Wild type p73 overexpression and high-grade malignancy in breast cancer

The overexpression of wild type p73 is the most frequent alteration of p73 in malignancies. We investigated, in 70 breast carcinomas, p73 mRNA expression and its relationship to p53 mutations, determined by an immunohistochemical method, and loss heterozygosity (LOH) status of the 1p36 region, together with its possible implication in the pathogenesis of breast carcinomas. LOH, amplifying DNA by PCR using 5 markers, of 1p36 region (one intragenic to p73 gene) was found in 17% of cases but no significant correlation was observed with p73 overexpression. p53 positive immunostaining was present in 33% of breast carcinomas, and these exhibited a statistically significant relation with p73 overexpressed tumors. Overexpression of p73 mRNA was observed in 19 tumors (27%). The analysis of cases with p73 overexpression and cases with normal mRNA expression, in terms of age and pathologic characteristics of the tumors showed a significant association of p73 overexpression and tumors with lymph node metastases, vascular invasion and higher pathologic stage. These results suggest that p73 overexpression is a molecular alteration that could be implicated in the tumorigenesis of breast carcinomas and, eventually, in a poor clinical behavior.     

FHIT gene and flanking region on chromosome 3p are subjected to extensive allelic loss in Egyptian breast cancer patients

The fragile histidine triad gene (FHIT) is a candidate tumor suppressor gene at chromosome 3p14.2. Deletions in FHIT gene were reported in different types of cancer including breast cancer. In this study, we investigated the loss of heterozygosity (LOH) incidence that target FHIT genomic structure and chromosome 3p in cancerous and pre-neoplastic lesions of Egyptian breast patients. Genomic DNA was isolated from tumor tissues and their normal counterparts of 55 Egyptian patients diagnosed with breast cancer and 11 patients diagnosed with preneoplastic breast lesions. LOH was detected in 51% of breast cancer cases in at least one microsatellite marker of the four investigated markers. While, none of the markers showed LOH among the pre-neoplastic breast lesions. We also observed a significant association between LOH and invasive ductal carcinoma (IDC) histopathological type while no association observed between LOH and patients' age, tumor grade, or lymph node involvement. We also investigated FHIT gene expression profiles in breast cancer using Oncomine database. We found that FHIT is significantly reduced in all investigated studies. We conclude that, FHIT is underexpressed in breast cancer tissues compared to their normal counterparts due to the extensive allelic loss that is observed in its gene structure.     

P73 functionally replaces p53 in Adriamycin-treated, p53-deficient breast cancer cells

p53-Related genes, p73 and p63, encode 2 classes of proteins, TA-p73/p63 and DeltaN-p73/p63. TA-p73/p63 demonstrate p53-like properties including gene transactivation and cell death promotion, whereas DeltaN-p73/p63 lack these p53-like functions. Although p53-deficient cancer cells are often less responsive to chemotherapy, they are not completely drug resistant, suggesting that other apoptotic pathways are at work. Here, we compared for the first time to our knowledge p73 and p63 activation in various breast cancer (BC) cell lines after Adriamycin (ADR) treatment, an agent considered as mandatory in breast cancer chemotherapy. Our study was carried out using 1 p53-proficient BC cell line (MCF7 cells) and 3 BC cell lines deficient in p53 response (MCF7/ADR(IGR), MDA-MB157 and T47D) after ADR-induced genotoxic stress. We report that in cells with no p53 response after ADR treatment, TAp73, but not TAp63 or DeltaN-p73/p63, may replace p53 in triggering not only apoptosis but also cell cycle arrest or DNA repair effectors such as p21, GADD45, 14-3-3sigma and p53R2. We also demonstrate that TAp73 siRNA inhibits the accumulation of TAp73 in response to ADR treatment in MDA-MB157 cells and confers protection against ADR. ADR-induced downregulation of the DeltaNp73 isoform in the T47D cell line with nonfunctional mutant p53 further supports anti-apoptotic function of the isoform antagonistic to both p53 and TA-p73/p63. Exogenous TAp73 and DeltaNp73 overexpression in p53-response-deficient cell lines further confirms these results. cDNA microarray techniques demonstrated that the cellular response induced by p73 during ADR treatment could involve specific genes.     

p73 mutations are not detected in sporadic and hereditary breast cancer

Recently, a novel tumor suppressor gene, p73, was isolated and mapped to chromosome 1p36, a region commonly associated with loss of heterozygosity in neuroblastoma and other human malignancies, including breast cancer. The p73 gene shares considerable homology with the common tumor suppressor gene p53, both in composition and function. This study examines the potential participation of p73 in the pathogenesis of sporadic and hereditary breast cancers. Mutation analysis of 29 hereditary breast cancer cases revealed five independent silent mutations in the hereditary cases that are unlikely to play a role in tumor development. Mutation analysis of 48 sporadic breast tumors did not identify any unique variants. Eleven common polymorphisms scattered throughout the gene were also detected. Thus, mutations in the p73 gene appear to play little if any role in hereditary or sporadic breast cancer.     

