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Frequent Loss of PTEN Expression Is Linked to Elevated Phosphorylated Akt Levels, but Not Associated with p27 and Cyclin D1 Expression, in Primary Epithelial Ovarian Carcinomas

2001; Elsevier BV; Volume: 158; Issue: 6 Linguagem: Inglês

10.1016/s0002-9440(10)64681-0

ISSN

1525-2191

Autores

Keisuke Kurose, Xiao-Ping Zhou, Tsutomu Araki, Stephen A. Cannistra, Eamonn R. Maher, Charis Eng,

Tópico(s)

Polyamine Metabolism and Applications

Resumo

PTEN (MMAC1/TEP1), a tumor suppressor gene on chromosome subband 10q23.3, is variably mutated and/or deleted in a variety of human cancers. Germline mutations in PTEN, which encode a dual-specificity phosphatase, have been implicated in at least two hamartoma tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Among several series of ovarian cancers, the frequency of loss of heterozygosity (LOH) of markers flanking and within PTEN, is ∼30 to 50%, and the somatic intragenic PTEN mutation frequency is <10%. In this study, we screened primary adenocarcinomas of the ovary for LOH of polymorphic markers within and flanking the PTEN gene and for intragenic mutations of the PTEN gene and compared them to PTEN expression using immunohistochemistry. Furthermore, we sought to detect the expression of the presumed downstream targets of PTEN, such as P-Akt, p27, and cyclin D1 by immunohistochemistry. LOH at 10q23 was observed in 29 of 64 (45%) cases. Of the 117 samples, 6 somatic intragenic PTEN mutations, 1 germline mutation, and 1 novel polymorphism were found in 7 (6%) patients. Immunostaining of 49 ovarian cancer samples revealed that 13 (27%) were PTEN immunostain-negative, 25 (51%) had reduced staining, and the rest (22%) were PTEN expression-positive. Among the 44 informative tumors assessed for 10q23 LOH and PTEN immunostaining, there was an association between 10q23 LOH and decreased or absent staining (P = 0.0317). Of note, there were five (11%) tumors with neither mutation nor deletion that exhibited no PTEN expression and 10 (25%) others without mutation or deletion but had decreased PTEN expression. Among the 49 tumors available for immunohistochemistry, 28 (57%) showed P-Akt-positive staining, 24 (49%) had decreased p27 staining, and cyclin D1 was overexpressed in 35 (79%) cases. In general, P-Akt expression was inversely correlated with PTEN expression (P = 0.0083). These data suggest that disruption of PTEN by several mechanisms, allelic loss, intragenic mutation, or epigenetic silencing, all contribute to epithelial ovarian carcinogenesis, and that epigenetic silencing is a significant mechanism. The Akt pathway is prominently involved, but clearly not in all cases. Surprisingly, despite in vitro demonstration that p27 and cyclin D1 lies downstream of PTEN and Akt, there was no correlation between p27 and cyclin D1 expression and PTEN or P-Akt status. Thus, in vivo, although PTEN and Akt play a prominent role in ovarian carcinogenesis, p27 and cyclin D1 might not be the primary downstream targets. Alternatively, these observations could also suggest that pathways involving other than Akt, p27 and cyclin D1 that lie downstream of PTEN play roles in ovarian carcinogenesis. PTEN (MMAC1/TEP1), a tumor suppressor gene on chromosome subband 10q23.3, is variably mutated and/or deleted in a variety of human cancers. Germline mutations in PTEN, which encode a dual-specificity phosphatase, have been implicated in at least two hamartoma tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Among several series of ovarian cancers, the frequency