The nerve growth factor alters calreticulin translocation from the endoplasmic reticulum to the cell surface and its signaling pathway in epithelial ovarian cancer cells
- Authors:
- Published online on: February 28, 2017 https://doi.org/10.3892/ijo.2017.3892
- Pages: 1261-1270
Abstract
Introduction
Ovarian cancer is the seventh most common cancer among women worldwide and the first cause of death due to a gynecological malignancy (1). This disease causes ~150,000 deaths every year (1) and it has a mortality rate between 17 and 47% 5 years after the diagnosis (2,3). Approximately 90% of ovarian cancers are of epithelial origin, known as epithelial ovarian cancer (EOC) (4). EOC is typically asymptomatic at early stages, and therefore it is usually diagnosed at advanced stages, when it responds poorly to therapy (4). Although many patients respond to the first line of treatment, surgery and chemotherapy, recurrence is developed in ~90% of advanced cases (5). Due to this poor response, it is important to find new and more effective treatments. Immunotherapy is designed to boost the body's natural defenses against cancer (6) and it appears to be a promising option against EOC (7). However, a deeper understanding of the underlying mechanisms that govern immune detection of cancer cells is needed. In the present study, we aimed to increase our understanding of calreticulin (CRT) translocation, a necessary event for cancer cells to be recognized by the immune system (8), and how the nerve growth factor (NGF), a pro-angiogenic molecule overexpressed in EOC (9), participates in this process.
Several processes and signaling pathways are altered in a cancer cell, including angiogenesis (10). Angiogenesis is induced to provide the tumor with neo-vasculature, giving cancer cells access to sustenance and oxygen. During tumor progression, cancer cells undergo an 'angiogenic switch', characterized by increased expression of pro-angiogenic molecules (11). The normal active ovary undergoes angiogenesis at regular intervals; in ovarian cancer, there are disturbances in the normal angiogenic regulation, including alteration of the proangiogenic molecules such as NGF and the vascular endothelial growth factor (VEGF) expression. Besides, the normal ovary expresses NGF and TRKA in granulosa cells, and NGF induces the proliferation of granulosa and theca cells (12). On the other hand, NGF levels are elevated and induce angiogenesis through its high affinity receptor TRKA by acting directly on endothelial cells and by stimulating VEGF production in epithelial cells in EOC (12). Besides, NGF induces proliferation and survival of EOC cells (13).
Tumor development is also accompanied by changes in the tumor's microenvironment (14,15). These changes can lead to the accumulation of misfolded and unfolded proteins in the endoplasmic reticulum (ER) lumen, inducing ER stress and eliciting the unfolded protein response (UPR) (16). In solid tumors the UPR promotes proliferation, survival, growth, migration and metastasis (17,18). Several ER stress sensors are responsible for UPR implementation, including PERK. Under ER stress conditions, PERK is phosphorylated and induces the phosphorylation of eIF2α (19).
The PERK/eIF2α branch of the UPR is important in CRT translocation (20). CRT is an ER resident that can also be present in other cell compartments, such as the cytosol, the nucleus (21), the cell membrane (22) and the extracellular space (23). CRT levels are increased in several types of cancer (23). This protein appears to have a dual role in cancer: on the one hand, it has a direct tumorigenic effect by inducing proliferation, migration and metastasis (24–26) and on the other, CRT is a potent anti-angiogenic molecule (27). Besides, when present on the cell surface, CRT triggers an anticancer immune response (8). Calreticulin translocation is induced by several chemotherapeutic agents through a pathway that involves the activation of the PERK/eIF2α branch of the UPR (28).
