Alberto Selman

Tyrosine kinase A receptor (trkA): a potential marker in epithelial ovarian cancer.

OBJECTIVES To evaluate the role of trkA receptor as a potential tumor marker in serous epithelial ovarian cancer and its relationship with the angiogenic factors expression as vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). Additionally, to examine whether NGF and VEGF secreted by epithelial ovarian cancer (EOC) explants and from epithelial ovarian cancer cell line (A2780) are involved in the process of angiogenesis, such as cellular proliferation, migration and differentiation of the human endothelial cell line (EA.hy926). METHODS The mRNA levels of VEGF, NGF and trkA receptors were measured using PCR in 60 ovarian samples. Cellular localization and semi-quantitative estimation of VEGF, NGF, total trkA and p-trkA was performed using IHC in epithelial cells. NGF, total trkA and p-trkA protein were also evaluated in endothelial cells from the same tissues. Human endothelial cell line EA.hy926 was cultured with conditioned media obtained from both EOC explants and from the A2780 cell line, with or without NGF stimulus. RESULTS Significantly higher levels of NGF, total trkA and p-trkA protein expressions were observed in epithelial and endothelial cells in poorly differentiated EOC versus normal ovary. Interestingly, the p-trkA receptor expression level showed the most significant difference and its presence was only found in borderline tumor and EOC samples indicating the importance of trkA receptor in EOC as a potential tumor marker. A significant increase in proliferation, migration and differentiation of EA.hy926 cells was observed with NGF, and this effect was significantly reverted when NGF was immuno-blocked and when a trkA inhibitor was used, showing that NGF is an important angiogenic factor in EOC by activating its trkA receptor. CONCLUSION These results indicate that p-trkA may be considered as a new potential tumor marker in EOC, and that NGF may also act as a direct angiogenic factor in EOC.

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.

Caesarean hysterectomy for placenta praevia/accreta using an approach via the pouch of Douglas

Placenta praevia/accreta is associated with significant maternal morbidity and mortality and is a common cause of obstetric hysterectomy. This paper describes posterior retrograde abdominal hysterectomy, a new surgical technique for caesarean hysterectomy, in 11 women with placenta percreta, increta or accreta There were no intraoperative or postoperative maternal complications, and only one fetus required admission to the neonatal unit, for prematurity. Our technique in placenta praevia/accreta allows easy identification of the vagina and early uterine devascularisation, as well as safe resection of the involved urinary bladder in women with placenta percreta showing bladder penetration. Analytical studies are needed to confirm our findings.

Nerve Growth Factor Stimulates Cellular Proliferation of Human Epithelial Ovarian Cancer

Due to its ability to induce vascular endothelial growth factor expression and proliferation, migration, and vasculogenesis of endothelial cells, nerve growth factor (NGF) has been considered as an angiogenic factor in epithelial ovarian cancer (EOC). In this work, we evaluated the angiogenic and proliferative mRNA expression profiles of EOC and addressed the responsiveness of EOC explants to NGF stimulation. Twenty EOC samples were obtained from Obstetrics and Gynecology Department, University of Chiles Clinical Hospital. Global gene expression profiles of selected poorly differentiated serous EOC samples were obtained with DNA oligonucleotide microarrays. In addition, EOC explants were subjected to NGF stimulation and levels of p-AKT, BAX, BCL2, Ki-67, c-MYC, and FOXL2 proteins were determined by immunohistochemistry. Results showed that mRNAs coding for specific transcriptional regulators and antiapoptotic components of the NGF signaling pathway were upregulated in EOC cells. At the protein level, key members of the NGF pathway including p-AKT, BCL2/BAX, Ki-67, and c-MYC were found increased, while FOXL2 was decreased in response to NGF stimulation. These findings strongly suggest that NGF stimulates cellular proliferation of human EOC.

Secondary cytoreductive surgery in the patient with recurrent ovarian cancer is often beneficial

The majority of women with ovarian cancer will present at an advanced stage and ultimately experience a recrudescence of tumor. Recent data indicates that secondary cytoreductive surgery is feasible, well tolerated and associated with significant prolongation of survival in selected patients with recurrent ovarian cancer.

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.

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.

Abstract A26: Levels of phospho-Connexin 43 and phospho-TRKB in epithelial ovarian cancer.

