Somatic mutation of PTEN in bladder carcinoma.

The tumour suppressor gene PTEN/MMAC1, which is mutated or homozygously deleted in glioma, breast and prostate cancer, is mapped to a region of 10q which shows loss of heterozygosity (LOH) in bladder cancer. We screened 123 bladder tumours for LOH in the region of PTEN. In 53 informative muscle invasive tumours (> or = pT2), allele loss was detected in 13 (24.5%) and allelic imbalance in four tumours (overall frequency 32%). LOH was found in four of 60 (6.6%) informative, non-invasive tumours (pTa/pT1). We screened 63 muscle invasive tumours for PTEN mutations by single-strand conformation polymorphism (SSCP) analysis and for homozygous deletion by duplex quantitative polymerase chain reaction (PCR). Two homozygous deletions were identified but no mutations. Of 15 bladder tumour cell lines analysed, three showed homozygous deletion of all or part of the PTEN gene, but none had mutations detectable by SSCP analysis. Our results indicate that PTEN is involved in the development of some bladder tumours. The low frequency of mutation of the retained allele in tumours with 10q23 LOH suggests that there may be another predominant mechanism of inactivation of the second allele, for example small intragenic deletions, that hemizygosity may be sufficient for phenotypic effect, or that there is another target gene at 10q23.     

Rare-type Mutations of MMAC1 Tumor Suppressor Gene in Human Glioma Cell Lines and Their Tumors of Origin

A total of 10 glioma cell lines were examined to evaluate the status of the MMAC1 gene, a candidate tumor suppressor gene. Six cell lines showed mutations with presumed loss of heterozygosity and 1 cell line showed no mRNA expression. The 6 mutations consisted of 3 3-bp deletions (codons 17, 101 or 199), 1 missense mutation (codon 252) and 2 truncation mutations (1 nonsense mutation at codon 233 and 1 2-bp insertion at codon 241). Among them, the 3-bp deletions, which are a rare type of mutation in MMAC1 gene, were located in the N-terminal half (codons 1–212) of the coding region, which is considered important in MMAC1 function. The missense mutation was located unusually in the C-terminal half (codons 212–403), but it was in a small region in which some other reported missense mutations are clustered. Thus, these 4 mutations were suggested to have functional effects on the MMAC1 activity, like the other 2 mutations with predicted protein truncations. By sequence analysis of cDNA clones, we confirmed that all the mutations including these 4 rare ones were in the MMAC1 gene, not in the PTH2 pseudogene. In 2 cases, we also examined the primary glioma tissues from which the cell lines had been derived and found the same mutations as in the cell lines in both cases. This suggested that the mutations in these cell lines were derived from the primary glioma tissues, but not from artifacts arising during long-term in vitro cultivation.     

Rare-type mutations of MMAC1 tumor suppressor gene in human glioma cell lines and their tumors of origin.

A total of 10 glioma cell lines were examined to evaluate the status of the MMAC1 gene, a candidate tumor suppressor gene. Six cell lines showed mutations with presumed loss of heterozygosity and 1 cell line showed no mRNA expression. The 6 mutations consisted of 3 3-bp deletions (codons 17, 101 or 199), 1 missense mutation (codon 252) and 2 truncation mutations (1 nonsense mutation at codon 233 and 12-bp insertion at codon 241). Among them, the 3-bp deletions, which are a rare type of mutation in MMAC1 gene, were located in the N-terminal half (codons 1-212) of the coding region, which is considered important in MMAC1 function. The missense mutation was located unusually in the C-terminal half (codons 212-403), but it was in a small region in which some other reported missense mutations are clustered. Thus, these 4 mutations were suggested to have functional effects on the MMAC1 activity, like the other 2 mutations with predicted protein truncations. By sequence analysis of cDNA clones, we confirmed that all the mutations including these 4 rare ones were in the MMAC1 gene, not in the PTH2 pseudogene. In 2 cases, we also examined the primary glioma tissues from which the cell lines had been derived and found the same mutations as in the cell lines in both cases. This suggested that the mutations in these cell lines were derived from the primary glioma tissues, but not from artifacts arising during long-term in vitro cultivation.     

