Carolina Vera

Role of nerve growth factor and its TRKA receptor in normal ovarian and epithelial ovarian cancer angiogenesis.

In normal ovarian function a controlled angiogenesis is essential. Several growth factors are involved in this process, such as the vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). The angiogenesis process in the normal ovary is a tightly controlled process that occurs in each ovarian cycle. Also, angiogenesis is critical for ovarian cancer development and it is responsible for tumor spread, metastasis and its peritoneal dissemination. Ovarian cancer is the fifth leading cause of cancer death in women and it is distinguished as the most lethal gynecologic cancer. In recent years angiogenesis has been given considerable attention in order to identify targets for developing effective anti-tumor therapies. Several molecules have been reported to promote angiogenesis, such as platelet-derived growth factor (PDGF) and its receptors, the angiopoietin/Tie ligand/receptor system and fibroblast growth factor (FGF). Primarily, VEGF has been identified to play key roles in driving angiogenesis. The above-mentioned molecules are candidate drug targets. Used in combination with other treatments, anti-angiogenic therapies have managed to reduce disease progression. The present review is focused in NGF and its high affinity receptor tyrosine kinase A (TRKA). The expression of VEGF, proliferation and the angiogenesis process in ovarian cancer is importantly induced by NGF, among other molecules.

Nerve growth factor induces the expression of chaperone protein calreticulin in human epithelial ovarian cells.

Epithelial ovarian cancer is highly angiogenic and high expression of Nerve Growth Factor (NGF), a proangiogenic protein. Calreticulin is a multifunctional protein with anti-angiogenic properties and its translocation to the tumor cell membrane promotes recognition and engulfment by dendritic cells. The aim of this work was to evaluate calreticulin expression in human normal ovaries, benign and borderline tumors, and epithelial ovarian cancer samples and to evaluate whether NGF regulates calreticulin expression in human ovarian surface epithelium and in epithelial ovarian cancer cell lines. Calreticulin mRNA and protein levels were analyzed using RT-PCR, Western blot and immunohistochemistry in 67 human ovarian samples obtained from our Institution. Calreticulin expression induced by NGF stimulation in cell lines was evaluated using RT-PCR, Western blot and immunocytochemistry. We found a significant increase of calreticulin mRNA levels in epithelial ovarian cancer samples as compared to normal ovaries, benign tumors, and borderline tumors. Calreticulin protein levels, evaluated by Western blot, were also increased in epithelial ovarian cancer with respect to benign and borderline tumors. When HOSE and A2780 cell lines were stimulated with Nerve Growth Factor, we found an increase in calreticulin protein levels compared to controls. This effect was reverted by GW441756, a TRKA specific inhibitor. These results suggest that NGF regulates calreticulin protein levels in epithelial ovarian cells through TRKA receptor activation.

Proinflammatory environment and role of TNF-α in endometrial function of obese women having polycystic ovarian syndrome

Background/Objectives:A high percentage of women having polycystic ovarian syndrome (PCOS) exhibit hyperinsulinemia and obesity. Transforming necrosis factor-α (TNF-α) is an adipokine that increases in obesity and negatively affects insulin action in several tissues, including the endometrium. In fact, it has been reported that insulin signaling is altered in the endometrium of PCOS women, affecting its reproductive function. The aim of this study was to determine the proinflammatory environment and TNF-α signaling in endometrium from obese women with PCOS, and also to evaluate the effect of TNF-α on endometrial cell energy homeostasis.Methods:Serum and endometrial tissues were obtained from four study groups: normal-weight, normal-weight-PCOS, obese and obese-PCOS (hyperandrogenemia/hyperinsulinemia) (n=7 per group). Serum TNF-α level was assayed by enzyme-linked immunosorbent assay (ELISA); endometrial TNF-α level and its receptors (TNFR1/TNFR2) as well as nuclear factor (NF)-κB content were determined by immunohistochemistry. Finally, we evaluated TNF-α effect on glucose uptake in cultured human endometrial stromal cells (T-HESC) treated or not with testosterone/insulin resembling partially the PCOS condition.Results:TNF-α plasma levels were similar between groups, whereas cytokine levels and macrophage number increased in endometrium from obese-PCOS women (P<0.001). Both receptor types were higher in obese vs normal-weight women, particularly TNFR2 content in the obese-PCOS group (P<0.001). Furthermore, an increased NF-κB nuclear content in endometrium from obese-PCOS was observed (P<0.001). Finally, TNF-α treatment of T-HESC cultures exhibited a decrease of glucose uptake (P<0.05), although similar to cells treated with testosterone or testosterone/insulin/TNF-α.Conclusions:These results suggest that the PCOS condition induces an inflammatory state exacerbated when obesity is present, where a higher TNF-α signaling is observed, all of which could affect glucose uptake in the tissue and may cause fertility failures in these women.

