Somatic mutations of the PPP2R1B candidate tumor suppressor gene at chromosome 11q23 are infrequent in ovarian carcinomas

Previous studies have demonstrated frequent allelic losses of distal chromosome 11q in ovarian carcinomas. The tumor suppressor gene(s) presumably targeted by these losses have not yet been identified. PPP2R1B is a candidate tumor suppressor gene at 11q23 that has recently been shown to be mutated in a subset of colorectal and lung cancers. We evaluated 5 ovarian carcinoma cell lines and 27 primary ovarian carcinomas for allelic losses of 11q23 and for mutations in the open reading frame of PPP2R1B. We also evaluated the primary tumors for allelic losses at 17p13, another chromosomal region frequently affected by losses of heterozygosity (LOH) in ovarian cancers. 11q23 and 17p13 allelic losses were identified in 25% and 74% of the carcinomas, respectively. No mutations within PPP2R1B coding sequences were found. These findings indicate that mutations of the PPP2R1B gene are infrequent in ovarian cancer and that deletions affecting the distal portion of chromosome 11q in ovarian cancer likely target inactivation of other genes.     

The two human homologues of yeast UFD2 ubiquitination factor, UBE4A and UBE4B, are located in common neuroblastoma deletion regions and are subject to mutations in tumours

Chromosomes 11q and 1p are commonly deleted in advanced-stage neuroblastomas and are therefore assumed to contain tumour suppressor genes involved in the development of this cancer. The two UFD2 yeast gene human homologues, UBE4A and UBE4B, involved in the ubiquitin/proteasome pathway, are located in 11q and 1p, respectively. UBE4B has previously been analysed for mutations and one mutation in the splice donor site of exon 9, c.1439 + 1G > C, was found in a neuroblastoma tumour with fatal outcome. We speculated that the homologue UBE4A might be involved in an alternative tumourigenesis pathway. The coding exons of UBE4A were therefore sequenced. One putative missense mutation (1028T > C, leading to I343T, residing in exon 8) was found in neuroblastoma tumour 20R8; this finding was confirmed by sequencing in both directions. The change, isoleucine (non-polar) to threonine (polar), was situated in a highly conserved amino acid region. In addition, two novel variants were also found in intronic sequences of UBE4A. It might be speculated that the proteins generated from UBE4B and UBE4A are involved in protecting the cell from environmental stress and that inactivation of either of them could contribute to malignancy.     

Refinement and distinction of the chromosome 10p candidate tumor suppressor region in human gliomas

Our recent studies have shown that telomeric 10p is frequently deleted in both low and high grade gliomas. Other studies have shown an invariant association of erb-b amplification and loss of chromosome 10. It has not been determined whether this association was specific for a particular locus on chromosome 10. We sought to assess this by examining tumors, for both 10p loss and gene amplification. 55% of tumors showed some degree of allelic imbalance at 10p15. Amplification of erb-b, c-myc, and a mitochondrial cDNA probe, recently found to be amplified in diverse grades of gliomas was assessed. erb-b and the mitochondrial cDNA probe showed amplification in 28% of the tumors; c-myc gene was amplified in only one astrocytoma. No significant association was noted when comparing the presence of a specific amplified gene, and loss of loci on chromosome. Chromosome 10p15 shows significant loss in glial tumors, and is independent of the chromosome 10 region associated with erb-b amplification. The region at 10p15 is distinct in harboring a putative tumor suppressor gene in gliomas, and is involved earlier in the multi-step pathway to glial tumor malignancy.     

Localization of a putative tumor suppressor gene in the sub-telomeric region of chromosome 8p.

Several regions of chromosome arm 8p are frequently deleted in a variety of human malignancies including those of the prostate, head and neck, lung, and colon, suggesting that there is more than one tumor suppressor gene on this chromosome arm. Both laryngeal and oral squamous cell carcinomas exhibit three distinct and nonoverlapping regions of deletion on 8p. We have further refined the localization of the putative suppressor in 8p23 by using eight microsatellite loci to create a high resolution deletion map of 150 squamous cell carcinomas of the larynx and oral cavity. These new data demonstrate that there are two distinct classes of deletion within this relatively small region of the chromosome and suggest two possible locations for the gene within the D8S264 to D8S1788 interval. We also determined that there is little difference between the allelic loss frequencies of microsatellites mapping near the telomeric ends of other chromosome arms and loci mapping to more centromere proximal regions of the same arm. These data suggest that the high allelic loss frequencies seen at 8p23 loci are not the result of a generalized instability of chromosome ends and are instead consistent with the activation of a specific suppressor gene.     