Mutation and expression of the DCC gene in human lung cancer

Chromosome 18q is frequently deleted in lung cancers, and a common region of 18q deletions was mapped to chromosome 18q21. Since the DCC candidate tumor suppressor gene has been mapped in this region, mutation and expression of the DCC gene were examined in 46 lung cancer cell lines, consisting of 14 small cell lung carcinomas (SCLCs) and 32 non-small cell lung carcinomas (NSCLCs), to elucidate the pathogenetic significance of DCC alterations in human lung carcinogenesis. A heterozygous missense mutation was detected in a NSCLC cell line, Ma26, while homozygous deletion was not detected in any of the cell lines. The DCC gene was expressed in 11 (24%) of the 46 cell lines, and the incidence of DCC expression was significantly higher in SCLCs (7/14, 50%) than in NSCLCs (4/32, 13%) (P = .01, Fisher's exact test). Therefore, genetic alterations of DCC are infrequent; however, the levels of DCC expression vary among lung cancer cells, in particular, between SCLCs and NSCLCs. The present result does not implicate DCC as a specific mutational target of 18q deletions in human lung cancer; however, it suggests that DCC is a potential target of inactivation by genetic defects including intron or promoter mutations and/or epigenetic alterations. The present result also suggests that DCC expression is associated with some properties of SCLCs, such as a neuroendocrine (NE) feature.     

Evidence for two candidate tumour suppressor loci on chromosome 9q in transitional cell carcinoma (TCC) of the bladder but no homozygous deletions in bladder tumour cell lines.

The most frequent genetic alterations in transitional cell carcinoma (TCC) of the bladder involve loss of heterozygosity (LOH) on chromosome 9p and 9q. The LOH on chromosome 9p most likely targets the CDKN2 locus, which is inactivated in about 50% of TCCs. Candidate genes that are the target for LOH on chromosome 9q have yet to be identified. To narrow the localization of one or more putative tumour suppressor genes on this chromosome that play a role in TCC of the bladder, we examined 59 tumours with a panel of microsatellite markers along the chromosome. LOH was observed in 26 (44%) tumours. We present evidence for two different loci on the long arm of chromosome 9 where potential tumour suppressor genes are expected. These loci are delineated by interstitial deletions in two bladder tumours. Our results confirm the results of others and contribute to a further reduction of the size of these regions, which we called TCC1 and TCC2. These regions were examined for homozygous deletions with EST and STS markers. No homozygous deletions were observed in 17 different bladder tumour cell lines.     

Abnormal structure and expression of PTEN/MMAC1 gene in human uterine cancers

The PTEN/MMAC1 gene, located on human chromosome 10q23, has recently been implicated as a candidate tumor suppressor gene in human cancers. In the present study, 12 uterine cancer cell lines and 87 uterine cancers of various grades and histological type were analyzed for PTEN/MMAC1 gene. Three of 44 endometrial carcinoma (7%) showed no PTEN/MMAC1 mRNA expression by RT-PCR analysis. Sequencing analysis of entire coding region of PTEN/MMAC1 gene revealed mutations in three of six endometrial cancer cell lines (50%) and 17 of 44 endometrial cancer tissues (39%). In contrast, for cervical cancers, only one of six cancer cell lines (2%) showed mutation, and one of 43 cancer tissues (2%) had an abnormality. Overall, 36% of the abnormal spots were located in exon 5, 24% were in exon 8, 16% were in exon 3, and 8% were in exon 6, and single cases of abnormality were found in exons 1, 4, and 7. Our results revealed that, in total, 60% of abnormalities were clustered in exons 5 and 8. Exon 5 is a functional domain of the PEN/MMAC1 gene, and therefore, abnormalities in this region may be important for loss of PTEN/MMAC1 gene function. Finally, we found a high frequency of PTEN/MMAC1 gene abnormalities in endometrial carcinomas but a low frequency in cervical carcinomas. These findings suggest that disruption of PTEN/MMAC1 by mutation or absence of expression may contribute to the pathogenesis or neoplastic evolution in a large proportion of endometrial carcinomas but in a small proportion of cervical carcinomas.     