of loss of heterozygosity (LOH) of markers flanking and within PTEN, is ∼30 to 50%, and the somatic intragenic PTEN mutation frequency is <10%. In this study, we screened primary adenocarcinomas of the ovary for LOH of polymorphic markers within and flanking the PTEN gene and for intragenic mutations of the PTEN gene and compared them to PTEN expression using immunohistochemistry. Furthermore, we sought to detect the expression of the presumed downstream targets of PTEN, such as P-Akt, p27, and cyclin D1 by immunohistochemistry. LOH at 10q23 was observed in 29 of 64 (45%) cases. Of the 117 samples, 6 somatic intragenic PTEN mutations, 1 germline mutation, and 1 novel polymorphism were found in 7 (6%) patients. Immunostaining of 49 ovarian cancer samples revealed that 13 (27%) were PTEN immunostain-negative, 25 (51%) had reduced staining, and the rest (22%) were PTEN expression-positive. Among the 44 informative tumors assessed for 10q23 LOH and PTEN immunostaining, there was an association between 10q23 LOH and decreased or absent staining (P = 0.0317). Of note, there were five (11%) tumors with neither mutation nor deletion that exhibited no PTEN expression and 10 (25%) others without mutation or deletion but had decreased PTEN expression. Among the 49 tumors available for immunohistochemistry, 28 (57%) showed P-Akt-positive staining, 24 (49%) had decreased p27 staining, and cyclin D1 was overexpressed in 35 (79%) cases. In general, P-Akt expression was inversely correlated with PTEN expression (P = 0.0083). These data suggest that disruption of PTEN by several mechanisms, allelic loss, intragenic mutation, or epigenetic silencing, all contribute to epithelial ovarian carcinogenesis, and that epigenetic silencing is a significant mechanism. The Akt pathway is prominently involved, but clearly not in all cases. Surprisingly, despite in vitro demonstration that p27 and cyclin D1 lies downstream of PTEN and Akt, there was no correlation between p27 and cyclin D1 expression and PTEN or P-Akt status. Thus, in vivo, although PTEN and Akt play a prominent role in ovarian carcinogenesis, p27 and cyclin D1 might not be the primary downstream targets. Alternatively, these observations could also suggest that pathways involving other than Akt, p27 and cyclin D1 that lie downstream of PTEN play roles in ovarian carcinogenesis. Ovarian cancer is the most common cancer in women to be diagnosed at an advanced stage and is the fifth leading cause of cancer deaths among women in the United States.1Landis SH Murray T Bolden S Wingo PA Cancer statistics, 1999.CA Cancer J Clin. 1999; 49: 8-31Crossref PubMed Scopus (3136) Google Scholar Various genetic alterations have been associated with ovarian cancer, such as somatic amplification of the oncogenes HER-2/neu, KRAS, and C-MYC, somatic mutation of the TP53 tumor suppressor gene and germline mutations of BRCA1 and BRCA2.2Piver MS Baker TR Piedmonte M Sandecki AM Epidemiology and etiology of ovarian cancer.Semin Oncol. 1991; 18: 177-185PubMed Google Scholar, 3Pejovic T Genetic changes in ovarian cancer.Ann Med. 1995; 27: 73-78Crossref PubMed Scopus (64) Google Scholar, 4Berchuck A Carney M Lancaster JM Marks J Futreal AP Familial breast-ovarian cancer syndromes: BRCA1 and BRCA2.Clin Obstet Gynecol. 