Carmela Ricciardelli

Androgen receptor inhibits estrogen receptor-alpha activity and is prognostic in breast cancer

There is emerging evidence that the balance between estrogen receptor-alpha (ER(alpha)) and androgen receptor (AR) signaling is a critical determinant of growth in the normal and malignant breast. In this study, we assessed AR status in a cohort of 215 invasive ductal breast carcinomas. AR and (ER(alpha)) were coexpressed in the majority (80-90%) of breast tumor cells. Kaplan-Meier product limit analysis and multivariate Cox regression showed that AR is an independent prognostic factor in (ER(alpha))-positive disease, with a low level of AR (less than median of 75% positive cells) conferring a 4.6-fold increased risk of cancer-related death (P = 0.002). Consistent with a role for AR in breast cancer outcome, AR potently inhibited (ER(alpha))transactivation activity and 17beta-estradiol-stimulated growth of breast cancer cells. Transfection of MDA-MB-231 breast cancer cells with either functionally impaired AR variants or the DNA-binding domain of the AR indicated that the latter is both necessary and sufficient for inhibition of (ER(alpha)) signaling. Consistent with molecular modeling, electrophoretic mobility shift assays showed binding of the AR to an estrogen-responsive element (ERE). Evidence for a functional interaction of the AR with an ERE in vivo was provided by chromatin immunoprecipitation data, revealing recruitment of the AR to the progesterone receptor promoter in T-47D breast cancer cells. We conclude that, by binding to a subset of EREs, the AR can prevent activation of target genes that mediate the stimulatory effects of 17beta-estradiol on breast cancer cells.

Androgens induce divergent proliferative responses in human breast cancer cell lines

Although the majority of primary human breast cancers express the androgen receptor (AR), the role of androgens in breast cancer growth and progression is poorly understood. We have investigated the effects of the naturally occurring androgen, dihydrotestosterone (DHT), and a synthetic non-metabolizable androgen, mibolerone, on the proliferation of six human breast cancer cell lines. The anti-proliferative and proliferative effects of androgens were only observed in cell lines that expressed the AR. Two of the AR-positive cell lines, T47-D and ZR-75-1 were growth inhibited in the presence of either DHT or mibolerone, while the proliferation of MCF-7 and MDA-MB-453 cells was increased by both androgens. Co-incubation of cultures with 1 nM DHT and a 100-fold excess of the androgen receptor antagonist, hydroxyflutamide, resulted in reversal of both inhibitory and stimulatory effects of DHT on T47-D, MCF-7 and MDA-MB-453 cell proliferation, indicating that DHT action is mediated by the AR in these lines. Hydroxyflutamide only partially reversed the DHT-induced growth inhibition of ZR-75-1 cultures, which suggests that growth inhibition of these cells may be mediated by non-AR pathways of DHT (or DHT metabolite) action. Mibolerone action on breast cancer cell growth was similar to that of DHT, with the exception that growth stimulation of MCF-7 and MDA-MB-453 cells was only partially reversed in the presence of a 100-fold excess of hydroxyflutamide. Anandron, another androgen receptor antagonist, was able to reverse all inhibitory and stimulatory actions of the androgens. AR antisense oligonucleotides reduced the level of immunoreactive AR expression in MDA-MB-453 and ZR-75-1 cells by more than 60%, but only reversed the growth inhibitory action of mibolerone in ZR-75-1 cultures. The results suggest that androgen action in breast cancer cell lines may not be solely mediated by binding of androgen to the AR. For example, metabolites of DHT with oestrogenic activity, or androgen binding to receptors other than the AR, may explain the divergent responses to androgens observed in different breast cancer cell lines.

The biological role and regulation of versican levels in cancer

Increased expression of the proteoglycan, versican is strongly associated with poor outcome for many different cancers. Depending on the cancer type, versican is expressed by either the cancer cells themselves or by stromal cells surrounding the tumor. Versican plays diverse roles in cell adhesion, proliferation, migration and angiogenesis, all features of invasion and metastasis. These wide ranging functions have been attributed to the central glycosaminoglycan-binding region of versican, and to the N-(G1) and C-(G3) terminal globular domains which collectively interact with a large number of extracellular matrix and cell surface structural components. Here we review the recently identified mechanisms responsible for the regulation of versican expression and the biological roles that versican plays in cancer invasion and metastasis. The regulation of versican expression may represent one mechanism whereby cancer cells alter their surrounding microenvironment to facilitate the malignant growth and invasion of several tumor types. A greater understanding of the regulation of versican expression may contribute to the development of therapeutic methods to inhibit versican function and tumor invasion.

