The oncogene phosphatidylinositol 3'-kinase catalytic subunit alpha promotes angiogenesis via vascular endothelial growth factor in ovarian carcinoma

The gene of phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) has been implicated as an oncogene in ovarian cancer [L. Shayesteh et al., Nat. Genet., 21: 99-102, 1999]. In this study, we examined the expression of PIK3CA mRNA and its p110alpha protein product in human ovarian carcinoma and investigated its role in regulating angiogenesis via vascular endothelial growth factor (VEGF). PIK3CA mRNA was detected in 66.6% of stage I and 93.9% of advanced stage ovarian cancer specimens and in all 17 ovarian cancer cell lines. PIK3CA mRNA levels were significantly higher in invasive carcinomas compared with benign and low malignant potential neoplasms (P = 0.007), but no significant difference was seen between early and advanced stage carcinomas (P = 0.812). Strong expression of immunoreactive p110alpha was detected in tumor cells and/or stroma endothelium. PIK3CA expression in vivo positively correlated, both at the mRNA and the protein level, with the expression of VEGF as well as with the extent of microvascular development. Furthermore, PIK3CA mRNA overexpression positively correlated with increased proliferation and decreased apoptosis of tumor cells in vivo. In vitro, PIK3CA expression positively correlated with the expression of VEGF in ovarian cancer cells, whereas the phosphatidylinositol 3'-kinase inhibitor Ly294002 reduced both the constitutive and inducible expression of hypoxia-inducible factor-1alpha at the mRNA and protein levels and abrogated VEGF up-regulation by glucose starvation. Furthermore, Ly294002 suppressed cell proliferation and, at higher doses, induced marked apoptosis in ovarian cancer cells. Collectively, these data strongly indicate that PIK3CA supports ovarian cancer growth through multiple and independent pathways affecting cell proliferation, apoptosis and angiogenesis, and plays an important role in ovarian cancer progression.     

人垂体肿瘤转化基因1在卵巢癌组织中的表达及其意义

背景与目的:人垂体肿瘤转化基因1(humanpituitarytumortransforminggene1,hPTTG1)是新近发现的一个癌基因。我们用基因表达谱芯片对晚期卵巢低分化浆液性癌进行卵巢癌相关基因的筛查,发现该基因在这种卵巢癌中明显高表达。本研究对不同临床分期、不同病理类型及不同组织学分级的上皮性卵巢癌组织hPTTG1的表达进行检测,探讨该基因在卵巢癌发生发展中的作用。方法:用非竞争性RT-PCR方法半定量检测27例卵巢癌及4例正常卵巢组织中hPTTG1mRNA的表达,用免疫组化方法检测该27例卵巢癌及18例正常卵巢组织中hPTTG1蛋白的表达。结果:在正常卵巢组织中hPTTG1mRNA有低水平的表达,而卵巢癌组织hPTTG1mRNA表达高于正常,两者之间有显著性差异(P<0.01),卵巢癌组织较正常卵巢组织表达倍增1.1～4.8,中位倍增2.4。进一步分析卵巢癌组织中hPTTG1mRNA表达水平与临床分期及病理分级的关系,未发现hPTTG1mRNA表达水平的高低与临床分期具有相关性,但发现hPTTG1mRNA倍增与肿瘤分化程度密切相关(r=0.686,P<0.05)。hPTTG1蛋白在所有卵巢癌组织中表达,而在正常卵巢组织中未检测到hPTTG1蛋白的表达。结论:hPTTG1表达在早期卵巢癌即发生改变,其表达可能与不良分化有关;hPTTG1在卵巢癌组织中高表达,可能是卵巢癌的一个分子标志。     

Human kallikrein gene 11 (KLK11) mRNA overexpression is associated with poor prognosis in patients with epithelial ovarian cancer.