1998; 41: 157-166Crossref PubMed Scopus (19) Google Scholar Because carcinogenesis is a multistep process,5Fearon ER Vogelstein B A genetic model for colorectal tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (10136) Google Scholar it is likely that several as yet unknown genes play a role in the development and/or progression of ovarian cancer. Previous studies focusing on genetic changes occurring during ovarian cancer development have revealed clonal abnormalities in many chromosomal regions. Earlier allelotyping studies of ovarian cancer found loss of heterozygosity (LOH) at 10q in 11 to 21% of ovarian cancers, although only three markers, not in the 10q23 region, were used.6Sato T Saito H Morita R Koi S Lee JH Nakamura Y Allelotype of human ovarian cancer.Cancer Res. 1991; 51: 5118-5122PubMed Google Scholar, 7Cliby W Ritland S Hartmann L Dodson M Halling KC Keeney G Podratz KC Jenkins RB Human epithelial ovarian cancer allelotype.Cancer Res. 1993; 53: 2393-2398PubMed Google Scholar Recent reports using microsatellite markers that were specifically chosen for 10q23 identified a LOH frequency of 31 to 52%.8Teng DH Hu R Lin H Davis T Iliev D Frye C Swedlund B Hansen KL Vinson VL Gumpper KL Ellis L El-Naggar A Frazier M Jasser S Langford LA Lee J Mills GB Pershouse MA Pollack RE Tornos C Troncoso P Yung WK Fujii G Berson A Bookstein R Bolen JB Tavtigian SV Steck PA MMAC1/PTEN mutations in primary tumor specimens and tumor cell lines.Cancer Res. 1997; 57: 5221-5225PubMed Google Scholar, 9Obata K Morland SJ Watson RH Hitchcock A Chenevix-Trench G Thomas EJ Campbell IG Frequent PTEN/MMAC mutations in endometrioid but not serous or mucinous epithelial ovarian tumors.Cancer Res. 1998; 58: 2095-2097PubMed Google Scholar, 10Saito M Okamoto A Kohno T Takakura S Shinozaki H Isonishi S Yasuhara T Yoshimura T Ohtake Y Ochiai K Yokota J Tanaka T Allelic imbalance and mutations of the PTEN gene in ovarian cancer.Int J Cancer. 2000; 85: 160-165PubMed Google Scholar The tumor suppressor gene PTEN/MMAC1/TEP1, encoding a dual-specificity phosphatase, has been cloned and mapped to chromosome subband 10q23.3.11Li DM Sun H TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta.Cancer Res. 1997; 57: 2124-2129PubMed Google Scholar, 12Li J Yen C Liaw D Podsypanina K Bose S Wang SI Puc J Miliaresis C Rodgers L McCombie R Bigner SH Giovanella BC Ittmann M Tycko B Hibshoosh H Wigler MH Parsons R PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer.Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4329) Google Scholar, 13Steck PA Pershouse MA Jasser SA Yung WK Lin H Ligon AH Langford LA Baumgard ML Hattier T Davis T Frye C Hu R Swedlund B Teng DH Tavtigian SV Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers.Nat Genet. 1997; 15: 356-362Crossref PubMed Scopus (2534) Google Scholar Germline PTEN mutations have been found in the inherited autosomal-dominant Cowden and Bannayan-Riley-Ruvalcaba syndromes, which are characterized by multiple hamartomas and by an increased risk of malignant and benign breast and thyroid tumors.14Liaw D Marsh DJ Li J Dahia PL Wang SI Zheng Z Bose S Call KM Tsou HC Peacocke M Eng C Parsons R Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome.Nat Genet. 1997; 16: 64-67Crossref PubMed Scopus (1713) Google Scholar, 15Marsh DJ Dahia PL Zheng Z Liaw D Parsons R Gorlin RJ Eng C Germline mutations in PTEN are present in Bannayan-Zonana syndrome.Nat Genet. 1997; 16: 333-334Crossref PubMed Scopus (575) Google Scholar, 16Marsh DJ Coulon V Lunetta KL Rocca-Serra P Dahia PL Zheng Z Liaw D Caron S Duboue B Lin AY Richardson AL Bonnetblanc JM Bressieux JM Cabarrot-Moreau A Chompret A Demange L Eeles RA Yahanda AM Fearon ER Fricker JP Gorlin RJ Hodgson SV Huson S Lacombe D LePrat F Odent S Toulouse C Olopade OI Sobol H Tishler S Woods CG Robinson BG Weber HC Parsons R Peacocke M Longy M Eng C Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation.Hum Mol Genet. 