Mutation analysis of the p73 gene in nonastrocytic brain tumours.

Loss of heterozygosity (LOH) involving the distal chromosome 1 p36 region occurs frequently in nonastrocytic brain tumours, but the tumour suppressor gene targeted by this deletion is unknown. p73 is a novel gene that has high sequence homology and similar gene structure to the p53 gene; it has been mapped to 1 p36, and may thus represent a candidate for this tumour suppressor gene. To determine whether p73 is involved in nonastrocytic brain tumour development, we analysed 65 tumour samples including 26 oligodendrogliomas, 4 ependymomas, 5 medulloblastomas, 10 meningiomas, 2 meningeal haemangiopericytomas, 2 neurofibrosarcomas, 3 primary lymphomas, 8 schwannomas and 5 metastatic tumours to the brain, for p73 alterations. Characterization of allelic loss at 1 p36-p35 showed LOH in about 50% of cases, primarily involving oligodendroglial tumours (22 of 26 cases analysed; 85%) and meningiomas (4 of 10; 40%). PCR-SSCP and direct DNA sequencing of exons 2 to 14 of p73 revealed a missense mutation in one primary lymphoma: a G-to-A transition, with Glu291Lys change. 8 additional cases displayed no tumour-specific alterations, as 3 distinct polymorphic changes were identified: a double polymorphic change of exon 5 was found in one ependymoma and both samples derived from an oligodendroglioma, as follows: a G-to-A transition with no change in Pro 146, and a C-to-T variation with no change in Asn 204: a delG at exon 3/+12 position was identified in 4 samples corresponding to 2 oligodendrogliomas, 1 ependymoma and 1 meningioma, and a C-to-T change at exon 2/+10 position was present in a metastatic tumour. Although both LOH at 1 p36 and p73 sequence changes were evidenced in 4 cases, it is difficult to establish a causal role of the p73 variations and nonastrocytic brain tumours development.     

Clinicopathological features of hereditary breast cancer

The possible role of germline mutations ofBRCA1 andBRCA2 as causative agents of familial breast cancer was assessed. Their possible involvement in the carcinogenesis of hereditary breast cancer was investigated using 63 clinically suspect families. Twenty-one lineages (33.3%) had mutations in one of the twoBRCA genes. This relatively low incidence suggested that germline mutations in unknown genes are involved in the carcinogenesis of hereditary breast cancer in the Japanese population. However, the clinicopathological features characteristic of hereditary breast cancer, such as early disease onset, a high incidence of bilateral breast cancer, and a high incidence of multiple primary carcinomas in other organs were confirmed in the present study.     

p73在宫颈癌和宫颈上皮内瘤变中的表达

目的探讨p73在宫颈肿瘤的发生发展过程中的表达情况及其意义。方法采用免疫组织化学染色的方法对12例慢性宫颈炎、15例低度宫颈上皮内瘤变、25例高度宫颈上皮内瘤变和65例宫颈浸润癌进行检测,确定染色强度,计算阳性率并进行χ2检验分析。结果按照上述病变顺序,p73染色阳性率分别是75.0%、93.3%、68.0%、41.5%。宫颈炎及宫颈上皮内瘤变与宫颈癌相比,差异显著,P〈0.005。但宫颈炎及宫颈上皮内瘤变间比较,无显著性差异。结论随着宫颈肿瘤发展程度的增加,p73的表达逐渐降低,进一步支持p73是宫颈肿瘤的抑癌基因。     

Allelic loss of the PTEN region (10q23) in breast carcinomas of poor pathophenotype

