Exploring the Control of PARP1 Levels in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is a leading cause of mortality from gynecologic malignancies worldwide. Although a transformative improvement has been shown with the introduction of PARP (poly(ADP-ribose) polymerase) inhibitors, the emergence of resistance to these drugs represents a therapeutic challenge. Hence, expanding our understanding of mechanisms behind the control of PARP1 expression can provide strategic guidance for the translation of novel therapeutic strategies. The Signal Transducer and Activator of Transcription (STAT) family of proteins consists of transcription factors critically involved in the regulation of important cellular functions. Notably, we recently demonstrated that, in cervical cancer cells, STAT1 controls PARP1 levels through multiple mechanisms, possibly involving also STAT3. Here, we tested the hypothesis that a similar mechanism might be operative in HGSOC. To this end, the impact of STAT1/STAT3 modulation on PARP1 expression was assessed in established and primary HGSOC cells, and molecular biology studies proved that STAT1 might act at both transcriptional and post-transcriptional levels to modulate the PARP1 level. Notably, bioinformatics analysis of TCGA databases demonstrated that increased STAT1 mRNA expression levels are associated with a favorable prognosis and with response to chemotherapy in HGSOC patients. Our findings suggest an alternative strategy for targeting HGSOC cells based on their dependency on PARP1.

Dual inhibition of CDK12 and CDK13 uncovers actionable vulnerabilities in patient-derived ovarian cancer organoids.

BACKGROUND: High grade serous ovarian cancer (HGSOC) is highly lethal, partly due to chemotherapy resistance and limited availability of targeted approaches. Cyclin dependent kinases 12 and 13 (CDK12/13) are promising therapeutic targets in human cancers, including HGSOC. Nevertheless, the effects of their inhibition in HGSOC and the potential synergy with other drugs are poorly known. METHODS: We analyzed the effects of the CDK12/13 inhibitor THZ531 in HGSOC cells and patient-derived organoids (PDOs). RNA sequencing and quantitative PCR analyses were performed to identify the genome-wide effects of short-term CDK12/13 inhibition on the transcriptome of HGSOC cells. Viability assays with HGSOC cells and PDOs were performed to assess the efficacy of THZ531 as single agent or in combination with clinically relevant drugs. RESULTS: The CDK12 and CDK13 genes are deregulated in HGSOC and their concomitant up-regulation with the oncogene MYC predicts poor prognosis. HGSOC cells and PDOs display high sensitivity to CDK12/13 inhibition, which synergizes with drugs in clinical use for HGSOC. Transcriptome analyses revealed cancer-relevant genes whose expression is repressed by dual CDK12/13 inhibition through impaired splicing. Combined treatment with THZ531 and inhibitors of pathways regulated by these cancer relevant genes (EGFR, RPTOR, ATRIP) exerted synergic effects on HGSOC PDO viability. CONCLUSIONS: CDK12 and CDK13 represent valuable therapeutic targets for HGSOC. We uncovered a wide spectrum of CDK12/13 targets as potential therapeutic vulnerabilities for HGSOC. Moreover, our study indicates that CDK12/13 inhibition enhances the efficacy of approved drugs that are already in use for HGSOC or other human cancers.

What's beyond BRCA Mutational Status in High Grade Serous Ovarian Cancer? The Impact of Hormone Receptor Expression in a Large BRCA-Profiled Ovarian Cancer Patient Series: A Retrospective Cohort Study.

Several studies have explored the prognostic role of hormone receptor status in high-grade serous ovarian cancer (HGSOC) patients. However, few reports have investigated their expression according to BRCA mutational status. The aim of this single-center, observational, retrospective study was to explore the hormone receptor pattern and its potential prognostic role in a cohort of 207 HGSOC women stratified for BRCA mutational status. To this end, ERα, ERß1, ERß2, ERß5, PR, and AR expression were assessed by immunohistochemistry in 135 BRCA-wild type (BRCA-wt) and 72 BRCA1/2 mutation carriers (BRCA-mut). No significant difference emerged in hormone receptor expression between the two sub-samples, except for a significantly lower ERα expression observed in pre-menopausal BRCA1/2-mut as compared to BRCA-wt patients (p = 0.02). None of the examined hormone receptors has revealed a significant prognostic role in the whole sample, apart from the ratio ERα/ERß5 nuclear, for which higher values disclosed a positive role on the outcome in BRCA-wt subgroup (HR 0.77; CI 0.61-0.96; p = 0.019). Conversely, it negatively affected overall survival in the presence of BRCA1/2-mut (HR 1.41; CI 1.06-1.87; p = 0.020). Finally, higher PR levels were associated with platinum sensitivity in the whole sample (p = 0.019). Our data, though needing further validation, suggest a potential role of oestrogen-mediated pathways in BRCA1/2-associated HGSOC tumorigenesis, thus revealing a possible therapeutic potential for targeting this interaction.

