Mutant p53 interactome identifies nardilysin as a p53R273H-specific binding partner that promotes invasion

The invasiveness of tumour cells depends on changes in cell shape, polarity and migration. Mutant p53 induces enhanced tumour metastasis in mice, and human cells overexpressing p53R273H have aberrant polarity and increased invasiveness, demonstrating the 'gain of function' of mutant p53 in carcinogenesis. We hypothesize that p53R273H interacts with mutant p53-specific binding partners that control polarity, migration or invasion. Here we analyze the p53R273H interactome using stable isotope labelling by amino acids in cell culture and quantitative mass spectrometry, and identify at least 15 new potential mutant p53-specific binding partners. The interaction of p53R273H with one of them--nardilysin (NRD1)--promotes an invasive response to heparin binding-epidermal growth factor-like growth factor that is p53R273H-dependant but does not require Rab coupling protein or p63. Advanced proteomics has thus allowed the detection of a new mechanism of p53-driven invasion.     

Cooperation among Numb, MDM2 and p53 in the development and progression of pancreatic cancer

We study the expression of Numb, MDM2 and p53 for clinical significance in pancreatic cancer (PC) and their functional relationship in regulating biological behaviors of PC cells. IHC, IB and qRT-PCR were used to detect Numb, MDM2 and p53 expression in PC. Transfection and drug intervention were used to investigate their functional relationship in PC cells. IHC showed that Numb expression was negatively associated with tumor size, differentiation and UICC stage, while expression of MDM2 and p53 was positively associated with tumor T and UICC stages, respectively (P?<?0.05). Numb was an independent prognostic indicator in PC (P?<?0.05). Patients with Numb-positive expression or combined with MDM2-negative expression had a significantly better overall survival (P?<?0.05). Altered expression of Numb can regulate wild-type but not mutant p53 expression, while MDM2 knockdown increased Numb but not mutant p53 protein level. Meanwhile, Numb knockdown increased chemoresistance but decreased activated p53 and cleaved-caspase-3 protein expression in gemcitabine-treated Capan-2 cells. Moreover, Numb co-immunoprecipitated with p53 to prevent p53 ubiquitin-dependent protein degradation and this ubiquitin-dependent regulation plays an important role in the coordinate function of these three proteins on cell invasion and migration in PC cells. Our study is the first to demonstrate the clinical significance and functional cooperation among Numb, MDM2 and p53 involved in the development and progression of PC.     

Diacylglycerol kinase α controls RCP-dependent integrin trafficking to promote invasive migration

Inhibition of αvβ3 integrin or expression of oncogenic mutants of p53 promote invasive cell migration by enhancing endosomal recycling of α5β1 integrin under control of the Rab11 effector Rab-coupling protein (RCP). In this paper, we show that diacylglycerol kinase α (DGK-α), which phosphorylates diacylglycerol to phosphatidic acid (PA), was required for RCP to be mobilized to and tethered at the tips of invasive pseudopods and to allow RCP-dependent α5β1 recycling and the resulting invasiveness of tumor cells. Expression of a constitutive-active mutant of DGK-α drove RCP-dependent invasion in the absence of mutant p53 expression or αvβ3 inhibition, and conversely, an RCP mutant lacking the PA-binding C2 domain was not capable of being tethered at pseudopod tips. These data demonstrate that generation of PA downstream of DGK-α is essential to connect expression of mutant p53s or inhibition of αvβ3 to RCP and for this Rab11 effector to drive the trafficking of α5β1 that is required for tumor cell invasion through three-dimensional matrices.     