Loss of heterozygosity (LOH) in loci of the 10q23 region that harbor the PTEN gene and mutations in the sequence of this gene have been found in several primary human tumors including breast carcinomas, suggesting that this gene could be implicated in their pathogenesis. We investigated allelic losses in microsatellites of the 10q23 region, and their correlations with nine pathologic parameters in 105 breast carcinomas. The LOH analysis was performcd by amplifying DNA by PCR, using five markers of the 10q23 region (D10S1687, D10S541, D10S2491, D10S583 and D10S571). LOH in at least one marker of the PTEN region was found in 29.5% of tumors. The statistical comparison between carcinomas with and without LOH in terms of the pathologic parameters showed significant differences in age (p=0.03), lymph node metastases (p=0.02), and higher histological grade (p=0.02); a trend toward significance was found for progesterone receptors (p=0.05). LOH in an individual marker and statistically significant relationships to tumor characteristics were observed at locus D10S541 for lymph node metastases (p=0.04), at D10S2491 (intragenic to the PTEN gene) for lymph node metastases (p=0.02), and at D10S583 for progesterone receptors (p=0.01) and for high grade (p=0.03). These results suggest the PTEN gene, or other genes of the 10q23 region, could be functionally related to breast cancer, probably influencing the development of histological features associated with poor prognosis.     

鼻咽癌组织中p73蛋白表达及其与临床病理特征的关系

目的：探讨鼻咽癌组织中p73蛋白的表达及其与临床病理特征的关系。方法：应用免疫组化S－P法，检测p73蛋白在83例鼻咽癌组织和20例鼻咽黏膜慢性炎症组织中的表达以及p53蛋白在鼻咽癌组织中的表达情况。结果：83例鼻咽癌组织中p73蛋白表达阳性率（50．6％）较20例鼻咽 黏膜慢性炎症组织（15．0％）明显增高（P＜0．05），I，Ⅱ期鼻咽癌组织p73蛋白表达阳性率（27．8％）低于Ⅲ，Ⅳ期（56．9％）P＜0．05，P73蛋白表达阳性率与患者性别，颈部淋巴结转移，局部区域复发，远处转移放疗后5年生存率无关，P53蛋白阳性率为51．8％，其表达与P73蛋白表达无相关性（P＞0．05），结论：P73蛋白在鼻咽癌组织中的表达水平明显上调，其表达在肿瘤的发生发展中起重要作用。     

Transcriptional dysregulation of the p73L / p63 / p51 / p40 / KET gene in human squamous cell carcinomas: expression of Delta Np73L, a novel dominant-negative isoform, and loss of expression of the potential tumour suppressor p51

We have recently identified a second p53 -related p73L gene, also referred to as p63 / p51 / p40 / KET gene, which encodes the 2 major isoforms p73L and p51 resulting from different exon usage at their amino terminal regions. Although p73L and p51 are suspected to play oncogenic and tumour suppressive roles in mammalian cells, respectively, no evidence of linkage between the expression of these isoforms and human cancers has been reported so far. In this study, we first investigated the expression profile of p51 and p73L in various human tumour cell lines and found that a novel isoform, termed DeltaNp73L, was predominantly expressed in squamous cell carcinomas. The expression profile of DeltaNp73L/p73L/p51 in primary human skin cancer specimens showed that the expression of p51 was frequently lost (62%) but was detected in all normal skin samples. In p51-expressing skin cancers, DeltaNp73L expression was associated at a high frequency (75%) though it was not detected in normal skin tissues. Transient co-transfection data indicate the possibility that DeltaNp73L can inhibit p53-, and more preferentially, p51-mediated transactivation. These data suggest that the loss of expression of p51 and/or the expression of DeltaNp73L might contribute to the pathogenesis of human squamous cell carcinomas.     

Allelic expression of p73 in human ovarian cancers

Background: The p73 gene is structurally related to the tumor suppressor gene p53. The role of p73 in tumor development is still unclear and no data on ovarian cancer are so far available. For this reason we have analyzed, in a panel of ovarian cancers, the allelic distribution and expression of p73.Patients and methods: Fifty-one samples from ovarian cancers and five human ovarian cancer cell lines growing in culture were analyzed. Allelic origin was analyzed by PCR after digestion with the restriction enzyme Sty I. Heterozygous, informative cases were selected for studies aimed at evaluating allelic expression of p73.Results: We found an allelic distribution similar to that previously reported. LOH was found in two patients with ovarian cancer. In one case in which normal ovarian tissue was available biallelic expression of p73 was found.Conclusion: In comparisons of ovarian cancers and borderline tumors, no differences in allelic distribution and/or expression were found, suggesting that p73 does not play an important role in the pathogenesis and development of ovarian cancer.     