1998; 7: 507-515Crossref PubMed Scopus (558) Google Scholar, 17Eng C Genetics of Cowden syndrome: through the looking glass of oncology.Int J Oncol. 1998; 12: 701-710PubMed Google Scholar, 18Eng C Ji H Molecular classification of the inherited hamartoma polyposis syndromes: clearing the muddied waters.Am J Hum Genet. 1998; 62: 1020-1022Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 19Marsh DJ Kum JB Lunetta KL Bennett MJ Gorlin RJ Ahmed SF Bodurtha J Crowe C Curtis MA Dasouki M Dunn T Feit H Geraghty MT Graham JMJ Hodgson SV Hunter A Korf BR Manchester D Miesfeldt S Murday VA Nathanson KL Parisi M Pober B Romano C Tolmie JL Trembath R Winter RM Zackai EH Zori RT Weng LP Dahia PL Eng C PTEN mutation spectrum and genotype-phenotype correlations in Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden syndrome.Hum Mol Genet. 1999; 8: 1461-1472Crossref PubMed Scopus (483) Google Scholar Recently, a Proteus-like syndrome was found to result from germline and germline mosaic PTEN mutations.20Zhou XP Marsh DJ Hampel H Mulliken JB Gimm O Eng C Germline and germline mosaic PTEN mutations associated with a Proteus-like syndrome of hemihypertrophy, lower limb asymmetry, arteriovenous malformations and lipomatosis.Hum Mol Genet. 2000; 9: 765-768Crossref PubMed Scopus (175) Google Scholar Ovarian cancer, in general, is not considered part of these syndromes. Somatic mutation and/or deletion of PTEN occurs to a greater or lesser extent in a wide variety of human cancers that show LOH in this region, including glioblastoma, endometrial cancer, prostate cancer, and breast cancer.12Li J Yen C Liaw D Podsypanina K Bose S Wang SI Puc J Miliaresis C Rodgers L McCombie R Bigner SH Giovanella BC Ittmann M Tycko B Hibshoosh H Wigler MH Parsons R PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer.Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4329) Google Scholar, 13Steck PA Pershouse MA Jasser SA Yung WK Lin H Ligon AH Langford LA Baumgard ML Hattier T Davis T Frye C Hu R Swedlund B Teng DH Tavtigian SV Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers.Nat Genet. 1997; 15: 356-362Crossref PubMed Scopus (2534) Google Scholar, 21Duerr EM Rollbrocker B Hayashi Y Peters N Meyer-Puttlitz B Louis DN Schramm J Wiestler OD Parsons R Eng C von Deimling A PTEN mutations in gliomas and glioneuronal tumors.Oncogene. 1998; 16: 2259-2264Crossref PubMed Scopus (313) Google Scholar, 22Feilotter HE Nagai MA Boag AH Eng C Mulligan LM Analysis of PTEN and the 10q23 region in primary prostate carcinomas.Oncogene. 1998; 16: 1743-1748Crossref PubMed Scopus (180) Google Scholar, 23Kong D Suzuki A Zou TT Sakurada A Kemp LW Wakatsuki S Yokoyama T Yamakawa H Furukawa T Sato M Ohuchi N Sato S Yin J Wang S Abraham JM Souza RF Smolinski KN Meltzer SJ Horii A PTEN1 is frequently mutated in primary endometrial carcinomas.Nat Genet. 1997; 17: 143-144Crossref PubMed Scopus (311) Google Scholar, 24Mutter GL Lin MC Fitzgerald JT Kum JB Baak JP Lees JA Weng LP Eng C Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers.J Natl Cancer Inst. 2000; 92: 924-930Crossref PubMed Google Scholar Genetic, functional, and animal modeling studies have substantiated the tumor suppressor function of PTEN. PTEN is a lipid phosphatase whose major substrate is phosphatidylinositol-3,4,5-triphosphate [PtdIns(3,4,5)P3], downstream of which lies the Akt (PKB) pathway.25Maehama T Dixon JE The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate.J Biol Chem. 1998; 273: 13375-13378Crossref PubMed Scopus (2621) Google Scholar, 26Stambolic V Suzuki A de la Pompa JL Brothers GM Mirtsos C Sasaki T Ruland J Penninger JM Siderovski DP Mak TW Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN.Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2127) Google Scholar, 27Li J Simpson L Takahashi M Miliaresis C Myers MP Tonks N Parsons R The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene.Cancer Res. 1998; 58: 5667-5672PubMed Google Scholar, 28Furnari FB Huang HJ Cavenee WK The phosphoinositol phosphatase activity of PTEN mediates a serum-sensitive G1 growth arrest in glioma cells.Cancer Res. 1998; 58: 5002-5008PubMed Google Scholar, 29Dahia PL Aguiar RC Alberta J Kum JB Caron S Sill H Marsh DJ Ritz J Freedman A Stiles C Eng C PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanisms in haematological malignancies.Hum Mol Genet. 1999; 8: 185-193Crossref PubMed Scopus (268) Google Scholar The serine-threonine kinase Akt, when phosphorylated, protects cells from apoptosis.30Dudek H Datta SR Franke TF Birnbaum MJ Yao R Cooper GM Segal RA Kaplan DR Greenberg ME Regulation of neuronal survival by the serine-threonine protein kinase Akt.Science. 1997; 275: 661-665Crossref PubMed Scopus (2230) Google Scholar, 31Datta SR Dudek H Tao X Masters S Fu H Gotoh Y Greenberg ME Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4969) Google Scholar PTEN may also be involved in cell migration, spreading, and focal adhesion formation through dephosphorylating focal adhesion kinase, presumably through its protein phosphatase activity.32Tamura M Gu J Matsumoto K Aota S Parsons R Yamada KM Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN.Science. 1998; 280: 1614-1617Crossref PubMed Scopus (1084) Google Scholar, 33Gu J Tamura M Pankov R Danen EH Takino T Matsumoto K Yamada KM Shc and FAK differentially regulate cell motility and directionality modulated by PTEN.J Cell Biol. 1999; 146: 389-403Crossref PubMed Scopus (376) Google Scholar Ectopic expression of PTEN results in cell-cycle arrest at G1 and/or apoptosis at least in the glioma and breast cancer cell line models.27Li J Simpson L Takahashi M Miliaresis C Myers MP Tonks N Parsons R The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene.Cancer Res. 1998; 58: 5667-5672PubMed Google Scholar, 28Furnari FB Huang HJ Cavenee WK The phosphoinositol phosphatase activity of PTEN mediates a serum-sensitive G1 growth arrest in glioma cells.Cancer Res. 1998; 58: 5002-5008PubMed Google Scholar, 34Weng LP Smith WM Dahia PL Ziebold U Gil E Lees JA Eng C PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death.Cancer Res. 1999; 59: 5808-5814PubMed Google Scholar It has been demonstrated that this function of PTEN is Akt-dependent, and can be rescued by expression of phosphorylated Akt.27Li J Simpson L Takahashi M Miliaresis C Myers MP Tonks N Parsons R The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene.Cancer Res. 1998; 58: 5667-5672PubMed Google Scholar, 35Ramaswamy S Nakamura N Vazquez F Batt DB Perera S Roberts TM Sellers WR Regulation of G1 progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway.Proc Natl Acad Sci USA. 1999; 96: 2110-2115Crossref PubMed Scopus (512) Google Scholar One of the targets of PTEN in its ability to block cell-cycle progression at the G1 phase has been suggested to be the cyclin-dependent kinase inhibitor p27.28Furnari FB Huang HJ Cavenee WK The phosphoinositol phosphatase activity of PTEN mediates a serum-sensitive G1 growth arrest in glioma cells.Cancer Res. 1998; 58: 5002-5008PubMed Google Scholar, 36Li DM Sun H PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells.Proc Natl Acad Sci USA. 1998; 95: 15406-15411Crossref PubMed Scopus (441) Google Scholar, 37Cheney IW Neuteboom ST Vaillancourt MT Ramachandra M Bookstein R Adenovirus-mediated gene transfer of MMAC1/PTEN to glioblastoma cells inhibits S phase entry by the recruitment of p27Kip1 into cyclin E/CDK2 complexes.Cancer Res. 1999; 59: 2318-2323PubMed Google Scholar, 38Bruni P Boccia A Baldassarre G Trapasso F Santoro M Chiappetta G Fusco A Viglietto G PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells mediated by p27kip1.Oncogene. 