靶向突变p53蛋白的抗肿瘤药物

p53蛋白是人体内十分重要的肿瘤抑制因子,通过调节细胞周期阻滞、诱导细胞凋亡等作用发挥肿瘤抑制功能。突变后的p53蛋白不仅具有显性负性效应（dominant negative effect,DN）抑制野生型p53蛋白功能,而且还通过功能获得性效应（gain of function,GOF）调节细胞代谢、侵袭、迁移等方式促进肿瘤的发生。p53蛋白在超过50%的肿瘤组织中发生突变,是肿瘤细胞区别于正常细胞的一个特异性药物靶点。因此,针对突变p53蛋白开发新型抗癌药物一直是研究的热点。长期以来,由于突变p53蛋白表面较为光滑,缺乏药物结合口袋,使其被认为是一个不可成药的靶点。随着高通量筛选技术的发展以及对突变p53蛋白结构的深入了解,许多靶向突变p53蛋白的小分子化合物被报道并在体外展现出较好的抗肿瘤活性,多款基于突变p53蛋白研发的化合物已经进入临床试验阶段。本文就靶向p53蛋白治疗肿瘤的直接和间接策略进行综述,重点针对突变p53蛋白重激活剂与降解突变p53蛋白的小分子化合物作用机制进行梳理,以期为后续开发靶向突变p53蛋白药物的创新提供帮助。     

结直肠癌mP53 和PCNA 表达的相关性及临床意义

目的:探讨结直肠癌中突变型P53基因(mP53)和增殖细胞核抗原(PCNA)表达的相关性及临床意义。方法:应用免疫组化二步法,检测60例结直肠癌组织及20例正常肠粘膜中mP53、PCNA的表达,结合临床病理资料进行统计分析。结果:60例结直肠癌中mP53阳性表达率65.0%,PCNA阳性表达率78.3%,20例正常肠粘膜中mP53、PCNA表达均为阴性(P<0.05)。mP53和PCNA阳性表达率在低分化组、浆膜层浸润组、淋巴结转移组均较高(P均<0.05)。mP53和PCNA表达呈正相关(r=0.58,P<0.05)。结论:mP53和PCNA在结直肠癌中表达均增高,二者与结直肠癌病理学分级、浸润深度和淋巴结转移有关,可作为判断结直肠癌预后的参考指标。     

Id1-induced inhibition of p53 facilitates endothelial cell migration and tube formation by regulating the expression of beta1-integrin

The Id1 protein is critical for endothelial cell angiogenesis, and this function is particularly relevant to cancer development, cardiovascular disease, and wound healing. We hypothesized that Id1 enhanced migration and tubulogenesis by controlling the expression and function of p53. In this study, we examined cell migration following Id1 overexpression and silencing endothelial cells. The results showed that overexpression of Id1 enhanced cell migration and increased beta1-integrin expression, but inhibition of beta1-integrin blocked motility even in clones overexpressing Id1, suggesting that Id1 regulated motility through beta1-integrin. Further analysis revealed that p53, whose expression and distribution is regulated by Id1, was critical for cell migration, and may be involved in regulating the expression of beta1-integrin. Inhibiting p53 function using PFT-α, a functional inhibitor of p53, increased the expression of beta1-integrin and promoted cell migration even in Id1-silencing endothelial cells, demonstrating that the Id1 knockdowns induced inhibition of endothelial cell migration and the expression of beta1-integrin were controlled by p53. In addition, Id1-p53 pathway regulated the cytoskeleton formation and tubulogenesis. These results demonstrate that Id1-induced beta1-integrin expression in endothelial cells and the function of Id1 in cell migration and tubulogenesis are dependent on p53.     

Inhibition of Breast Cancer Metastasis Suppressor 1 Promotes a Mesenchymal Phenotype in Lung Epithelial Cells That Express Oncogenic K-RasV12 and Loss of p53

