Novel therapeutic interventions for p53-altered tumors through manipulation of its family members,p63 and p73

TP53 is highly mutated in human cancers, thus targeting this tumor suppressor pathway is highly desirable and will impact many cancer patients.^1,2 Iwakuma T, Lozano G, Flores ER. Li-Fraumeni syndrome: a p53 family affair. Cell Cycle 2005; 4:865-7; PMID:15917654; http://dx.doi.org/10.4161/cc.4.7.1800 Muller PA, Vousden KH. p53 mutations in cancer. Nat Cell Biol 2013; 15:2-8; PMID:23263379; http://dx.doi.org/10.1038/ncb2641  Therapeutic strategies to reactivate the p53-pathway have been challenging,^3,4 Martins CP, Brown-Swigart L, Evan GI. Modeling the therapeutic efficacy of p53 restoration in tumors. Cell 2006; 127:1323-34; PMID:17182091; http://dx.doi.org/10.1016/j.cell.2006.12.007 Ventura A. Restoration of p53 function leads to tumour regression in vivo. Nature 2007; 445:661-5; PMID:17251932; http://dx.doi.org/10.1038/nature05541  and no effective treatment exists.⁵ Khoo KH, Verma CS, Lane DP. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat Rev Drug Discov 2014; 13:217-36; PMID:24577402; http://dx.doi.org/10.1038/nrd4288[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] We utilized the p53-family members, p63 and p73, which are not frequently mutated in cancer, to treat p53-defective cancers. The N-terminal splice variants of p63 and p73 are denoted as the TA and ΔN isoforms. We recently demonstrated that deletion of either ΔNp63 or ΔNp73 in p53-deficient mouse tumors results in tumor regression mediated by metabolic programming. Using this strategy, we identified pramlintide, a synthetic analog of amylin, as an effective treatment for p53 deficient and mutant tumors. Here, we show the utility of using pramlintide, as a potential cancer preventive option for p53-deficient tumors in mouse models. Additionally, we found that in vivo inhibition of both ΔNp63 and ΔNp73 in combination accelerates tumor regression and increases survival of p53-deficient mice. We report that inhibition of both ΔNp63 and ΔNp73 in combination results in upregulation of 3 key metabolic regulators, IAPP, GLS2, and TIGAR resulting in an increase in apoptosis and tumor regression in ΔNp63/ΔNp73/p53 deficient thymic lymphomas. These data highlight the value of generating inhibitors that will simultaneously target ΔNp63 and ΔNp73 to treat cancer patients with alterations in p53.     

Distribution of GSTM1, GSTT1, GSTP1 and TP53 disease-associated gene variants in native and urban Venezuelan populations

The contemporary Venezuelan population is the product of major admixture process across various historical events, which has provided it a particular genetic background. The aim of this study concerns the analysis of glutathione S-transferase (GST) GSTM1, GSTP1 and GSTT1 genetic variants and five polymorphisms at the TP53 gene, which are related to cancer susceptibility, in an urban/admixed population and five Amerindian tribes (Bari, Panare, Pemon, Warao and Wayuu) from Venezuela. Genotyping was carried out in 120 individuals from an urban sample and 188 Amerindians. The analysis performed on TP53 haplotype and GST allele distribution showed a close correlation for Pemon and Warao populations, while Bari group appears isolated from the other populations. GSTT1 null variant frequency in our admixed (11%) and native samples (0.0–11.4%) was lower when compared with Caucasians, Africans and Asians. Frequency of the GSTP1*Val cancer-associated allele found in Bari (88.6%) and Panare (63.0%) is of the highest so far reported. Fourteen TP53 haplotypes were observed in the admixed populations, whereas only 3 to 5 in Amerindians. To our knowledge this is the first report of GST polymorphisms and TP53 haplotype distribution in Venezuelans. The distribution of most of analyzed polymorphisms in the urban sample is consistent with the admixed origin of the present-day population of Venezuela. While, the inter-ethnic variations in genetic polymorphisms found in Native American tribes seem to be the result of the influence of demographic factors. These results provide additional data for undertaking ethnographic and disease association studies in Venezuela.     

The p53 tumor suppressor network in cancer and the therapeutic modulation of cell death

The molecular subversion of cell death is acknowledged as a principal contributor to the development and progression of cancer. The p53 tumor suppressor protein is among the most commonly altered proteins in human cancer. The p53 protein mediates critical functions within cells including the response to genotoxic stress, differentiation, senescence, and cell death. Loss of p53 function can result in enhanced rates of cell proliferation, resistance to cell death stimuli, genomic instability, and metastasis. The community of cancer scientists is now in possession of a vast repository of information regarding the frequency, specific mechanisms, and clinical context of cell death deregulation in cancer. This information has enabled the design of therapeutic agents to target proteins, including p53. The feasibility and impact of targeting cell death signaling proteins has been established in preclinical models of human cancer. The appropriate application of these targeted agents is now being established in clinical trials.     