2000; 19: 3146-3155Crossref PubMed Scopus (129) Google Scholar It has been shown that up-regulation of p27 by PTEN has occurred in different cell lines.36Li DM Sun H PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells.Proc Natl Acad Sci USA. 1998; 95: 15406-15411Crossref PubMed Scopus (441) Google Scholar, 37Cheney IW Neuteboom ST Vaillancourt MT Ramachandra M Bookstein R Adenovirus-mediated gene transfer of MMAC1/PTEN to glioblastoma cells inhibits S phase entry by the recruitment of p27Kip1 into cyclin E/CDK2 complexes.Cancer Res. 1999; 59: 2318-2323PubMed Google Scholar However, it remains to be established whether the increase of p27 expression is truly a direct effect of PTEN′s action in vivo and which intermediate steps are involved in synthesis of this cell-cycle inhibition signal. Another potential mechanism of cell-cycle control by PTEN may be through inhibition of cyclin D1 accumulation. AKT phosphorylates GSK3 (glycogen synthase kinase 3), leading to its inactivation.39Cross DA Alessi DR Cohen P Andjelkovich M Hemmings BA Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B.Nature. 1995; 378: 785-789Crossref PubMed Scopus (4426) Google Scholar Active GSK3 phosphorylates cyclin D1, targeting it for degradation.40Diehl JA Cheng M Roussel MF Sherr CJ Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization.Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1873) Google Scholar Therefore, Akt seems to promote cyclin D1 accumulation.41Coffer PJ Jin J Woodgett JR Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation.Biochem J. 1998; 335: 1-13Crossref PubMed Scopus (972) Google Scholar, 42Cantley LC Neel BG New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway.Proc Natl Acad Sci USA. 1999; 96: 4240-4245Crossref PubMed Scopus (1761) Google Scholar Although only rare mutations of the PTEN gene were reported in ovarian cancer,8Teng DH Hu R Lin H Davis T Iliev D Frye C Swedlund B Hansen KL Vinson VL Gumpper KL Ellis L El-Naggar A Frazier M Jasser S Langford LA Lee J Mills GB Pershouse MA Pollack RE Tornos C Troncoso P Yung WK Fujii G Berson A Bookstein R Bolen JB Tavtigian SV Steck PA MMAC1/PTEN mutations in primary tumor specimens and tumor cell lines.Cancer Res. 1997; 57: 5221-5225PubMed Google Scholar, 9Obata K Morland SJ Watson RH Hitchcock A Chenevix-Trench G Thomas EJ Campbell IG Frequent PTEN/MMAC mutations in endometrioid but not serous or mucinous epithelial ovarian tumors.Cancer Res. 1998; 58: 2095-2097PubMed Google Scholar, 10Saito M Okamoto A Kohno T Takakura S Shinozaki H Isonishi S Yasuhara T Yoshimura T Ohtake Y Ochiai K Yokota J Tanaka T Allelic imbalance and mutations of the PTEN gene in ovarian cancer.Int J Cancer. 2000; 85: 160-165PubMed Google Scholar, 43Sakurada A Suzuki A Sato M Yamakawa H Orikasa K Uyeno S Ono T Ohuchi N Fujimura S Horii A Infrequent genetic alterations of the PTEN/MMAC1 gene in Japanese patients with primary cancers of the breast, lung, pancreas, kidney, and ovary.Jpn J Cancer Res. 1997; 88: 1025-1028Crossref PubMed Scopus (101) Google Scholar, 44Tashiro H Blazes MS Wu R Cho KR Bose S Wang SI Li J Parsons R Ellenson LH Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignancies.Cancer Res. 1997; 57: 3935-3940PubMed Google Scholar, 45Maxwell GL Risinger JI Tong B Shaw H Barrett JC Berchuck A Futreal PA Mutation of the PTEN tumor suppressor gene is not a feature of ovarian cancers.Gynecol Oncol. 