Expression of Extracellular Matrix Components Versican, Chondroitin Sulfate, Tenascin, and Hyaluronan, and Their Association with Disease Outcome in Node-Negative Breast Cancer

Purpose: The purpose is to determine whether the levels of expression of extracellular matrix components in peritumoral stroma are predictive of disease outcome for women with node-negative breast cancer. Experimental Design: Tumor tissue from 86 patients with node-negative breast cancer was examined by immunohistochemical staining for the expression of versican, chondroitin sulfate (CS), tenascin, and hyaluronan (HA). With the exception of HA, the expression of the extracellular matrix components was measured by video image analysis. Statistical correlation of the immunohistochemical data with clinicopathological characteristics and disease outcome was performed. Results: All of the extracellular matrix components were present in the peritumoral stroma of the entire study cohort. In contrast, immunoreactivity within the cancer cell was observed in 82% of tumors for HA, 12% for CS, and 4% for tenascin; no immunostaining of cancer cells for versican was observed for any of the tumors. Cox regression and Kaplan-Meier analyses indicated that elevated expression of stromal versican predicted increased risk and rate of relapse in this cohort. Elevated expression of tenascin was predictive of increased risk and rate of death only. Although neither CS nor HA were predictive of disease outcome in this cohort, tumor size was predictive of increased risk and rate of both relapse and survival. Conclusions: Elevated expression within peritumoral stromal matrix of versican and tenascin was predictive of relapse-free and overall survival, respectively, in women with node-negative breast cancer.

The Role of Annexin A2 in Tumorigenesis and Cancer Progression

Annexin A2 is a calcium-dependent, phospholipid-binding protein found on various cell types. It is up-regulated in various tumor types and plays multiple roles in regulating cellular functions, including angiogenesis, proliferation, apoptosis, cell migration, invasion and adhesion. Annexin A2 binds with plasminogen and tissue plasminogen activator on the cell surface, which leads to the conversion of plasminogen to plasmin. Plasmin is a serine protease which plays a key role in the activation of metalloproteinases and degradation of extracellular matrix components essential for metastatic progression. We have recently found that both annexin A2 and plasmin are increased in conditioned media of co cultured ovarian cancer and peritoneal cells. Our studies suggest that annexin A2 is part of a tumor-host signal pathway between ovarian cancer and peritoneal cells which promotes ovarian cancer metastasis. Accumulating evidence suggest that interactions between annexin A2 and its binding proteins play an important role in the tumor microenvironment and act together to enhance cancer metastasis. This article reviews the current knowledge on the biological role of annexin A2 and its binding proteins in solid malignancies including ovarian cancer.

Formation of Hyaluronan- and Versican-rich Pericellular Matrix by Prostate Cancer Cells Promotes Cell Motility

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.

Chick Chorioallantoic Membrane (CAM) Assay as an In Vivo Model to Study the Effect of Newly Identified Molecules on Ovarian Cancer Invasion and Metastasis

The majority of ovarian cancer patients present with advanced disease and despite aggressive treatment, prognosis remains poor. Significant improvement in ovarian cancer survival will require the development of more effective molecularly targeted therapeutics. Commonly, mouse models are used for the in vivo assessment of potential new therapeutic targets in ovarian cancer. However, animal models are costly and time consuming. Other models, such as the chick embryo chorioallantoic membrane (CAM) assay, are therefore an attractive alternative. CAM assays have been widely used to study angiogenesis and tumor invasion of colorectal, prostate and brain cancers. However, there have been limited studies that have used CAM assays to assess ovarian cancer invasion and metastasis. We have therefore developed a CAM assay protocol to monitor the metastatic properties of ovarian cancer cells (OVCAR-3, SKOV-3 and OV-90) and to study the effect of potential therapeutic molecules in vivo. The results from the CAM assay are consistent with cancer cell motility and invasion observed in in vitro assays. Our results demonstrate that the CAM assay is a robust and cost effective model to study ovarian cancer cell metastasis. It is therefore a very useful in vivo model for screening of potential novel therapeutics.

