Carmen Ruggiero

Potential of olive oil phenols as chemopreventive and therapeutic agents against cancer: A review of in vitro studies

Olive oil is a common component of Mediterranean dietary habits. Epidemiological studies have shown how the incidence of various diseases, including certain cancers, is relatively low in the Mediterranean basin compared to that of other European or North America countries. Current knowledge indicates that the phenolic fraction of olive oil has antitumor effects. In addition to the ability to be chemopreventive, with its high antioxidant activity, the antitumor effects of olive oil phenols (OO-phenols) has been studied because of their capacity to inhibit proliferation and promote apoptosis in several tumor cell lines, by diverse mechanisms. This review will summarize and discuss the most recent relevant results on the antitumor effect of OO-phenols on leukemia tumor cells, colorectal carcinoma cells, and breast cancer (BC) cells. In particular, very recent data will be reported and discussed showing the molecular signaling pathways activated by OO-phenols in different histopathological BC cell types, suggesting the potential use of OO-phenols as adjuvant treatment against several subsets of BC. Data summarized here represent a good starting point for more extensive studies for better insight into the molecular mechanisms induced by OO-phenols and to increase the availability of chemopreventive or therapeutic drugs to fight cancer.

17β-Estradiol activates GPER- and ESR1-dependent pathways inducing apoptosis in GC-2 cells, a mouse spermatocyte-derived cell line

In mammals, spontaneous apoptosis is observed particularly in differentiating spermatogonia and in spermatocytes. 17β-Estradiol (E2) in primary rat pachytene spermatocytes (PS) binds estrogen receptor α (ESR1) and GPER to activate EGFR/ERK/c-Jun pathway leading to up regulation of proapoptotic factor bax. Aim of this study was to clarify the effector pathway(s) controlling spermatocytes apoptosis using as model GC-2 cells, an immortalized mouse pachytene spermatocyte-derived cell line, which reproduces primary cells responses to E2. In fact, in GC-2 cells we observed that ESR1 and GPER activation caused rapid ERK and c-Jun phosphorylation, bax up-regulation, events associated with apoptosis. We further investigated the apoptotic mechanism demonstrating that E2, as well as ESR1 and GPER specific agonists, induced sustained ERK, c-Jun and p38 phosphorylation, Cytochrome c release, caspase 3 and endogenous substrate Poly (ADP-ribose) polymerase (PARP) activation and increased expression of cell cycle inhibitor p21. When ESR1 or GPER expression was silenced, E2 was still able to decrease cell proliferation, only the concomitant silencing abolished E2 effect. These results indicate that GC-2 cells are a valid cell model to study E2-dependent apoptosis in spermatocytes and show that E2, activating both ESR1 and GPER, is able to induce an ERK1/2, c-Jun and p38-dependent mitochondrion apoptotic pathway in this cell type.

Targeting Estrogen Receptor-α Reduces Adrenocortical Cancer (ACC) Cell Growth in Vitro and in Vivo: Potential Therapeutic Role of Selective Estrogen Receptor Modulators (SERMs) for ACC Treatment

CONTEXT Adrenocortical carcinoma (ACC) is a rare tumor with a very poor prognosis and no effective treatment. ACC is characterized by an increased production of IGF-II and by estrogen receptor (ER)-α up-regulation. OBJECTIVE The objective of this study was to define the role played by ERα in 17β-estradiol (E2)- and IGF-II-dependent ACC growth and evaluate whether selective estrogen receptor modulators are effective in controlling ACC growth in vivo. EXPERIMENTAL DESIGN The human adrenocortical cell line H295R was used as an in vitro model and to generate xenograft tumors in athymic nude mice. RESULTS In H295R cells IGF-II controlled expression of steroidogenic factor-1 that, in turn, increased aromatase transcription and, consequently, estrogen production, inducing cell proliferation. ERα silencing significantly blocked E2- and IGF-II-dependent cell proliferation. This effect was dependent on the regulation of cyclin D1 expression by ERα, activated in response to both E2 and IGF-II. In fact, IGF-II induced ERα activation by phosphorylating serine 118 and 167. Furthermore, we demonstrated that ERα mediated E2-induced nongenomic signaling that stimulated IGF-I receptor (IGF1R), ERK1/2, and AKT phosphorylation, resulting in a ligand-independent activation of the IGF1R-induced pathway. In addition, E2 potentiated this pathway by up-regulating IGF1R expression as a consequence of increased cAMP-responsive element binding protein activation and binding to IGF1R promoter. The estrogen antagonist, hydroxytamoxifen, the active metabolite of tamoxifen, reduced IGF1R protein levels and both E2- and IGF-II-induced cell proliferation. Moreover, H295R xenograft growth was strongly reduced by tamoxifen. CONCLUSION These findings establish a critical role for ERα in E2- and IGF-II-dependent ACC proliferation and provide a rationale for targeting ERα to control the proliferation of ACC.

Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion

Inhibiting the guanine nucleotide exchange factor VAV2 may prevent metastatic progression of adrenocortical carcinoma. Target VAV2 for adrenocortical carcinoma Adrenocortical carcinoma (ACC) is an aggressive cancer of the steroid hormone–producing tissue of the adrenal gland. Patients with ACC have few clinical options and poor prognosis, given that invasion and distant metastasis are often already present at the time of diagnosis. The molecular cause is unknown, but various genes exhibit increased expression in ACC tumors, including the gene encoding the transcription factor SF-1. Ruggiero et al. found that the expression of an SF-1 target gene encoding guanine exchange factor VAV2 was a critical mediator of changes in ACC cell morphology that enabled invasion in both culture and in vivo models. VAV2 is associated with progression in other tumor types, and pharmacological inhibitors against VAV2 are under development. The findings suggest that targeting VAV2 may be a future clinical option for ACC patients. Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a dismal prognosis. Genomic studies have enabled progress in our understanding of the molecular bases of ACC, but factors that influence its prognosis are lacking. Amplification of the gene encoding the transcription factor steroidogenic factor-1 (SF-1; also known as NR5A1) is one of the genetic alterations common in ACC. We identified a transcriptional regulatory mechanism involving increased abundance of VAV2, a guanine nucleotide exchange factor for small GTPases that control the cytoskeleton, driven by increased expression of the gene encoding SF-1 in ACC. Manipulating SF-1 and VAV2 abundance in cultured ACC cells revealed that VAV2 was a critical factor for SF-1–induced cytoskeletal remodeling and invasion in culture (Matrigel) and in vivo (chicken chorioallantoic membrane) models. Analysis of ACC patient cohorts indicated that greater VAV2 abundance robustly correlated with poor prognosis in ACC patients. Because VAV2 is a druggable target, our findings suggest that blocking VAV2 may be a new therapeutic approach to inhibit metastatic progression in ACC patients.

Establishment of a mouse xenograft model of metastatic adrenocortical carcinoma

Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Very important advances have been made in the identification of the genetic determinants of adrenocortical carcinoma pathogenesis but our understanding is still limited about the mechanisms that determine cancer spread and metastasis. One major problem hindering preclinical experimentation for new therapies for adrenocortical carcinoma is represented by the lack of suitable animal models for metastatic disease. With the aim to overcome these limitations, in this study we tested several protocols in order to establish a mouse xenograft model of metastatic adrenocortical carcinoma. The most efficient method, based upon intrasplenic injection followed by splenectomy, produced metastases with high efficiency, whose development could be followed over time by bioluminescence measurements. We expect that the availability of this model will greatly improve the possibilities for preclinical testing of new treatments for advanced-stage disease.Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Very important advances have been made in the identification of the genetic determinants of adrenocortical carcinoma pathogenesis but our understanding is still limited about the mechanisms that determine cancer spread and metastasis. One major problem hindering preclinical experimentation for new therapies for adrenocortical carcinoma is represented by the lack of suitable animal models for metastatic disease. With the aim to overcome these limitations, in this study we tested several protocols in order to establish a mouse xenograft model of metastatic adrenocortical carcinoma. The most efficient method, based upon intrasplenic injection followed by splenectomy, produced metastases with high efficiency, whose development could be followed over time by bioluminescence measurements. We expect that the availability of this model will greatly improve the possibilities for preclinical testing of new treatments for advanced-stage disease.