1998; 70: 13-16Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 46Yokomizo A Tindall DJ Hartmann L Jenkins RB Smith DI Liu W Mutation analysis of the putative tumor suppressor PTEN/MMAC1 in human ovarian cancer.Int J Oncol. 1998; 13: 101-105PubMed Google Scholar because of PTEN′s role in the cell cycle and cell death as well as the gene's localization to 10q23, PTEN is an excellent candidate to play an important role in ovarian carcinogenesis. Therefore, we sought to determine whether structural alterations in PTEN occurred with any frequency in ovarian cancer, if loss of PTEN expression, detected by immunohistochemistry, is a major mechanism of loss of function, and if there is a correlation between structural alterations of PTEN gene and PTEN protein expression. Further, we looked for any alteration in the expression of the presumed downstream targets of PTEN, such as P-Akt, p27, and cyclin D1 by immunohistochemistry, and investigated the correlation among all these variables. One hundred seventeen epithelial ovarian tumors were obtained from patients undergoing surgery for primary epithelial ovarian cancer. Thirty-eight tumors were obtained from The Ohio State University, Columbus, OH (OSU), 25 were from the Beth Israel-Deaconess Medical Center, Boston, MA (BOS), 31 were from the University of Birmingham, UK (UK), and 23 were from Nippon Medical School, Tokyo, Japan (NMS). Apart from the 31 ovarian carcinomas from the UK that are of unknown histological sub-type, 32 were serous tumors, 28 were endometrioid cancers, 7 were clear cell cancers, 6 were mucinous tumors, 6 were mixed epithelial tumors, 6 were undifferentiated tumors, and 1 was a squamous cell carcinoma. Corresponding noncancerous tissues were available only from OSU, BOS, and NMS samples. Corresponding paraffin blocks were available only from OSU and BOS samples. Genomic DNA was extracted from tumor and matched normal tissue with a QIAamp DNA Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions, or by microdissection of normal and tumor areas of paraffin-embedded archival tissue using standard protocols.47Eng C Thomas GA Neuberg DS Mulligan LM Healey CS Houghton C Frilling A Raue F Williams ED Ponder BA Mutation of the RET proto-oncogene is correlated with RET immunostaining in subpopulations of cells in sporadic medullary thyroid carcinoma.J Clin Endocrinol Metab. 1998; 83: 4310-4313PubMed Google Scholar Sixty-eight ovarian cancers (35 from OSU, 11 from BOS, and 22 from NMS) in which both tumor and corresponding normal DNA were available were analyzed for LOH at five polymorphic markers flanking and within the PTEN gene. The markers are ordered from centromere to telomere: D10S579-D10S1765-IVS4 + 109ins/delTCTTA-IVS8 + 32T/G-D10S541. PTEN lies between D10S1765 and D10S541, a genetic distance of 1 cM but a physical distance of only several hundred kilobase pairs. D10S1765 is within 500 kb upstream of the transcriptional start site of PTEN and D10S541 is within 300 kb of the translational stop site. IVS4 + 109ins/delTCTTA and IVS8 + 32T/G lie within PTEN.19Marsh DJ Kum JB Lunetta KL Bennett MJ Gorlin RJ Ahmed SF Bodurtha J Crowe C Curtis MA Dasouki M Dunn T Feit H Geraghty MT Graham JMJ Hodgson SV Hunter A Korf BR Manchester D Miesfeldt S Murday VA Nathanson KL Parisi M Pober B Romano C Tolmie JL Trembath R Winter RM Zackai EH Zori RT Weng LP Dahia PL Eng C PTEN mutation spectrum and genotype-phenotype correlations in Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden syndrome.Hum Mol Genet. 