EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human orthologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs' and encodes a HECT domain E3 ubiquitin protein-ligase. To investigate the possible involvement of EDD in human cancer, several cancers from diverse tissue sites were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus using an EDD-specific microsatellite, CEDD, and other polymorphic microsatellites mapped in the vicinity of the 8q22.3 locus. Of 143 cancers studied, 38 had AI at CEDD (42% of 90 informative cases). In 14 of these cases, discrete regions of imbalance encompassing 8q22.3 were present, while the remainder had more extensive 8q aberrations. AI of CEDD was most frequent in ovarian cancer (22/47 informative cases, 47%), particularly in the serous subtype (16/22, 73%), but was rare in benign and borderline ovarian tumours. AI was also common in breast cancer (31%), hepatocellular carcinoma (46%), squamous cell carcinoma of the tongue (50%) and metastatic melanoma (18%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers, while among breast cancer cell lines EDD overexpression and increased gene copy number were correlated. These results demonstrate that AI at the EDD locus is common in a diversity of carcinomas and that the EDD gene is frequently overexpressed in breast and ovarian cancer, implying a potential role in cancer progression.

Down-regulation of the candidate tumor suppressor gene PAR-4 is associated with poor prognosis in breast cancer

Substantial experimental evidence indicates that PAWR gene (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) is a central player in cancer cell survival and a potential target for cancer-selective targeted therapeutics. However, little is known about the role of PAR-4 in breast cancer. We investigated the possible role of PAR-4 expression in breast cancer. IHC results on tissue microarrays containing 1,161 primary breast tumor samples showed that 57% (571/995) of analyzable cases were negative for PAR-4 nuclear staining. Down-regulation of nuclear PAR-4 protein expression predicted a poor prognosis for breast cancer patients (OS; P=0.041, log-rank test). PAR-4 down-regulation also correlates with poor survival in the group of patients with luminal A subtype breast cancer (P=0.028). Additionally, in this large series of breast cancer patients, we show that ERBB2/HER2, EGFR and pAKT protein expression are significantly associated with shorter disease-free survival and overall survival, but the prognosis was even worse for HER2-positive, EGFR-positive or pAKT-positive breast cancer patients with tumors negative for nuclear PAR-4 expression. Furthermore, using three-dimensional (3D) cell culture we provide preliminary results showing that PAR-4 is highly expressed in the MCF10A cells inside the acini structure, suggesting that PAR-4 might have a role in the lumen acini formation. Taken together, our results provide, for the first time, evidence that PAR-4 may have a role in the process of the mammary eland morphogenesis and its functional inactivation is associated with tumor aggressive phenotype and might represent an additional prognostic and predictive marker for breast cancer.

The severity of epithelial dysplasia is associated with loss of maspin expression in actinic cheilitis

Background Prolonged exposure of the lip to sunlight may cause actinic cheilitis (AC) and squamous cell carcinoma (SCC). Maspin is a serpin with tumor suppressor functions. This work analyzed the presence and distribution of maspin in AC and lip SCC. Methods Sections from 36 cases diagnosed as AC (18 cases with mild epithelial dysplasia, 11 with moderate and 7 with severe), 18 cases diagnosed as lip SCC and 7 specimens containing normal lip vermillion epithelium were submitted for immunohistochemical analysis to detect maspin. Results All AC cases with mild and two cases with moderate dysplasia were scored 3. The remaining nine cases with moderate dysplasia were identified as score 2, whereas all cases with severe dysplasia were scored 1. Positive staining for maspin decreased from the basal layer to the surface. Among the 18 lip SCCs studied, 15 cases showed abundant staining for maspin. Epithelium adjacent to the SCCs also showed intense positive staining in all cells. Conclusions Our results suggest that the loss of maspin expression occurs from the basal layer to the surface. Lip SCCs related to solar radiation show an intense presence of maspin protein in almost all tumor cells as well as the neighboring epithelium. Fontes A, Sousa SM, Santos E, Martins MT. The severity of epithelial dysplasia is associated with loss of maspin expression in actinic cheilitis.

A case of myoepithelial carcinoma displaying biallelic inactivation of the tumour suppressor gene APC in a patient with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by mutation of the APC gene. It is characterised by the appearance of hundreds to thousands of colorectal adenomas in adolescence and the subsequent development of colorectal cancer. Various extracolonic malignancies are associated with FAP, including desmoids and neoplasms of the stomach, duodenum, pancreas, liver, and brain. We present a family affected by FAP with an exon 14 APC mutation displaying two rare extracolonic lesions, a hepatoblastoma and a myoepithelial carcinoma. The hepatoblastoma was found in a male patient aged 2 years. The second lesion, a myoepithelial carcinoma of the right cheek, was found in a female patient aged 14 years. Inactivation of the normal APC allele was demonstrated in this lesion by loss of heterozygosity analysis, thus implicating APC in the initiation or progression of this neoplasm. This is the first reported case of this lesion in a family affected by FAP.