Preclinical models of epithelial ovarian cancer: practical considerations and challenges for a meaningful application.

Despite many improvements in ovarian cancer diagnosis and treatment, until now, conventional chemotherapy and new biological drugs have not been shown to cure the disease, and the overall prognosis remains poor. Over 90% of ovarian malignancies are categorized as epithelial ovarian cancers (EOC), a collection of different types of neoplasms with distinctive disease biology, response to chemotherapy, and outcome. Advances in our understanding of the histopathology and molecular features of EOC subtypes, as well as the cellular origins of these cancers, have given a boost to the development of clinically relevant experimental models. The overall goal of this review is to provide a comprehensive description of the available preclinical investigational approaches aimed at better characterizing disease development and progression and at identifying new therapeutic strategies. Systems discussed comprise monolayer (2D) and three-dimensional (3D) cultures of established and primary cancer cell lines, organoids and patient-derived explants, animal models, including carcinogen-induced, syngeneic, genetically engineered mouse, xenografts, patient-derived xenografts (PDX), humanized PDX, and the zebrafish and the laying hen models. Recent advances in tumour-on-a-chip platforms are also detailed. The critical analysis of strengths and weaknesses of each experimental model will aid in identifying opportunities to optimize their translational value.

Identification of a novel gene signature predicting response to first-line chemotherapy in BRCA wild-type high-grade serous ovarian cancer patients.

BACKGROUND: High-grade serous ovarian cancer (HGSOC) has poor survival rates due to a combination of diagnosis at advanced stage and disease recurrence as a result of chemotherapy resistance. In BRCA1 (Breast Cancer gene 1) - or BRCA2-wild type (BRCAwt) HGSOC patients, resistance and progressive disease occur earlier and more often than in mutated BRCA. Identification of biomarkers helpful in predicting response to first-line chemotherapy is a challenge to improve BRCAwt HGSOC management. METHODS: To identify a gene signature that can predict response to first-line chemotherapy, pre-treatment tumor biopsies from a restricted cohort of BRCAwt HGSOC patients were profiled by RNA sequencing (RNA-Seq) technology. Patients were sub-grouped according to platinum-free interval (PFI), into sensitive (PFI > 12 months) and resistant (PFI < 6 months). The gene panel identified by RNA-seq analysis was then tested by high-throughput quantitative real-time PCR (HT RT-qPCR) in a validation cohort, and statistical/bioinformatic methods were used to identify eligible markers and to explore the relevant pathway/gene network enrichments of the identified gene set. Finally, a panel of primary HGSOC cell lines was exploited to uncover cell-autonomous mechanisms of resistance. RESULTS: RNA-seq identified a 42-gene panel discriminating sensitive and resistant BRCAwt HGSOC patients and pathway analysis pointed to the immune system as a possible driver of chemotherapy response. From the extended cohort analysis of the 42 DEGs (differentially expressed genes), a statistical approach combined with the random forest classifier model generated a ten-gene signature predictive of response to first-line chemotherapy. The ten-gene signature included: CKB (Creatine kinase B), CTNNBL1 (Catenin, beta like 1), GNG11 (G protein subunit gamma 11), IGFBP7 (Insulin-like growth factor-binding protein 7), PLCG2 (Phospholipase C, gamma 2), RNF24 (Ring finger protein 24), SLC15A3 (Solute carrier family 15 member 3), TSPAN31 (Tetraspanin 31), TTI1 (TELO2 interacting protein 1) and UQCC1 (Ubiquinol-cytochrome c reductase complex assembly factor). Cytotoxicity assays, combined with gene-expression analysis in primary HGSOC cell lines, allowed to define CTNNBL1, RNF24, and TTI1 as cell-autonomous contributors to tumor resistance. CONCLUSIONS: Using machine-learning techniques we have identified a gene signature that could predict response to first-line chemotherapy in BRCAwt HGSOC patients, providing a useful tool towards personalized treatment modalities.

