Progress in the research of p53 tumour suppressor activity controlled by Numb in triple‐negative breast cancer

Numb is known as a cell fate determinant as it identifies the direction of cell differentiation via asymmetrical partitioning during mitosis. It is considered as a tumour suppressor, and a frequent loss of Numb expression in breast cancer is noted. Numb forms a tri‐complex with p53 and E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing the ubiquitination and degradation of p53. In this study, we examined Numb expression in 125 patients with triple‐negative breast cancer (TNBC). The results showed that 61 (48.8%) patients presented with a deficient or decreased Numb expression. The percentage of Ki67 > 14% in the retained Numb group was significantly lower than that in the decreased and deficient Numb groups (86.00% vs. 98.40%, P = .0171). This study aimed to detect the expression and migration of Numb, HDM2 and p53 in the membrane, cytoplasmic and nuclear fractions of normal mammary epithelial cell line MCF‐10A and basal‐like TNBC cell line MDA‐MB‐231. We obtained the cell fractions to identify changes in these three protein levels after the re‐expression of NUMB in the MDA‐MB‐231 cells and the knocking down of NUMB in the MCF‐10A cells. Results showed that Numb regulates p53 levels in the nucleus where the protein levels of Numb are positively correlated with p53 levels, regardless if it is re‐expressed in the MDA‐MB‐231 cells or knocked down in the MCF‐10A cells. Moreover, HDM2 was remarkably decreased only in the membrane fraction of NUMB knock‐down cells; however, its mRNA levels were increased significantly. Our results reveal a previously unknown molecular mechanism that Numb can migrate into the nucleus and interact with HDM2 and p53.     

Stabilization of the MDM2 oncoprotein by mutant p53

MDM2 is a short-lived protein that regulates p53 degradation. We report here that transient coexpression of MDM2 and several p53 hotspot mutants resulted in stabilization and increased expression of MDM2. Ectopic expression of the mutant p53(175H) allele by recombinant adenovirus infection or stable transfection also stabilized endogenous MDM2 in p53-null cells. A panel of human tumor cell lines expressing different endogenous mutant p53 alleles also contained stabilized nuclear MDM2 at elevated levels when compared with p53-null cells. MDM2 was present in complexes with mutant p53 in tumor cells, and stabilization of MDM2 required direct binding to mutant p53. These results reveal a novel property of mutant p53 and a unique feature of tumors with p53 missense mutations. Accumulation of stable MDM2 may contribute to tumorigenesis through its p53-independent transforming functions.     

MDM2 regulates vascular endothelial growth factor mRNA stabilization in hypoxia

Expression of vascular endothelial growth factor (VEGF) increases in cancer cells during hypoxia. Herein, we report that the MDM2 oncoprotein plays a role in hypoxia-mediated VEGF upregulation. In studying the characteristics of MDM2 and VEGF expression in neuroblastoma cells, we found that hypoxia induced significantly higher upregulation of both VEGF mRNA and protein in MDM2-positive cells than in the MDM2-negative cells, even in cells without wild-type (wt) p53. We found that hypoxia induced translocation of MDM2 from the nucleus to the cytoplasm, which was associated with increased VEGF expression. Enforcing overexpression of cytoplasmic MDM2 by transfection of the mutant MDM2/166A enhanced expression of VEGF mRNA and protein production, even without hypoxia. The results of mechanistic studies demonstrated that the C-terminal RING domain of the MDM2 protein bound to the AU-rich sequence within the 3' untranslated region (3'UTR) of VEGF mRNA; this binding increased VEGF mRNA stability and translation. In addition, knockdown of MDM2 by small interfering RNA (siRNA) in MDM2-overexpressing cancer cells resulted in inhibition of VEGF protein production, cancer cell survival, and angiogenesis. Our results suggest that MDM2 plays a p53-independent role in the regulation of VEGF, which may promote tumor growth and metastasis.     

Ribosomal protein S3: A multi-functional protein that interacts with both p53 and MDM2 through its KH domain

The p53 protein responds to cellular stress and regulates genes involved in cell cycle, apoptosis, and DNA repair. Under normal conditions, p53 levels are kept low through MDM2-mediated ubiquitination and proteosomal degradation. In search for novel proteins that participate in this regulatory loop, we performed an MDM2 peptide pull-down assay and mass spectrometry to screen for potential interacting partners of MDM2. We identified ribosomal protein S3 (RPS3), whose interaction with MDM2, and notably p53, was further established by His and GST pull-down assays, fluorescence resonance energy transfer and an in situ proximity ligation assay. Additionally, in cells exposed to oxidative stress, p53 levels increased slightly over 24 h, whereas MDM2 levels declined after 6 h exposure, but rose over the next 18 h of exposure. Conversely, in cells exposed to oxidative stress and harboring siRNA to knockdown RPS3 expression, decreased p53 levels and loss of the E3 ubiquitin ligase domain possessed by MDM2 were observed. DNA pull-down assays using a 7,8-dihydro-8-oxoguanine duplex oligonucleotide as a substrate found that RPS3 acted as a scaffold for the additional binding of MDM2 and p53, suggesting that RPS3 interacts with important proteins involved in maintaining genomic integrity.     