PURPOSE: The purpose of this study was to examine expression levels of the human tissue kallikrein 11 gene (KLK11) in epithelial ovarian tumors and to identify the relationship between KLK11 expression and patient survival. EXPERIMENTAL DESIGN: KLK11 mRNA expression was examined by semiquantitative PCR in 64 epithelial ovarian tumors (7 adenomas, 6 low malignant potential tumors, and 51 adenocarcinomas) and in 10 normal ovaries. Semiquantitative PCR results were correlated with clinicopathologic variables and overall survival. cDNA from human normal tissues and tumor tissues was also analyzed. RESULTS: KLK11 mRNA expression was detected in various human cancer tissues including breast, lung, colon, prostate, pancreas, and ovarian carcinoma. The mean value of relative KLK11 expression ratio was significantly higher in ovarian tumor samples than in normal ovary samples (compared with normal samples: adenoma, P = 0.0006; low malignant potential tumor, P = 0.0049; and carcinoma, P < 0.0001). No statistically significant associations between KLK11 mRNA expression level and clinical stage, histological type, or histological grade were observed. The log-rank test showed that high KLK11 mRNA expression and advanced clinical stage significantly correlated with poor patient survival (P = 0.0185 and P = 0.0043, respectively). High KLK11 mRNA expression and clinical stage remained significantly associated with overall survival (P = 0.0225 and P = 0.0202, respectively) after multivariate analysis. CONCLUSIONS: KLK11 expression may play an important role in ovarian cancer development and act as an independent prognostic marker in ovarian cancer patients.     

Increased expression of IGF II mRNA-binding protein 1 mRNA is associated with an advanced clinical stage and poor prognosis in patients with ovarian cancer

The purpose of this study was to examine the expression of IGF II mRNA-binding protein (IMP-1, -2, and -3) mRNA in epithelial ovarian tumors, and to identify the association of IMP-1, -2, and -3 expression levels with patient survival. IMP mRNA expression levels were examined by semi-quantitative PCR in 59 epithelial ovarian tumors (8 adenomas, 5 LMP tumors, and 46 adenocarcinomas) and in 7 normal ovaries. Results of semiquantitative PCR were correlated with clinicopathological variables and overall survival. Human normal and tumor tissue cDNAs were included in all of the analyses. The IMP family mRNA expression was detected in almost all cancer tissues examined, including breast, lung, colon, prostatic, and ovarian carcinoma with the exception of pancreatic carcinoma. The mean value of the relative IMP-1 mRNA expression ratio was significantly higher in both ovarian cancer and adenoma samples compared to normal ovarian samples (p<0.05). IMP-2 and IMP-3 expression levels were significantly higher in both ovarian cancer and ovarian LMP tumor samples compared to either ovarian adenomas or to normal ovary samples (p<0.05). A significantly higher IMP-1 mRNA expression level was observed in patients with an advanced clinical stage (p=0.015) and high histological grade (p=0.023). Log-rank testing showed that IMP-1 overexpression (p=0.0398) and an advanced clinical stage (p=0.0050) were significantly correlated with poor patient survival, whereas neither IMP-2 nor IMP-3 overexpression were associated with poor prognoses. In multivariate analysis, IMP-1 overexpression lost its significance, whereas the clinical stage (p=0.0432) remained significantly associated with overall survival. IMP mRNA expression levels might play an important role in ovarian cancer development and progression, and IMP-1 overexpression is a prognostic marker for patients with ovarian cancer.     

Increased MCL–1 Expression Is Associated with Poor Prognosis in Ovarian Carcinomas

