PPP2R1B gene alterations inhibit interaction of PP2A-Abeta and PP2A-C proteins in colorectal cancers

The chromosome region 11q is frequently deleted in colorectal cancers. The PPP2R1B tumor suppressor gene, encoding the beta isoform of the A subunit of serine/threonine-specific protein phosphatase 2A (PP2A-Abeta), located at 11q22-23, is inactivated in patients with cancer. The present study investigated whether or not PP2A-Abeta is altered in colorectal cancers. We searched for alterations of the PPP2R1B gene and interactions between PP2A-Abeta and PP2A-C proteins in 50 surgically resected colorectal cancer tissues. Missense mutations and homozygous deletions of the PPP2R1B gene were found in 4 of 50 patients (8%) and in 1 of 50 patients, respectively, with colorectal cancers. Deletions and/or point mutations within 412-601 amino acid sequences (binding regions of PP2A-C protein) of the PPP2R1B gene derived from colorectal cancer tissues inhibited co-immunoprecipitation of PP2A-Abeta and PP2A-C proteins. These finding suggested that the PPP2R1B gene functions as a tumor suppressor gene and acts as a molecular switch that becomes active in response to specific up-stream signals. Upon activation, the gene alters the activities of specific downstream target proteins for the cell cycle regulations and/or metabolism in some colorectal cancers.     

Alterations of the PPP1R3 gene in human cancer.

Recently, the PTEN/MMAC1 gene encoding a protein phosphatase (PP) and the PPP2R1B gene encoding a regulatory subunit of PP2A have been identified as being genetically altered in several types of human cancers, indicating that aberrations of intracellular signaling pathways via PPs are involved in human carcinogenesis. Here we report genetic alterations of the PPP1R3 gene located at chromosome 7q31, which encodes regulatory subunit 3 of PP1, in various types of human cancers. Mutations of the PPP1R3 gene were detected in 5 of 33 (15%) non-small cell lung cancer cell lines and 2 of 38 (5%) primary non-small cell lung cancers and were also observed in cell lines derived from a small cell lung cancer, an ovarian cancer, a colorectal cancer, and a gastric cancer. Mutations were widely dispersed in the coding region of the PPP1R3 gene. Three of the 11 detected mutations were nonsense mutations, whereas the remaining ones were missense mutations, most of which caused substitutions of evolutionarily conserved amino acids. These findings suggest that PPP1R3 alteration plays a role in the development of human cancers and that PPP1R3 could act as a tumor suppressor gene.     

Absence of PPP2R1B gene alterations in primary ovarian cancers

The PPP2R1B gene has recently been implicated as a tumor suppressor based on the finding of somatic alterations in lung and colon cancers. PPP2R1B is located on chromosome 11q22-24 which coincides with the site of frequent loss of heterozygosity (LOH) in ovarian cancer. We investigated if the PPP2R1B gene was inactivated in ovarian cancer by single strand conformational polymorphism (SSCP) and heteroduplex (HD) analysis of 99% of the coding region. LOH at the PPP2R1B locus was detected in 32% of the malignant tumors but no somatic alterations were detected in any of 65 malignant, five borderline or six benign tumors. A germline G > A transition (GGC > GAC) in codon 90 was detected in 4/76 tumors. This alteration has previously been described as a mutation but on further investigation we found that the frequency of this variant among 167 ovarian cancers (4.2%) was not statistically significantly different from that observed in 247 non-cancer random controls (2.4%). We conclude that the PPP2R1B gene is not involved in the pathogenesis of ovarian cancer. The codon 90 Gly > Asp alteration may represent a non-pathological polymorphism and consequently the mutation frequency reported in lung cancers may have been overstated and the designation of PPP2R1B as a tumor suppressor gene should be regarded with caution.     