Evaluation of Angiogenesis-Related Genes as Prognostic Biomarkers of Bevacizumab Treated Ovarian Cancer Patients: Results from the Phase IV MITO16A/ManGO OV-2 Translational Study.

BACKGROUND: Epithelial ovarian cancer (EOC) is a rare, highly lethal disease. In a subset of high grade EOC patients, maintenance therapy with the antiangiogenic drug Bevacizumab (BEV) is a valuable option. To date, no validated predictive or prognostic biomarkers exist for selecting EOC patients that might benefit from BEV treatment. METHODS: Immunohistochemistry and RT-qPCR evaluated the expression of seven angiogenesis-related proteins and of a twelve microRNAs angio-signature in EOC patients, treated in first line with chemotherapy plus BEV (MITO16A/ManGO OV-2 phase IV trial). Centralized statistical analyses assessed the associations between each biomarker, clinical prognostic factors and survival outcomes. RESULTS: High miR-484 expression was associated with longer progression-free and overall survival. Notably, the combined expression of miR-484 and its target VEGFB identified a subset of patients that might mostly benefit from BEV treatment. No other significant correlations were found between the other analyzed biomarkers and patients' survival. The application of a shrinkage procedure to adjust for over-fitting hazard ratio estimates reduced the association significance. CONCLUSIONS: The analysis of angiogenesis related biomarkers in EOC patients homogenously treated with BEV in first line provides novel insight in their prognostic value and suggests that some of them might merit to be tested as predictive markers of drug activity in dedicated randomized trials.

In search for biomarkers and potential drug targets for uterine serous endometrial cancer.

OBJECTIVE: Serous endometrial cancer (USC) is a challenging malignancy associated with metastasis, recurrence and poor outcome. To identify clinically relevant prognostic biomarkers, we focused on a panel of proteins selected after a comprehensive literature review, for tumour profiling of a homogeneous cohort of USC patients. METHODS: Protein levels and localization were assessed by immunohistochemistry analysis in 36 hysterectomy samples. Tissue sections were stained with the following antibodies: Aurora A, phospho (T288) Aurora A, BRCA1, CHK1, CIP2A, Cyclin B1, Cyclin E, E2F-1, phospho (S364) E2F-1, FBXW7, FOXM1, phospho (S9) GSK3Beta, PLK1, phospho (T210) PLK1, PPP2R1B, p73, RAD51. Each marker was evaluated as a continuously-scaled variable for association with disease progression and death, using Cox proportional hazards models. The sample consisted of 36 patients with USC, half with stage III or IV disease. RESULTS: Results showed that higher CHK1 (Checkpoint kinase 1) expression was associated with a decreased risk of progression and death, after adjusting for stage. Interestingly, analysis of a TCGA data set of 109 USC patients corroborates our results showing a favourable prognostic role of CHEK1 after adjusting for stage. Higher FBXW7 (F-box and WD repeat domain containing 7) expression and higher cytoplasmic expression of PPP2R1B (Protein Phosphatase 2 A, Scaffold Subunit Abeta) were each associated with a decreased risk of progression, after adjusting for stage. CONCLUSIONS: In conclusion, results from the present study identify new clinically relevant biomarkers and potential drug targets for uterine serous endometrial cancer.

KLF7: a new candidate biomarker and therapeutic target for high-grade serous ovarian cancer.

BACKGROUND: In spite of great progress in the surgical and clinical management, until now no significant improvement in overall survival of High-Grade Serous Ovarian Cancer (HGSOC) patients has been achieved. Important aspects for disease control remain unresolved, including unclear pathogenesis, high heterogeneity and relapse resistance after chemotherapy. Therefore, further research on molecular mechanisms involved in cancer progression are needed to find new targets for disease management. The Krüppel-like factors (KLFs) are a family of transcriptional regulators controlling several basic cellular processes, including proliferation, differentiation and migration. They have been shown to play a role in various cancer-relevant processes, in a context-dependent way. METHODS: To investigate a possible role of KLF family members as prognostic biomarkers, we carried out a bioinformatic meta-analysis of ovarian transcriptome datasets in different cohorts of late-stage HGSOC patients. In vitro cellular models of HGSOC were used for functional studies exploring the role of KLF7 in disease development and progression. Finally, molecular modelling and virtual screening were performed to identify putative KLF7 inhibitors. RESULTS: Bioinformatic analysis highlighted KLF7 as the most significant prognostic gene, among the 17 family members. Univariate and multivariate analyses identified KLF7 as an unfavourable prognostic marker for overall survival in late-stage TCGA-OV and GSE26712 HGSOC cohorts. Functional in vitro studies demonstrated that KLF7 can play a role as oncogene, driving tumour growth and dissemination. Mechanistic targets of KLF7 included genes involved in epithelial to mesenchymal transition, and in maintaining pluripotency and self-renewal characteristics of cancer stem cells. Finally, in silico analysis provided reliable information for drug-target interaction prediction. CONCLUSIONS: Results from the present study provide the first evidence for an oncogenic role of KLF7 in HGSOC, suggesting it as a promising prognostic marker and therapeutic target.