p53基因家族的新成员 p73和 p63

p53作为广泛存在的肿瘤抑制基因,最近发现了其家族成员：p73和p63。它们在结构和功能上具有相似性,均能触发细胞周期停滞,诱导凋亡,但它们在肿瘤抑制和组织发育中起着截然不同的作用,深入了解它们彼此之间的相似性和差异将对理解肿瘤发生的机制产生重要的影响。本文总结了最近几年来此领域内的最新进展。另外,p73和p63在C端的SAM结构说明它们可能参与组织的发育过程。     

p53 expression is decreased in primary breast carcinomas with microsatellite instability

p53 and p185 expression in primary breast cancer with microsatellite instability (MSI) is still largely unexplored. To investigate the relationship between these oncoproteins and the pathways of genomic instability, we examined 52 primary invasive breast cancers stratified by the presence and absence of MSI. We determined the status of eight microsatellite loci using radioactive and silver staining methods, and evaluated the immunohistochemical expression of p53 and p185 in a consecutive series of Italian cancer patients characterized by clinical-pathological and biological parameters. Nineteen cases (36.5%) were MSI-positive in at least two loci. p53 was expressed in 15 cases (28.8%) and p185 in eight (15.4%). MSI-positive tumors were inversely correlated with p53 expression (p=0.0007); in addition, the percent of p53-expressing cells decreased as the number of MSI-positive loci increased. MSI-positive tumors were correlated with a larger tumor size (p=0.04), lymph-node metastasis (p=0.001), and advanced clinical stage (p=0.0006). These data demonstrate the existence of two subsets of primary breast cancers: one characterized by MSI, the other by p53 expression. MSI-positive patients had a more advanced and/or aggressive disease.     

Prognostic significance of loss of heterozygosity on chromosome 9p21–22 in human esophageal cancer

Background Deletions involving chromosome 9p21, on which the tumor suppressor genep16/MTS1 is located, have been noted in esophageal cancer. We investigated the relationship between the deletion of chromosome 9p21–22 and the clinical features of esophageal cancer. Methods We examined the loss of heterozygosity (LOH) on chromosome 9p21–22 in 56 esophageal cancers using polymerase chain reaction (PCR) analysis and 2 microsatellite markers (RPS6 and IFNA). Results In 18 out of 50 informative cases (36%), LOH had occurred at 1 or 2 loci on chromosome 9p21–22. We found no relationship between LOH on chromosome 9p21–22 and patient sex, age tumor length, location, histologic differentiation, depth of tumor invasion, the extent of lymph node metastasis, histologic stage, or curability. Among 35 patients without an absolute noncurative resection, the mean survival of 11 patients with LOH on chromosome 9p21–22 was 19.3 months, compared with 42.3 months for 24 patients with a normal allele; thus, the survival rate of those with LOH was significantly lower than that of patients without LOH on chromosome 9p21–22 (log-rank test;P=0.03). Conclusion These data suggest that LOH on chromosome 9p21–22, on which the cell-cycle regulatorp16/MTS1 gene is located, may be related to cancer development, and probably can serve as a clinical marker for evaluating a patient's prognosis.     

大肠癌组织中p73、p51的相关性研究

目的：研究大肠癌组织中p73,p51基因的表达情况及其临床意义。方法：采用逆转录聚合酶链反应技术（RT-PCR）,检测30例大肠癌组织及邻近正常大肠粘膜组织p73及p51的两个转录p51A、p51BmRNA表达情况。结果：30例大肠癌组织中有22例p73表达阳性,相对应的正常大肠组织仅有3例表达阳性,两者具有显著性差异（P〈0.01）,大肠癌组织的p73阳性表达率与大肠癌的分化程度及TNM分期均无关（P〉0.05）;p51A、p51B在30例大肠癌组织和相对应的正常大肠组织呈低水平表达,其中大肠癌组织中p51A表达量明显高于正常大肠组织（P〈0.01）,与细胞分化程度及TNM分期无关（P〉0.05）,而p51B在大肠癌组织和正常大肠组织中的表达量无明显差异（P〉0.05）。结论：大肠癌组织中存在p73、p51A的过度转录表达,p73、p51A的过度表达与大肠癌的发生发展存在一定的关系。     

p73 is over-expressed in vulval cancer principally as the Delta 2 isoform.

