1) ゲノム不安定性・遺伝子変異に基づく子宮体癌の浸潤能獲得と分子生物学的予後不良因子の検討(シンポジウム1:腫瘍「婦人科癌の浸潤・転移機構の解明と臨床応用」,第65回日本産科婦人科学会・学術講演会講演要旨)
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Background: Genomic instability, such as chromosomal instability (CI) and microsatellite instability (MSI), is known to be associated with cancer progression in various tumors. Endometrial cancer is one of the tumor types, in which either CI or MSI may occur. MSI is caused by deficiency of DNA mismatch repair genes (MMR), including MLH1 and MSH2, and is associated with mutator phenotype. MSI is commonly observed in endometrial and colorectal cancer. Indeed, Lynch syndrome (HNPCC or Hereditary nonpolyposis colorectal cancer) has a high risk of colon cancer and other cancers including endometrium, ovary and stomach, due to inherited mutations in MMR. Chromosomal instability (CI) is measured by the number of chromosomal copy alterations within entire tumor genomes, and the CI phenotypes can be classified according to the level of alterations, such as CI-high, CI-Intermediate and CI-Negative. It is not fully understood whether these two genomic instabilities are correlated each other. Endometrial cancer is also known to possess mutations frequently in Ras-PI3K (phosphatidylinositol 3'-kinase) pathway, such as PTEN and K-Ras. The aims of this study are (i) to characterize genomic instability, (ii) to identify biomarker(s) to predict prognosis, (iii) to understand profiles of genetic mutations (alterations), (iv) to clarify the invasionrelated functions of genetic mutations and (v) to explore molecular targeted therapies on basis of genetic mutations with identification of biomarkers to predict the sensitivity in endometrial cancer. Methods: A comprehensive genomic survey was performed in 47 endometrial carcinomas with paired DNA for chromosomal imbalances by SNP (Single Nucleotide Polymorphism) typing array and screened 32 samples for MSI status. Mutational analysis, including PIK3CA, AKT1 genes, was also performed in 89 endometrial cancer specimens. Anchorage-independent growth was analyzed by soft agar assays. Immortalized human epithelial cells were transfected with mutant K-Ras and/or mutant PIK3CA. The antitumor effect of NVP-BEZ235 (a dual PI3K/mTOR inhibitor) and RAD001 (an mTOR inhibitor) was analyzed on a panel of 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). Results: Five or more copy number alterations (CNAs), classified as CI-high, were observed in 13 (28%), one to four CNAs (CI-intermediate) in 23 (49%) and no CNAs (CI-negative) in 11 (23%) tumors. MSI-high with 3 or more alterations among 5 microsatellite markers was identified in11 (35%) patients. MSI-high was less common in CI-high tumors (11%), compared with CI-intermediate/negative tumors (43%). Multivariate analysis showed that CI-high is an independent poor prognostic factor. Copy number losses at the locus of PTEN and NF1 (negative regulator of Ras) were observed at 29% and 13%, respectively. Copy number gains at the locus of K-Ras and PIK3CA were also found at 13% and 13%, respectively. Mutations of PIK3CA and AKT1 were reported in other types of tumors, but not in endometrial cancers. In this study, mutations of PIK3CA were identified in of 24 (36%) of 66 endometrial carcinomas. Mutations of AKT1 were also observed in 2 of 89 endometrial carcinomas. Coexistence of PIK3 CA/PTEN mutations occurred at high frequency (26%). PTEN and K-Ras were mutated at 73% and 30%, respectively, in PIK3CA mutant tumors, whereas they were mutated at 51% and 12%, respectively, in PIK3CA wild-type tumors. Combined with CNAs, Ras-PI3K pathway activating mutations were detected at 96% in endometrial carcinomas. PIK3CA mutations were almost exclusively detected in invasive tumors, although the other PI3K activating alterations were also common in non-invasive tumors, such as atypical endometrial hyperplasia. Therefore, PIK3CA mutation is a later event than the other alterations. In soft agar assay, introduction of either mutant PIK3CA alone or mutant K-Ras mutation alone failed to transform the immortalized epithelial cells, however, introduction of mutant PIK3CA with mutant K-Ras transformed the cells, suggesting that mutant PIK3CA cooperates with mutant K-Ras to cause tumor invasion. PTEN mutant cell lines without K-Ras alterations (n=9) were more sensitive to NVP-BEZ235 than were cell lines with K-Ras alterations (n=4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD 001 in the sensitive cell lines. G1 arrest was strongly induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or U0126, sensitized the K-Ras mutant cells to NVP-BEZ235. Conclusions: 1. Genomic instability, such as MSI and CI, is a tumor-specific characteristic in endometrial cancer. 2. Ras-PI3K pathway is broadly activated in endometrial cancer through various types of alterations (genetic mutations and CNAs in PTEN, PIK3CA, AKT1, K-Ras and NF1). 3. Mutant PIK3CA cooperates with other PI3K pathway mutations, such as mutant K-Ras, to effect oncogenic transformation from non-invasive tumor to invasive cancer. 4. CI status can be easily evaluated by genome-wide copy number analysis, and CI-high is a promising biomarker to predict poor prognosis in endometrial cancer. 5. A dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Alterations of PTEN and PIK3CA might be useful biomarkers for sensitivity, whereas alterations of K-Ras might be for resistance to a dual PI3K/mTOR inhibitor.
- 2013-10-01
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- 1) ゲノム不安定性・遺伝子変異に基づく子宮体癌の浸潤能獲得と分子生物学的予後不良因子の検討(シンポジウム1:腫瘍「婦人科癌の浸潤・転移機構の解明と臨床応用」,第65回日本産科婦人科学会・学術講演会講演要旨)