Clinical Value of lncRNA MEG3 in High-Grade Serous Ovarian Cancer.

Long non-coding RNAs (lncRNAs) are emerging as regulators in cancer development and progression, and aberrant lncRNA profiles have been reported in several cancers. Here, we evaluated the potential of using the maternally expressed gene 3 (MEG3) tissue level as a prognostic marker in high-grade serous ovarian cancer (HGSOC), the most common and deadliest gynecologic malignancy. To the aim of the study, we measured MEG3 transcript levels in 90 pre-treatment peritoneal biopsies. We also investigated MEG3 function in ovarian cancer biology. We found that high MEG3 expression was independently associated with better progression-free (p = 0.002) and overall survival (p = 0.01). In vitro and in vivo preclinical studies supported a role for MEG3 as a tumor suppressor in HGSOC, possibly through modulation of the phosphatase and tensin homologue (PTEN) network. Overall, results from this study demonstrated that decreased MEG3 is a hallmark for malignancy and tumor progression in HGSOC.

Estrogens Counteract Platinum-Chemosensitivity by Modifying the Subcellular Localization of MDM4.

Estrogen activity towards cancer-related pathways can impact therapeutic intervention. Recent omics data suggest possible crosstalk between estrogens/gender and MDM4, a key regulator of p53. Since MDM4 can either promote cell transformation or enhance DNA damage-sensitivity, we analysed in vivo impact of estrogens on both MDM4 activities. In Mdm4 transgenic mouse, Mdm4 accelerates the formation of fibrosarcoma and increases tumor sensitivity to cisplatin as well, thus confirming in vivo Mdm4 dual mode of action. Noteworthy, Mdm4 enhances chemo- and radio-sensitivity in male but not in female animals, whereas its tumor-promoting activity is not affected by mouse gender. Combination therapy of transgenic females with cisplatin and fulvestrant, a selective estrogen receptor degrader, was able to recover tumor cisplatin-sensitivity, demonstrating the relevance of estrogens in the observed sexual dimorphism. Molecularly, estrogen receptor-α alters intracellular localization of MDM4 by increasing its nuclear fraction correlated to decreased cell death, in a p53-independent manner. Importantly, MDM4 nuclear localization and intra-tumor estrogen availability correlate with decreased platinum-sensitivity and apoptosis and predicts poor disease-free survival in high-grade serous ovarian carcinoma. These data demonstrate estrogen ability to modulate chemo-sensitivity of MDM4-expressing tumors and to impinge on intracellular trafficking. They support potential usefulness of combination therapy involving anti-estrogenic drugs.

A combined ANXA2-NDRG1-STAT1 gene signature predicts response to chemoradiotherapy in cervical cancer.

BACKGROUND: A better understanding of locally advanced cervical cancer (LACC) is mandatory for further improving the rates of disease control, since a significant proportion of patients still fail to respond or undergo relapse after concurrent chemoradiation treatment (CRT), and survival for these patients has generally remained poor. METHODS: To identify specific markers of CRT response, we compared pretreatment biopsies from LACC patients with pathological complete response (sensitive) with those from patients showing macroscopic residual tumor (resistant) after neoadjuvant CRT, using a proteomic approach integrated with gene expression profiling. The study of the underpinning mechanisms of chemoradiation response was carried out through in vitro models of cervical cancer. RESULTS: We identified annexin A2 (ANXA2), N-myc downstream regulated gene 1 (NDRG1) and signal transducer and activator of transcription 1 (STAT1) as biomarkers of LACC patients' responsiveness to CRT. The dataset collected through qPCR on these genes was used as training dataset to implement a Random Forest algorithm able to predict the response of new patients to this treatment. Mechanistic investigations demonstrated the key role of the identified genes in the balance between death and survival of tumor cells. CONCLUSIONS: Our results define a predictive gene signature that can help in cervical cancer patient stratification, thus providing a useful tool towards more personalized treatment modalities.