P53 and Rb tumor suppressor gene alterations in gastric cancer

Inactivation of tumor suppressor genes has been frequently observed in gastric carcinogenesis. Our purpose was to study the involvement of p53, APC, DCC, and Rb genes in gastric carcinoma. METHOD: Loss of heterozygosity of the p53, APC, DCC and Rb genes was studied in 22 gastric cancer tissues using polymerase chain reaction; single-strand conformation polymorphism of the p53 gene exons 5-6 and exons 7-8 was studied using 35S-dATP, and p53 expression was detected using a histological immunoperoxidase method with an anti-p53 clone. RESULTS AND DISCUSSION: No loss of heterozygosity was observed in any of these tumor suppressor genes; homozygous deletion was detected in the Rb gene in 23% (3/13) of the cases of intestinal-type gastric carcinoma. Eighteen (81.8%) cases showed band mobility shifts in exons 5-6 and/or 7-8 of the p53 gene. The presence of the p53 protein was positive in gastric cancer cells in 14 cases (63.6%). Normal gastric mucosa showed negative staining for p53; thus, the immunoreactivity was likely to represent mutant forms. The correlation of band mobility shift and the immunoreactivity to anti-p53 was not significant (P = .90). There was no correlation of gene alterations with the disease severity. CONCLUSIONS: The inactivation of Rb and p53 genes is involved in gastric carcinogenesis in our environment. Loss of the Rb gene observed only in the intestinal-type gastric cancer should be further evaluated in association with Helicobacter pylori infection. The p53 gene was affected in both intestinal and diffuse histological types of gastric cancer.

Transcriptional control of the Notch1 tumour suppressor gene

Abstract : The Notch pathway is an important regulator of differentiation and carcinogenesis. In keratinocytes and possibly other specific epithelial cell types, it acts as tumour suppressor. Expression of endogenous Notch1 gene is markedly reduced in keratinocyte-derived squamous cell carcinoma (SCC) and cervical cancer cells, as well as in prostate cancer cell lines, and this difference is, at least in part, at the transcriptional level. Little is known on transcriptional control of the Notch1 gene with the exception that it is a p53-target. Our work focused on the mechanisms involved in the different transcription level of the Notch1 gene in normal versus cancer cells. We show that the fully active minimal Notch1 promoter is differentially controlled in normal versus cancer cells. It consists of two distinct regions, one downstream of the transcription start site, which is likely to bind the basic transcription apparatus, and one upstream region characterized by highly GC-rich sequence. This latter region binds Sp/KLF family members, specifically Spa and KLF4, which is upregulated in cancer cells. This is functionally significant as KLF4 overexpression is sufficient to downmodulate Notchl gene transcription, while KLF4 knockdown, in combination with Spa, results in Notch1 upregulation. Control of Notch1 by KLF4/Sp3 is independent of p53. Biochemically, KLF4/Sp3 seem to affect preferentially the initiation step of Notch1 gene transcription, while p53 controls both initiation and elongation steps. Thus, the Notch1 gene is a negative Sp3/KLF4-target and this mechanism contributes, in parallel with p53, to Notch1 downregulation in cancer. Résumé : La voie de signalisation induite par Notch est considérablement impliquée dans la différenciation des cellules et dans la carcinogénèse. Dans les kératinocytes ainsi que dans d'autres types cellulaires de l'épithelium, il agit comme suppresseur de tumeur. L'expression endogène de Notch1 est remarquablement réduite dans les cellules du carcinome spino-cellulaire et du cancer du col de l'utérus ou dans les lignées cellulaires du cancer de la prostate. Cette différence s'explique, du moins en partie, par le niveau de transcription. Peu de choses sont connues sur le contrôle transcriptionnel de Notch1 à l'exception du fait qu'il soit une cible de p53. Notre travail s'est concentré sur les mécanismes impliqués dans la transcription de Notch1, mécanismes qui diffèrent entre les cellules normales et les cellules cancéreuses. Nous avons trouvé la plus petite région du promoteur de Notch1 qui est suffisante pour induire un haut niveau transcriptionnel et qui est contrôlée différemment dans les cellules normales et les cellules cancéreuses. Elle est constituée de deux régions distinctes: une en aval du site de départ de la transcription, qui lie probablement le complexe de base pour la transcription, et une en amont caractérisée par une séquence riche en GC. Cette région lie les membres de la famille Sp/KLF, spécifiquement Sp3 et KLF4, qui sont surexprimés dans les cellules cancéreuses. Ceci est fonctionnellement significatif car la surexpression de KLF4 dans les kératinocytes est suffisante pour diminuer la transcription de Notch1, alors que l'inhibition de KLF4 et de Spa, résulte en une augmentation de Notch1. En outre, le contrôle de Notch1 par KLF4 et Spa est indépendant de p53. Biochimiquement, KLF4 et Spa semblent plutôt affecter l'initiation de la transcription de Notch1 alors que p53 contrôle aussi bien l'initiation que l'élongation. En conclusion, le gène Notch1 est inhibé par Spa et KLF4: ce mécanisme contribue, en parallèle à p53, à diminuer l'expression de Notch1 dans les cellules cancéreuses.

