNF2 knockdown-induced apoptosis is partially dependent on p53. Various p53-targeted genes were increased in RNF2 knockdown cells. Further studies revealed that in RNF2 knockdown cells, the p53 protein level was increased, the half-life of p53 was prolonged and p53 ubiquitination was decreased. In contrast, cells overexpressing RNF2 showed a decreased p53 protein level, a shorter p53 half-life and increased p53 ubiquitination. Importantly, we found that RNF2 directly binds with both p53 and MDM2 and promotes MDM2-mediated p53 ubiquitination. RNF2 over-expression could also increase the half-life of MDM2 and inhibit its ubiquitination. The regulation on p53 and MDM2 stability by RNF2 was observed during the etoposide-induced DNA damage response. These results provide a possible mechanism explaining the oncogenic function of RNF2, and because RNF2 is important for cancer cell survival and proliferation, it might be an ideal target for cancer therapy or prevention. Keywords: RNF2, p53, apoptosis Citation Format: Weihong Wen, Cong Peng, Myoung Ok Kim, Chul Ho Jeong, Feng Zhu, Ke Yao, Tatyana Zykova, Wei-Ya Ma, Andria Carper, Alyssa Langfald, Ann M. Bode, Zigang Dong. Knockdown of RNF2 induces apoptosis by regulating MDM2 and p53 stability. [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 3509.

Abstract 1810: Heterogeneous activation of PP-1 and Akt determines AR-v7 protein expression in prostate cancer cells under AR inhibition

Abstract Background: Blocking the androgen receptor (AR) signaling is the first- and second-line treatment for metastatic prostate cancers. Although the newly developed AR pathway inhibitor (ARPI) improves patient survival, tumors invariably recurs and are most often driven by reactivation of the AR signaling. Enhanced expression of the AR splice variant, AR-v7, had been demonstrated to be one of the key mechanisms that confer prostate tumors with therapy resistance. However, molecular mechanisms by which control AR-v7 protein expression remain unclear. Experimental Design: Multiple prostate cancer lines were challenged with androgen depletion and/or Enzalutamide treatment. Protein phosphatase-1 (PP-1) and Akt kinase activation were measured. AR and AR-v7 serine phosphorylation, ubiquitination, protein degradation as well as subcellular localization were determined. Results: Enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the genetic background of prostate cancer cell lines. PP-1 activation prevents AR-v7 from serine213 phosphorylation, Mdm2-mediated ubiquitination modification, cytoplasmic localization and proteasomal protein degradation. By contrast, Akt activation functions in the opposite to PP-1 and stimulates AR-v7 degradation. Therefore, the balance of PP-1 and Akt activations under ARPI treatment determines AR-v7 protein expression in prostate cancer cells. Conclusion: Our studies highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. It also provides an explanation on heterogeneous AR-v7 protein expression in prostate tumor biopsies. Citation Format: Yinan Li, Ning Xie, Martin E. Gleave, Paul Rennie, Xuesen Dong. Heterogeneous activation of PP-1 and Akt determines AR-v7 protein expression in prostate cancer cells under AR inhibition. [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 1810.

HBP1-mediated Regulation of p21 Protein through the Mdm2/p53 and TCF4/EZH2 Pathways and Its Impact on Cell Senescence and Tumorigenesis

The activity of the CDK inhibitor p21 is associated with diverse biological activities, including cell proliferation, senescence, and tumorigenesis. However, the mechanisms governing transcription of p21 need to be extensively studied. In this study, we demonstrate that the high-mobility group box-containing protein 1 (HBP1) transcription factor is a novel activator of p21 that works as part of a complex mechanism during senescence and tumorigenesis. We found that HBP1 activates the p21 gene through enhancing p53 stability by inhibiting Mdm2-mediated ubiquitination of p53, a well known positive regulator of p21. HBP1 was also found to enhance p21 transcription by inhibiting Wnt/β-catenin signaling. We identified histone methyltransferase EZH2, the catalytic subunit of polycomb repressive complex 2, as a target of Wnt/β-catenin signaling. HBP1-mediated repression of EZH2 through Wnt/β-catenin signaling decreased the level of trimethylation of histone H3 at lysine 27 of overall and specific histone on the p21 promoter, resulting in p21 transactivation. Although intricate, the reciprocal partnership of HBP1 and p21 has exceptional importance. HBP1-mediated elevation of p21 through the Mdm2/p53 and TCF4/EZH2 pathways contributes to both cellular senescence and tumor inhibition. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence and tumorigenesis with an impact on protein ubiquitination and overall histone methylation state.

USP7 Enforces Heterochromatinization of p53 Target Promoters by Protecting SUV39H1 from MDM2-Mediated Degradation.

The H3K9me3 repressive histone conformation of p53 target promoters is abrogated in response to p53 activation by MDM2-mediated SUV39H1 degradation. Here, we present evidence that the USP7 deubiquitinase protects SUV39H1 from MDM2-mediated ubiquitination in the absence of p53 stimulus. USP7 occupies p53 target promoters in unstressed conditions, a process that is abrogated with p53 activation associated with loss of the H3K9me3 mark on these same promoters. Mechanistically, USP7 forms a trimeric complex with MDM2 and SUV39H1, independent of DNA, and modulates MDM2-dependent SUV39H1 ubiquitination. Furthermore, we show that this protective function of USP7 on SUV39H1 is independent of p53. Finally, USP7 blocking cooperates with p53 in inducing apoptosis by enhancing p53 promoter occupancy and dependent transactivation of target genes. These results uncover a layer of the p53 transcriptional program mediated by USP7, which restrains relaxation of local chromatin conformation at p53 target promoters.

RNF12 promotes p53-dependent cell growth suppression and apoptosis by targeting MDM2 for destruction

The oncoprotein MDM2 is an E3 ubiquitin ligase that targets tumor suppressor p53 for ubiquitination and proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Dysregulation of MDM2-p53 axis was frequently observed in human cancers. Originally, it is proposed that MDM2 degradation was mainly achieved by destructive self-ubiquitination. However, recent study suggests that MDM2 may be targeted for degradation by an external E3 ubiquitin ligase(s) under physiological levels. Here, we identified E3 ubiquitin ligase RNF12 as an MDM2-interacting protein through yeast two hybrid methods. We demonstrated that RNF12 targets MDM2 for ubiquitination and proteasomal-dependent degradation, which is independent of MDM2's self-ubiquitination activity. Accordingly, RNF12 elevates p53 protein level by abrogating MDM2-mediated p53 degradation and ubiquitination. Finally, we showed that RNF12 regulates cell growth suppression and DNA damage-induced apoptosis in a p53-dependent manner. Taken together, we establish RNF12 as a novel positive regulator of p53 pathway and an external E3 ubiquitin ligase for MDM2 destruction. These data shed light on the potential roles of RNF12 in MDM2-p53 axis and tumor suppression.