Estrogen receptor ß: Potential target for therapy in adult granulosa cell tumors?

OBJECTIVE: Adult granulosa cell tumor (AGCT) is a rare form of sex-cord stromal ovarian tumors. Due to their origin, AGCTs secrete estrogens, and thus, estrogen receptor (ER)-mediated signaling has been considered as a possible target for therapy. The aim of the present study was to get insights into estrogen receptor status and activity in AGCTs, as a strategy to provide molecular support for personalized hormonal treatments. METHODS: We evaluated by immunohistochemistry the expression of ERα, ERß isoforms (i.e. ERß1, ERß2 and ERß5), progesterone and androgen receptor (PR, AR) in 20 untreated AGCTs and 12 unmatched recurrent lesions. Thereafter, we visualized by immunofluorescence, the subcellular distribution of cytoplasmic receptors, and by the proximity ligation assays (PLA) we characterized in situ their ability to interact with other proteins involved in the apoptotic cascade. RESULTS: Primary AGCTs predominantly expressed ERß isoforms, along with PR and AR, while only 30% of patients showed ERα expression. Recurrent tumors were associated with a decrease in AR levels. From mechanistic studies it emerges that ERß2, and to a lesser extent ERß1 and AR, are mitochondrial components in cancer cells and that ERß2 can act as a binding partner of proteins involved in the apoptotic cascade, in turn potentially inhibiting apoptosis. CONCLUSIONS: As in other endocrine tumors, ERß may play a role in the pathogenesis of AGCT; it is crucial to understand estrogen receptor-mediated pathways before planning hormonal treatment strategies in AGCT.

Hormone receptor expression profile of low-grade serous ovarian cancers.

OBJECTIVE: Low-grade serous ovarian carcinomas (LGSOCs) are a histological subtype of epithelial ovarian tumors, accounting for fewer than 5% of all cases of ovarian carcinoma. Due to the chemoresistant nature of this subtype a search for more effective systemic therapies is actively ongoing, hormonal therapy showing some degree of activity in this clinical setting. The present study ought to investigate the hormone receptor status of LGSOCs, as a strategy to provide molecular support for patient-tailored hormonal treatments. METHODS: Estrogen receptor α (ERα), ERß isoforms (i.e. ERß1, ERß2 and ERß5), progesterone and androgen receptor (PR, AR) expression was evaluated by immunohistochemistry in 25 untreated LGSOC primary tumors, 6 matched metastases and 6 micropapillary variant of serous borderline tumors (micropapillary SBOTs). In vitro cellular models were used to provide insights into clinical observations. RESULTS: Our results showed prominent expression of nuclear ERα, ERß2, ERß5 and PR in LGSOC primary tissues, while metastatic lesions also exhibit considerable cytoplasmic ERß2 levels. Notably, a higher expression of ERß1 protein was determined in micropapillary SBOTs compared to LGSOCs. In vitro experiments on LGSOC cell lines (i.e. HOC-7 and VOA-1056) revealed low/absent ERα, PR and AR protein expression, whereas the three ERß isoforms were all present. Proliferation of HOC-7 and VOA-1056 was not modulated by either the endogenous or the selective synthetic ligands. CONCLUSIONS: These novel findings highlight the need of assessing relative levels of ERα and ERß isoforms in the total receptor pool in future clinical studies investigating molecular predictors of response to hormonal therapy in LGSOC.

Ovarian low and high grade serous carcinomas: hidden divergent features in the tumor microenvironment.