Functional studies of the chromosome 3p21.3 candidate tumor suppressor gene BLU/ZMYND10 in nasopharyngeal carcinoma

Chromosome 3p plays an important role in tumorigenesis in many cancers, including nasopharyngeal carcinoma (NPC). We have previously shown chromosome 3p can suppress tumor growth in vivo by using the monochromosome transfer approach, which indicated the chromosome 3p21.3 region was critical for tumor suppression. BLU/ZMYND10 is one of the candidate tumor suppressor genes mapping in the 3p21.3 critical region and is a candidate TSG for NPC. By quantitative RT-PCR, it is frequently downregulated in NPC cell lines (83%) and NPC biopsies (80%). However, no functional studies have yet verified the functional role of BLU/ZMYND10 as a tumor suppressor gene. In the current study, a gene inactivation test (GIT) utilizing a tetracycline regulation system was used to study the functional role of BLU/ZMYND10. When BLU/ZMYND10 is expressed in the absence of doxycycline, the stable transfectants were able to induce tumor suppression in nude mice. In contrast, downregulation of BLU/ZMYND10 in these tumor suppressive clones by doxycycline treatment restored the tumor formation ability. This study provides the first significant evidence to demonstrate BLU/ZMYND10 can functionally suppress tumor formation in vivo and is, therefore, likely to be one of the candidate tumor suppressor genes involved in NPC.     

Expression of TSLC1, a candidate tumor suppressor gene mapped to chromosome 11q23, is downregulated in unfavorable neuroblastoma without promoter hypermethylation

Although it has been well documented that loss of human chromosome 11q is frequently observed in primary neuroblastomas, the smallest region of overlap (SRO) has not yet been precisely identified. Previously, we performed array-comparative genomic hybridization (array-CGH) analysis for 236 primary neuroblastomas to search for genomic aberrations with high-resolution. In our study, we have identified the SRO of deletion (10-Mb or less) at 11q23. Within this region, there exists a TSLC1/IGSF4/CADM1 gene (Tumor suppressor in lung cancer 1/Immunoglobulin superfamily 4/Cell adhesion molecule 1), which has been identified as a putative tumor suppressor gene for lung and some other cancers. Consistent with previous observations, we have found that 35% of primary neuroblastomas harbor loss of heterozygosity (LOH) on TSLC1 locus. In contrast to other cancers, we could not detect the hypermethylation in its promoter region in primary neuroblastomas as well as neuroblastoma-derived cell lines. The clinicopathological analysis demonstrated that TSLC1 expression levels significantly correlate with stage, Shimada's pathological classification, MYCN amplification status, TrkA expression levels and DNA index in primary neuroblastomas. The immunohistochemical analysis showed that TSLC1 is remarkably reduced in unfavorable neuroblastomas. Furthermore, decreased expression levels of TSLC1 were significantly associated with a poor prognosis in 108 patients with neuroblastoma. Additionally, TSLC1 reduced cell proliferation in human neuroblastoma SH-SY5Y cells. Collectively, our present findings suggest that TSLC1 acts as a candidate tumor suppressor gene for neuroblastoma.     

Identification of Fat4 as a candidate tumor suppressor gene in breast cancers

Fat, a candidate tumor suppressor in Drosophila, is a component of Hippo signaling pathway involved in controlling organ size. We found that a ～3 Mbp deletion in mouse chromosome 3 caused tumorigenesis of a non‐tumorigenic mammary epithelial cell line. The expression of Fat4 gene, one member of the Fat family, in the deleted region was inactivated, which resulted from promoter methylation of another Fat4 allele following the deletion of one Fat4 allele. Re‐expression of Fat4 in Fat4‐deficient tumor cells suppressed the tumorigenecity whereas suppression of Fat4 expression in the non‐tumorigenic mammary epithelial cell line induced tumorigenesis. We also found that Fat4 expression was lost in a large fraction of human breast tumor cell lines and primary tumors. Loss of Fat4 expression in breast tumors was associated with human Fat4 promoter methylation. Together, these findings suggest that Fat4 is a strong candidate for a breast tumor suppressor gene. © 2008 Wiley‐Liss, Inc.     

Beta-catenin mutations and expression, 249serine p53 tumor suppressor gene mutation, and hepatitis B virus infection in southern African Blacks with hepatocellular carcinoma

BACKGROUND AND OBJECTIVES: To ascertain the prevalence of deregulating mutations of beta-catenin gene, and to correlate this with the occurrence of 249(serine) p53 gene mutation and hepatitis B virus infection in southern African Blacks with hepatocellular carcinoma. METHODS: Paired cancer/non-cancerous liver tissues from 21 and cancer tissues alone from 20 Black Africans with hepatocellular carcinoma were studied. RT-PCR-SSCP and sequencing were used to detect mutations in exon 3 of the beta-catenin gene, and PCR, restriction endonuclease analysis, and sequencing to detect the p53 gene mutation. Immunostaining was used to identify beta-catenin protein expression in hepatocytes. RESULTS: No mutations in exon 3 of the beta-catenin gene were found in tumor or non-tumorous tissues. Immunohistochemical staining showed beta-catenin protein expression in membranes and cytoplasm of hepatocytes but not in the nuclei. The 249serine p53 gene mutation was detected in 27.2% of the hepatocellular carcinoma tissues but not in non-cancerous tissues. No correlation was found between beta-catenin mutation and over-expression and 249serine p53 gene mutations or hepatitis B virus surface antigenemia. CONCLUSIONS: Unlike hepatocellular carcinomas in China, Japan, and Europe, deregulating beta-catenin gene mutations do not appear to occur in southern African Blacks with this tumor and do not therefore interact with either the 249serine p53 gene mutation or hepatitis B virus infection in its pathogenesis.     