To investigate the potential role of the BCL–2 gene family (BAX, BCL–2, MCL–1 , and BCL-XL) in ovarian cancer development and progression, mRNA expression levels of these genes were measured using semi-quantitative PCR in epithelial ovarian tumor tissues and normal ovaries. The immunohistochemical expression of MCL–1 in ovarian tumors was also examined. The expression levels of BAX and MCL–1 mRNA were significantly higher in ovarian cancers and in adenomas than in normal ovaries ( P <0.05). In contrast, the BCL–2 mRNA expression level in ovarian cancers was significantly lower than in ovarian adenomas and in normal ovaries ( P <0.05). Expression of BCL-XL mRNA was no different between normal ovaries and ovarian tumors. Log-rank testing showed that low BAX mRNA expression and high MCL–1 mRNA expression significantly correlate with poor survival for patients with stage III ovarian carcinomas (BAX, P =0.05; MCL–1, P =0.02). Immunohistochemical analysis showed that diffuse-positive expression of MCL–1 protein in mucinous carcinomas was significantly higher than in mucinous low malignant potential (LMP) tumors ( P =0.03). In ovarian cancer cases, diffuse-positive expression of MCL–1 protein significantly correlates with advanced clinical stage, high histologic grade, and poor survival (stage, P <0.01; grade, P =0.01; survival, P =0.01). These results suggest that increased MCL–1 expression may play an important role in replacing the functions of increased BAX and decreased BCL–2 in ovarian carcinoma cells, thereby promoting cell survival, and resulting in a poor prognosis for patients with ovarian cancer.     

Increased MCL-1 expression is associated with poor prognosis in ovarian carcinomas.

To investigate the potential role of the BCL-2 gene family (BAX, BCL-2, MCL-1, and BCL-XL) in ovarian cancer development and progression, mRNA expression levels of these genes were measured using semi-quantitative PCR in epithelial ovarian tumor tissues and normal ovaries. The immunohistochemical expression of MCL-1 in ovarian tumors was also examined. The expression levels of BAX and MCL-1 mRNA were significantly higher in ovarian cancers and in adenomas than in normal ovaries (P < 0.05). In contrast, the BCL-2 mRNA expression level in ovarian cancers was significantly lower than in ovarian adenomas and in normal ovaries (P < 0.05). Expression of BCL-XL mRNA was no different between normal ovaries and ovarian tumors. Log-rank testing showed that low BAX mRNA expression and high MCL-1 mRNA expression significantly correlate with poor survival for patients with stage III ovarian carcinomas (BAX, P = 0.05; MCL-1, P = 0.02). Immunohistochemical analysis showed that diffuse-positive expression of MCL-1 protein in mucinous carcinomas was significantly higher than in mucinous low malignant potential (LMP) tumors (P = 0.03). In ovarian cancer cases, diffuse-positive expression of MCL-1 protein significantly correlates with advanced clinical stage, high histologic grade, and poor survival (stage, P < 0.01; grade, P = 0.01; survival, P = 0.01). These results suggest that increased MCL-1 expression may play an important role in replacing the functions of increased BAX and decreased BCL-2 in ovarian carcinoma cells, thereby promoting cell survival, and resulting in a poor prognosis for patients with ovarian cancer.     

The transmembrane protease serine (TMPRSS3/TADG-12) D variant: a potential candidate for diagnosis and therapeutic intervention in ovarian cancer.

The purpose of this study was to examine the expression of splice variants of the TADG-12 (TMPRSS3) gene in normal ovarian epithelial tissue and ovarian carcinoma and further to associate the expression of TADG-12 variant with clinicopathologic characteristics if such an association exists. TADG-12D variant expression was examined by semiquantitative PCR in 50 ovarian tumors [41 adenocarcinomas, 3 low malignant potential (LMP) tumors, and 6 adenomas] and 7 normal ovaries. In carcinomas as well as LMP tumors and adenomas, TADG-12D variant mRNA expression was significantly elevated compared to that in normal ovary samples. TADG-12 has several splice variants, one of which we originally identified and 3 others identified by Scott et al. [Nat Genet 2001;27:59-63]. We previously examined the expression of TADG-12V variant and here we confirm the overexpression of TADG-12D variant in ovarian carcinomas. Moreover, TADG-12D variant mRNA expression level in carcinomas was significantly elevated compared to that in adenomas and TADG-12D variant mRNA expression level in advanced clinical stage diseases was significantly higher than that in early stage diseases in ovarian carcinomas. With regard to histological type, TADG-12D variant mRNA expression level in mucinous adenocarcinomas was significantly higher than those in the other tissue subtypes. These features imply that TADG-12D variant expression may play an important role in ovarian cancer development and progression, and this variant may be useful both as a molecular target for therapy and/or a diagnostic marker.     