Only recently low-grade serous carcinoma (LGSOC) of the ovary has been recognized as a disease entity distinct from the more common high-grade serous carcinoma (HGSOC), with significant differences in pathogenesis and clinical and pathologic features. The present study aimed at evaluating whether the different natural histories and patterns of response to therapy demonstrated for LGSOC and HGSOC, along with a diverse genomic landscape, may also reside in the supporting tumor stroma, specifically in the state of differentiation and activation of tumor associated macrophages (TAMs). TAMs play complex roles in tumorigenesis since they are believed to possess both tumor rejecting (M1 macrophages) and tumor promoting (M2 macrophages) activities. Here we showed that, when compared to HGSOC (n = 55), LGSOC patients (n = 25) exhibited lower density of tumor-infiltrating CD68+ macrophage, along with an attenuated M2-skewed (CD163+) phenotype. Accordingly, assessment of intratumoral vascularization and of matrix metalloproteinase 9 expression (a key protein involved in tumor invasion and metastasis) revealed lower expression in LGSOC compared to HGSOC patients, in line with emerging evidence supporting a role for TAMs in all aspects of tumor initiation, growth, and development. In conclusion, results from the present study demonstrate that microenvironmental factors contribute greatly to determine clinical and pathological features that differentiate low and high grade serous ovarian carcinomas. This understanding may increase possibilities and opportunities to improve disease control and design new therapeutic strategies.

Multiple direct and indirect mechanisms drive estrogen-induced tumor growth in high grade serous ovarian cancers.

The notion that menopausal estrogen replacement therapy increases ovarian cancer risk, but only for the two more common types (i.e. serous and endometrioid), while possibly decreasing risk for clear cell tumors, is strongly suggestive of causality. However, whether estradiol (E2) is tumorigenic or promotes development of occult preexisting disease is unknown. The present study investigated molecular and cellular mechanisms by which E2 modulates the growth of high grade serous ovarian cancer (HGSOC). Results showed that ERα expression was necessary and sufficient to induce the growth of HGSOC cells in in vitro models. Conversely, in vivo experimental studies demonstrated that increasing the levels of circulating estrogens resulted in a significant growth acceleration of ERα-negative HGSOC xenografts, as well. Tumors from E2-treated mice had significantly higher proliferation rate, angiogenesis, and density of tumor-associated macrophage (TAM) compared to ovariectomized females. Accordingly, immunohistochemical analysis of ERα-negative tissue specimens from HGSOC patients showed a significantly greater TAM infiltration in premenopausal compared to postmenopausal women. This study describes novel insights into the impact of E2 on tumor microenvironment, independently of its direct effect on tumor cell growth, thus supporting the idea that multiple direct and indirect mechanisms drive estrogen-induced tumor growth in HGSOC.

Sexual dimorphism in medulloblastoma features.

AIMS: Male sex is a risk factor for medulloblastoma (MB), and is also a negative predictor for clinical outcome. The aim of this study was to assess sex differences in tumour biological features and hormone receptor profiles in a cohort of MB patients. METHODS AND RESULTS: Sixty-four MBs and five normal cerebella were included in the study. Cell proliferation (Ki67), apoptosis (cleaved caspase-3) and microvessel density (CD31) were evaluated in tumours by immunohistochemistry. Tissues were analysed for oestrogen receptor (ER)α, ERß1, ERß2, ERß5 and androgen receptor (AR) expression. The results demonstrated sex-specific features in MBs, with tumours from females showing a higher apoptosis/proliferation ratio and less tumour vascularization than tumours from males. MBs were negative for ERα and AR, but expressed ERß isoforms at similar levels between the sexes. Altogether, these findings indicate that signalling mechanisms that control cell turnover and angiogenesis operate more efficiently in females than in males. The lack of sex differences in the hormone receptor profiles suggests that circulating oestrogens could be the major determinants of the sexual dimorphism observed in MB features. CONCLUSIONS: Here, we provide molecular support for epidemiological data showing sex differences in MB incidence and outcome, completely defining the hormone receptor profile of the tumours.

Mitochondrial estrogen receptor ß2 drives antiapoptotic pathways in advanced serous ovarian cancer.