Global Levels of Specific Histone Modifications and an Epigenetic Gene Signature Predict Prostate Cancer Progression and Development

Background: Epigenetic alterations are common in prostate cancer, yet how these modifications contribute to carcinogenesis is poorly understood. We investigated whether specific histone modifications are prognostic for prostate cancer relapse, and whether the expression of epigenetic genes is altered in prostate tumorigenesis. Methods: Global levels of histone H3 lysine-18 acetylation (H3K18Ac) and histone H3 lysine-4 dimethylation (H3K4diMe) were assessed immunohistochemically in a prostate cancer cohort of 279 cases. Epigenetic gene expression was investigated in silico by analysis of microarray data from 23 primary prostate cancers (8 with biochemical recurrence and 15 without) and 7 metastatic lesions. Results: H3K18Ac and H3K4diMe are independent predictors of relapse-free survival, with high global levels associated with a 1.71-fold (P < 0.0001) and 1.80-fold (P = 0.006) increased risk of tumor recurrence, respectively. High levels of both histone modifications were associated with a 3-fold increased risk of relapse (P < 0.0001). Epigenetic gene expression profiling identified a candidate gene signature (DNMT3A, MBD4, MLL2, MLL3, NSD1, and SRCAP), which significantly discriminated nonmalignant from prostate tumor tissue (P = 0.0063) in an independent cohort. Conclusions: This study has established the importance of histone modifications in predicting prostate cancer relapse and has identified an epigenetic gene signature associated with prostate tumorigenesis. Impact: Our findings suggest that targeting the epigenetic enzymes specifically involved in a particular solid tumor may be a more effective approach. Moreover, testing for aberrant expression of epigenetic genes such as those identified in this study may be beneficial in predicting individual patient response to epigenetic therapies. Cancer Epidemiol Biomarkers Prev; 19(10); 2611–22. ©2010 AACR.

Androgen receptor signaling: Mechanism of interleukin-6 inhibition

Nonsteroidal signaling via the androgen receptor (AR) plays an im-portant role in hormone-refractory prostate cancer. Previously, we have reported that the pleiotropic cytokine, interleukin (IL)-6, inhibited dihydrotestosterone-mediated expression of prostate-specific antigen in LNCaP cells (Jia et al., Mol Can Res 2003;1:385–92). In the present study, we explored the mechanisms involved in this inhibition and considered possible effects on AR nuclear translocation, recruitment of transcription cofactors, and the signaling pathways that may mediate this inhibitory effect. IL-6 neither induced nuclear localization of the AR nor inhibited dihydrotestosterone-induced nuclear translocation of the receptor. IL-6 did not affect AR or p160 coactivator recruitment to the transcription initiation complex on the prostate-specific antigen enhancer and promoter. Moreover, it did not lead to the recruitment of the corepressor silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) or histone deacetylase 1 (HDAC1) at the same sites. IL-6 did, however, prevent the recruitment of the secondary coactivator, p300, to the complex and partially inhibited histone H3 acetylation at the same loci. Furthermore, inhibition by IL-6 was not mediated by the mitogen-activated protein kinase or the Akt pathways and was partially abrogated by signal transducers and activators of transcription-3 knock-down using small interfering RNA. Our results show that IL-6 modulates androgen action through the differential recruitment of cofactors to target genes. These findings may account for the pleiotropic actions of IL-6 in malignant prostate cells.

Role of versican, hyaluronan and CD44 in ovarian cancer metastasis.

There is increasing evidence to suggest that extracellular matrix (ECM) components play an active role in tumor progression and are an important determinant for the growth and progression of solid tumors. Tumor cells interfere with the normal programming of ECM biosynthesis and can extensively modify the structure and composition of the matrix. In ovarian cancer alterations in the extracellular environment are critical for tumor initiation and progression and intra-peritoneal dissemination. ECM molecules including versican and hyaluronan (HA) which interacts with the HA receptor, CD44, have been shown to play critical roles in ovarian cancer metastasis. This review focuses on versican, HA, and CD44 and their potential as therapeutic targets for ovarian cancer.