Dihydropyrimidine dehydrogenase mRNA level correlates with the response to 5-fluorouracil-based chemo-immuno-radiation therapy in human oral squamous cell cancer

Reduced expression of a cyclin-dependent kinase inhibitor, p27, has been reported to be associated with poor prognosis in several human cancers. The aim of this study was to investigate the potential role of p27 in ovarian cancer development and progression. Immunohistochemical expression of p27 was determined using 117 epithelial ovarian tumor tissues and 8 normal ovaries. p27 mRNA expression was examined by semi-quantitative PCR amplification using 26 ovarian cancer samples. Nuclear staining of p27 was commonly observed in the normal ovarian surface epithelium and the epithelial cells of germinal inclusion cysts. Positive p27 staining rates were significantly higher in serous adenomas (p=0.006) and in serous LMP tumors (p=0.013) than that in serous carcinomas (Fisher's exact test). In serous ovarian cancers, positive p27 staining rate was significantly higher in early stage (stage1/2) than that in advanced stage (stage 3/4) diseases (p=0.030, Fisher's exact test). Log-rank testing showed that negative p27 expression significantly correlates with poor survival in serous ovarian cancer patients (p=0.041). Considerable levels of p27 mRNA were detected in all ovarian cancer samples examined. These results suggest that the underexpression of p27 caused by post-translational mechanism may contribute to the development and progression and result in poor prognosis of serous ovarian cancers.     

Frequent gene dosage alterations in stromal cells of epithelial ovarian carcinomas

Stromal cells are an active and integral part of epithelial neoplasms. We have previously observed allelic imbalance on chromosome 3p21 in both stromal and epithelial cells of ovarian tumors. This study was designed to explore gene dosage alterations throughout human chromosomes from stromal and epithelial cells of epithelial ovarian carcinomas. Thirteen stromal and 24 epithelial samples, microdissected from epithelial ovarian carcinomas, were analyzed using multiplex ligation-dependent probe amplification technique. Analysis covered 110 cancer related genes. Frequent genetic alterations were detected both in the stroma and epithelium of ovarian carcinomas. The mean number of altered genes per tumor was 10.8 in stroma and 23.6 in epithelium. In the stroma, the mean number of gains was 6.6 and of losses 4.2 and in the epithelium 13.7 and 9.9. The high number of changes associated with advanced tumor stage (p = 0.035) and death due to ovarian cancer (p = 0.032). The most frequent alteration was the deletion of the deleted in colorectal carcinoma (DCC) on chromosome 18q21.3 in 62% of samples. Loss of DCC was related to endometrioid subtype (p = 0.033). Large chromosomal aberrations were detected on the basis of alterations in adjacent genes. Most importantly, 38 genes showed similar genetic alterations (gain-gain or loss-loss) in stromal and epithelial compartments of 11 tumor pairs. Thus, frequent genetic alterations in stromal cells of epithelial ovarian carcinomas resembled those of malignant epithelial cells and may indicate a common precursor cell type. Epithelial-mesenchymal transition may generate transformed cancer cells and modify the tumor microenvironment with distinct properties.     

