Ilaria De Stefano

Antiproliferative and antiangiogenic effects of the benzophenanthridine alkaloid sanguinarine in melanoma

This study was aimed at evaluating the potential application of benzophenanthridine alkaloids, sanguinarine and cheleritrine, in the therapy of melanoma cancer. In vitro antiproliferative activity of sanguinarine was higher than that of cheleritrine against the B16 melanoma 4A5 cells. Both agents were able to produce DNA breaks, and the DNA unwinding assay showed that they act as DNA intercalating agents. Sanguinarine was selected for determination of its in vivo preclinical efficacy. Oral treatment with sanguinarine reduced the tumor burden in a transplantable murine tumor grown in a syngeneic host (B16 melanoma 4A5 in C57BL/6 mice), and in a human tumor xenograft grown in immunodeficient mice (A375 human melanoma in athymic nude mice). In A375 tumors a significant decrease in the proliferation marker Ki67, and a reduction in the activated mitogen-activated protein kinases (p-p44/42 MAPK), and in protein kinase B (pAKT) were also observed. Three out of eleven A375-bearing treated mice were tumor-free at the end of treatment, and did not develop any tumor after a further, treatment-free, observation period of 60 days. Sanguinarine also showed a striking antiangiogenic activity in mice. Data from the present study support the concept that sanguinarine can be effective in melanoma skin cancer.

Cytoplasmic expression of estrogen receptor beta (ERβ) predicts poor clinical outcome in advanced serous ovarian cancer.

OBJECTIVE In this study we investigated the prognostic value of estrogen receptor α (ERα), ERβ and progesterone receptor (PR) expression in 58 untreated advanced serous ovarian cancer patients. The study also included 12 macroscopically and histopathologically normal ovaries. MATERIALS AND METHODS Protein expression was evaluated by immunohistochemistry, and antibody staining detected in both the nuclear and cytoplasmic compartments was taken into account. Immunopositivity was analyzed in relation to tumor clinicopathological variables, disease-free survival (DFS), and overall survival (OS). RESULTS Epithelial cells in ovarian cancer tissue showed significantly lower levels of nuclear ERβ and PR, but not ERα, than in normal ovarian tissue. In the case of ERβ, however, while normal ovarian epithelium exhibited almost exclusively strong nuclear staining, ovarian cancer tissue mostly showed cytoplasmic immunopositivity. Nuclear ERα and ERβ expression were not associated with clinical outcome. Conversely, any cytoplasmic ERβ expression was an independent unfavorable prognostic factor for DFS, a finding approaching statistical significance also for OS. These data suggest that, in advanced serous ovarian cancer, cytoplasmic ERβ signaling may be more important for patient survival than its nuclear signaling. In the case of PR, positivity was an independent favorable prognostic factor for DFS. CONCLUSIONS These novel findings, that need to be confirmed in a large prospective trial, suggest that additional prognostic, and possibly therapeutic opportunities may be available in advanced serous ovarian cancer.

Estrogen receptor beta in cancer: an attractive target for therapy.

While it is well documented that the mitogenic actions of estrogens are critical in the development and progression of human breast and some gynecologic cancers, only latest data demonstrate a crucial involvement of estrogen-signaling in the carcinogenesis of non-classical estrogen target tissues, as colon, prostate, lung, skin, and brain. Only recently it has also been found out that the biological effects of estrogens are mediated by two distinct estrogen receptors (ERs), ERα and ERβ, and that their relative levels in a given cell are important determinants of response to estradiol and selective estrogen receptor modulators. Indeed, although ERα and ERβ have similar structure, they produce different effects, and there is currently increasing evidence that, for some tumors, an imbalanced ERβ expression might play a pivotal role in tumor development and progression. However, the prognostic value, the potential significance in predicting response to endocrine therapy, and, eventually, the utility of ERβ as a therapeutic target need to be assessed in large-scale and prospective clinical studies. This review examines the experimental and clinical evidences for a role of ERβ in carcinogenesis of classical and nonclassical estrogen target tissues. If anomalies of ERβ expression could be demonstrated to represent a critical step in the development and progression of some types of cancers, its re-expression by genetic engineering, as well as the use of targeted ERβ therapies would constitute new important therapeutic approaches.

Changes in the expression of oestrogen receptors and E‐cadherin as molecular markers of progression from normal epithelium to invasive cancer in elderly patients with vulvar squamous cell carcinoma

Zannoni G F, Prisco M G, Vellone V G, De Stefano I, Scambia G & Gallo D
(2011) Histopathology58, 265–275
Changes in the expression of oestrogen receptors and E‐cadherin as molecular markers of progression from normal epithelium to invasive cancer in elderly patients with vulvar squamous cell carcinoma

The expression ratios of estrogen receptor α (ERα) to estrogen receptor β1 (ERβ1) and ERα to ERβ2 identify poor clinical outcome in endometrioid endometrial cancer

The prognostic relevance of estrogen (ER) and progesterone receptor (PR) expression in endometrioid endometrial cancer is still controversially discussed. The present study has focused on the evaluation of the prognostic value of ERα, ERβ1, ERβ2, and PR in this histotype. Specifically, we were interested in evaluating whether the relative level of ER subtype-specific expression (in terms of a ratio ERα/ERβ1 and ERα/ERβ2) would predict clinical outcome better than their absolute levels in patients with endometrioid endometrial cancer. To this end, protein content was assessed by immunohistochemistry in a group of 121 cases and staining was analyzed in relation to clinicopathologic variables, disease-free survival and overall survival. Results obtained have demonstrated that none of the biological markers analyzed possess an independent prognostic role with regard to disease-free survival. Multivariate analysis of overall survival has shown that ERα alone is not an independent prognostic indicator in patients with endometrioid endometrial cancer (hazard ratio [HR]; 0.5; 95% confidence interval [CI], 0.09-3.0; P = .5). On the other hand, an ERα/ERβ1 ratio of 1 or less or an ERα/ERβ2 ratio of 1 or less has proved to be independently associated with a higher risk of death (HR, 6.4 [95% CI, 1.0-40.6; P = .04] and 9.7 [95% CI, 1.1-85.3; P = .04], respectively) along with age, tumor stage, and Ki-67. In conclusion, we report here that the ERα/ERβ1 and ERα/ERβ2 expression ratios are independent prognostic markers of survival in endometrioid endometrial cancer; these findings suggest that phenotyping these interacting markers conjointly may better predict patient survival than each individual marker alone.