PRDM1 is a tumor suppressor gene in natural killer cell malignancies.

Natural killer cell lymphoma (NKCL) constitutes a rare and aggressive form of non-Hodgkin lymphoma, and there is little insight into its pathogenesis. Here we show that PRDM1 is a tumor suppressor gene in NKCLs that is inactivated by a combination of monoallelic deletion and promoter CpG island hypermethylation. We observed monoallelic deletion of PRDM1 loci in 8 of 18 (44%) NKCL cases. The other allele showed significant promoter methylation in 12 of 17 (71%) cases. In support of its role as a tumor suppressor gene, the reconstitution of PRDM1 in PRDM1-null NK cell lines led to G2/M cell cycle arrest, increased apoptosis, and a strong negative selection pressure with progressive elimination of PRDM1-expressing cells, which was enhanced when IL-2 concentration is limiting. We observed a progressive increase in PRDM1 expression-in particular, PRDM1α-in normal NK cells in response to IL-2 and in normal NK cells activated with an engineered NK cell target, K562-Cl9-mb21, suggesting its role in NK cell homeostasis. In support of this role, knockdown of PRDM1 by shRNA in normal NK cells resulted in the positive selection of these cells. We identified MYC and 4-1BBL as targets of PRDM1 in NK cells. Disruption of homeostatic control by PRDM1 may be an important pathogenetic mechanism for NKCL.

Transition mutation in codon 248 of the p53 tumor suppressor gene induced by reactive oxygen species and a nitric oxide-releasing compound.

Exposing the human bronchial epithelial cell line BEAS-2B to the nitric oxide (NO) donor sodium 1-(N,N-diethylamino)diazen-1-ium-1, 2-diolate (DEA/NO) at an initial concentration of 0.6 mM while generating superoxide ion at the rate of 1 microM/min with the hypoxanthine/xanthine oxidase (HX/XO) system induced C:G-->T:A transition mutations in codon 248 of the p53 gene. This pattern of mutagenicity was not seen by 'fish-restriction fragment length polymorphism/polymerase chain reaction' (fish-RFLP/PCR) on exposure to DEA/NO alone, however, exposure to HX/XO led to various mutations, suggesting that co-generation of NO and superoxide was responsible for inducing the observed point mutation. DEA/NO potentiated the ability of HX/XO to induce lipid peroxidation as well as DNA single- and double-strand breaks under these conditions, while 0.6 mM DEA/NO in the absence of HX/XO had no significant effect on these parameters. The results show that a point mutation seen at high frequency in certain common human tumors can be induced by simultaneous exposure to reactive oxygen species and a NO source.

Oncogene-mediated downregulation of RECK, a novel transformation suppressor gene

The RECK gene was initially isolated as a transformation suppressor gene encoding a novel membrane-anchored glycoprotein and later found to suppress tumor invasion and metastasis by regulating matrix metalloproteinase-9. Its expression is ubiquitous in normal tissues, but undetectable in many tumor cell lines and in fibroblastic lines transformed by various oncogenes. The RECK gene promoter has been cloned and characterized. One of the elements responsible for the oncogene-mediated downregulation of mouse RECK gene is the Sp1 site, where the Sp1 and Sp3 factors bind. Sp1 transcription factor family is involved in the basal level of promoter activity of many genes, as well as in dynamic regulation of gene expression; in a majority of cases as a positive regulator, or, as exemplified by the oncogene-mediated suppression of RECK gene expression, as a negative transcription regulator. The molecular mechanisms of the downregulation of mouse RECK gene and other tumor suppressor genes are just beginning to be uncovered. Understanding the regulation of these genes may help to develop strategies to restore their expression in tumor cells and, hence, suppress the cells' malignant behavior.

