Similar protein expression profiles of ovarian and endometrial high-grade serous carcinomas

Background:

Ovarian and endometrial high-grade serous carcinomas (HGSCs) have similar clinical and pathological characteristics; however, exhaustive protein expression profiling of these cancers has yet to be reported.

Methods:

We performed protein expression profiling on 14 cases of HGSCs (7 ovarian and 7 endometrial) and 18 endometrioid carcinomas (9 ovarian and 9 endometrial) using iTRAQ-based exhaustive and quantitative protein analysis.

Results:

We identified 828 tumour-expressed proteins and evaluated the statistical similarity of protein expression profiles between ovarian and endometrial HGSCs using unsupervised hierarchical cluster analysis (P<0.01). Using 45 statistically highly expressed proteins in HGSCs, protein ontology analysis detected two enriched terms and proteins composing each term: IMP2 and MCM2. Immunohistochemical analyses confirmed the higher expression of IMP2 and MCM2 in ovarian and endometrial HGSCs as well as in tubal and peritoneal HGSCs than in endometrioid carcinomas (P<0.01). The knockdown of either IMP2 or MCM2 by siRNA interference significantly decreased the proliferation rate of ovarian HGSC cell line (P<0.01).

Conclusions:

We demonstrated the statistical similarity of the protein expression profiles of ovarian and endometrial HGSC beyond the organs. We suggest that increased IMP2 and MCM2 expression may underlie some of the rapid HGSC growth observed clinically.     

术前血浆D-二聚体水平与浆液性卵巢癌一线化疗疗效及预后的相关性评价

目的：探讨术前血浆D －二聚体水平与浆液性卵巢癌一线化疗疗效及预后的相关性。方法：回顾性分析天津医科大学肿瘤医院2008年1 月至2010年5 月143 例浆液性卵巢癌患者的术前血浆D-二聚体水平,并依据血浆D-二聚体正常水平分为血浆D-二聚体水平（≤ 0.3 mg/L ）正常组（100 例）和血浆D-二聚体水平（> 0.3 mg/L ）升高组（43例）,比较血浆D-二聚体不同水平与临床病理因素、化疗疗效及预后的相关性。结果：血浆D-二聚体水平与FIGO分期、残余病灶大小、恶性腹水、术前CA125 水平及新辅助化疗密切相关。化疗后血浆D-二聚体水平升高组的完全缓解率34.88%（15/ 43）明显低于血浆D-二聚体正常组73.00%（73/ 100）,两者比较差异具有统计学意义（P < 0.001）。 血浆D-二聚体水平升高组的无进展生存率与总生存率明显低于血浆D-二聚体正常组（25.58% vs . 50.00% 与32.56% vs . 65.00%）,两者比较差异具有统计学意义（P < 0.05）。 Cox 多因素分析显示血浆D －二聚体水平是患者预后的独立预测因素。结论：术前血浆D-二聚体水平可作为有效预测浆液性卵巢癌一线化疗疗效及预后的生物学指标。     

Expression profiles of PRKG1, SDF2L1 and PPP1R12A are predictive and prognostic factors for therapy response and survival in high-grade serous ovarian cancer

High-grade serous ovarian cancer (HGS-EOCs) is generally sensitive to front-line platinum (Pt)-based chemotherapy although most patients at an advanced stage relapse with progressive resistant disease. Clinical or molecular data to identify primary resistant cases at diagnosis are not yet available. HGS-EOC biopsies from 105 Pt-sensitive (Pt-s) and 89 Pt-resistant (Pt-r) patients were retrospectively selected from two independent tumor tissue collections. Pathway analysis was done integrating miRNA and mRNA expression profiles. Signatures were further validated in silico on a cohort of 838 HGS-EOC cases from a published dataset. In all, 131 mRNAs and 5 miRNAs belonging to different functionally related molecular pathways distinguish Pt-s from Pt-r cases. Then, 17 out of 23 selected elements were validated by orthogonal approaches (SI signature). As resistance to Pt is associated with a short progression-free survival (PFS) and overall survival (OS), the prognostic role of the SI signature was assessed, and 14 genes associated with PFS and OS, in multivariate analyses (SII signature). The prognostic value of the SII signature was validated in a third extensive cohort. The expression profiles of SDF2L1, PPP1R12A and PRKG1 genes (SIII signature) served as independent prognostic biomarkers of Pt-response and survival. The study identified a prognostic molecular signature based on the combined expression profile of three genes which had never been associated with the clinical outcome of HGS-EOC. This may lead to early identification, at the time of diagnosis, of patients who would not greatly benefit from standard chemotherapy and are thus eligible for novel investigational approaches.     