Frequent inactivation of the BAP1 gene in epithelioid-type malignant mesothelioma

In the present study, we analyzed genomic alterations of BRCA1-associated protein 1 (BAP1) in 23 malignant mesotheliomas (MMs), 16 epithelioid and seven non-epithelioid, consisting of 18 clinical specimens and five established cell lines. In examining these samples for homozygous deletions and sequence-level mutations, we found biallelic BAP1 gene alterations in 14 of 23 MMs (61%). Seven of these 14 MMs had homozygous deletions of the partial or entire BAP1 gene, another five had sequence-level mutations, including small deletions, a nonsense mutation, and missense mutations with additional monoallelic deletions, and the remaining two had homozygous mutations without allelic loss. All but one of the 14 BAP1 gene mutations were found in the epithelioid-type MMs; BAP1 mutations were found in 13 of 16 epithelioid-type MMs, but in only one of seven non-epithelioid-type MMs (13/16 vs 1/7; P = 0.005). There was no BAP1 mRNA expression in MMs with biallelic deletion and repressed expression was confirmed in MM specimens with deletion/mutation as compared with Met5a, SV40-transformed normal mesothelial cells. Western blot showed that seven of eight epithelioid MMs analyzed were BAP1 negative. Immunostaining with anti-BAP1 antibody in normal lung tissues revealed clear nuclear staining of normal mesothelial cells. No nuclear staining was observed among BAP1 mutation-positive MM tumors, whereas nuclear staining was observed among BAP1 mutation-negative MM tumors. These results suggest that the lack of the tumor suppressor BAP1 may be more specifically involved in the pathogenesis of epithelioid MM rather than non-epithelioid MM, and would be useful for diagnosis of epithelioid-type MM.     

Relative reciprocity of NRAS and PTEN/MMAC1 alterations in cutaneous melanoma cell lines

Both inactivation of the tumor suppressor gene, PTEN/MMAC1, and oncogenic activation of RAS have been described in human cutaneous melanoma. In mice, activation of a RAS-containing pathway is a necessary step in the pathogenesis of murine melanomas. Because PTEN negatively regulates on the downstream effects of phosphatidylinositol-3-kinase (PI3-K), we hypothesized that the loss of PTEN/MMAC1 and the activation of RAS may be largely equivalent because RAS is a known positive upstream regulator of PI3-K. We expanded our previous survey of PTEN/MMAC1 mutations and analyzed the RAS status of 53 cutaneous melanoma cell lines, 18 glioma cell lines, and 17 uncultured cutaneous melanoma metastasis. Overall, 51% of the cell lines had alterations in either PTEN/MMAC1 or RAS. We found 16 cell lines (30%) with alterations in PTEN/MMAC1 and 11 cell lines (21%) with activating NRAS mutations; only 1 cell line had concurrent alterations in both genes. Moreover, glioma cell lines with a high frequency of PTEN/MMAC1 inactivation had no identifiable RAS alterations. Ectopic expression of PTEN in several cutaneous melanoma cell lines suppressed colony formation irrespective of PTEN/MMAC1 status; furthermore, PTEN expression in cell lines carrying activated RAS also suppressed colony formation. The relative reciprocity of PTEN/MMAC1 abrogation and NRAS activation suggests that the two genetic changes, in a subset of cutaneous melanomas, are functionally overlapping.     

p16 involvement in primary bladder tumors: analysis of deletions and mutations

Different studies have proposed that genetic alterations leading to inactivation of a tumor suppressor gene on chromosome 9 is an important early event in bladder tumorigenesis. Recent reports have described the p16 gene as the main target. In order to better define its role, we studied 9p21 deletions by microsatellite analysis and its coding sequence. Forty-eight percent of the 44 samples we studied showed LOH surrounding p16. Three of these 44 samples displayed point mutations in p16 and three others were suspected of homozygous deletion. These results suggest that simultaneous loss of both p16 alleles, by point mutation or homozygous deletion, seems to be infrequent in bladder tumors.     