Role of Nerve Growth Factor (NGF) and miRNAs in Epithelial Ovarian Cancer

Ovarian cancer is the eighth most common cancer in women worldwide, and epithelial ovarian cancer (EOC) represents 90% of cases. Nerve growth factor (NGF) and its high affinity receptor tyrosine kinase A receptor (TRKA) have been associated with the development of several types of cancer, including EOC; both NGF and TRKA levels are elevated in this pathology. EOC presents high angiogenesis and several molecules have been reported to induce this process. NGF increases angiogenesis through its TRKA receptor on endothelial cells, and by indirectly inducing vascular endothelial growth factor expression. Other molecules controlled by NGF include ciclooxigenase-2, disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) and calreticulin (CRT), proteins involved in crucial processes needed for EOC progression. These molecules could be modified through microRNA regulation, which could be regulated by NGF. MicroRNAs are the widest family of non-coding RNAs; they bind to 3′-UTR of mRNAs to inhibit their translation, to deadenilate or to degraded them. In EOC, a deregulation in microRNA expression has been described, including alterations of miR-200 family, cluster-17-92, and miR-23b, among others. Since the NGF-microRNA relationship in pathologies has not been studied, this review proposes that some microRNAs could be associated with NGF/TRKA activation, modifying protein levels needed for EOC progression.

Molecular Mechanisms of Androstenediol in the Regulation of the Proliferative Process of Human Endometrial Cells

Proliferation in endometria of women with polycystic ovarian syndrome (PCOS) is increased, similar to the biosynthesis of androstenediol (estrogenic metabolite). As previously shown, in human endometrial cells, androstenediol increases CYCLIN D1 levels and KI67 and decreases P27 content. The objective of the present investigation was to determine the mechanisms by which androstenediol promotes endometrial cell-cycle progression. Estrogen receptor α (ERα) activation and changes in CYCLIN D1 and P27 levels were evaluated by Western blot in T-HESC and St-T1b endometrial cell lines, using receptor antagonists; activation of PI3K-protein kinase B (AKT) and mitogen-activated protein kinases-extracellular signal–regulated kinases (MAPK-ERK)1/2 pathways was evaluated using PI3K, MAPK/ERK kinase (MEK)1/2, and RNA-polymerase II inhibitors. The data showed that androstenediol treatment significantly increases CYCLIN D1 and decreases P27 levels through ERα activation (P < .05). In addition, an increase in AKT/ERK1/2 phosphorylations was determined (P < .05). In the presence of RNA-polymerase II inhibitor, phosphorylation of AKT/ERK1/2 decreased (P < .05), meaning that endometrial cells need transcriptional activity to activate the kinases involved. It was also observed that PI3K action is required for P27 and CYCLIN D1 changes. Therefore, the action of androstenediol in endometria depends on PI3K-AKT and MAPK-ERK1/2 pathways activation, together with cell transcriptional machinery. This could be of clinical significance, as in pathologies such as PCOS, increased endometrial levels of androstenediol together with a high prevalence of endometrial hyperplasia and adenocarcinoma have been reported.