Targeting the multidrug transporter Patched potentiates chemotherapy efficiency on adrenocortical carcinoma in vitro and in vivo : Patched inhibition improves cancer therapy

One of the crucial challenges in the clinical management of cancer is the resistance to chemotherapeutics. We recently demonstrated that the Hedgehog receptor Patched, which is overexpressed in many recurrent and metastatic cancers, is a multidrug transporter for chemotherapeutic agents such as doxorubicin. The present work provides evidences that Patched is expressed in adrenocortical carcinoma (ACC) patients, and is a major player of the doxorubicin efflux and the doxorubicin resistance in the human ACC cell line H295R. We discovered that methiothepin inhibits the doxorubicin efflux activity of Patched. This drug‐like molecule enhances the cytotoxic, pro‐apoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells which endogenously overexpress Patched, and thereby mitigates the resistance of these cancer cells to doxorubicin. Moreover, we report that in mice the combination of methiothepin with doxorubicin prevents the development of xenografted ACC tumors more efficiently than doxorubicin alone by enhancing the accumulation of doxorubicin specifically in tumors without obvious undesirable side effects. Our results suggest that the use of an inhibitor of Patched drug efflux such as methiothepin in combination with doxorubicin could be a promising therapeutic option for adrenocortical carcinoma, and most likely also for other Patched‐expressing cancers.

VAV2: a novel prognostic marker and a druggable target for adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a dismal prognosis. Current polychemotherapeutic cytotoxic regimens or targeted therapies have a limited efficacy in the advanced metastatic stage of the disease. The molecular mechanisms underlying adrenocortical tumorigenesis remain to be fully elucidated. An important player involved in ACC pathogenesis is the transcription factor steroidogenic factor-1 (SF-1), which has a pivotal role in the development of adrenal glands and gonads and in the control of steroidogenesis [1]. Increased SF-1 dosage is associated with enhanced adrenocortical tumor cell proliferation, development of adrenocortical tumors in mice and confers a more severe prognosis to adult adrenocortical cancers [2, 3]. We analyzed the mechanisms by which SF-1 controls gene expression in ACC cells and showed that SF-1 regulates multiple gene categories according to its dosage [2]. SF-1 dosage-dependent target gene products are implicated in a variety of cellular processes and control pivotal signalling pathways [2]. In our recent study “Dosage-dependent transcriptional regulation of VAV2 by Steroidogenic Factor-1 drives tumor cell invasion” [4], we focused on one of the SF-1 dosage-dependent target genes identified in our previous genomic studies, VAV2. It encodes a guanine nucleotide exchange factor (GEF) for Rho small GTPases, which has an important function in many aspects of tumor biology [5]. Our findings revealed a transcriptional regulatory loop involving upregulation of VAV2, driven by overexpression of SF-1 in ACC (Figure 1). VAV2 is an essential factor driving the invasive phenotype of ACC cells, whose expression is robustly correlated with prognosis in ACC patients and represents a potentially druggable target [4]. Our findings put in direct relationship dosagedependent transcriptional regulation by SF-1 with VAV2, providing the first example of direct transcriptional regulation of a GEF by an oncogenic transcription factor [4]. Classically, GEF activation in cancer rather involves activation by upstream tyrosine kinases or overexpression due to genomic imbalances. Indeed, the known mechanism of VAV2 activation in cancer consists in the phosphorylation of regulatory tyrosine residues by oncogenic kinases [5]. VAV2 is hyperactivated in head and neck squamous cell carcinoma through an epidermal growth factor (EGFR)-dependent autocrine loop [5]. In turn, it is responsible for slowing down receptor internalization and degradation, further increasing EGF signalling. In addition to modulate Rho small GTPase activity and cytoskeleton remodeling, VAV2 and VAV3 control a transcriptional program that regulates specific steps of metastatic dissemination of breast cancer cells to the lungs [5]. Moreover, Vav2−/−Vav3−/− double knockout mice display reduced xenograft tumor growth when transplanted with lung or melanoma cells. This suggests a role for VAV proteins in the host microenvironment, highlighting the multiple ways by which those GEFs participate to tumorigenesis. Elevation of Rho small GTPase GEF expression and/or activity is a common phenomenon during cancer progression [6]. Thus, Rho-GEFs represent attractive therapeutic targets for cancer [6]. Targeting the binding of Rho small GTPases to GEFs is a rational strategy to inhibit Rac activity [6]. Because of their important role in tumor spreading and metastasis, VAV proteins have been proposed to be potentially druggable by small molecules and amenable to development of novel tools for cancer therapy. The recent development of inhibitors of RAC1VAV2 association [7] has provided the proof of principle that this interaction represents a druggable target in cancer. The extensive analysis of the mechanism of action of Brefeldin A, the first known GEF inhibitor, has led to the general concept of « interfacial inhibition » [6]. It refers to inhibitors, like the recently developed Vav2-Rac interaction inhibitor Ehop-016 [7], that act by stabilizing protein complexes and targeting regions in or near interfaces. Our findings open the way to the development Editorial