Allelic loss of chromosome 17p distinguishes high grade from low grade transitional cell carcinomas of the bladder

Forty-three transitional cell carcinomas of the bladder of differing grades and stages were examined for reduction to homozygosity for chromosomes 9q, 11p, and 17p. Allelic loss of chromosome 9q was seen in 24 of 38 informative grades II, III, and IV tumors providing further evidence for a bladder tumor suppressor gene on this chromosome. In contrast to the grade-independent involvement of chromosome 9q, allelic losses of chromosomes 11p and 17p were seen only in grade III and IV tumors. The results with chromosome 17p were particularly striking and showed that 0 of 10 grade II versus 20 of 31 grade III and IV tumors had allelic losses for this chromosome harboring the p53 tumor suppressor gene often mutated in other human cancers. The data suggest that cumulative genetic damage is sustained in transitional cell carcinomas and that one of the underlying molecular mechanisms distinguishing low grade from high grade tumors involves chromosome 17p.     

Frequent allelic losses on chromosome 13q in human male breast carcinomas

Loss of genetic material on chromosomes 13q and 17 has been suggested to be of importance in the initiation and progression of female breast cancer, but their involvement is less well illustrated in male breast carcinomas. The present study was designed to investigate the incidence of allelic loss and microsatellite instability for chromosomes 13q, 17p and 17q in 13 sporadic male breast carcinomas using matched normal-tumour DNA samples and seven polymorphic microsatellite markers. Genetic imbalance was found in one or more informative markers in 85% of the patients, with more frequent loss of heterozygosity and microsatellite instability at loci on chromosome 13q. Thus, a high incidence of allelic losses was observed at the retinoblastoma gene (4/6) and likewise at the D13S263 locus (7/12), which also exhibited the highest frequency of microsatellite instability. The intragenic microsatellite in intron 1 of the TP53 gene on chromosome 17p revealed loss of heterozygosity in 3 of 8 informative patients. The investigated proximal region of chromosome 13q is postulated to harbour several potential tumour suppressor genes associated with female breast cancer. The high incidence of allelic losses at the D13S263 microsatellite, located distal to both the BRCA2 and the Brush-1 loci but proximal to the retinoblastoma gene, possibly indicates the presence of an additional tumour suppressor gene which may be involved in male breast carcinomas. However, this hypothesis needs verification in an extended study of male breast carcinomas.     

PTEN/MMAC1 Mutation and Frequent Loss of Heterozygosity Identified in Chromosome 10q in a Subset of Hepatocellular Carcinomas

Frequent allelic losses on chromosome 10q have been reported in several types of cancers, suggesting the presence of a putative tumor suppressor gene(s) on the chromosomal arm. We examined loss of heterozygosity (LOH) on chromosome 10q in 37 hepatocellular carcinomas (HCC) using eleven dinucleotide microsatellite markers, spanning the entire chromosome arm of 10q. Twelve (32%) out of 37 informative cases showed allelic losses of at least one locus on 10q and eight tumors showed a partial deletion of 10q. Analysis of deletion mapping of these eight cases identified two commonly deleted regions within the distal part of 10q (10q24-q26), a 20-cM interval flanked by D10S597 and D10S216 and a 24-cM interval flanked by D10S216 and D10S590. Moreover, we detected a somatic missense mutation (Met→Val) of a candidate tumor suppressor gene PTEN/MMAC1, located at 10q23.3, in one HCC with LOH of 10q. Our findings indicated the presence of putative tumor suppressor gene(s) in the distal region of 10q that might be involved in the development and progression of HCC. Inactivation of PTEN/MMAC1 gene located outside the commonly deleted region of 10q might also play an important role in a subset of HCCs.     

PTEN / MMAC1 mutation and frequent loss of heterozygosity identified in chromosome 10q in a subset of hepatocellular carcinomas.

Frequent allelic losses on chromosome 10q have been reported in several types of cancers, suggesting the presence of a putative tumor suppressor gene(s) on the chromosomal arm. We examined loss of heterozygosity (LOH) on chromosome 10q in 37 hepatocellular carcinomas (HCC) using eleven dinucleotide microsatellite markers, spanning the entire chromosome arm of 10q. Twelve (32%) out of 37 informative cases showed allelic losses of at least one locus on 10q and eight tumors showed a partial deletion of 10q. Analysis of deletion mapping of these eight cases identified two commonly deleted regions within the distal part of 10q (10q24-q26), a 20-cM interval flanked by D10S597 and D10S216 and a 24-cM interval flanked by D10S216 and D10S590. Moreover, we detected a somatic missense mutation (Met --> Val) of a candidate tumor suppressor gene PTEN / MMAC1, located at 10q23.3, in one HCC with LOH of 10q. Our findings indicated the presence of putative tumor suppressor gene(s) in the distal region of 10q that might be involved in the development and progression of HCC. Inactivation of PTEN / MMAC1 gene located outside the commonly deleted region of 10q might also play an important role in a subset of HCCs.     