Expression and sequence analysis of candidates for the 1p36.31 tumor suppressor gene deleted in neuroblastomas

Neuroblastomas are characterized by 1p deletions, suggesting that a tumor suppressor gene (TSG) resides in this region. We have mapped the smallest region of deletion (SRD) to a 2 Mb region of 1p36.31 using microsatellite and single nucleotide polymorphisms. We have identified 23 genes in this region, and we have analysed these genes for mutations and RNA expression patterns to identify candidate TSGs. We sequenced the coding exons of these genes in 30 neuroblastoma cell lines. Although rare mutations were found in 10 of the 23 genes, none showed a pattern of genetic change consistent with homozygous inactivation. We examined the expression of these 23 genes in 20 neuroblastoma cell lines, and most showed readily detectable expression, and no correlation with 1p deletion. However, 7 genes showed uniformly low expression in the lines, and 2 genes (CHD5, RNF207) had virtually absent expression, consistent with the expected pattern for a TSG. Our mutation and expression analysis in neuroblastoma cell lines, combined with expression analysis in normal tissues, putative function and prior implication in neuroblastoma pathogenesis, suggests that the most promising TSG deleted from the 1p36 SRD is CHD5, but TNFRSF25, CAMTA1 and AJAP1 are also viable candidates.     

PTEN/MMAC1 mutations in hepatocellular carcinomas: somatic inactivation of both alleles in tumors.

Allelic loss of loci on chromosome 10q occurs frequently in hepatocellular carcinomas. Somatic mutations of the PTEN/MMAC1 gene on this chromosome at 10q23 were recently identified in sporadic cancers of the uterus, brain, prostate and breast. To investigate the potential role of PTEN/MMAC1 gene in the genesis of hepatocellular carcinomas, we examined 96 tumors for allelic loss on 10q and also for subtle mutations anywhere within the coding region of PTEN/MMAC1 gene. Allelic loss was identified in 25 of the 89 (27%) tumors that were informative for polymorphic markers in the region. Somatic mutations were identified in five of those tumors: three frameshift mutations, a 1-bp insertion at codon 83-84 in exon 4 and two 4-bp deletions, both at codon 318-319 in exon 8; two C-to-G transversion mutation, both at -9 bp from the initiation codon in the 5' non-coding region of exon 1. No missense mutation was observed in this panel of tumors. In most of the informative tumors carrying intragenic mutations of one allele, we were able to detect loss of heterozygosity as well. These findings suggest that two alleles of the PTEN/MMAC1 gene may be inactivated by a combination of intragenic point mutation on one allele and loss of chromosomal material on the other allele in some of these tumors.     

Genetic alterations of the tumor suppressor gene PTEN/MMAC1 in human brain metastases.

The high mutation rate in advanced brain tumors, recent functional studies, and the high frequency of mutations in prostate metastases all strongly suggest that PTEN/MMAC1 alterations are involved in the formation of metastases. We searched for genetic alterations in the PTEN/MMAC1 gene in 56 consecutive brain metastases from various primary tumors by loss of heterozygosity (LOH), direct sequence analysis, and differential PCR analysis. The highest LOH rates were detected in metastases deriving from lung (67%) and breast (64%) cancers. Three (25%) of the eight detected inactivating mutations (one nonsense mutation, one splice-site mutation, one 11-bp deletion, and five homozygous deletions) were found in metastases originating from 12 different lung carcinomas, suggesting that PTEN/MMAC1 alterations may play a role in the progression of this tumor. With the exception of lung carcinomas, our findings indicate that genetic abnormalities of the PTENM/MMAC1 gene are only involved in a relatively small subset of brain metastases. However, the discrepancy between the high overall LOH rate (50%) and the low frequency of PTEN/MMAC1 mutation detection rate (14%) suggests the presence of one or more additional tumor suppressor genes on chromosome 10q.     

