Frequent inactivation of PTEN in prostate cancer cell lines and xenografts

Loss of chromosome 10q is a frequently observed genetic defect in prostate cancer. Recently, the PTEN/MMAC1 tumor suppressor gene was identified and mapped to chromosome 10q23.3. We studied PTEN structure and expression in 4 in vitro cell lines and 11 in vivo xenografts derived from six primary and nine metastatic human prostate cancers. DNA samples were allelotyped for eight polymorphic markers within and surrounding the PTEN gene. Additionally, the nine PTEN exons were tested for deletions. In five samples (PC3, PC133, PCEW, PC295, and PC324), homozygous deletions of the PTEN gene or parts of the gene were detected. PC295 contained a small homozygous deletion encompassing PTEN exon 5. In two DNAs (PC82 and PC346), nonsense mutations were found, and in two (LNCaP and PC374), frame-shift mutations were found. Missense mutations were not detected. PTEN mRNA expression was clearly observed in all cell lines and xenografts without large homozygous deletions, showing that PTEN down-regulation is not an important mechanism of PTEN inactivation. The high frequency (60%) of PTEN mutations and deletions indicates a significant role of this tumor suppressor gene in the pathogenesis of prostate cancer.     

Identification of PTEN/MMAC1 alterations in uncultured melanomas and melanoma cell lines

A novel tumor suppressor gene, PTEN/MMAC1, has been recently shown to be mutated in gliomas, breast, prostate, kidney cancers and melanomas. Loss-of-heterozygosity studies in melanoma have suggested the presence of at least one chromosome 10q locus lost early in tumor progression. In this study, we screened 45 melanoma cell lines and 17 paired uncultured metastatic melanoma and peripheral blood specimens for PTEN/ MMAC1 alterations using PCR-SSCP and direct sequencing. We found nine melanoma cell lines with homozygous deletions (five with intragenic loss) and four cell lines with mutations (one nonsense and one frameshift; two intronic); from among our uncultured melanoma specimens, we found one tumor with a somatic 17 bp duplication in exon 7 leading to a premature stop codon and one tumor with a possible homozygous deletion. Furthermore, we have identified a novel intragenic polymorphism within intron 4 of PTEN/MMAC1. Taken together, these data suggest that PTEN/MMAC1 may be a chromosome 10q tumor suppressor important in melanoma tumor formation or progression.     

Point mutation and homozygous deletion of PTEN/MMAC1 in primary bladder cancers

A new tumor suppressor gene PTEN/MMAC1 was recently isolated at chromosome 10q23 and found to be inactivated by point mutation or homozygous deletion in glioma, prostate and breast cancer. PTEN/MMAC1 was also identified as the gene predisposing to Cowden disease, an autosomal dominant cancer predisposition syndrome associated with an increased risk of breast, skin and thyroid tumors and occasional cases of other cancers including bladder and renal cell carcinoma. We screened 345 urinary tract cancers by microsatellite analysis and found chromosome 10q to be deleted in 65 of 285 (23%) bladder and 15 of 60 (25%) renal cell cancers. We then screened the entire PTEN/MMAC1 coding region for mutation in 25 bladder and 15 renal cell primary tumors with deletion of chromosome 10q. Two somatic point mutations, a frameshift and a splicing variant, were found in the panel of bladder tumors while no mutation was observed in the renal cell carcinomas. To screen for homozygous deletion, we isolated two polymorphic microsatellite repeats from genomic BAC clones containing the PTEN/MMAC1 gene. Using these new informative markers, we identified apparent retention at the gene locus indicative of homozygous deletion of PTEN/MMAC1 in four of 65 bladder and 0 of 15 renal cell tumors with LOH through chromosome 10q. Identification of the second inactivation event in six bladder tumors with LOH of 10q implies that the PTEN/MMAC1 gene is occasionally involved in bladder tumorigenesis. However, the low frequency of biallelic inactivation suggests that either PTEN/MMAC1 is inactivated by other mechanisms or it is not the only target of chromosome 10q deletion in primary bladder and renal cell cancer.     