RRM2在上皮性卵巢肿瘤组织中的表达及其与血管生成的关系

目的探讨核糖核苷酸还原酶小亚基(RRM2)在上皮性卵巢肿瘤中的表达及其与血管生成的关系。方法采用免疫组织化学PV-6000二步法和RT-PCR法,检测RRM2、CD105 (Endoglin)在正常卵巢、良性卵巢肿瘤、低度潜在恶性上皮性卵巢肿瘤和上皮性卵巢癌组织中的表达,用CD105（Endoglin）标记微血管密度（MVD）,并分析RRM2 mRNA和CD 105 mRNA表达的关系。结果(1) 低度潜在恶性上皮性卵巢肿瘤组织(1.586±0.650,66.67% )和上皮性卵巢癌组织(1.870±0.618, 69.35%)的RRM2 mRNA表达量和蛋白阳性表达率均高于正常卵巢组织(0.771±0.495,0)和良性卵巢肿瘤组织(0.952±0.601, 26.67%) (P＜0.05)。（2）低度潜在恶性上皮性卵巢肿瘤组织(2.190±0.512, 23.15±4.38)和上皮性卵巢癌组织(2.735±0.636, 25.27±6.91)中的CD105 mRNA表达量和MVD均高于正常卵巢组织(0.686±0.637, 3.40±1.78)和良性卵巢肿瘤组织(0.763±0.547,12.15±2.29) (P＜0.05)。（3）RRM2 mRNA表达量和蛋白阳性表达率在低度潜在恶性组和FIGOⅠ~Ⅱ期卵巢癌组高于正常卵巢组和良性卵巢肿瘤组(P＜0.05),在FIGOⅢ~Ⅳ期高于Ⅰ~Ⅱ期（t=-2.370,χ²=5.937,P＜0.05)。(4)RRM2 mRNA和CD105 mRNA之间表达呈正相关(r=0.713,P＜0.05)。结论RRM2可能参与上皮性卵巢癌发生的早期事件,对上皮性卵巢癌的血管生成可能有一定促进作用,有望成为一个新的早期诊断指标。     

Cyr61在卵巢上皮性癌中的表达及其与血管生成关系的研究

目的 探讨高半胱氨酸蛋白61(Cysteine-rich 61,Cyr61)在卵巢上皮性癌中的表达及其与血管生成的关系。方法 用逆转录PCR的方法半定量检测40例卵巢癌及10例正常卵巢组织中Cyr61 mRNA的表达,用免疫组化方法检测该40例卵巢上皮性癌及10例正常卵巢组织中Cyr61蛋白及微血管密度(MVD)的表达。结果 卵巢上皮性癌中,Cyr61mRNA及蛋白表达均高于正常卵巢组织,MVD蛋白表达也高于正常卵巢组织,差异均有显著性,并与肿瘤浸润发展有一定相关性,且MVD随Cyr61表达增强而升高。结论 Cyr61的过度表达在卵巢上皮性癌的发生、发展、浸润过程中起重要的作用,而且生物学行为与血管生成密切相关。     

卵巢上皮癌组织中活性氧与乏氧诱导因子-1α的表达及其相关性

目的 检测卵巢上皮癌组织中乏氧诱导因子-1α（HIF-1α）mRNA、蛋白以及活性氧（ROS）的表达,探讨卵巢上皮癌组织中氧代谢的异常,以及两者在卵巢上皮癌发生发展中的相互作用.方法 应用实时RT-PCR法和免疫组织化学Elivision plus二步法分别测定卵巢上皮癌组织中HIF-1αmRNA及蛋白的表达;应用DCFH-DA法检测组织中ROS的表达.结果 卵巢上皮癌组织HIF-1 αmRNA相对含量（4.466±0.754）明显高于良性卵巢肿瘤（6.073±1.053）（t=-7.701,P=0.000）,良性卵巢肿瘤、早期（5.048±0.645）及晚期卵巢上皮癌（4.146 ±0.632）HIF-1α mRNA表达呈递增趋势（t=5.301,P=0.000）;HIF-1α mRNA表达与组织学分级及淋巴结转移呈正相关（t=3.724、-3.851,均P=0.000）.HIF-1 α蛋白在卵巢上皮癌组织中阳性表达率[63.49％（40/63）1明显高于良性对照[36.36％（8/22）]（U=433,P=0.005）,HIF-1 α蛋白阳性表达率与淋巴结转移及临床分期呈正相关（x2=4.289、12.360,P=0.038、0.000）.卵巢上皮癌组织中ROS表达水平为110.239±18.414,较良性对照（58.366±20.360）明显升高（t=11.068,P=0.000）;ROS表达与卵巢上皮癌组织学分级、淋巴结转移及临床分期呈正相关（t=-2.055、2.816、-4.138,P=0.044、0.007、0.000）.在卵巢上皮癌组织中HIF-1 α蛋白表达与ROS呈中度正相关（r=0.572,P=0.000）;在中低分化组及晚期组中两者呈中度正相关（r=0.540,P=O.002;r=0.572,P=0.000）;在淋巴结转移组中两者呈高度正相关（r=0.703,P=0.001）;HIF-1α mRNA与ROS表达呈低度相关（r=0.376,P=0.002）.结论 乏氧是卵巢上皮癌的基本特征,随着肿瘤进展乏氧程度逐渐加重;HIF-1α的异常表达与卵巢上皮癌潜在的恶性生物学行为密切相关,可能影响卵巢上皮癌预后.在乏氧状态下,ROS与HIF-1 α可能参与肿瘤的发生过程.在卵巢上皮癌侵袭转移过程中,ROS对HIF-1α的正向调节可能发挥重要作用.肿瘤乏氧状态下,ROS对HIF-1 α的调节可以发生在转录和蛋白等水平,主要是蛋白水平的改变.     