Alterations of the PPP1R3 gene in hematological malignancies

PPP1R3 (protein phosphatase 1, regulatory subunit 3) is a candidate tumor suppressor gene at chromosome 7q31, since nonsense and missense mutations of the PPP1R3 gene have been detected in a variety of human cancers. Loss of chromosome 7q is a recurrent abnormality in hematological malignancies, especially of myeloid lineage, and a common region of 7q deletions has been mapped to 7q31. Thus, it has been suggested that 7q31 harbors a tumor suppressor gene whose functional loss contributes to leukemogenesis. To evaluate the possible involvement of the PPP1R3 gene in the development of hematological malignancies, we examined 72 leukemia and lymphoma cell lines for alterations of the PPP1R3 gene by PCR-SSCP and direct sequence analyses. Mutations were detected in 1 (2.8%) of 36 myeloid cell lines, 4 (20.0%) of 20 B-lineage lymphoid cell lines and none of 16 T-lineage lymphoid cell lines. All the mutations were heterozygous, and they consisted of two missense mutations and three silent mutations. The PPP1R3 gene was expressed in cell lines of various cell lineages. These results indicate that PPP1R3 is not a major target of 7q deletions in myeloid leukemia, however, alterations of the PPP1R3 gene may contribute to the development of a subset of hematological malignancies.     

Somatic mutations and genetic polymorphisms of the PPP1R3 gene in patients with several types of cancers

Recently, we found nonsense and missense mutations of the PPP1R3 (protein phosphatase 1, regulatory subunit 3) gene in diverse human cancer cell lines and primary lung carcinomas, indicating that PPP1R3 functions as a tumor suppressor in human carcinogenesis. In this study, to assess the prevalence of PPP1R3 mutations in human primary cancers and the genetic diversity of the PPP1R3 gene in the human population, somatic mutations and genetic polymorphisms in the PPP1R3 gene were examined in 137 pairs of cancerous and non-cancerous tissues of patients with cancers of colon, ovary, and liver. Five somatic mutations including two missense mutations were detected in three cancerous tissues consisting of two colorectal carcinomas and one ovarian carcinoma. Five novel single nucleotide polymorphisms (SNPs) associated with the substitution of amino acids were also identified in cancer patients, in addition to five known nonsynonymous SNPs, including three previously reported ones as having an impact on the susceptibility to insulin resistant disorders. Differences in the activities and properties of multiple PPP1R3 proteins, which are produced in human cells due to variable somatic mutations and genetic polymorphisms in the PPP1R3 gene, can be involved in human carcinogenesis and susceptibility to diseases.     

Frequent allelic imbalance at the ATM locus in DNA multiploid colorectal carcinomas

DNA multiploidy may involve specific DNA ploidy states with respect to genetic alterations such as oncogenes, tumor suppressor gene mutation and microsatellite instability. To clarify the role of DNA multiploidy in colorectal cancer, we analysed allelic imbalance involving the ATM gene, localized to chromosome 11q22-23 and thought to be involved in genetic stability, in a series of multiploid colorectal carcinomas. In addition, p53 gene mutation (exons 5-8) and allelic imbalance at 11q24 loci distal to the ATM locus were also examined. The crypt isolation technique coupled with DNA cytometric sorting and polymerase chain reaction assay using 10 microsatellite markers tightly linked to the ATM gene were used to study ATM allelic imbalance in 55 colorectal carcinomas (15 diploid, 13 aneuploid, 27 multiploid). While allelic imbalance at the ATM locus was rarely observed in diploid and aneuploid carcinomas, multiploid carcinomas exhibited a high frequency of ATM allelic imbalance. In multiploid carcinoma samples, diploid subpopulations showed a smaller range of allelic imbalance at the loci tested compared to aneuploid subpopulations that demonstrated allelic imbalance over a relatively large region. Also, the frequency of AI at 11q24 showed a similar tendency to that at the ATM locus for each DNA ploidy state. An association between p53 gene mutation and ATM allelic imbalance in multiploid carcinoma was also observed. Our results suggest that ATM allelic imbalance and p53 gene mutations occur during the progression from diploid to aneuploid cell populations in multiploid colorectal carcinomas.     