Mutations in the p16INK4/MTS1/CDKN2, p15INK4B/MTS2, and p18 genes in primary and metastatic lung cancer

We examined the genomic status of cyclin-dependent kinase-4 and -6 inhibitors, p16INK4,p15INK4B, and p18, in 40 primary lung cancers and 31 metastatic lung cancers. Alterations of the p16INK4 gene were detected in 6 (2 insertions and 4 homozygous deletions) of 22 metastatic non-small cell lung cancers (NSCLCs; 27%), but none were detected in 25 primary NSCLCs, 15 primary small cell lung cancers (SCLCs), or 9 metastatic SCLCs, indicating that mutation in the p16INK4 gene is a late event in NSCLC carcinogenesis. Although three intragenic mutations of the p15INK4B gene were detected in 25 primary NSCLCs (12%) and five homozygous deletions of the p15INK4B gene were detected in 22 NSCLCs (23%), no genetic alterations of the p15INK4B gene were found in primary and metastatic SCLCs. The p18 gene was wild type in these 71 lung cancers, except 1 metastatic NSCLC which showed loss of heterozygosity. We also examined alterations of these three genes and expression of p16INK4 in 21 human lung cancer cell lines. Alterations of the p16INK4 and p15INK4B genes were detected in 71% of the NSCLC cell lines (n = 14) and 50% of the NSCLC cell lines (n = 14), respectively, but there were none in the 7 SCLC cell lines studied. No p18 mutations were detected in these 21 cell lines. These results indicate that both p16INK4 and p15INK4B gene mutations are associated with tumor progression of a subset of NSCLC, but not of SCLC, and that p15INK4B mutations might also be an early event in the molecular pathogenesis of a subset of NSCLC.     

PTEN gene alterations in lymphoid neoplasms

Recently, a novel phosphatase designated PTEN/MMAC1/TEP1 and located on chromosome 10q23.3 has been implicated as a new tumor suppressor gene in human cancer. Allelic loss and mutation of this gene has been reported in epithelial derived tumors, including breast cancer and prostate cancer, and in glioblastoma multiforme. The present study was designed to evaluate the potential involvement of PTEN in the pathogenesis of lymphoid neoplasms. We analyzed 27 hematopoietic cell lines (representing a variety of lymphoid lineages), 65 primary lymphoid tumors (including 24 lymphoblastic leukemia/lymphoma [LBL], 30 large B-cell lymphoma [LBCL], 7 Burkitt's lymphoma [BL], and 4 anaplastic large cell lymphoma [ALCL]), and 25 nonmalignant lymph node controls. Gene deletion and gross rearrangement were evaluated using Southern blot analysis, and mutations were studied by polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) (PCR-SSCP) and sequencing. Six of 27 cell lines (22.2%) and 3 of 65 primary lymphomas (4.6%) contained alterations of this gene. A large homozygous deletion spanning exons 2 through 5 was detected in one LBL cell line, and two insertions potentially resulting in premature termination, were detected in a second LBL cell line. Nonconservative nucleotide variations were found in two other cell lines (one LBCL and one BL) and in one primary case of LBCL. In addition, two other cell lines (one BL and one myeloma) and two primary lymphomas, both LBCL, contained small deletions within intron 7. These deletions mapped to a poly-T-rich tract just 5' to the intron 7/exon 8 spice site. Their significance is unclear, as they may represent polymorphisms. Overall, our results suggest that abnormalities of the PTEN gene can contribute to pathogenesis in a small percentage of malignant lymphomas.     

Genotypic analysis of tumor suppressor genes PTEN/MMAC1 and p53 in head and neck squamous cell carcinomas

OBJECTIVES: Tumor suppressor gene mutations in both p53 and PTEN/MMAC1 genomic DNA have been detected in many types of cancer. The purpose of this study was to investigate the presence and importance of PTEN/MMAC1 mutations in squamous cell carcinomas. METHODS: Exons of each gene were amplified after polymerase chain reaction (PCR) using genomic DNA derived from cell lines of squamous cell carcinoma of the head and neck (SCCHN) and snap-frozen biopsy specimens from primary established head and neck tumors. The amplified and purified DNA was then sequenced directly. RESULT: As anticipated, point mutations of the p53 gene were found in 80% of cell lines examined. A single base mutation in codon 151 was found in six of 10 cell lines studied. PTEN/MMAC1 gene mutations were found in neither the cell lines tested nor the tumor biopsy samples. CONCLUSION: This study, as well as a large volume of data, confirms that mutations of the p53 gene are frequent events in head and neck cancer cell lines. Although PTEN/MMAC1 gene mutations have been found in a variety of carcinomas, this gene was not found to be mutated in SCCHN cell lines or in primary squamous cell carcinomas of the head and neck. This information is useful for further studies of mutations in these cell lines.     