p73 was studied in squamous cancers and precursor lesions of the vulva. Over-expression of p73 occurred commonly in both human papillomavirus (HPV)-positive and -negative squamous cell cancers (SCC) and high-grade premalignant lesions. Whereas expression in normal vulval epithelium was detected only in the basal and supra-basal layers, expression in neoplastic epithelium increased with grade of neoplasia, being maximal at both protein and RNA levels in SCC. p73 Delta 2 was the principal over-expressed isoform in the majority of cases of vulval SCC and often the sole form expressed in SCC. Over-expression of p73 was associated with expression of HPV-encoded E7 or with hypermethylation or mutation of p16(INK4a) in HPV-negative cases. There was a close correlation between expression of p73 and p14(ARF) in cancers with loss of p53 function. The frequent over-expression of p73 Delta 2 in neoplastic but not normal vulval epithelium, and its co-ordinate deregulation with other E2F-1 responsive genes suggests a role in the oncogenic process.     

Frequent inactivation of the p73 gene by abnormal methylation or LOH in non-Hodgkin's lymphomas

p73 is a candidate tumor suppressor and imprinted gene that shares significant homology with the p53 gene. It is located on 1p36, a region frequently deleted in neuroblastoma and other tumors. To investigate the pattern of inactivation of this gene in human lymphomas, we studied 59 tumors to identify abnormal methylation in exon 1 and loss of heterozygosity (LOH) at this locus. p73 was methylated in 13/50 (26%) B cell lymphomas. There was no evidence of p73 methylation in the 9 T cell lymphomas analyzed. Burkitt's lymphomas showed the highest proportion of methylated cases (36%), although this alteration also affected other aggressive lymphomas such as diffuse large cell and some marginal zone lymphomas. LOH at the p73 locus was detected in 4/34 (11%) B and 1/9 (11%) T cell lymphomas. The p73 expression analysis showed absence or low level of p73 product in methylated lymphomas, whereas p73 was always detected in unmethylated tumors. We found monoallelic expression in normal peripheral blood samples, consistent with imprinting. None of the tumors showed LOH and methylation of the remaining allele simultaneously, suggesting that alteration of the expressed allele could lead to the total inactivation of the gene. Our results show that deletion or methylation of the p73 gene could be important mechanisms in suppressing p73 expression in B cell non-Hodgkin's lymphomas.     

淋巴-浆细胞恶性血液病患者p73基因表达及与预后的关系

目的：探讨p73基因在急性淋巴细胞白血病（ALL）和多发性骨髓瘤（MM）发生发展中的机制。方法：应用RT-PCR技术检测58例ALL患者和40例多发性骨髓瘤的p73基因mRNA表达情况,并结合患者的临床资料进行分析。结果：58例ALL患者中,p73mRNA阴性表达率为32.8%（19/58）,40例MM患者p73mRNA均阳性表达。p73mRNA阴性表达与ALL患者首次化疗既获完全缓解及平均缓解时间的降低有明显关系。结论：p73mRNA检测对判断ALL患者预后有一定的意义。p73基因异常可能不是MM发病的重要分子事件。     

Mutant p53 mediates survival of breast cancer cells

Background:

p53 is the most commonly mutated tumour-suppressor gene in human cancers. Unlike other tumour-suppressor genes, most p53 cancer mutations are missense mutations within the core domain, leading to the expression of a full-length mutant p53 protein. Accumulating evidence has indicated that p53 cancer mutants not only lose tumour suppression activity but also gain new oncogenic activities to promote tumourigenesis.

Methods:

The endogenous mutant p53 function in human breast cancer cells was studied using RNA interference (RNAi). Gene knockdown was confirmed by quantitative PCR and western blotting. Apoptosis was evaluated by morphological changes of cells, their PARP cleavage and annexin V staining.

Results:

We show that cancer-associated p53 missense mutants are required for the survival of breast cancer cells. Inhibition of endogenous mutant p53 by RNAi led to massive apoptosis in two mutant p53-expressing cell lines, T47D and MDA-MB-468, but not in the wild-type p53-expressing cells, MCF-7 and MCF-10A. Reconstitution of an RNAi-insensitive mutant p53 in MDA-MB-468 cells completely abolished the apoptotic effects after silencing of endogenous mutant p53, suggesting the specific survival effects of mutant p53. The apoptotic effect induced by mutant p53 ablation, however, is independent of p63 or p73 function.

Conclusion:

These findings provide clear evidence of a pro-survival ‘gain-of-function'' property of a subset of p53 cancer mutants in breast cancer cells.