Loss of heterozygosity on chromosome 13q12-q14, BRCA-2 mutations and lack of BRCA-2 promoter hypermethylation in sporadic epithelial ovarian tumors

BACKGROUND: BRCA-1 and BRCA-2 are tumor suppressor genes in familial breast-ovarian carcinoma syndrome. BRCA-1 is also a tumor suppressor gene in sporadic ovarian carcinomas. However, the role of BRCA-2 in sporadic ovarian tumors remains unclear. METHODS: DNA from 52 patients with clinically apparent sporadic ovarian tumors was extracted from blood and from fresh-frozen tumor tissue and normal tissue (10 benign, 7 borderline, and 35 malignant). Loss of heterozygosity (LOH) was analyzed in six microsatellite loci on chromosome 13q. BRCA-2 mutations were detected by single-strand conformation polymorphism analysis and the protein truncation test. BRCA-2 promoter methylation was evaluated by methylation specific polymerase chain reaction analysis. RESULTS: LOH on chromosome 13q12-q14 was identified in 16 tumors (30.8%): Fifteen of these tumors were carcinomas (15 of 35 tumors; 42.8%) and one was a borderline tumor. LOH was frequent in carcinomas with serous differentiation (12 of 16 tumors; 75%). LOH on chromosome 13q12-q14 coexisted with LOH on chromosome 17q in 10 carcinomas. BRCA-2 methylation was not detected in any tumor. BRCA-2 mutations were found in three tumors (one somatic nonsense and two germline frameshift). BRCA-2 fulfilled the two hits for a tumor suppressor gene in these three tumors; in one of them, a BRCA-1 tumor suppressor role had been demonstrated previously. CONCLUSIONS: The results suggest that BRCA-1 and BRCA-2 may act synergically in sporadic ovarian carcinomas with serous differentiation. The demonstration of BRCA-2 germline mutations in patients with ovarian carcinoma with LOH on chromosome 13q12-q14 and lack of a remarkable family history of cancer suggest that the proportion of ovarian carcinomas that result from hereditary predisposition may be higher than previously estimated.     

Absence of PPP2R1A mutations in Wilms tumor

Evidence from genetic linkage analysis indicates that a gene located at 19q13.4, FWT2, is responsible for predisposition to Wilms tumor in many Wilms tumor families. This region has also been implicated in the etiology of sporadic Wilms tumor through loss of heterozygosity analyses. The PPP2R1A gene, encoding the alpha isoform of the heterotrimeric serine/threonine protein phosphatase 2A (PP2A), is located within the FWT2 candidate region and is altered in breast and lung carcinomas. PPP2R1B, encoding the beta isoform, is mutated in lung, colon, and breast cancers. These findings suggested that both PPP2R1A and PPP2R1B may be tumor suppressor genes. Additionally, PP2A is important in fetal kidney growth and differentiation and has an expression pattern similar to that of the Wilms tumor suppressor gene WT1. Since PPP2R1A was therefore a compelling candidate for the FWT2 gene, we analysed the coding region of PPP2R1A in DNA and RNA samples from affected members of four Wilms tumor families and 30 sporadic tumors and identified no mutations in PPP2R1A in any of these 34 samples. We conclude that PPP2R1A is not the 19q familial Wilms tumor gene and that mutation of PPP2R1A is not a common event in the etiology of sporadic Wilms tumor.     

Frequent Allelic Loss at 6q26-27 in Breast Carcinomas of the Solid-tubular Histologic Type

To characterize the effect that inactivation of a putative tumor suppressor gene on 6q appears to have on breast carcinogenesis, we examined loss of heterozygosity in 528 primary breast carcinomas with a polymorphic marker located at 6q26-27, a frequent target region of allelic loss in ovarian and breast cancers. Of the 56 informative tumors of the solid-tubular type, 29 (52%) showed allelic loss at 6q26-27; however, only 10 of 42 papillotubular carcinomas (23%) and 34 of 86 scirrhous carcinomas (39%) showed allelic loss (p=0.0215). These results are consistent with reported alterations at 6q26-27 in serous and mucinous types of ovarian cancers, in that they suggest that inactivation of one or more genes in that region may affect carcinogenic mechanisms in a histologic type-specific manner in neoplasms of the female reproductive organs.     