The nerve growth factor alters calreticulin translocation from the endoplasmic reticulum to the cell surface and its signaling pathway in epithelial ovarian cancer cells

Ovarian cancer is the seventh most common cancer among women worldwide, causing approximately 120,000 deaths every year. Immunotherapy, designed to boost the bodys natural defenses against cancer, appears to be a promising option against ovarian cancer. Calreticulin (CRT) is an endoplasmic reticulum (ER) resident chaperone that, translocated to the cell membrane after ER stress, allows cancer cells to be recognized by the immune system. The nerve growth factor (NGF) is a pro-angiogenic molecule overexpressed in this cancer. In the present study, we aimed to determine weather NGF has an effect in CRT translocation induced by cytotoxic and ER stress. We treated A2780 ovarian cancer cells with NGF, thapsigargin (Tg), an ER stress inducer and mitoxantrone (Mtx), a chemotherapeutic drug; CRT subcellular localization was analyzed by immunofluorescence followed by confocal microscopy. In order to determine NGF effect on Mtx and Tg-induced CRT translocation from the ER to the cell membrane, cells were preincubated with NGF prior to Mtx or Tg treatment and CRT translocation to the cell surface was determined by flow cytometry. In addition, by western blot analyses, we evaluated proteins associated with the CRT translocation pathway, both in A2780 cells and human ovarian samples. We also measured NGF effect on cell apoptosis induced by Mtx. Our results indicate that Mtx and Tg, but not NGF, induce CRT translocation to the cell membrane. NGF, however, inhibited CRT translocation induced by Mtx, while it had no effect on Tg-induced CRT exposure. NGF also diminished cell death induced by Mtx. NGF effect on CRT translocation could have consequences in immunotherapy, potentially lessening the effectiveness of this type of treatment.

Metformin prevents nerve growth factor-dependent proliferative and proangiogenic effects in epithelial ovarian cancer cells and endothelial cells

Background: Epithelial ovarian cancer (EOC) is characterized by exacerbated angiogenesis regulated by proangiogenic and growth factors. Nerve growth factor (NGF) is overexpressed in EOC where it promotes proliferation as well as survival and is considered a proangiogenic factor. Metformin, a drug commonly used in the treatment of diabetes, is attributed to antineoplastic effects, but the underlying mechanisms remain unknown. Given that current therapies yield modest results in EOC patients, the aim of this study was to determine the effects of metformin on NGF-enhanced proliferation of EOC cells and the angiogenic potential of endothelial cells. Methods: A2780 (EOC), HOSE (human ovarian surface epithelial) and EA.hy926 (endothelial) cells were treated with NGF and metformin. Cell viability, cell proliferation and cell cycle were evaluated in all three cell lines, and the angiogenic potential in endothelial EA.hy926 cells. Results: NGF enhanced cell proliferation in A2780, HOSE and EA.hy926 cells (p < 0.05), while metformin treatment decreased cell proliferation in A2780 and EA.hy926 cells (p < 0.05). Moreover, the NGF-enhanced angiogenic score in EA.hy926 cells was prevented by metformin (p < 0.05). Conclusions: Given that NGF plays a significant role in EOC progression, our current findings suggest that metformin holds considerable promise as an adjuvant treatment in ovarian cancer.

Effect of TNF-α on Molecules Related to the Insulin Action in Endometrial Cells Exposed to Hyperandrogenic and Hyperinsulinic Conditions Characteristics of Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) affects not only ovarian functions but is also able to affect endometrium metabolism. Around 80% of women with PCOS are obese. High tumor necrosis factor (TNF)-α production and low adiponectin levels are characteristics of obesity. Interestingly, endometrium from obese women with PCOS presents an insulin-resistance condition, high TNF-α levels, and low adiponectin levels. However, TNF-α effect on molecules associated with insulin action in endometrial cells remains unclear. Therefore, the objective of this work was to evaluate TNF-α effect on expression of molecules associated with adiponectin (insulin sensitizing) and TNF-α signaling pathways and on Glucose Transporter type 4 (GLUT-4) levels in human endometrial cells under the characteristic conditions of hyperandrogenic/hyperinsulinic (HA/HI) PCOS. Two human endometrial stromal cell lines (T-HESC/St-T1b) under HA/HI conditions were used to assay the effect of high TNF-α concentration (100 ng/mL) on adiponectin, AdipoR1-AdipoR2 receptors, Adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1), Phospho-AMP-activated protein kinase T172 (p-AMPKT172), GLUT-4, Tumor necrosis factor receptor 1 (TNFR1)-Tumor necrosis factor receptor 2 (TNFR2) receptors protein levels, and nuclear factor κB (NFκB) nuclear content, by Western blot or immunocytochemistry. The NFκB participation in TNF-α effect on adiponectin expression was assayed using an NFκB inhibitor (pyrrolidine dithiocarbamate). The TNF-α increases the expression of molecules associated with its own signaling pathway (P < .05) and decreases the protein levels of adiponectin and its associated molecules (P < .05). Moreover, TNF-α increases NFκB nuclear content (P < .001), whereas with NFκB inhibition the decrease in adiponectin content induced by TNF-α was not observed. GLUT-4 levels were lower with TNF-α treatment (P < .01). Thus, in human endometrial stromal cells, high TNF-α levels negatively affect the insulin action through decreased adiponectin signaling and GLUT-4 protein. This could explain the failures observed in endometrial function of obese women with PCOS.