肺癌组织中PCNA、p63和p53蛋白的表达及临床意义

目的:检测蛋白增殖细胞核抗原(PCNA)、p63和p53在肺癌组织中的表达情况,以探讨三者在肺癌的发生、发展中的生物学作用和临床意义。方法:选取195例肺癌组织(其中57例有癌旁组织),应用组织芯片技术和免疫组织化学方法观察三种蛋白的表达情况,并研究三者之间及其与临床病理参数的关系。结果:PCNA、p63和p53蛋白在肺癌组织中的阳性表达率分别为96.41%、38.46%及58.46%,但三者在癌旁组织中均无表达,差异有统计学意义(均P0.05);在肺癌组织中,PCNA、p63和p53蛋白的表达情况均与组织分型有关(P0.05),且PCNA、p53蛋白表达与分化程度有关(P0.05),分化越差,表达越高;p53表达与PCNA表达呈正相关(r=0.352,P=0.043),p63与p53、PCNA的表达不相关(P0.05)。结论:肺癌组织中PCNA、p63和p53蛋白的表达升高,三者均在肺癌的发生、发展中发挥着重要作用,并且临床可通过检测三者的蛋白水平,作为鉴别肺鳞状细胞癌与其他类型癌的重要参考指标,为病理诊断提供依据。     

Co‐expression of mouse TMEM63A,TMEM63B and TMEM63C confers hyperosmolarity activated ion currents in HEK293 cells

Osmoreception is essential for systemic osmoregulation, a process to stabilize the tonicity and volume of the extracellular fluid through regulating the ingestive behaviour, sympathetic outflow and renal function. The sensation of osmotic changes by osmoreceptor neurons is mediated by ion channels that detect the change of osmolarity in extracellular fluid. However, the molecular identity of these channels remains mysterious. AtCSC1and OSCA1,two closely related paralogues from Arabidopsis, have been demonstrated to form hyperosmolarity activated ion channels, which makes their mammalian orthologues—the members of TMEM63 proteins, possible candidates for osmoreceptor transduction channel. To test this possibility, we cloned the cDNAs of all the three members of the mouse TMEM63 family, TMEM63A, TMEM63B and TMEM63C from the mRNA from mouse brain. When all of the three subtypes of TMEM63 proteins were co‐expressed in HEK293 cells, we recorded membrane currents evoked by hypertonic stimulation in these cells. However, the cells expressing the combinations of any two subtypes of TMEM63 proteins could not exhibit any hyperosmolarity evoked currents. Thus, all the three members of TMEM63 proteins are required to constitute a hyperosmolarity activated ion channel. We propose that the TMEM63 proteins may serve as an osmolarity sensitive ion channel for the osmoreception. Copyright © 2016 John Wiley & Sons, Ltd.     

The lncRNA TP73‐AS1 promotes ovarian cancer cell proliferation and metastasis via modulation of MMP2 and MMP9

Ovarian cancer is one of the most common gynecologic malignancy with poor prognosis. Recently, long noncoding RNAs (lncRNAs) have been identified as key regulators in cancer development. The current study investigated the role of lncRNA P73 antisense RNA 1T (TP73‐AS1) in ovarian cancer. Quantitative real‐time polymerase chain reaction determined the expression levels of TP‐73AS1, matrix metallopeptidases (MMPs) messenger RNA. Cell proliferative ability, cell invasion, and migration were CCK‐8 and colony formation, and transwell invasion and migration assays, respectively. The protein levels of matrix metallopeptidase 2 (MMP2) and MMP9 were measured by Western blot. TP73‐AS1 was upregulated in the ovarian cancer tissues and ovarian cancer cells, and upregulation of TP73‐AS1 was associated with poor prognosis. Knockdown of TP73‐AS1 significantly suppressed cell proliferation, invasion, and migration of SKOV3 cells, and overexpression of TP73‐AS1 promoted cell proliferation, invasion, and migration of OVCA429 cells. In addition, knockdown of TP73‐AS1 suppressed the in vivo tumor growth. Tumor metastasis RT² profiler polymerase chain reaction array showed that MMP2 and MMP9 was significantly upregulated by TP73‐AS1 overexpression in ovarian cancer cells. TP73‐AS1 overexpression enhanced the expression of MMP2 and MMP9 in ovarian cancer cells. Knockdown of MMP2 and MMP9 attenuated the effects of TP73‐AS1 overexpression on cell invasion and migration. The clinical data showed that MMP2 and MMP9 were upregulated and positively correlated with TP73‐AS1 expression in ovarian cancer tissues. Collectively, our results demonstrated the oncogenic role of TP73‐AS1 in ovarian cancer, and targeting TP73‐AS1 may represent a novel approach in battling against ovarian cancer.     

A novel c.1037C > G (p.Ala346Gly) mutation in TP63 as cause of the ectrodactyly-ectodermal dysplasia and cleft lip/palate (EEC) syndrome

Ectrodactyly – ectodermal dysplasia and cleft lip/palate (EEC) syndrome (OMIM 604292) is a rare disorder determined by mutations in the TP63 gene. Most cases of EEC syndrome are associated to mutations in the DNA binding domain (DBD) region of the p63 protein. Here we report on a three-generation Brazilian family with three individuals (mother, son and grandfather) affected by EEC syndrome, determined by a novel mutation c.1037C > G (p.Ala346Gly). The disorder in this family exhibits a broad spectrum of phenotypes: two individuals were personally examined, one presenting the complete constellation of EEC syndrome manifestations and the other presenting an intermediate phenotype; the third affected, a deceased individual not examined personally and referred to by his daughter, exhibited only the split-hand/foot malformation (SHFM). Our findings contribute to elucidate the complex phenotype-genotype correlations in EEC syndrome and other related TP63-mutation syndromes. The possibility of the mutation c.1037C > G being related both to acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome and SHFM is also raised by the findings here reported.     