PPP2R1B gene in chronic lymphocytic leukemias and mantle cell lymphomas

Deletion of chromosome bands 11q22-q23 is one of the most common structural chromosome alterations in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). The PPP2R1B gene is located very close to the minimal common deletion region of 11q22-q23 in CLL and MCL. Recently, the PPP2R1B gene was found to be mutated in human lung and colon cancers. To evaluate the role of the PPP2R1B gene in the pathogenesis of CLL and MCL, we performed RT-PCR analysis and cDNA sequencing on 10 CLL RNA samples and SSCP analysis on 26 CLL and 37 MCL genomic DNA samples. A deletion of exon 3 was found in one CLL sample. No mutation was detected in the SSCP analysis. To exclude the possibility of large genomic deletions we performed Southern blotting analysis. One MCL sample showed abnormal bands. Our results do not suggest that the PPP2R1B gene has a major pathogenic role in CLL and MCL.     

Somatic Mutations of the PTEN/MMAC1 Gene in Fifteen Japanese Endometrial Cancers: Evidence for Inactivation of Both Alleles

Loss of heterozygosity (LOH) of chromosome 10q is observed in approximately 40% of endometrial cancers. Mutations in PTEN/MMAC1 , a gene recently isolated from the 10q23 region, are responsible for two dominantly inherited neoplastic syndromes, Cowden disease and Bannayan-Zonana syndrome. Somatic mutations of this gene have also been detected in sporadic cancers of the brain, prostate and breast. To investigate the potential role of this putative tumor suppressor gene in endometrial carcinogenesis as well, we examined 46 primary endometrial cancers for LOH at the 10q23 region, and for mutations in the entire coding region and exon-intron boundaries of the PTEN/MMAC1 gene. LOH was identified in half of the 38 informative cases, and subtle somatic mutations were detected in 15 tumors (33%). Our results suggest that of the genes studied so far in endometrial carcinomas, PTEN/MMAC1 is the most commonly mutated one, and that inactivation of both copies by allelic loss and/or mutation, a pattern that defines genes as "tumor suppressors,'contributes to tumorigenesis in endometrial cancers.     

Frequent allelic losses and mutations of the p53 gene in human ovarian cancer.

The p53 gene on chromosome 17p is considered to be a tumor suppressor gene, and frequent mutations of the p53 gene have been found in a wide variety of human cancers. We examined 31 ovarian cancers for allelic losses and mutations of the p53 gene by polymerase chain reaction-single strand conformation polymorphism analysis as well as restriction fragment length polymorphism analysis. Allelic loss of the p53 gene was detected in 16 of 20 cases (80%). Mutations were detected in 9 of 31 cases (29%): 2 cases in exon 4; 5 cases in exons 5-6; and 2 cases in exons 7-8. In 8 of 9 cases, p53 mutations were accompanied by losses of the normal allele. These alterations of the p53 gene were commonly detected from stage I to stage IV. These results suggest that alterations of the p53 gene play an important role in the development of human ovarian cancers.     

Refinement of an ovarian cancer tumour suppressor gene locus on chromosome arm 22q and mutation analysis of CYP2D6, SREBP2 and NAGA

Loss of heterozygosity on chromosome 22q was detected in 53% of 123 ovarian carcinomas, suggesting the presence of at least one tumour suppressor gene. We have refined the location of one possible tumour suppressor gene to the region between the microsatellite markers D22S299 and CYP2D. Located within this region are the genes SREBP2 (sterol regulatory element binding protein 2) and NAGA (N-acetyl-alpha-D-galactosaminidase). Investigation of the coding exons of these genes by single stranded conformational polymorphism/heteroduplex analysis failed to identify any somatic genetic alterations in 57 ovarian tumours which exhibited LOH on 22q13. The CYP2D gene locus straddles the distal boundary of the candidate region. Germline variants of the active CYP2D6 gene with differing abilities to metabolise specific substrates have been implicated in the development of various cancers. Comparison of the frequency of the two common germline mutations among 258 ovarian tumours and 231 non-cancer controls did not reveal any significant differences between the two groups. This suggests that the known polymorphic variants of CYP2D6 are not involved in ovarian cancer predisposition. We also conclude that neither NAGA nor SREBP2 are likely to be mutated in ovarian carcinomas.     