Expression of the breast cancer metastasis suppressor 1 (BRMS1) protein is dramatically reduced in non-small cell lung cancer (NSCLC) cells and in primary human tumors. Although BRMS1 is a known suppressor of metastasis, the mechanisms through which BRMS1 functions to regulate cell migration and invasion in response to specific NSCLC driver mutations are poorly understood. To experimentally address this, we utilized immortalized human bronchial epithelial cells in which p53 was knocked down in the presence of oncogenic K-Ras^V12 (HBEC3-p53KD-K-Ras^V12). These genetic alterations are commonly found in NSCLC and are associated with a poor prognosis. To determine the importance of BRMS1 for cytoskeletal function, cell migration and invasion in our model system we stably knocked down BRMS1. Here, we report that loss of BRMS1 in HBEC3-p53KD-K-Ras^V12 cells results in a dramatic increase in cell migration and invasion compared to controls that expressed BRMS1. Moreover, the loss of BRMS1 resulted in additional morphological changes including F-actin re-distribution, paxillin accumulation at the leading edge of the lamellapodium, and cellular shape changes resembling mesenchymal phenotypes. Importantly, re-expression of BRMS1 restores, in part, cell migration and invasion; however it does not fully reestablish the epithelial phenotype. These finding suggests that loss of BRMS1 results in a permanent, largely irreversible, mesenchymal phenotype associated with increased cell migration and invasion. Collectively, in NSCLC cells without p53 and expression of oncogenic K-Ras our study identifies BRMS1 as a key regulator required to maintain a cellular morphology and cytoskeletal architecture consistent with an epithelial phenotype.     

p53 in cell invasion,podosomes, and invadopodia

Cell invasion of the extracellular matrix is prerequisite to cross tissue migration of tumor cells in cancer metastasis, and vascular smooth muscle cells in atherosclerosis. The tumor suppressor p53, better known for its roles in the regulation of cell cycle and apoptosis, has ignited much interest in its function as a suppressor of cell migration and invasion. How p53 and its gain-of-function mutants regulate cell invasion remains a puzzle and a challenge for future studies. In recent years, podosomes and invadopodia have also gained center stage status as veritable apparatus specialized in cell invasion. It is not clear, however, whether p53 regulates cell invasion through podosomes and invadopodia. In this review, evidence supporting a negative role of p53 in podosomes formation in vascular smooth muscle cells will be surveyed, and signaling nodes that may mediate this regulation in other cell types will be explored.     

p53 in cell invasion,podosomes, and invadopodia

Cell invasion of the extracellular matrix is prerequisite to cross tissue migration of tumor cells in cancer metastasis, and vascular smooth muscle cells in atherosclerosis. The tumor suppressor p53, better known for its roles in the regulation of cell cycle and apoptosis, has ignited much interest in its function as a suppressor of cell migration and invasion. How p53 and its gain-of-function mutants regulate cell invasion remains a puzzle and a challenge for future studies. In recent years, podosomes and invadopodia have also gained center stage status as veritable apparatus specialized in cell invasion. It is not clear, however, whether p53 regulates cell invasion through podosomes and invadopodia. In this review, evidence supporting a negative role of p53 in podosomes formation in vascular smooth muscle cells will be surveyed, and signaling nodes that may mediate this regulation in other cell types will be explored.     

Mutant p53 promotes RCP-dependent chemoresistance coinciding with increased delivery of P-glycoprotein to the plasma membrane

TP53 is the most frequently mutated gene in cancers. Mutations lead to loss of p53 expression or expression of a mutant protein. Mutant p53 proteins commonly lose wild-type function, but can also acquire novel functions in promoting metastasis and chemoresistance. Previously, we uncovered a role for Rab-coupling protein (RCP) in mutant p53-dependent invasion. RCP promotes endosomal recycling and signalling of integrins and receptor tyrosine kinases. In a screen to identify novel RCP-interacting proteins, we discovered P-glycoprotein (P-gp). Thus, we hypothesised that mutant p53 could promote chemoresistance through RCP-dependent recycling of P-gp. The interaction between RCP and P-gp was verified endogenously and loss of RCP or mutant p53 rendered cells more sensitive to cisplatin and etoposide. In mutant p53 cells we detected an RCP-dependent delivery of P-gp to the plasma membrane upon drug treatment and decreased retention of P-gp substrates. A co-localisation of P-gp and RCP was seen in mutant p53 cells, but not in p53-null cells upon chemotherapeutic exposure. In conclusion, mutant p53 expression enhanced co-localisation of P-gp and RCP to allow for rapid delivery of P-gp to the plasma membrane and increased resistance to chemotherapeutics.Subject terms: Tumour-suppressor proteins, Preclinical research     

Characterization of p53 in Chinese hamster cell lines CHO-K1, CHO-WBL, and CHL: implications for genotoxicity testing.