内皮抑素和血管内皮生长因子在卵巢癌组织中的表达及其临床意义

背景与目的:血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)能够诱导血管生成,导致肿瘤的生长、侵袭及远处转移;内皮抑素(endostatin)强效抑制肿瘤血管生成,进而抑制肿瘤生长、侵袭和转移。为探讨内皮抑素和VEGF在卵巢癌组织中的表达及其与卵巢癌发生发展的关系,本研究拟从基因和蛋白水平研究二者在卵巢癌组织中的表达情况。方法:运用逆转录聚合酶链反应(RT-PCR)和免疫组织化学染色法检测内皮抑素和VEGF在正常卵巢组织、卵巢癌组织中的表达情况。结果:卵巢癌组织中内皮抑素和VEGFmRNA表达水平及蛋白阳性率均高于正常卵巢组织(P<0.05);临床Ⅲ、Ⅳ期卵巢癌组织中内皮抑素和VEGFmRNA表达水平及蛋白阳性率均高于Ⅰ、Ⅱ期(P<0.05);内皮抑素mRNA≤0.5组和mRNA>0.5组,其蛋白阳性率分别为29.4%和77.8%(P<0.05);VEGFmRNA≤0.5组和mRNA>0.5组,其蛋白阳性率分别为20.0%和72.0%(P<0.05)。结论:正常卵巢组织和卵巢癌组织中内皮抑素、VEGFmRNA和蛋白含量的变化是一致的,内皮抑素与VEGF比例失调可能是卵巢癌发生发展的重要机制之一。     

Human kallikrein 8 (hK8/TADG-14) expression is associated with an early clinical stage and favorable prognosis in ovarian cancer

The purpose of this study was to examine human kallikrein 8 (hK8/TADG-14) expression in epithelial ovarian tumors and to investigate the association of hK8 expression levels with patient survival. Human kallikrein 8 protein (hK8) expression was examined by immunohistochemistry in 74 ovarian adenocarcinomas and 6 normal ovaries. Results of immunostaining were correlated with clinicopathological variables and overall survival of the patients. Human kallikrein 8 gene (KLK8) mRNA expression was examined by semi-quantitative PCR in 35 ovarian tumors and 7 normal ovaries. Expression of hK8 was not detected on the surface epithelium of normal ovaries. In contrast, hK8 expression was detected in 51.4% (38/74) of carcinomas with a significantly higher detection rate of hK8 expression being observed in early stage disease compared to advanced stage disease (p=0.0192). Data analysis using the log-rank test showed hK8 expression correlated significantly with favorable patient survival (p=0.0328). Younger age (p=0.0008), early clinical stage (p<0.0001), and low histological grades of the tumors (p=0.0018) were also associated significantly with a favorable prognosis. In a multivariate model, age (p=0.0186) and clinical stage (p<0.0001) remained associated significantly with overall survival, whereas hK8 expression and histological grade lost their significance. There was significant relationship between the hK8 expression status and KLK8 mRNA expression levels (p=0.0304). Expression of hK8 is increased during the development of ovarian cancer and down-regulated during ovarian cancer progression. Expression of hK8 is a favorable prognostic marker in patients with ovarian cancer.