Evidence of epigenetic regulation of the tumor suppressor gene cluster flanking RASSF1 in breast cancer cell lines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Downregulation of the RECK-tumor and metastasis suppressor gene in glioma invasiveness

Invasive behavior is the pathological hallmark of malignant gliomas, being responsible for the failure of surgery, radiation, and chemotherapy. Matrix metalloproteinases (MMPs) are essential for proper ECM remodeling and invasion. The tumor and metastasis suppressor RECK protein regulates at least three members of the MMPs family: MMP-2, MMP-9, and MT1-MMP. In order to mimic the in vivo invasion process, A172 and T98G, respectively, non-invasive and invasive human glioblastoma cell lines, were cultured onto uncoated (control) or type I collagen gel-coated surface, and maintained for up to 7 days to allow establishment of the invasive process. We show that the collagen substrate causes decreased growth rates and morphological alterations correlated with the invasive phenotype. Electronic transmission microscopy of T98G cells revealed membrane invaginations resembling podosomes, which are typically found in cells in the process of crossing tissue boundaries, since they constitute sites of ECM degradation. Real time PCR revealed higher RECK mRNA expression in A172 cells, when compared to T98G cells and, also, in samples obtained from cultures where the invasive process was fully established. Interestingly, the collagen substrate increases RECK expression in A172 cells and the same tendency is displayed by T98G cells. MMPs-2 and -9 displayed higher levels of expression and activity in T98G cells, and their activities are also upregulated by collagen. Therefore, we suggest that: (1) RECK down regulation is critical for the invasiveness process displayed by T98G cells; (2) type 1 collagen could be employed to modulate RECK expression in glioblastoma cell lines. Since a positive correlation between RECK expression and patients survival has been noted in several types of tumors, our results may contribute to elucidate the complex mechanisms of malignant gliomas invasiveness.

Occurrence and expression of p53 suppressor gene and c-Myc oncogene in dog eyelid tumors

Purpose: To detect the occurrence and expression of the suppressor gene p53 and of the oncogene c-Myc in eyelid tumors of dogs using the PCR, RT-PCR, PCR-ELISA and RT-PCR-ELISA techniques. These genes have not been described in dog eyelid tumors before. Methods: Nine samples of eyelid or third eyelid epithelial tumors were obtained from the archives of the Department of Veterinary Pathology. Tumor diagnosis was confirmed by evaluation of hematoxylin-eosin stained sections, and immunohistochemistry for cytokeratin AE1/AE3 and vimentin V9. A canine mammary tumor was used for positive control. Agarose gel electrophoresis, PCR-ELISA and RT-PCR-ELISA were used to detect p53 and c-Myc genes. Results: The occurrence of p53 was detected in most of the eyelid tumors and third eyelid tumors studied (88.8%, n = 8) and was expressed in 75% of the positive samples, as indicated by ELISA. The c-Myc gene was found in 77.7% (n = 7) of the samples and was expressed in eight samples. Conclusions: Eyelid and third eyelid tumors of dogs express both the p53 and the c-Myc genes as shown by PCR and RT-PCR. However, PCR ELISA and RT-PCR ELISA were more efficient in assessing occurrence and expression of these genes because they identified amplified products that were not detected by agarose gel electrophoresis. © 2010 American College of Veterinary Ophthalmologists.

PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of the tumour suppressor gene TP53 and expression of p53, Bcl-2 and p63 proteins in canine transmissible venereal tumour

Canine transmissible venereal tumour (CTVT) is a neoplasm transmitted among healthy dogs by direct contact with injured skin and/or mucous tissue. This study aimed to identify the TP53 gene, messenger RNA (mRNA) as well as the expression of p53, Bcl-2 and p63 proteins in histological sections of 13 CTVT samples at different stages of evolution. The in situ hybridization (ISH) and in situ reverse transcriptase polymerase chain reaction (RT-PCR) assays were used, which showed the DNA homologous to TP53 and its respective mRNA in 92.3% of the samples. We detected p53, p63 and Bcl-2 proteins in most of the cell samples in different grades of intensity. In addition, 46% of the samples were in the progressive and 54% in the regression phase. This is the first description of these proteins and a detailed study of their role in CTVT cells needs to be addressed in or to verify how these cells undergo apoptosis.