Expression of Ki-67, p53 and p63 proteins in keratocyst odontogenic tumours: an immunohistochemical study

Aim To investigate the immunohistochemical expression of Ki-67, p53 and p63 in Keratocyst Odontogenic Tumours (KOTs) in order to contribute to the biological profile of this tumor. Methods Immunohistochemical technique was performed using the EnVision™ System in 37 cases of KOTs. Results Ki-67- and p53-immunostained cells were mainly located in the suprabasal layers. p63-positive cells were found throughout the lining cystic epithelium. No difference in the immunostaining for these proteins was observed between primary and recurrent KOTs (Ki-67: P = 0.5591; p53: P = 0.9847; p63: P = 0.9127), or between KOTs associated with Nevoid Basal Cell Carcinoma Syndrome (NBCCS) and sporadic KOTs (Ki-67: P = 0.7013; p53: P = 0.3197; p63: P = 0.2427). Conclusions It is possible that biological behavior of KOTs may be related to suprabasal proliferative compartment in the cystic epithelium as observed by high levels of Ki-67, p53 and p63. In addition, p63 immunostaining may represent immaturity of keratinocytes in KOTs, and suggests that this protein may participate in the regulation of epithelial cell differentiation. Taken together, these data may favor tumorigenesis on KOTs.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Simultaneous haploinsufficiency of Pten and Trp53 tumor suppressor genes accelerates tumorigenesis in a mouse model of prostate cancer

Tumor suppressor gene PTEN is important in the initiation and progression of human prostate carcinoma, whereas the role of TP53 remains controversial. Since Pten/Trp53 double conditional knockout mice show earlier onset and fast progression of prostate cancer when compared to Pten knockout mice, we asked whether heterozygosity of these two tumor suppressor genes was sufficient to accelerate prostatic tumorigenesis. To answer this question we examined prostatic lesion progression of Pten/Trp53 double heterozygous mice and a series of controls such as Pten heterozygous, Pten conditional knockout, Trp53 heterozygous and Trp53 knockout mice. Tissue recombination of adult prostatic epithelium coupled with embryonic rat seminal vesicle mesenchyme was used as a tool to stimulate prostatic epithelial proliferation. In our study, high-grade prostatic intraepithelial neoplasia (PIN) was found with high frequency at 8 weeks post-tissue recombination transplantation. PIN lesions in Pten/Trp53 double heterozygous mice were more severe than those seen in Pten heterozygous alone. Furthermore, morphologic features attributable to Pten or Trp53 loss appeared to be enhanced in double heterozygous tissues. LOH analysis of Pten and Trp53 in genomic DNA collected from high-grade PIN lesions in Pten heterozygous and Pten/Trp53 double heterozygous mice showed an intact wild-type allele for both genes in all samples examined. In conclusion, simultaneous heterozygosity of Pten and Trp53 accelerates prostatic tumorigenesis in this mouse model of prostate cancer independently of loss of heterozygosity of either gene.     

Cross‐talk between T‐Ag presence and pRb family and p53/p73 signaling in mouse and human medulloblastoma

The formation and progression of mudulloblastoma (MB) is poorly understood. However, somatic inactivation of pRb/p105, in combination with a somatic or a germ‐line TP53 inactivation, leads to MB in a mouse model. Presently, there is no specific evidence of pathway/s alterations for the other two members of the retinoblastoma family, pRb2/p130 and/or p107 in MB. JC virus (JCV) is a human polyomavirus. Although there is no firm evidence that this virus plays a causal role in human neoplasia, it has been clearly proven that JCV is highly oncogenic when injected into the brain of experimental animals. The mechanism of JCV‐induced tumorigenesis is not entirely clear. However, several studies relate the oncogenic properties of JCV mainly to its early protein large T‐antigen (T‐Ag), which is able to bind and inactivate both TP53 and Rb family proteins. Here, we compared the protein expression profiles of p53, p73, pRb family proteins, and PCNA, as main regulators of cell proliferation and death, in different cell lines of mouse primitive neuroectodermal tumors (PNET), either T‐Ag‐positive or ‐negative, and in human MB cell lines. Our goal was to determine if changes in the relative expression of these regulators could trigger molecular perturbations underlying MB pathogenesis in mouse and human cells. Our results support that the presence of JCV T‐Ag may interfere with the expression of pRb family proteins, specific p73 isoforms, and p53. In turn, this “perturbation” may trigger a network of signals strictly connected with survival and apoptosis. J. Cell. Biochem. 110: 182–190, 2010. © 2010 Wiley‐Liss, Inc.