PTEN/MMAC1 Mutations in Hepatocellular Carcinomas: Somatic Inactivation of Both Alleles in Tumors

Allelic loss of loci on chromosome 10q occurs frequently in hepatocellular carcinomas. Somatic mutations of the PTEN/MMAC1 gene on this chromosome at 10q23 were recently identified in sporadic cancers of the uterus, brain, prostate and breast. To investigate the potential role of PTEN/MMAC1 gene in the genesis of hepatocellular carcinomas, we examined 96 tumors for allelic loss on 10q and also for subtle mutations anywhere within the coding region of PTEN/MMAC1 gene. Allelic loss was identified in 25 of the 89 (27%) tumors that were informative for polymorphic markers in the region. Somatic mutations were identified in five of those tumors: three frameshift mutations, a 1-bp insertion at codon 83–84 in exon 4 and two 4-bp deletions, both at codon 318–319 in exon 8; two C-to-G transversion mutation, both at -9 bp from the initiation codon in the 5'non-coding region of exon 1. No missense mutation was observed in this panel of tumors. In most of the informative tumors carrying intragenic mutations of one allele, we were able to detect loss of heterozygosity as well. These findings suggest that two alleles of the PTEN/MMAC1 gene may be inactivated by a combination of intragenic point mutation on one allele and loss of chromosomal material on the other allele in some of these tumors.     

Rare mutations and no hypermethylation at the CDKN2A locus in epithelial ovarian tumours

The tumour-suppressor gene CDKN2A (p16, MTS1, CDK4I) encodes a cell cycle-regulatory protein and is located on chromosome 9p21, a region deleted in a wide variety of human cancers. To determine the role of the CDKN2A gene in the development of ovarian adenocarcinomas, we examined a large series of benign, low malignant potential (LMP) and invasive ovarian neoplasms for evidence of loss of heterozygosity (LOH), homozygous deletions, point mutations and hypermethylation of the CDKN2A locus. We have previously reported LOH on 9p in 45% of malignant ovarian neoplasms and a smaller percentage of benign and LMP tumours. In the current study, 6 malignant tumours were identified with partial deletions of 9p21. In 5 of these, the CDKN2A gene lays within the minimal deleted region. Homozygous deletions of CDKN2A were observed in only 2/88 invasive ovarian tumours and in 5/11 ovarian cancer cell lines. Of 15 primary ovarian tumours analyzed, one nonsense mutation was identified in a mucinous LMP tumour. No evidence of hypermethylation of the CDKN2A gene was found in 50 primary ovarian adenocarcinomas nor in 3 ovarian cancer cell lines. In conclusion, homozygous deletions, mutations and the de novo methylation of 5′ CpG island are not frequent modes of inactivation of the CDKN2A gene in ovarian cancer. The target of 9p LOH in ovarian adenocarcinomas is therefore unknown. Int. J. Cancer 70:508–511. © 1997 Wiley-Liss Inc.     

An intronic variant of the TGFBR1 gene is associated with carcinomas of the kidney and bladder