Multisite analysis of high-grade serous epithelial ovarian cancers identifies genomic regions of focal and recurrent copy number alteration in 3q26.2 and 8q24.3

High-grade serous epithelial ovarian cancer (HGS-EOC) is a systemic disease, with marked intra and interpatient tumor heterogeneity. The issue of spatial and temporal heterogeneity has long been overlooked, hampering the possibility to identify those genomic alterations that persist, before and after therapy, in the genome of all tumor cells across the different anatomical districts. This knowledge is the first step to clarify those molecular determinants that characterize the tumor biology of HGS-EOC and their route toward malignancy. In our study, -omics data were generated from 79 snap frozen matched tumor biopsies, withdrawn before and after chemotherapy from 24 HGS-EOC patients, gathered together from independent cohorts. The landscape of somatic copy number alterations depicts a more homogenous and stable genomic portrait than the single nucleotide variant profile. Genomic identification of significant targets in cancer analysis identified two focal and minimal common regions (FMCRs) of amplification in the cytoband 3q26.2 (region α, 193 kb long) and 8q24.3 (region β, 495 kb long). Analysis in two external databases confirmed regions α and β are features of HGS-EOC. The MECOM gene is located in region α, and 15 genes are in region β. No functional data are yet available for the genes in the β region. In conclusion, we have identified for the first time two FMCRs of amplification in HGS-EOC, opening up a potential biological role in its etiopathogenesis.     

Extracellular microRNA profiling for prognostic prediction in patients with high-grade serous ovarian carcinoma

High-grade serous ovarian carcinoma is a leading cause of death in female patients worldwide. MicroRNAs (miRNAs) are stable noncoding RNAs in the peripheral blood that reflect a patient’s condition, and therefore, they have received substantial attention as noninvasive biomarkers in various diseases. We previously reported the usefulness of serum miRNAs as diagnostic biomarkers. Here, we investigated the prognostic impact of the serum miRNA profile. We used the GSE106817 dataset, which included preoperative miRNA profiles of patients with ovarian malignancies. Excluding patients with other malignancy or insufficient prognostic information, we included 175 patients with high-grade serous ovarian carcinoma. All patients except four underwent surgery and received chemotherapy as initial treatment. The median follow-up period was 54.6 months (range, 3.5-144.1 months). Univariate Cox regression analysis revealed that higher levels of miR-187-5p and miR-6870-5p were associated with both poorer progression-free survival (PFS) and overall survival (OS), and miR-1908-5p, miR-6727-5p, and miR-6850-5p were poor prognostic indicators of PFS. The OS and PFS prognostic indices were then calculated using the expression values of three prognostic miRNAs. Multivariate Cox regression analysis showed that both indices were significantly independent poor prognostic factors (hazard ratio for OS and PFS, 2.343 [P = .015] and 2.357 [P = .005], respectively). In conclusion, circulating miRNA profiles can potentially provide information to predict the prognosis of patients with high-grade serous ovarian carcinoma. Therefore, there is a strong demand for early clinical application of circulating miRNAs as noninvasive biomarkers.     