Cancer is the second cause of death in Chile, and one of the most aggressive and worse prognosis is ovarian cancer. Between 70 and 80% of ovarian cancer cases correspond to epithelial ovarian cancer (EOC). EOC has a poor treatment response, so that is necessary to study new targets in order to improve cancer therapy. In this context, the neurotrophin receptors (for instance TRK receptors) and cell adhesion and communication molecules such as connexins are very important. Researches undertaken by our group have found that Nerve Growth Factor and its high affinity receptor TRKA are involved in cell proliferation and angiogenesis in EOC. TRKB, another subtype of TRK receptor, by interacting with its high affinity ligand brain-derived neurotrophic factor or NGF has been strongly associated with metastatic processes and resistance to cancer therapies in many kinds of cancers. Specially, phosphorylation of TRKB on residue tyrosine 812 (pTRKB) is capable of activating FLCy-PKC pathway, which has an important role on proliferation, apoptosis and protein modifications, like connexins. On the other hand, cells are anchored and connected into tissues through gap junctions, among others. These are channels that connect cytoplasmic contents of neighbor cells and also regulate many physiological processes such as cell cycle, permeability and migration. Connexins are proteins that form these channels. Connexin 43 (Cx43) has been widely studied in cell lines and tissues from cancer, being present in ovarian cancer. Phosphorylation of Cx43 on serine 368 (pCx43) disrupts gap junctions and intercellular junctional communication. This could be an advantage for malignant cells to survive in processes such as therapy resistance, proliferation and metastasis. This phosphorylation is induced by several tyrosine kinase receptors, for instance platelet-derived growth factor receptor. The main purpose of this study is to describe the presence and levels of pTRKB and pCx43 on women´s ovary samples and to associate them with patients´ survival through a retrospective study. Samples of female patients (age 26 -79, average 52 years) who underwent surgical procedures at University of Chile Clinical Hospital were used. Each participant signed an informed consent approved by the institutional ethics committee. Fresh tissue stored at -80°C and paraffin-fixed tissues were employed, jointly with retrospectively analysis of medical records. Biopsy and classification of samples were performed in the Pathology Department of the same Hospital, and the samples were classified in inactive or normal ovaries (N=17), ovarian tumor (N=18) and epithelial ovarian cancer (N=33). pCx43 and pTRKB levels were determined by immunohistochemistry (IHQ) and levels of Cx43 and pCx43 were measured by Western-blot. The semi-quantitative analysis was performed by ImageProPlus 6.0.250 and UnScan-It gel 6.1. These results were associated with patients9 survival. Kruskal Wallis and Mann Whitney tests were performed. The results showed that pTRKB and pCx43 were found in epithelial cells of ovarian tissues which levels increased during the progression of EOC; in fact, pTRKB content was evaluated by IHQ being 1.7 times in tumors and 3,2 times in EOC (p These results suggest that the studied molecules play a role in the progression of EOC and should be considered as potential targets to be evaluated in future research. Grants: FONDECYT 1110372, CONICYT-FONDAP 15130011 and OAIC University of Chile Clinical Hospital project 603/13 Citation Format: Maritza P. Garrido, Alberto Selman, Fernando Gabler, Margarita Vega, Carmen Romero. Levels of phospho-Connexin 43 and phospho-TRKB in epithelial ovarian cancer. [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 A26.

Author's reply re: Caesarean hysterectomy for placenta praevia/accreta using an approach via the pouch of Douglas.

occlusion of the iliac internal artery does not have a firm recommendation to reduce blood loss, so it is not clear why he used it. Additionally, he interrupted the blood supply in the cervicovaginal junction, a fact that demonstrates the poor efficacy of major arterial ligature in abnormal invasive placentation. After appraise a video (supporting by the author), we cannot realise the advantages of this technique to solve the dissection of the vesicouterine plane because we cannot find any evidence of anterior abnormal placentation, such as bulging, myometrial thinning or newly formed vessels between the bladder, the uterus and the placenta. Unfortunately, there is no viewing of the invaded area before or after the surgery; for this reason, it is not possible to conclude if this case is a true placenta accreta or percreta. The author should be able to demonstrate that in his experience this technique reduces maternal morbidity (bleeding and bladder lesions), compared with traditional hysterectomy or previously published conservative techniques. We hope that obstetricians think deeply about this technique before using it, mainly to understand that is not an easy solution for abnormal placentation cases, and also that Dr Matsuzaki be recognised as the original author of this approach.&