TTC4, a novel candidate tumor suppressor gene at 1p31 is often mutated in malignant melanoma of the skin

A novel candidate tumor suppressor gene, TTC4, on chromosome 1p31 has been described recently. Since aberrations in this region have been detected in malignant melanoma, we investigated DNA of paraffin-embedded sections from 16 typical naevi, 19 atypical naevi, 32 primary melanomas (15 superficial spreading melanomas, 17 nodular melanomas) and 25 metastases and DNA from four melanoma cell lines by PCR and direct sequencing analysis for mutations in all exons of TTC4. Tumors comprised a wide range of thickness (Breslow index) and Clark levels. No mutations could be detected in typical or atypical naevi, but we found seven different point mutations in the tumor samples, six of them causing an amino acid change. Ten melanoma samples belonging to nine patients showed one or more of these mutations. In detail, in six of 25 metastases, in two of 17 nodular melanomas and in two of 15 superficial spreading melanomas point mutations could be detected. In two cell lines, a loss of a whole exon could be demonstrated and in one cell line we found a point mutation. In addition, three polymorphisms were found. Our findings indicate that TTC4 may participate in the pathogenesis of malignant melanomas of the skin.     

Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C

Initial analysis identified the NPRL2/G21 gene located in 3p21.3C, the lung cancer region, as a strong candidate tumor suppressor gene. Here we provide additional evidence of the tumor suppressor function of NPRL2/G21. The gene has highly conserved homologs/orthologs ranging from yeast to humans. The yeast ortholog, NPR2, shows three highly conserved regions with 32 to 36% identity over the whole length. By sequence analysis, the main product of NPRL2/G21 encodes a soluble protein that has a bipartite nuclear localization signal, a protein-binding domain, similarity to the MutS core domain, and a newly identified nitrogen permease regulator 2 domain with unknown function. The gene is highly expressed in many tissues. We report inactivating mutations in a variety of tumors and cancer cell lines, growth suppression of tumor cells with tet-controlled NPRL2/G21 transgenes on plastic Petri dishes, and suppression of tumor formation in SCID mice. Screening of 7 renal, 5 lung, and 7 cervical carcinoma cell lines showed homozygous deletions in the 3' end of NPRL2 in 2 renal, 3 lung, and 1 cervical (HeLa) cell line. Deletions in the 3' part of NPRL2 could result in improper splicing, leading to the loss of the 1.8 kb functional NPRL2 mRNA. We speculate that the NPRL2/G21 nuclear protein may be involved in mismatch repair, cell cycle checkpoint signaling, and activation of apoptotic pathway(s). The yeast NPR2 was shown to be a target of cisplatin, suggesting that the human NPRL2/G21 may play a similar role. At least two homozygous deletions of NPRL2/G21 were detected in 6 tumor biopsies from various locations and with microsatellite instability. This study, together with previously obtained results, indicates that NPRL2 is a multiple tumor suppressor gene.     

The retinoic acid synthesis gene ALDH1a2 is a candidate tumor suppressor in prostate cancer

Prostate cancer is the most common cancer among men in the United States, and aberrant DNA methylation is known to be an early molecular event in its development. Here, we have used expression profiling to identify novel hypermethylated genes whose expression is induced by treatment of prostate cancer cell lines with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-aza-dC). Of the 271 genes that were induced by 5-aza-dC treatment, 25 also displayed reduced expression in primary prostate tumors compared with normal prostate tissue, and the decreased expression of only one gene, aldehyde dehydrogenase 1 family, member A2 (ALDH1a2), was also associated with shorter recurrence-free survival. ALDH1a2 encodes an enzyme responsible for synthesis of retinoic acid (RA), a compound with prodifferentiation properties. By immunohistochemistry, we observed that ALDH1a2 was expressed in epithelia from normal prostate but not prostate cancer. Using bisulfite sequencing, we determined that the ALDH1a2 promoter region was significantly hypermethylated in primary prostate tumors compared with normal prostate specimens (P = 0.01). Finally, transfection-mediated reexpression of wild-type ALDH1a2 (but not a presumptive catalytically dead mutant) in the prostate cancer cell line DU145 resulted in decreased colony growth (P < 0.0001), comparable with treatment with either 5-aza-dC or RA. Taken together, our findings implicate ALDH1a2 as a candidate tumor suppressor gene in prostate cancer and further support a role of retinoids in the prevention or treatment of prostate cancer.