卵巢浆液性肿瘤中OCT4蛋白的表达及与化疗耐药的关系研究

目的:探讨OCT4蛋白在卵巢浆液性肿瘤组织中的表达与卵巢肿瘤发生发展及与化疗耐药的关系。方法:采用免疫组织化学链霉素抗生物素蛋白-过氧化酶连接法（SP法）检测OCT4蛋白在10例正常卵巢组织、10例卵巢良性浆液性囊腺瘤、10例卵巢交界性浆液性囊腺瘤和67例卵巢浆液性腺癌（其中化疗耐药30例,化疗敏感37例）的石蜡病理切片中的表达情况。结果:OCT4蛋白的阳性表达率在正常卵巢组织、卵巢良性浆液性囊腺瘤、卵巢交界性浆液性囊腺瘤和卵巢浆液性腺癌组织中分别为0、40.00%、50.00%和62.69%,差异有显著统计学意义（P=0.002）。在卵巢浆液性腺癌化疗耐药和化疗敏感组织中分别为83.33%和45.95%,差异有显著统计学意义（P=0.002）。OCT4蛋白的阳性表达率与卵巢浆液性腺癌的临床病理特征无明显相关性（P＞0.05）。结论:OCT4蛋白可能在卵巢浆液性肿瘤发生发展和卵巢癌化疗耐药中发挥重要作用,可能作为卵巢癌化疗耐药预测的一个重要参考指标。     

Differential and divergent activity of insulin-like growth factor binding protein 6 in platinum-sensitive versus platinum-resistant high-grade serous ovarian carcinoma cell lines

Insulin-like growth factor binding protein 6 (IGFBP6) is a secreted protein with a controversial role in human malignancies, being downregulated in most types of human cancer, but upregulated in selected tumors. Ovarian cancer (OC) is a human malignancy characterized by IGFBP6 downregulation; however, the significance of its low expression during ovarian carcinogenesis is still poorly understood. In the present study, IGFBP6 expression and activation of its associated signaling pathway were evaluated in two matched OC cell lines derived from a high-grade serous OC before and after platinum resistance (PEA1 and PEA2 cells, respectively). A whole genome gene expression analysis was comparatively performed in both cell lines upon IGFBP6 stimulation using Illumina technology. IGFBP6 gene expression data from human OC cases were obtained from public datasets. Gene expression data from public datasets confirmed the downregulation of IGFBP6 in primary and metastatic OC tissues compared with in normal ovarian tissues. The comparative analysis of platinum-sensitive (PEA1) and platinum-resistant (PEA2) cell lines showed quantitative and qualitative differences in the activation of IGFBP6 signaling. Notably, IGFBP6 enhanced ERK1/2 phosphorylation only in PEA1 cells, and induced more evident and significant gene expression reprogramming in PEA1 cells compared with in PEA2 cells. Furthermore, the analysis of selected genes modulated by IGFBP6 (i.e., FOS, JUN, TNF, IL6, IL8 and EGR1) exhibited an inverse regulation in PEA1 versus PEA2 cells. In addition, selected hallmarks (TNFA_signaling_via_NFKB, TGF_beta_signaling, P53_pathway) and IL-6 signaling were positively regulated in PEA1 cells, whereas they were inhibited in PEA2 cells in response to IGFBP6. These data suggested that dysregulation of IGFBP6 signaling may serve a role in the progression of OC, and is likely associated with the development of platinum resistance.     

Rac1蛋白在人卵巢浆液性腺癌组织中的表达及其对卵巢癌细胞SKOV3迁移能力的影响

目的探讨Rac 1蛋白在人卵巢浆液性腺癌组织中的表达情况及其对卵巢癌细胞SKOV3迁移能力的影响。方法采用蛋白质印迹法(Western blot)检测59例卵巢浆液性腺癌组织、31例卵巢良性浆液性囊腺瘤组织和3 5例卵巢正常组织中Rac 1蛋白的相对表达量。筛选出抑制Rac 1蛋白表达效果最佳的载体,构建稳定转染细胞株SKOV3-Racli;采用细胞划痕实验检测SKOV3-Racli细胞的迁移能力。结果卵巢浆液性腺癌组织中Racl蛋白的相对表达量高于卵巢良性浆液性囊腺瘤组织和卵巢正常组织(P<0.05)。与阴性质粒比较,1号片段质粒的抑制率为12.59%,2号片段质粒的抑制率为56.48%,3号片段质粒的抑制率为73.68%。与野生型SKOV3组相比,SKOV3-Racli细胞组SKOV3-Racli细胞的迁移能力下降(P <0.05)。结论 Rac1蛋白对卵巢浆液性腺癌细胞迁移能力的影响可能与下调卵巢浆液性腺癌组织中Rac 1蛋白的表达有关。     