TGF-beta signaling is frequently perturbed in many human cancers, including renal cell carcinomas (RCCs) and transitional cell carcinomas (TCCs) of the bladder. Genetic alterations of the TGF-beta type 1 receptor (TGFBR1) may contribute to these perturbations. We therefore examined variations in the TGFBR1 gene by PCR, SSCP and RFLP in carcinomas of the urinary system and in tissues from noncancer, age-matched controls. A G-->A variant 24 bp downstream of the exon/intron 7 boundary of the TGFBR1 gene (Int7G24A) was evident in patients with RCC (46.5%, n = 86) and bladder and upper urinary tract TCC (49.2%, n = 65) significantly more frequently than in age-matched controls (28.3%, n = 113, p < 0.002 by chi2 test). Moreover, 8 homozygous variant carriers were found in the cancer groups, whereas not a single homozygous variant carrier was found in the control group. The Int7G24A allele (both heterozygous G/A and homozygous A/A carriers) was associated with increased RCC incidence (OR = 2.20, 95% CI 1.22-3.96) and TCC incidence (OR = 2.45, 95% CI 1.89-3.16). One somatic mutation of serine to phenylalanine at codon 57 of the TGFBR1 gene was confirmed in an upper urinary tract TCC. In conclusion, the Int7G24A variant in the TGFBR1 gene is significantly more frequent in patients with RCC and TCC than normal age-matched controls, suggesting that it may represent a risk factor for the development of kidney and bladder carcinomas.     

MMAC1/PTEN inhibits cell growth and induces chemosensitivity to doxorubicin in human bladder cancer cells

The development and progression of bladder cancer is associated with multiple alterations in the genome, including loss of chromosome 10. Recently, MMAC1/PTEN, a phosphatidylinositol phosphatase, has been mapped to chromosome 10q23. We previously demonstrated that MMAC1/PTEN has tumor suppressive properties in glioblastoma and prostate cancer. To investigate the efficacy of gene therapy with MMAC1/PTEN, we examined whether the exogenous introduction of MMAC1/PTEN via an adenoviral vector (Ad-MMAC) can inhibit tumor growth and reverse drug resistance to doxorubicin in human bladder cancer cells. Human bladder cancer cell lines UM-UC-3 and T24 were infected with Ad-MMAC to induce exogenous expression of MMAC1/PTEN. The cells were then analysed for cell growth and expression of phosphorylated protein kinase B (Akt/PKB) and MMAC1/PTEN. UM-UC-6dox, a doxorubicin resistant subline, was infected with Ad-MMAC to evaluate its role in reversing drug resistance to doxorubicin. We found that MMAC1/PTEN suppressed tumor growth in UM-UC-3 and T24 cells with arrest in the G1 phase of the cell cycle. We also showed that gene therapy with MMAC1/PTEN abrogated phosphorylated Akt/PKB expression in UM-UC-3, T24 and UMUC-6dox cells, and restored doxorubicin sensitivity in UM-UC-6dox. These data demonstrate that MMAC1/PTEN can induce growth suppression and increase sensitivity to doxorubicin in bladder cancer cells and suggest that the MMAC1/PTEN gene and its pathways can be therapeutic targets for bladder cancer.     

Loss of heterozygosity at 10q in tumors of the upper respiratory tract is associated with poor prognosis.

Frequent loss of a specific chromosomic region in cancers is often associated with inactivation of a tumor-suppressor gene. The long arm of chromosome 10 is deleted in several types of tumor, among them squamous-cell carcinomas of the head and neck (HNSCC). To determine the role of 10q deletions in the tumorigenesis of the upper respiratory tract, 47 HNSCCs were examined for loss of heterozygosity (LOH) at 10q: 43% of the cases analyzed showed LOH at 10q, and 2 distinct hot spots of deletion were identified, at 10q22-23 and 10q25-26. The possible involvement of pTEN/MMAC1, a tumor-suppressor gene mapped at 10q23, was also evaluated. No mutation, homozygous deletion or loss of expression of pTEN/MMAC1 was detected, indicating that inactivation of this gene plays a minor role in HNSCC development. Interestingly, the frequency of deletion at 10q was greater in invasive carcinoma than in adjacent carcinoma in situ, and a significant association between LOH and poor prognosis was observed. Taken together, our results suggest the presence in the long arm of chromosome 10 of (a) tumor-suppressor gene(s) other than pTEN/MMAC1 and presumably involved in the malignant progression of tumors of the upper respiratory tract. Int. J. Cancer (Pred. Oncol.) 84:432-436, 1999.