Since the p53 gene function is critical to how a cell responds to DNA damage, we investigated the p53 status in Chinese hamster cell lines commonly used in genotoxicity tests for cytogenetic damage around the world. These included: Chinese hamster ovary K1 (CHO-K1), Chinese hamster ovary WBL (CHO-WBL), and Chinese hamster lung (CHL) cells. The results of DNA sequencing, protein analysis, and cell cycle analysis demonstrate that the CHO-K1 and CHO-WBL cell lines have mutant p53 sequence [a mutation in codon 211 in exon 6 resulting in a change from Thr (ACA) to Lys (AAA)], mutant protein (high spontaneous levels that are non-inducible after X-irradiation), and mutant function (lack of G1 checkpoint). Interestingly, the CHL cell line has a completely wild-type p53 DNA sequence. However, the CHL cells have an abnormally high spontaneous level of wild-type p53 protein expression that is not inducible after X-irradiation, yet there is some evidence of G1 delay after irradiation. The protein data suggests that p53 in CHL cells is not being regulated normally, and thus is probably not functioning normally. The mechanism leading to this abnormal regulation of p53 in CHL cells clearly does not involve mutation in the p53 gene. Overall, the CHL cell line may be similar to the CHO cell lines, in that they all appear to have abnormal p53 function. Further work is needed to determine whether the presence of spontaneously high levels of wild-type p53 in CHL cells results in a difference in response to DNA damage (quantitatively or qualitatively) compared to the p53 mutant CHO cell lines.     

Mutant p53 disrupts MCF-10A cell polarity in three-dimensional culture via epithelial-to-mesenchymal transitions

Mutant p53 is not only deficient in tumor suppression but also acquires additional activity, called gain of function. Mutant p53 gain of function is recapitulated in knock-in mice that carry one null allele and one mutant allele of the p53 gene. These knock-in mice develop aggressive tumors compared with p53-null mice. Recently, we and others showed that tumor cells carrying a mutant p53 are addicted to the mutant for cell survival and resistance to DNA damage. To further define mutant p53 gain of function, we used the MCF-10A three-dimensional model of mammary morphogenesis. MCF-10A cells in three-dimensional culture undergo a series of morphological changes and form polarized and growth-arrested spheroids with hollow lumen, which resembles normal glandular architectures in vivo. Here, we found that endogenous wild-type p53 in MCF-10A cells was not required for acinus formation, but knockdown of endogenous wild-type p53 (p53-KD) led to partial clearance of cells in the lumen due to decreased apoptosis. Consistent with this, p53-KD altered expression patterns of the cell adhesion molecule E-cadherin, the cytoskeletal marker β-catenin, and the extracellular matrix protein laminin V. We also found that ectopic expression of the mutant G245S led to a phenotype similar to p53-KD, whereas a combination of ectopic expression of siRNA-resistant G245S with p53-KD led to a less cleared lumen. In contrast, ectopic expression of mutant R248W, R175H, and R273H disrupted normal acinus architectures with filled lumen and led to formation of irregular and multiacinus structures regardless of p53-KD. In addition, these mutants altered normal expression patterns and/or levels of E-cadherin, β-catenin, laminin V, and tight junction marker ZO-1. Furthermore, epithelial-to-mesenchymal transitions (EMT) markers, Snail, Slug, and Twist, were highly induced by mutant p53 and/or p53-KD. Together, we postulate that EMT represents a mutant p53 gain of function and mutant p53 alters cell polarity via EMT.