Tau protein as a potential predictive marker in epithelial ovarian cancer patients treated with paclitaxel/platinum first-line chemotherapy

Background

The aim of the study was to evaluate predictive and prognostic significance of microtubule-associated protein Tau in epithelial ovarian cancer (EOC) patients treated with paclitaxel and platinum-based chemotherapy.

Methods

74 patients with EOC (stage I-IV) who underwent cytoreductive surgery followed by standard paclitaxel/platinum chemotherapy were included in the retrospective analysis. Their formalin-fixed, paraffin-embedded tissue specimens were immunohistochemically stained for Tau protein, using semi-quantitative DAKO test. Tau expression was acknowledged as negative (0 and 1+) or positive (2+ and 3+). The correlation between Tau expression, progression free survival (PFS) and overall survival (OS) was evaluated. Statistical analysis included Kaplan-Meyer estimator, long rank test, Mann Whitney test and Cox proportional hazards model.

Results

25.7% (19/74) and 74.3% (55/74) of the patients were classified as Tau-negative and Tau-positive, respectively. Median PFS was 28.7 months for Tau-negative group and 15.9 months for Tau-positive group (p = 0.0355). In the univariate analysis 3-year OS in Tau-negative and Tau-positive groups was 80.2% and 52.4%, respectively (p = 0.0198). Low expression of protein Tau was associated with better OS, whereas an advanced stage at diagnosis, suboptimal surgery, serous histological type and resistance to first line chemotherapy were each correlated with worse OS (p <0,05). In multivariate analysis only resistance to first line chemotherapy remained significant (HR 22.59; 95% CI, 8.71-58.55; p <0.0001).

Conclusions

Negative tau protein seems to be both good prognostic factor and a predictor of response to paclitaxel/platinum-based chemotherapy in EOC patients.     

Boosting the apoptotic response of high-grade serous ovarian cancers with CCNE1 amplification to paclitaxel in vitro by targeting APC/C and the pro-survival protein MCL-1

Ovarian cancer exhibits the highest mortality rate among gynecological malignancies. Antimitotic agents, such as paclitaxel, are frontline drugs for the treatment of ovarian cancer. They inhibit microtubule dynamics and their efficiency relies on a prolonged mitotic arrest and the strong activation of the spindle assembly checkpoint (SAC). Although ovarian cancers respond well to paclitaxel, the clinical efficacy is limited due to an early onset of drug resistance, which may rely on a compromised mitosis exit associated with weakend intrinsic apoptosis. Accordingly, we aimed at overcoming SAC silencing that occurs rapidly during paclitaxel-induced mitotic arrest. To do this, we used a specific anaphase-promoting complex/cyclosome (APC/C) inhibitor to prevent a premature mitotic exit upon paclitaxel treatment. Furthermore, we investigated the role of the antiapoptotic BCL-2 family member MCL-1 in determining the fate of ovarian cancer cells lines with CCNE1 amplification that are challenged with clinically relevant dose of paclitaxel. Using time-laps microscopy, we demonstrated that APC/C and MCL-1 inhibition under paclitaxel prevents mitotic slippage in ovarian cancer cell lines and restores death in mitosis. Consistent with this, the combinatorial treatment reduced the survival of ovarian cancer cells in 2D and 3D cell models. Since a therapeutic ceiling has been reached with taxanes, it is of utmost importance to develop alternative strategies to improve the patient's survival. Thus, our study provides not only elements to understand the causes of taxane resistance in CCNE1-amplified ovarian cancers but also suggests a new combinatorial strategy that may improve paclitaxel-based efficacy in this highly lethal gynecological disease.     