We previously showed an unfavorable prognostic role of the cytoplasmic estrogen receptor ß2 (cERß2) in serous ovarian cancer. Here we aimed to investigate molecular determinants in cell survival function of cERß2 in this malignant disease. We used immunohistochemistry to evaluate differences in apoptosis (quantified by the expression of cleaved caspase-3) and cell proliferation (quantified by the expression of Ki-67) in 56 advanced serous ovarian cancer cases, stratified according to the absence or presence of estrogen receptor ß2 (ERß2) protein in the cytoplasmic compartment (31 cERß2- and 25 cERß2+ cases, respectively). Thereafter, by immunofluorescence, we visualized the subcellular distribution of ERß2, and by the proximity ligation assays, we characterized in situ its ability to interact with other proteins specifically involved in the apoptosis cascade. Finally, we assessed cytochrome c expression by immunohistochemistry. We demonstrated that, although not affecting tumor proliferation, cytoplasmic ERß2 expression was indeed associated to a lower apoptotic rate in ovarian cancer cases. Then, we proved that cERß2 is targeted to mitochondria where it interacts as a binding partner with BAD (B-cell lymphoma [Bcl] 2-associated death promoter). This interaction, precluding the Bcl-xL (B-cell lymphoma extra large)/BAD heterodimer formation, inhibited Bax (Bcl-2-like protein 4) oligomerization, the release of cytochrome c, and ultimately apoptosis. In conclusion, we provide in vivo mechanistic evidence for an antiapoptotic function of mitochondrial ERß2, a finding supporting the value of its cytoplasmic expression as an unfavorable prognostic biomarker for serous ovarian cancer.

Gender effect in experimental models of human medulloblastoma: does the estrogen receptor ß signaling play a role?

BACKGROUND: The male-to-female sex ratio for medulloblastoma (MB) is approximately 1.5∶1, female gender being also a favorable prognostic factor. This study aimed at evaluating the impact of gender on MB tumorigenesis. METHODS: In vitro activity of 17ß-estradiol (E2), DPN [2,3-bis(4-hydroxyphenyl)-propionitrile, a selective estrogen receptor ß (ERß)-agonist], PPT [4,4',4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, a selective ERα-agonist] or DHT (5 alpha-dihydrotestosterone) was evaluated in three human MB cell lines. D283 Med cells were transplanted into athymic mice. RESULTS: A significant expression of ERß, with little or no ERα, and low AR (androgen receptor) was found in MB cell lines. The compounds tested did not affect cell proliferation. In vivo, we observed a significantly lower growth of D283 Med in nude female mice compared to males. At microscopic examination, tumors from females showed a shift towards differentiation, as evaluated by lower nestin, and higher NSE (neuron-specific enolase) and GFAP (glial fibrillary acidic protein) expression compared to males. Tumors from females also showed lower Ki67 and p53 expression. The wild-type ERß protein (ERß1) was lost in male tumors, while it was a permanent feature in females, and a strong negative correlation was found between Ki67 and ERß1 expression. Conversely, tumor levels of ERß2 and ERß5 did not significantly differ between genders. Increased levels of cyclin-dependent kinase inhibitor p21 were observed in females, suggesting that estrogen may decrease tumor growth through blocking cell cycle progression. An inhibition of the insulin-like growth factor I (IGF-I) signaling was also evident in females. CONCLUSION: We provides mechanistic evidence supporting the idea that ERß1 signaling may have pro-differentiation and tumor suppressive function in medulloblastomas.

Prognostic significance of the estrogen receptor beta (ERß) isoforms ERß1, ERß2, and ERß5 in advanced serous ovarian cancer.

OBJECTIVE: In the present study we have examined the pattern of expression of the full length estrogen receptor ß (ERß1) and two ERß splice variant isoforms (ERß2, ERß5) in well-characterized advanced serous ovarian cancers. METHODS: Immunohistochemistry was performed with ERß1, ERß2, and ERß5 antibodies and results were correlated with pathological and clinical follow-up data. Expression of ERß isoforms in a panel of ovarian cancer cell lines and human tumor xenografts was also assessed. RESULTS: Immunohistochemical staining revealed cellular compartment-specific distribution for each isoform in malignant ovarian tissues exhibiting both nuclear staining and cytoplasmic staining. Patients with cytoplasmic ERß2 expression had significantly worse outcome (p = 0.006 at the multivariate analysis), the 5-year survival rate being nearly 28% for patients who did express cytoplasmic ERß2, and 60% in negative patients. Cytoplasmic ERß2 expression was also found to be significantly associated with chemoresistance. In concordance with clinical results both nuclear and cytoplasmic expressions were observed for the three isoforms in the cancer cell lines and human tumor xenografts tested. CONCLUSIONS: This is the first study to uncover an unfavorable prognostic role of ERß2 in advanced serous ovarian cancer. If anomalies of ERß2 cytoplasmic expression could be demonstrated to represent an independent unfavorable prognostic marker and/or a marker predicting chemoresistance in advanced serous ovarian cancer, its immunohistochemical assessment at the time of surgery, could help to recognize candidates for clinical trials of new interventions.