Abstract A45: Proteins involved in the Calreticulin translocation pathway are altered in human ovarian cancer samples.
Authors: Vera Carolina , García Paula , Selman Alberto , Gabler Fernando , Vega Margarita , Ferreira Arturo , Romero Carmen 
Published: May 2016

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&lt;0.05) and to normal inactive tissues (p &lt;0.05). We also found the presence of CD91 on both normal and pathologic ovarian samples by western-blot and immunohistochemistry. Interestingly, NGF didn't induce changes of eIF2α and PERK. Also, CRT kept a mainly peri-nuclear distribution with NGF treatment, while it changed with thapsigargin and mitoxantrone treatments, acquiring a distribution near the plasma membrane and in the cytoplasm.

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 García, 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.