Loss of heterozygosity and mutational analysis of the PTEN/MMAC1 gene in synchronous endometrial and ovarian carcinomas

Mutations of the human putative protein tyrosine phosphatase (PTEN/MMAC1) gene at chromosome 10q23 have been found frequently in type I endometrial carcinomas. Endometrioid adenocarcinoma is the most frequent histology seen in patients with clinically determined synchronous endometrial and ovarian carcinomas. We report a high incidence of PTEN/MMAC1 mutations and 10q23 loss of heterozygosity (LOH) in patients with synchronous endometrial and ovarian carcinomas. Paraffin-embedded precision microdissected tumors were analyzed for 10 matched synchronous endometrial and ovarian cancers and 11 matched control metastatic endometrial cancers. Single-stranded conformation polymorphism analysis was used to screen for mutations in all tumors and corresponding normal lymphocyte DNA. LOH was determined using a panel of four microsatellite markers within the PTEN/MMAC1 locus. PTEN/MMAC1 mutations were found in 43% (9 of 21) of the endometrial cancers studied, similarly represented in the clinically synchronous group (5 of 10 or 50%) and the advanced metastatic group (4 of 11; 36%; P = 0.53). In two of the five cases of clinically synchronous cancers, identical or progressive PTEN mutations were found in both the endometrial and ovarian cancers, suggesting that the ovarian tumor is a metastasis from the endometrial primary. PTEN/MMAC1 mutations in the advanced endometrial cancers were similar in the corresponding metastases. In one case, the mutation was seen in only one of two metastatic lymph nodes. The LOH analysis demonstrated 55% LOH in at least one PTEN/MMAC1 marker. These findings suggest that the putative tumor suppressor gene PTEN/MMAC1 may be a viable molecular marker to differentiate synchronous versus metastatic disease in a subset of clinically synchronous endometrial and ovarian carcinomas.     

PTEN mutations in gliomas and glioneuronal tumors

Cytogenetic and loss of heterozygosity studies have suggested the presence of at least one tumor suppressor gene on chromosome 10 involved in the formation of high grade gliomas. Recently, the PTEN gene, also termed MMAC1 or TEP1, on chromosomal band 10q23 has been identified. Initial studies revealed mutations of PTEN in limited series of glioma cell lines and glioblastomas. In order to systematically evaluate the involvement of PTEN in gliomas, we have analysed the entire PTEN coding sequence by SSCP and direct sequencing in a series of 331 gliomas and glioneuronal tumors. PTEN mutations were detected in 20/142 glioblastomas, 1/7 giant cell glioblastomas, 1/2 gliosarcomas, 1/30 pilocytic astrocytomas and 2/22 oligodendrogliomas. No PTEN mutations were detected in 52 astrocytomas, 37 oligoastrocytomas, three subependymal giant cell astrocytomas, four pleomorphic xanthoastrocytomas, 15 ependymomas, 16 gangliogliomas and one dysembryoplastic neuroepithelial tumor. In addition, all tumors were examined for the presence of homozygous deletions of the PTEN gene; these were detected in 7 glioblastomas that did not have PTEN mutations. Therefore, PTEN mutations occur in approximately 20% of glioblastomas but are rare in lower grade gliomas. These findings confirm that PTEN is one of the chromosome 10 tumor suppressor genes involved in the development of glioblastomas.     

Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma

The MMAC1/PTEN gene, located at 10q23.3, is a candidate tumor suppressor commonly mutated in glioma. We have studied the pattern of deletion, mutation, and expression of MMAC1/PTEN in 35 unrelated melanoma cell lines. Nine (26%) of the cell lines showed partial or complete homozygous deletion of the MMAC1/PTEN gene, and another six (17%) harbored a mutation in combination with loss of the second allele. Mutations could also be demonstrated in uncultured tumor specimens from which the cell lines had been established, and cell lines derived from two different metastases from one individual carried the same missense mutation. Collectively, these findings suggest that disruption of MMAC1/PTEN by allelic loss or mutation may contribute to the pathogenesis or neoplastic evolution in a large proportion of malignant melanomas.     

Genomic organization and mutation analysis of Hel-N1 in lung cancers with chromosome 9p21 deletions

Allelic loss of chromosome 9p21 is common in small cell lung cancer (SCLC), but inactivation of the tumor suppressor gene CDKN2a is rare, implying the existence of another target gene at 9p21. A recent deletion mapping study of chromosome 9p has also identified a site of deletion in non-small cell lung cancer (NSCLC) centered around D9S126. The Hel-N1 (human elav-like neuronal protein 1) gene encodes a neural-specific RNA binding protein that is expressed in SCLC. We have mapped this potentially important gene in lung tumorigenesis to within 100 kb of the D9S126 marker at chromosome band 9p21 by using homozygously deleted tumor cell lines and fluorescence in situ hybridization to normal metaphase spreads. Hel-N1 is, therefore, a candidate target suppressor gene in both SCLC and NSCLC. We have determined the genomic organization and intron/exon boundaries of Hel-N1 and have screened the entire coding region for mutations by sequencing 14 primary SCLCs and cell lines and 21 primary NSCLCs preselected for localized 9p21 deletion or monosomy of chromosome 9. A homozygous deletion including Hel-N1 and CDKN2a was found in a SCLC cell line, and a single-base polymorphism in exon 2 of Hel-N1 was observed in eight tumors. No somatic mutations of Hel-N1 were found in this panel of lung tumors. Hel-N1 does not appear to be a primary inactivation target of 9p21 deletion in lung cancer.     

The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway

The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.     

PTEN/MMAC1 mutations in endometrial cancers

Endometrial carcinomas represent the most common gynecological cancer in the United States, yet the molecular genetic events that underlie the development of these tumors remain obscure. Chromosome 10 is implicated in the pathogenesis of endometrial carcinoma based on loss of heterozygosity (LOH), comparative genomic hybridization, and cytogenetics. Recently, a potential tumor suppressor gene, PTEN/MMAC1, with homology to dual-specificity phosphatases and to the cytoskeletal proteins tensin and auxillin was identified on chromosome 10. This gene is mutated in several types of advanced tumors that display frequent LOH on chromosome 10, most notably glioblastomas. Additionally, germ-line mutations of PTEN/MMAC1 are responsible for several familial neoplastic disorders, including Cowden disease and Bannayan-Zonana syndrome. Because this locus is included in the region of LOH in many endometrial carcinomas, we examined 70 endometrial carcinomas for alterations in PTEN/MMAC1. Somatic mutations were detected in 24 cases (34%) including 21 cases that resulted in premature truncation of the protein, 2 tumors with missense alterations in the conserved phosphatase domain, and 1 tumor with a large insertion. These data indicate that PTEN/MMAC1 is more commonly mutated than any other known gene in endometrial cancers.     

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.     

Mutational analysis of CDKN2 (MTS1/p16ink4) in human breast carcinomas

The CDKN2 gene that encodes the cell cycle regulatory protein cyclin-dependent kinase-4 inhibitor (p16) has recently been mapped to chromosome 9p21. Frequent homozygous deletions of this gene have been documented in cell lines derived from different types of tumors, including breast tumors, suggesting that CDKN2 is a tumor suppressor gene involved in a wide variety of human cancers. To determine the frequency of CDKN2 mutations in breast carcinomas, we screened 37 primary tumors and 5 established breast tumor cell lines by single-strand conformation polymorphism analysis. In addition, Southern blot analysis was performed on a set of five primary breast carcinoma samples and five breast tumor cell lines. Two of the five tumor cell lines revealed a homozygous deletion of the CDKN2 gene, but no mutations were observed in any of the primary breast carcinomas. These results suggest that the mutation of the CDKN2 gene may not be a critical genetic change in the formation of primary breast carcinoma.