Alessandra Ciucci

Prognostic significance of the estrogen receptor beta (ERβ) isoforms ERβ1, ERβ2, and ERβ5 in advanced serous ovarian cancer

OBJECTIVE In the present study we have examined the pattern of expression of the full length estrogen receptor β (ERβ1) and two ERβ splice variant isoforms (ERβ2, ERβ5) in well-characterized advanced serous ovarian cancers. METHODS Immunohistochemistry was performed with ERβ1, ERβ2, and ERβ5 antibodies and results were correlated with pathological and clinical follow-up data. Expression of ERβ 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 ERβ2 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 ERβ2, and 60% in negative patients. Cytoplasmic ERβ2 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 ERβ2 in advanced serous ovarian cancer. If anomalies of ERβ2 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.

Expression of the glioma-associated oncogene homolog 1 (gli1) in advanced serous ovarian cancer is associated with unfavorable overall survival.

Recent evidence links aberrant activation of Hedgehog (Hh) signaling with the pathogenesis of several cancers including medulloblastoma, glioblastoma, melanoma as well as pancreas, colorectal, and prostate carcinomas. Here we investigated the role of the transcription factor Gli1 in ovarian cancer. To this end, the expression profile of Gli1 was examined in normal ovaries, ovarian tumors, and ovarian cancer cell lines, and the in vitro effects of a specific Hh-pathway blocker, KAAD-cyclopamine, or a specific Gli1 inhibitor (GANT58) on cell proliferation and on Hh target gene expression were also assessed. Results obtained showed that epithelial cells in ovarian cancer tissue express significantly higher levels of nuclear Gli1 than in normal ovarian tissue, where the protein was almost undetectable. In addition, multivariate analysis showed that nuclear Gli1 was independently associated to poor survival in advanced serous ovarian cancer patients (HR = 2.2, 95%CI 1.0–5.1, p = 0.04). In vitro experiments demonstrated Gli1 expression in the three ovarian carcinoma cell lines tested, A2780, SKOV-3 and OVCAR-3. Remarkably, although KAAD-cyclopamine led to decreased cell proliferation, this treatment did not inhibit hedgehog target gene expression in any of the three ovarian cancer cell lines, suggesting that the inhibition of cell proliferation was a nonspecific or toxic effect. In line with these data, no differences on cell proliferation were observed when cell lines were treated with GANT58. Overall, our clinical data support the role of Gli1 as a prognostic marker in advanced serous ovarian cancer and as a possible therapeutic target in this disease. However, our in vitro findings draw attention to the need for selection of appropriate experimental models that accurately represent human tumor for testing future therapies involving Hh pathway inhibitors.

HMGB1 and Cord Blood: Its Role as Immuno-Adjuvant Factor in Innate Immunity

In newborn the innate immune system provides essential protection during primary infections before the generation of an appropriate adaptive immune response that is initially not fully operative. Innate immune response is evoked and perpetuated by molecules derived from microorganisms or by the damage/death of host cells. These are collectively known as damage-associated molecular-pattern (DAMP) molecules. High-mobility group box 1 protein (HMGB1) or amphoterin, which previously was considered to be only a nuclear factor, has been recently identified as a DAMP molecule. When it is actively secreted by inflammatory cells or passively released from necrotic cells, HMGB1 mediates the response to infection, injury and inflammation, inducing dendritic cells maturation and T helper-1-cell responses. To characterize the role of HMGB1 in the innate and immature defense mechanisms in newborns, human cord blood (CB) mononuclear cells, in comparison to adult peripheral blood (PB) mononuclear cells, have been analyzed for its expression. By flow cytometry and western blot analysis, we observed that in CB and PB cells: i) HMGB1 is expressed on cell surface membranes of myeloid dendritic cell precursors, mostly, and lymphocytes (gamma/delta and CD4+ T cells) to a lesser extent; ii) different pro-inflammatory stimuli or molecules that mimic infection increased cell surface expression of HMGB1 as well as its secretion into extracellular environment; iii) the treatment with synthetic molecules such as aminobisphosphonates (ABs), identified to be γδ T cell antigens, triggered up-regulation of HMGB1 expression on mononuclear cells, as well γδ T lymphocytes, inducing its secretion. The modulation of its secretion and the HMGB1-mediated migration of monocytes indicated HMGB1 as regulator of immune response in an immature system, like CB, through engagement of γδ T lymphocytes and myeloid dendritic cell precursors, essential components of innate immunity. In addition, the increased HMGB1 expression/secretion triggered by ABs, previously characterized for their immuno-modulating and immune-adjuvant capabilities, indicated that immunomodulation might represent a new therapeutical approach for neonatal and adult pathologies.

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.

Gender Effect in Experimental Models of Human Medulloblastoma: Does the Estrogen Receptor β 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 17β-estradiol (E2), DPN [2,3-bis(4-hydroxyphenyl)-propionitrile, a selective estrogen receptor β (ERβ)-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 ERβ, 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 ERβ protein (ERβ1) was lost in male tumors, while it was a permanent feature in females, and a strong negative correlation was found between Ki67 and ERβ1 expression. Conversely, tumor levels of ERβ2 and ERβ5 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 ERβ1 signaling may have pro-differentiation and tumor suppressive function in medulloblastomas.

Mitochondrial estrogen receptor β2 drives antiapoptotic pathways in advanced serous ovarian cancer

We previously showed an unfavorable prognostic role of the cytoplasmic estrogen receptor β2 (cERβ2) in serous ovarian cancer. Here we aimed to investigate molecular determinants in cell survival function of cERβ2 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 β2 (ERβ2) protein in the cytoplasmic compartment (31 cERβ2- and 25 cERβ2+ cases, respectively). Thereafter, by immunofluorescence, we visualized the subcellular distribution of ERβ2, 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 ERβ2 expression was indeed associated to a lower apoptotic rate in ovarian cancer cases. Then, we proved that cERβ2 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 ERβ2, a finding supporting the value of its cytoplasmic expression as an unfavorable prognostic biomarker for serous ovarian cancer.

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), ERβ isoforms (i.e. ERβ1, ERβ2 and ERβ5), 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, ERβ2, ERβ5 and PR in LGSOC primary tissues, while metastatic lesions also exhibit considerable cytoplasmic ERβ2 levels. Notably, a higher expression of ERβ1 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 ERβ 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 ERβ isoforms in the total receptor pool in future clinical studies investigating molecular predictors of response to hormonal therapy in LGSOC.

Estrogen receptor β: 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, ERβ isoforms (i.e. ERβ1, ERβ2 and ERβ5), 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 ERβ 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 ERβ2, and to a lesser extent ERβ1 and AR, are mitochondrial components in cancer cells and that ERβ2 can act as a binding partner of proteins involved in the apoptotic cascade, in turn potentially inhibiting apoptosis. CONCLUSIONS As in other endocrine tumors, ERβ 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.

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.

Sexual dimorphism in medulloblastoma features

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.