Cyclin A as a marker for prognosis and chemotherapy response in advanced breast cancer

We wanted to study cyclin A as a marker for prognosis and chemotherapy response. A total of 283 women with metastatic breast cancer were initially enrolled in a randomised multicentre trial comparing docetaxel to sequential methotrexate-fluorouracil (MF) in advanced breast cancer after anthracycline failure. Paraffin-embedded blocks of the primary tumour were available for 96 patients (34%). The proportion of cells expressing cyclin A was determined by immunohistochemistry using a mouse monoclonal antibody to human cyclin A. Response evaluation was performed according to WHO recommendations. The median cyclin A positivity of tumour cells was 14.5% (range 1.2-45.0). Cyclin A correlated statistically significantly to all other tested proliferation markers (mitotic count, histological grade and Ki-67). A high cyclin A correlated significantly to a shorter time to first relapse, risk ratio (RR) 1.94 (95% CI 1.24-3.03) and survival from diagnosis, RR 2.49 (95% CI 1.45-4.29), cutoff point for high/low proliferation group 10.5%. Cyclin A did not correlate to chemotherapy response or survival after anthracycline, docetaxel or MF therapy. Of all tumour biological factors tested (mitotic count, histological grade and Ki-67), cyclin A seemed to have the strongest prognostic value. Cyclin A is a good marker for tumour proliferation and prognosis in breast cancer. In the present study, cyclin A did not predict chemotherapy response.     

A comprehensive analysis of the expression,epigenetic and genetic changes of HNF1B and ECI2 in 122 cases of high-grade serous ovarian carcinoma

High-grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer, with a poor prognosis; however, most studies concerning ovarian carcinoma have focused mainly on clear cell carcinoma. The involvement of hepatocyte nuclear factor 1β (HNF1B) in the carcinogenesis of HGSC has not yet been fully elucidated. To the best of our knowledge, the present study is the first to analyse the expression of the possible downstream target of HNF1B, enoyl-CoA (Δ) isomerase 2 (ECI2), in HGSC. The present study performed a comprehensive analysis of HNF1B mRNA and protein expression, and epigenetic and genetic changes, as well as an analysis of ECI2 mRNA and protein expression in 122 cases of HGSC. HNF1B protein expression was detected in 28/122 cases, and was positively associated with lymphovascular invasion (P=0.025). Protein expression of ECI2 was detected in 115/122 cases, but no associations with clinicopathological variables were revealed. Therefore, ECI2 does not seem to function as a suitable prognostic marker for HGSC. In the sample set, a positive correlation between HNF1B and ECI2 protein expression was detected (P=0.005). HNF1B mRNA was also positively correlated with HNF1B protein expression (P=0.001). HNF1B promoter methylation was detected in 26/67 (38.8%) of cases. A novel pathogenic somatic HNF1B mutation was detected in 1/61 (1.6%) of the analysed HGSC cases. No other correlations between the examined SNPs (rs4430796, rs757210 and rs7405776), HNF1B promoter methylation, HNF1B/ECI2 expression or clinicopathological characteristics were found.     

A distinct pre-existing inflammatory tumour microenvironment is associated with chemotherapy resistance in high-grade serous epithelial ovarian cancer

Background:

Chemotherapy resistance is a major determinant of poor overall survival rates in high-grade serous ovarian cancer (HGSC). We have previously shown that gene expression alterations affecting the NF-κB pathway characterise chemotherapy resistance in HGSC, suggesting that the regulation of an immune response may be associated with this phenotype.

Methods:

Given that intrinsic drug resistance pre-exists and is governed by both tumour and host factors, the current study was performed to examine the cross-talk between tumour inflammatory microenvironment and cancer cells, and their roles in mediating differential chemotherapy response in HGSC patients. Expression profiling of a panel of 184 inflammation-related genes was performed in 15 chemoresistant and 19 chemosensitive HGSC tumours using the NanoString nCounter platform.