Loss of heterozygosity at chromosome 1q loci in rat mammary tumors

To better characterize abnormalities affecting rat chromosome 1 during mammary carcinogenesis, tumors were induced by nitrosomethylurea in F₁ hybrid rats polymorphic at multiple chromosome 1 loci. By means of restriction fragment length polymorphism and microsatellite length polymorphism analyses, we observed loss of heterozygosity or allelic imbalance affecting various loci on the q arm of chromosome 1 in a high percentage of the 49 tumors analyzed. Fifty percent of the tumors showed loss or imbalance affecting the most distal (1q55) INS1 (rat insulin 1 gene) locus. The MT1PA (metallothionein-1 pseudogene a) locus was observed to be affected in 58% of tumors induced in BUF/NCr × ACI/Vsp rats. Most of the losses appeared to have occurred by mitotic recombination. No parental bias was observed on the affected chromosome 1. Tumors were also screened for mutations in codon 12 of the Ha-ras-1 gene, which is located on 1q. We observed an association between the presence of mutation and allelic imbalance. These studies confirm our previous cytogenetic observations of a high level of nonrandom instability affecting rat chromosome 1 during mammary carcinogenesis. The observed loss of heterozygosity may indicate the existence of a putative tumor suppressor gene within the distal half of the 1q arm. These abnormalities, however, could also be related to the early stages of Ha-ras amplification. © 1995 Wiley-Liss Inc.     

Common regions of deletion on chromosomes 5q, 6q, and 10q in renal cell carcinoma.

Relatively frequent losses of heterozygosity on chromosomes 5q, 6q, and 10q, in addition to loss of heterozygosity on the short arm of chromosome 3, have been observed in renal cell carcinomas. As the first step toward isolation of tumor suppressor genes on these three chromosomal arms, we used six restriction fragment length polymorphism markers for 5q, nine for 6q, and eight for 10q to identify regions commonly deleted in a panel of 64 renal cell carcinomas. Allelic losses were common at chromosome 5q21, the region where the MCC (mutated in colorectal cancer) gene was recently identified; at chromosome 6q27; and at chromosome 10q21-23. Furthermore, as association was observed between accumulation of allelic losses on these three chromosomal arms and progression of tumors. Loss of heterozygosity on chromosome 5 showed a correlation with the histopathological grade of a given tumor and the incidence of distant metastasis.     

Cytogenetic and molecular analyses of multiple endocrine neoplasias of the MEN1 syndrome

A pancreas carcinoid, an adrenocortical adenoma, three parathyroid adenomas and a parathyroid hyperplasia of 5 MEN1 patients were used for loss of heterozygosity (LOH) and comparative genomic hybridization (CGH) studies. The MEN1 gene is located in the region 11q13, approximately 30 kb distal to PGYM. Four tumors showed LOH on chromosome 11q13 (D11S11335, PGYM, D11S1883, FGF3, D11S937) however LOH was also found beyond 11q13. The pancreas carcinoid and adrenocortical adenoma, both from the same patient, showed LOH at marker 11q23.3 and 11q25. In the three parathyroid adenomas LOH was detected in five different markers: 11q21, 11q22.3, 11q23.2, 11q23.3 and 11q25. No LOH was found in parathyroid hyperplasia. CGH analysis showed in case of the pancreas carcinoid losses on chromosomes 1p, 2q, 3, 6, 9p, 11 and 12p. Gains were found at 4, 5, 7, 8, 13, 14, 15q, 18, 19. The parathyroid adenoma of the third patient showed losses only on chromosome 11 in the region 11p12-p15 and 11q12-q23. Our data indicate that other genes are involved in the tumorigenesis of the MEN1 syndrome. Especially the numerous allelic losses between markers 11q23 and 11q25 (D11S938 and D11S910) are a hint for further tumor suppressor genes on chromosome 11.