Tubular Numb promotes renal interstitial fibrosis via modulating HIF-1α protein stability

Tubulointerstitial fibrosis is the ultimate common pathway of all manners of chronic kidney disease. We previously demonstrated that specific deletion of Numb in proximal tubular cells (PTCs) prevented G2/M arrest and attenuated renal fibrosis. However, how Numb modulates cell cycle arrest remains unclear. Here, we showed that Numb overexpression significantly increased the protein level of hypoxia-inducible factor-1α (HIF-1α). Numb overexpression-induced G2/M arrest was blocked by silencing endogenous HIF-1α, subsequently downregulated the expression of cyclin G1 which is an atypical cyclin to promote G2/M arrest of PTCs. Further analysis revealed that Numb-augmented HIF-1α protein was blocked by simultaneously overexpressing MDM2. Moreover, silencing Numb decreased TGF-β1-induceded HIF-1α protein expression. While endogenous MDM2 was knocked down this reduction was reversed, indicating that Numb stabilized HIF-1α protein via interfering MDM2-mediated HIF-1α protein degradation. Interestingly, HIF-1α overexpression significantly upregulated the expression of Numb and silencing endogenous HIF-1α blocked CoCl₂ or TGF-β1-induced Numb expression. Chromatin immunoprecipitation (ChIP) assays demonstrated that HIF-1α binded to the promoter region of Numb. This binding was significantly increased by TGF-β1. Collectively, these data indicate that Numb and HIF-1α cooperates to promote G2/M arrest of PTCs, and thus aggravates tubulointerstitial fibrosis. Numb might be a potential target for the therapy of tubulointerstitial fibrosis.     

Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis

BackgroundP53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells.PurposeTo explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation.Study design and methodsDifferent mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53.ResultsThe triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation.ConclusionOur study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.     

MDM4 overexpression contributes to synoviocyte proliferation in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with features of inflammatory cell infiltration, synovial cell invasive proliferation, and ultimately, irreversible joint destruction. It has been reported that the p53 pathway is involved in RA pathogenesis. MDM4/MDMX is a major negative regulator of p53. To determine whether MDM4 contributes to RA pathogenesis, MDM4 mRNA and protein expression were assessed in fibroblast-like synoviocytes (FLS) by real-time PCR, western blotting, and in synovial tissues by immunohistochemistry. Furthermore, MDM4 was knocked down and overexpressed by lentivirus-mediated expression, and the proliferative capacity of FLS was determined by MTS assay. We found that cultured FLS from RA and osteoarthritis (OA) patients exhibited higher levels of MDM4 mRNA and protein expression than those from trauma controls. MDM4 protein was highly expressed in the synovial lining and sublining cells from both types of arthritis. Finally, MDM4 knockdown inhibited the proliferation of RA FLS by enhancing functional p53 levels while MDM4 overexpression promoted the growth of RA FLS by inhibiting p53 effects. Taken together, our results suggest that the abundant expression of MDM4 in FLS may contribute to the hyperplasia phenotype of RA synovial tissues.     

A novel nuclear interactor of ARF and MDM2 (NIAM) that maintains chromosomal stability

The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.     

Upregulation of heat shock protein 32 in Sertoli cells alleviates the impairments caused by heat shock-induced apoptosis in mouse testis

Heat stress results in apoptosis in testicular germ cells. A small heat shock protein (hsp), hsp32, is induced by heat stress in the testis, but little is known about its definitive function in physiological processes. To clarify the underlying role of hsp32, hsp32 expression and related signals in the heat shock pathway were analysed in mouse testes and Sertoli cells after heat stress in vivo and in vitro; meanwhile, expression of hsp32 was silenced only in the Sertoli cells using three different small interfering RNAs (siRNAs) to further verify the functional role of hsp32 in Sertoli cells, and hsp32-derived carbon monoxide (CO) contents in cultured media were analysed to clarify whether hsp32-derived CO involve in the apoptosis regulation mechanisms. The results from the in vivo experiment showed that the high expression levels of hsp32 (P?<?0.05) were observed whether chronic, moderate or acute, transient heat exposure. The in vitro experiment showed that acute, transient heat exposure resulted in increases in Sertoli cells apoptosis (P?<?0.01), the expression of hsp32 and caspase-3 activity; hsp32-siRNA knockdown of hsp32 expression resulted in upregulated apoptosis (P?<?0.01) and caspase-3 activity (P?<?0.01) in the Sertoli cells and hyperthermia increases CO (P?<?0.01) release by Sertoli cells. The results suggested that upregulating hsp32 in Sertoli cells inhibits caspase-3 activity and alleviates heat-induced impairments in mouse testis; hsp32-derived CO may involve in the regulation mechanism.     

Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination

To investigate the effect of mutations in the p53 C-terminal domain on MDM2-mediated degradation, we introduced single and multiple point mutations into a human p53 cDNA at four putative acetylation sites (amino acid residues 372, 373, 381, and 382). Substitution of all four lysine residues by alanines (the A4 mutant) and single lysine-to-alanine substitutions were functional in sequence-specific DNA binding and transactivation; however, the A4 mutant protein was resistant to MDM2-mediated degradation, whereas the single lysine substitutions were not. Although the A4 mutant protein and the single lysine substitutions both bound MDM2 reasonably well, the single lysine substitutions underwent normal MDM2-dependent ubiquitination, whereas the A4 protein was inefficiently ubiquitinated. In addition, the A4 mutant protein was found in the cytoplasm as well as in the nucleus of a subpopulation of cells, unlike wild-type p53, which is mostly nuclear. The partially cytoplasmic distribution of A4 mutant protein was not due to a defect in nuclear import because inhibition of nuclear export by leptomycin B resulted in nuclear accumulation of the protein. Taken together, the data suggest that mutations in the putative acetylation sites of the p53 C-terminal domain interfere with ubiquitination, thereby regulating p53 degradation.     

Numb在三阴乳腺癌中抑制HDM2泛素化降解p53

目的:探究Numb蛋白在三阴乳腺癌患者中的表达降低情况,及Numb蛋白在三阴乳腺癌中对抑癌因子p53蛋白水平的影响及调控机制,进一步研究Numb蛋白的降低与三阴乳腺癌发生发展的相关性,从而为缺乏有效治疗方法的三阴乳腺癌提供一个潜在的治疗新靶点。方法:40例三阴乳腺癌患者病理组织切片取自重庆医科大学临床病理诊断中心,采用免疫组化法检测Numb蛋白在三阴乳腺癌患者中的表达情况。MCF-10A细胞株和MDA-MB-231细胞株均为ATCC来源,采用qPCR和Western blot法检测对比Numb、HDM2、p53三者的转录水平和蛋白质水平在以上两个细胞株中差异。采用增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)质粒转染的方法在MDA-MB-231细胞中重表达Numb,采用q PCR和Western blot法验证Numb、HDM2、p53三者表达的变化。结果:转染NUMB-EGFP后MDA-MB-231细胞中Numb的mRNA和蛋白质水平均明显上调,HDM2无显著改变,p53在转录水平无明显变化,但在蛋白质水平显著升高。在231细胞中上调Numb蛋白可以在转录后水平调节p53水平,使p53蛋白随之显著升高。结论:Numb蛋白在三阴乳腺癌患者中表达降低的比列很高,为55%,且Numb蛋白在三阴乳腺癌细胞MDA-MB-231中可以调控抑癌因子p53蛋白水平,Numb蛋白水平与p53蛋白水平呈正相关。     

羽扇豆醇介导MDM2-p53通路对胃癌细胞生物学行为的影响

目的:探讨羽扇豆醇介导鼠双微基因2(Mouse double microgene 2,MDM2)-p53通路对胃癌细胞生物学行为的影响及相关机制。方法:对数生长期的胃癌小鼠MFC细胞株随机分为三组。实验1组与实验2组给予10 mg/L和20 mg/L的羽扇豆醇处理,对照组以等体积的1×磷酸盐缓冲液处理。对比三组MFC细胞细胞增殖、凋亡、迁移与侵袭,及MDM2-p53通路蛋白表达。结果:细胞处理后6 h与12 h,实验1组与实验2组的细胞增殖指数、细胞迁移与侵袭指数、MDM2蛋白相对表达水平显著低对于对照组,实验2组也低于实验1组,对比差异都有统计学意义(P<0.05)。细胞处理后6 h与12 h,实验1组与实验2组的细胞凋亡指数、p53蛋白相对表达水平显著高于对照组,实验2组也高于实验1组,对比差异都有统计学意义(P<0.05)。结论:羽扇豆醇能促进胃癌细胞p53蛋白的表达,抑制MDM2蛋白的表达,从而促进细胞凋亡,抑制胃癌的增殖、侵袭与转移,且具有剂量依赖性。