1999; 8: 1461-1472Crossref PubMed Scopus (483) Google Scholar, 48Dahia PL Marsh DJ Zheng Z Zedenius J Komminoth P Frisk T Wallin G Parsons R Longy M Larsson C Eng C Somatic deletions and mutations in the Cowden disease gene, PTEN, in sporadic thyroid tumors.Cancer Res. 1997; 57: 4710-4713PubMed Google Scholar, 49Carroll BT Couch FJ Rebbeck TR Weber BL Polymorphisms in PTEN in breast cancer families.J Med Genet. 1999; 36: 94-96PubMed Google Scholar The IVS4 + 109ins/delTCTTA and IVS8 + 32T/G polymorphisms were screened for by differential digestion with Afl II and Hin cII, respectively, according to the manufacturer's guidelines (New England Biolabs, Beverly, MA) as described previously.16Marsh DJ Coulon V Lunetta KL Rocca-Serra P Dahia PL Zheng Z Liaw D Caron S Duboue B Lin AY Richardson AL Bonnetblanc JM Bressieux JM Cabarrot-Moreau A Chompret A Demange L Eeles RA Yahanda AM Fearon ER Fricker JP Gorlin RJ Hodgson SV Huson S Lacombe D LePrat F Odent S Toulouse C Olopade OI Sobol H Tishler S Woods CG Robinson BG Weber HC Parsons R Peacocke M Longy M Eng C Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation.Hum Mol Genet. 1998; 7: 507-515Crossref PubMed Scopus (558) Google Scholar, 48Dahia PL Marsh DJ Zheng Z Zedenius J Komminoth P Frisk T Wallin G Parsons R Longy M Larsson C Eng C Somatic deletions and mutations in the Cowden disease gene, PTEN, in sporadic thyroid tumors.Cancer Res. 1997; 57: 4710-4713PubMed Google Scholar D10S579, D10S1765, and D10S541 were screened as documented previously.50Gimm O Perren A Weng LP Marsh DJ Yeh JJ Ziebold U Gil E Hinze R Delbridge L Lees JA Mutter GL Robinson BG Komminoth P Dralle H Eng C Differential nuclear and cytoplasmic expression of PTEN in normal thyroid tissue, and benign and malignant epithelial thyroid tumors.Am J Pathol. 2000; 156: 1693-1700Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar Polymerase chain reaction (PCR) conditions for these markers are described elsewhere.51Marsh DJ Roth S Lunetta KL Hemminki A Dahia PL Sistonen P Zheng Z Caron S van Orsouw NJ Bodmer WF Cottrell SE Dunlop MG Eccles D Hodgson SV Jarvinen H Kellokumpu I Markie D Neale K Phillips R Rozen P Syngal S Vijg J Tomlinson IP Aaltonen LA Eng C Exclusion of PTEN and 10q22-24 as the susceptibility locus for juvenile polyposis syndrome.Cancer Res. 1997; 57: 5017-5021PubMed Google Scholar, 52Marsh DJ Dahia PL Coulon V Zheng Z Dorion-Bonnet F Call KM Little R Lin AY Eeles RA Goldstein AM Hodgson SV Richardson AL Robinson BG Weber HC Longy M Eng C Allelic imbalance, including deletion of PTEN/MMACI, at the Cowden disease locus on 10q22-23, in hamartomas from patients with Cowden syndrome and germline PTEN mutation.Genes Chromosom Cancer. 1998; 21: 61-69Crossref PubMed Scopus (98) Google Scholar All samples were scanned for mutations by denaturing gradient gel electrophoresis (DGGE) as previously described.24Mutter GL Lin MC Fitzgerald JT Kum JB Baak JP Lees JA Weng LP Eng C Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers.J Natl Cancer Inst. 2000; 92: 924-930Crossref PubMed Google Scholar The entire PTEN- coding region, exon-intron boundaries, and flanking intronic sequences were PCR amplified and fractionated through denaturing gradient gels according to the conditions

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