Abstract A45: Proteins involved in the Calreticulin translocation pathway are altered in human ovarian cancer samples.

Ovarian cancer is the seventh most common cancer among women worldwide, with more than 200,000 new cases every year. The 5-year survival rate of advanced ovarian cancer ranges from 11 to 28 per cent. Thus, a better understanding of its pathogenesis at a molecular level is necessary to develop more effective therapies. Calreticulin (CRT) is a Ca+2 binding chaperone localized mainly in the endoplasmic reticulum (ER). However, it has been found in other cellular compartments, including the cytosol, nucleus, the plasma membrane and the extracellular space. At the cell surface, CRT acts as an “eat-me” signal, allowing the recognition of a cancer cell by the immune system and inducing an anti-cancer immune response. CRT translocation from the ER to the plasma membrane can be induced by several cytotoxic anti-cancer drugs; however, the exact mechanism is not completely understood. It involves the activation of an ER stress response, particularly the activation of PERK and eIF2αproteins. Once on the cell membrane, CRT would be able to interact with CD91. In previous work, we found that CRT levels increase in ovarian cancer samples compared to non-cancerous tumors and normal tissue. By immunohistochemistry, we detected CRT on the periphery of normal inactive cells, while it had a mainly perinuclear distribution on cancer cells, consistent with ER localization. We have also found that Nerve Growth Factor (NGF) is involved in ovarian cancer pathogenesis and it increases CRT protein levels in a human ovarian cancer cell line (A2780). The aim of this work was to evaluate the levels of proteins associated with CRT translocation, including eIF2α, PERK and CD91 in human ovarian samples and to evaluate CRT subcellular localization in A2780 cells treated with NGF, thapsigargin (ER inductor) and mitoxantrone (cytotoxic drug) for 2 and 4 hours. Human ovarian samples (six normal inactive ovarian tissues, twelve ovarian tumors and sixteen epithelial ovarian cancer samples) were obtained after written consent by patients from the Clinical Hospital University of Chile and with approval of its Ethics Committee. In ovarian tissues, levels of eIF2α, PERK and CD91 were evaluated by western blot and the localization of CD91 was evaluated by immunohistochemistry. CRT localization in treated A2780 cells was determined by confocal immunofluorescence microscopy. We found increased levels of phosphorylated eIF2α and phosphorylated PERK (active forms) relative to total protein, in ovarian cancer samples compared to tumors (p Several studies have shown that the ER stress response is elevated in several types of cancer. In this work, we found that this is also the case in epithelial ovarian cancer. These results suggest that ovarian cancer tissues have the machinery necessary to induce CRT translocation, including the presence of CD91, making it an attractive target for future immunotherapy developments. However, despite the activation of at least part of the pathway, CRT seems to remain on the endoplasmic reticulum in cancer cells. This could be indicating the presence of a mechanism that inhibits its mobilization, possibly involving NGF. Grants: FONDECYT 1110372, CONICYT-FONDAP 15130011 and CONICYT Scholarship 21120252 Citation Format: Carolina Vera, Paula Garcia, Alberto Selman, Fernando Gabler, Margarita Vega, Arturo Ferreira, Carmen Romero. Proteins involved in the Calreticulin translocation pathway are altered in human ovarian cancer samples. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A45.