Results:

A total of 11 significantly differentially expressed genes were found to distinguish the two groups. As STAT1 was the most significantly differentially expressed gene (P=0.003), we validated the expression of STAT1 protein by immunohistochemistry using an independent cohort of 183 (52 resistant and 131 sensitive) HGSC cases on a primary tumour tissue microarray. Relative expression levels were subjected to Kaplan–Meier survival analysis and Cox proportional hazard regression models.

Conclusions:

This study confirms that higher STAT1 expression is significantly associated with increased progression-free survival and that this protein together with other mediators of tumour–host microenvironment can be applied as a novel response predictive biomarker in HGSC. Furthermore, an overall underactive immune microenvironment suggests that the pre-existing state of the tumour immune microenvironment could determine response to chemotherapy in HGSC.     

Prognostic role of CIP2A expression in serous ovarian cancer

Background:

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein expressed in several solid cancers. Our purpose was to study its role in serous ovarian cancer patients, and the association to clinicopathological variables and molecular markers.

Methods:

We collected retrospectively 562 consecutive serous ovarian cancer patients treated at the Helsinki University Central Hospital. We stained tumour tissue microarrays for CIP2A by immunohistochemistry and constructed survival curves according to the Kaplan–Meier method. Associations to clinicopathological and molecular markers were assessed by the χ²-test.

Results:

We found strong cytoplasmic CIP2A immunoreactivity in 212 (40.4%) specimens, weak positivity in 222 (42.4%) specimens, and negative in 90 (17.2%). Immunopositive CIP2A expression was associated with high grade (P<0.0001), advanced stage (P=0.0005), and aneuploidy (P=0.001, χ²-test). Cancerous inhibitor of protein phosphatase 2A overexpression was also associated with EGFR protein expression (P=0.006) and EGFR amplification (P=0.043). Strong cytoplasmic CIP2A immunopositivity predicted poor outcome in ovarian cancer patients (P<0.0001, log-rank test).

Conclusion:

Our results show that CIP2A associates with reduced survival and parameters associated with high grade in ovarian cancer patients, and may thus be one of the factors that identify aggressive subtype (type II) of this disease.     

Insulin-like growth factor (IGF) and hepatocyte growth factor (HGF) in follicular fluid cooperatively promote the oncogenesis of high-grade serous carcinoma from fallopian tube epithelial cells: Dissection of the molecular effects

Incessant ovulation is believed to be a potential cause of epithelial ovarian cancer (EOC). Our previous investigations have shown that insulin-like growth factor (IGF2) and hepatocyte growth factor (HGF) in the ovulatory follicular fluid (FF) contributed to the malignant transformation initiated by p53 mutations. Here we examined the individual and synergistic impacts of IGF2 and HGF on enhancing the malignant properties of high-grade serous carcinoma (HGSC), the most aggressive type of EOC, and its precursor lesion, serous tubal intraepithelial carcinoma (STIC). In a mouse xenograft co-injection model, we observed that FF co-injection induced tumorigenesis of STIC-mimicking cells, FE25. Co-injection with IGF2 or HGF partially recapitulated the tumorigenic effects of FF, but co-injection with both resulted in a higher tumorigenic rate than FF. We analyzed the different transformation phenotypes influenced by these FF growth signals through receptor inhibition. The IGF signal was necessary for clonogenicity, while the HGF signal played a crucial role in the migration and invasion of STIC and HGSC cells. Both signals were necessary for the malignant phenotype of anchoring-independent growth but had little impact on cell proliferation. The downstream signals responsible for these HGF activities were identified as the tyrosine-protein kinase Met (cMET)/mitogen-activated protein kinase and cMET/AKT pathways. Together with the previous finding that the FF-IGF2 could mediate clonogenicity and stemness activities via the IGF-1R/AKT/mammalian target of rapamycin and IGF-1R/AKT/NANOG pathways, respectively, this study demonstrated the cooperation of the FF-sourced IGF and HGF growth signals in the malignant transformation and progression of HGSC through both common and distinct signaling pathways. These findings help develop targeted prevention of HGSC.