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.

Clinicopathologic and Immunohistochemical Features of Ovarian Clear Cell Carcinomas in Comparison With Type I and Type Ii Tumors

Two types of ovarian carcinomas are distinguished with respect to morphology, biology, and clinical course, and are designated as Type I and Type II tumors. However, placement of clear cell carcinomas into one of these 2 groups has been problematic as they exhibit morphologic, molecular, and clinical features that do not entirely resemble either Type I or Type II tumors. The present study aimed at better elucidating the clinicopathologic and immunohistochemical features of clear cell carcinomas, in comparison with the 2 main broad categories. To this end, a panel of classic clinicopathologic and immunohistochemical parameters, including estrogen receptor &agr; (ER&agr;), ER&bgr;, progesterone receptor, Ki67, p53, and HER2/neu was evaluated in 71 Type I, 157 Type II, and 21 clear cell carcinomas. Overall, findings from the present study support the idea that ovarian clear cell carcinomas are neither Type I nor Type II carcinomas of the ovary; indeed, results obtained showed that similarities between clear cell carcinomas and Type I were limited to the patient’s age, tumor dimension, incidence of lymph node and extranodal metastases, and p53 labeling index, whereas the patient’s age and incidence of extranodal metastases were the only parameters comparable with the Type II group. The hormonal receptor profile of clear cell carcinomas was characterized by low expression of nuclear ER&agr; and progesterone receptor, and by almost exclusively nuclear ER&bgr; immunopositivity, features significantly different from both Type I and II tumors. Finally, the percentage of HER2/neu-positive samples in clear cell carcinomas was 10- and 2.5-fold higher than Type I and Type II ovarian tumors, respectively. In conclusion, our study provides insights into clear cell carcinoma that could help in explaining its unique prognostic features, and, eventually, in orienting toward new therapeutic options.

Developmental and oncogenic radiation effects on neural stem cells and their differentiating progeny in mouse cerebellum

Neural stem cells are highly susceptible to radiogenic DNA damage, however, little is known about their mechanisms of DNA damage response (DDR) and the long‐term consequences of genotoxic exposure. Patched1 heterozygous mice (Ptc1+/−) provide a powerful model of medulloblastoma (MB), a frequent pediatric tumor of the cerebellum. Irradiation of newborn Ptc1+/− mice dramatically increases the frequency and shortens the latency of MB. In this model, we investigated the mechanisms through which multipotent neural progenitors (NSCs) and fate‐restricted progenitor cells (PCs) of the cerebellum respond to DNA damage induced by radiation, and the long‐term developmental and oncogenic consequences. These responses were assessed in mice exposed to low (0.25 Gy) or high (3 Gy) radiation doses at embryonic day 13.5 (E13.5), when NSCs giving rise to the cerebellum are specified but the external granule layer (EGL) has not yet formed, or at E16.5, during the expansion of granule PCs to form the EGL. We found crucial differences in DDR and apoptosis between NSCs and fate‐restricted PCs, including lack of p21 expression in NSCs. NSCs also appear to be resistant to oncogenesis from low‐dose radiation exposure but more vulnerable at higher doses. In addition, the pathway to DNA repair and the pattern of oncogenic alterations were strongly dependent on age at exposure, highlighting a differentiation‐stage specificity of DNA repair pathways in NSCs and PCs. These findings shed light on the mechanisms used by NSCs and PCs to maintain genome integrity during neurogenesis and may have important implications for radiation risk assessment and for development of targeted therapies against brain tumors. Stem Cells 2013;31:2506–2516

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.

Protective role of 17β‐estradiol on medulloblastoma development in Patched1 heterozygous mice

Medulloblastoma (MB) is the most common pediatric tumor of the CNS, representing ∼20% of all childhood CNS tumors. Although in recent years many molecular mechanisms that control MB development have been clarified, the effects of biological factors such as sex on this tumor remain to be explained. Epidemiological data, in fact, indicate a significant difference in the incidence of MB between the 2 sexes, with considerably higher susceptibility of males than females. Besides this different susceptibility, female sex is also a significant favorable prognostic factor in MB, with girls having a much better outcome. Despite these literature data, there has been little investigation into estrogen influence on MB development. In our study, we evaluated how hormone deficiency resulting from ovariectomy and hormone replacement influences the development of early and advanced MB stages in Patched1 heterozygous mice, a well‐characterized mouse model of radiation‐induced MB. Susceptibility to MB development was significantly increased in ovariectomized Ptch1+/− females and restored to levels observed in control mice after estrogen replacement. We next investigated the molecular mechanisms by which estrogen might influence tumor progression and show that ERβ, but not ERα, is involved in modulation of MB development by estrogens. Finally, our study shows that a functional interaction between estrogen‐ and IGF‐I‐mediated pathways may be responsible for the effects observed.