The GRP78/BiP inhibitor HA15 synergizes with mitotane action against adrenocortical carcinoma cells through convergent activation of ER stress pathways

Many types of cancer cells present constitutively activated ER stress pathways because of their significant burden of misfolded proteins coded by mutated and rearranged genes. Further increase of ER stress by pharmacological intervention may shift the balance towards cell death and can be exploited therapeutically. Recent studies have shown that an important component in the mechanism of action of mitotane, the only approved drug for the medical treatment of adrenocortical carcinoma (ACC), is represented by activation of ER stress through inhibition of the SOAT1 enzyme and accumulation of toxic lipids. Here we show that HA15, a novel inhibitor of the essential ER chaperone GRP78/BiP, inhibits ACC H295R cell proliferation and steroidogenesis and is able to synergize with mitotane action. These results suggest that convergent activation of ER stress pathways by drugs acting via different mechanisms represents a valuable therapeutic option for ACC.

The KDEL receptor signalling cascade targets focal adhesion kinase on focal adhesions and invadopodia

Membrane trafficking via the Golgi-localised KDEL receptor activates signalling cascades that coordinate both trafficking and other cellular functions, including autophagy and extracellular matrix degradation. In this study, we provide evidence that membrane trafficking activates KDEL receptor and the Src family kinases at focal adhesions of HeLa cells, where this phosphorylates ADP-ribosylation factor GTPase-activating protein with SH3 domain, ankyrin repeat and PH domain (ASAP)1 and focal adhesion kinase (FAK). Previous studies have reported extracellular matrix degradation at focal adhesions. Here, matrix degradation was not seen at focal adhesions, although it occurred at invadopodia, where it was increased by KDEL receptor activation. This activation of KDEL receptor at invadopodia of A375 cells promoted recruitment and phosphorylation of FAK on tyrosines 397 and 861. From the functional standpoint, FAK overexpression inhibited steady-state and KDEL-receptor-stimulated extracellular matrix degradation, whereas overexpression of the FAK-Y397F mutant only inhibited KDEL-receptor-stimulated matrix degradation. Finally, we show that the Src and FAK activated downstream of KDEL receptor are part of parallel signalling pathways. In conclusion, membrane-traffic-generated signalling via KDEL receptor activates Src not only at the Golgi complex, but also at focal adhesions. By acting on Src and FAK, KDEL receptor increases invadopodia-mediated matrix degradation.

NR5A1 (nuclear receptor subfamily 5, group A, member 1)

Review on NR5A1, with data on DNA/RNA, on the protein encoded and where the gene is implicated.