NGF suppresses ER stress-mediated responses (29,30), but there is no information on whether it can also alter CRT translocation. In a previous study, we found that NGF induces an increase in CRT levels in human ovarian cancer cells (31). In the present study, we first evaluated the ovarian cancer cells ability to translocate CRT under ER and chemotherapeutic stress conditions. We then evaluated whether NGF alters stress-induced CRT translocation. We found that cancer cells translocate CRT from the ER to the cell surface upon induction of both ER and chemotherapeutic stress, suggesting a potential benefit for cancer patients, given the ability of the immune system to recognize and destroy cancer cells with exposed CRT. Notably, we found that NGF can prevent CRT translocation induced by chemotherapeutic stress, but it has no effect on CRT translocation after induction of direct ER stress. Given the elevated levels of NGF in cancer patients, this result could yield valuable information for the design of novel cancer treatment options.
Materials and methods
Tissue samples
Ovarian tissues were obtained from Hospital Clínico, Universidad de Chile. Patients signed an informed consent, which was approved by the Hospital's Ethics Committee. Normal ovarian samples (N-Ov) were obtained from women subjected to hysterectomy with oophorectomy due to non-ovarian pathologies. Ovarian tissues were also obtained from women diagnosed with ovarian tumors, both benign (Be-T) and borderline (Bo-T). Differentiation of serous epithelial ovarian cancer tissues samples was classified as: well (EOC I), moderate (EOC II) or poor (EOC III). An experienced pathologist performed the classification of each sample.
Cell culture
A2780 epithelial ovarian cancer cells were maintained in Dulbecco's modified Eagle's essential medium supplemented with Ham's F12 Nutrient Mixture (DMEM/HamF12; Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (FBS; Hyclone™/Thermo Fisher Scientific, Rochester, NY, USA). All media contained 100 U/ml penicillin and 100 µg/ml streptomycin (Hyclone™/Thermo Fisher Scientific). Cells were kept in a humidified incubator with 5% CO2 at 37°C; medium was changed every 48 h.
Cell treatments
A2780 cells (1×106) were treated with 100 ng/ml of NGF (Sigma-Aldrich), 1 µM of thapsigargin (Abcam, Cambridge, UK) or 1 µM of mitoxantrone (Sigma-Aldrich) for 4 h, or with 100 ng/ml NGF for 2 h followed by thapsigargin or mitoxantrone. NGF concentration was chosen according to previous results by our group (9,31,32); thapsigargin and mitoxantrone concentrations were chosen because of previous publications showing their effect on CRT localization (33–35). Cells were either scrapped and collected with lysis buffer for western blot analysis, tripsinized and collected with sterile Dulbecco's phosphate-buffered saline (DPBS; Gibco-Invitrogen, Camarillo, CA, USA) for flow cytometry analysis, or the cells fixed with paraformaldehyde for immunofluorescence experiments.
Western blot analysis
Cells were scrapped and homogenized in RIPA lysis buffer (50 mM Tris-Base, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100 and 0.1% sodium dodecyl sulfate); 1X protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) was added. After 20 min of centrifugation at 10,000 × g at 4°C, the supernatant was collected and protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific). Proteins (50 µg) were mixed with an equal amount of loading buffer, boiled and loaded into an 8, 10 or 12% resolving SDS-PAGE gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane and blocked with Tris-buffered solution (20 mM Tris, pH 7.5; 137 mM NaCl; 0.1%) containing 0.1% Tween-20 (TTBS) and either 5% fat-free milk or 5% BSA. Afterwards, membranes were incubated overnight in primary antibody in either 5% milk in TTBS or 5% BSA in TTBS at 4°C, washed, and then incubated in HRP-labeled secondary antibody (1:5,000; KPL, Gaithersburg, MD, USA) for 1 h at room temperature. Signal detection was achieved with an enhanced chemiluminescence substrate, Western Lightning Plus-ECL (Perkin-Elmer, Waltham, MA, USA). Membranes were treated with stripping solution (25 mM glycine-HCl, pH 2.2, containing 1% w/v SDS and 1% v/v Tween-20) and re-probed with the primary antibody. Band intensities were measured densitometrically using ImageJ Image software (National Institutes of Health, Bethesda, MD, USA). Relative band densities were normalized to β-actin as a loading control or to the appropriate total protein for phosphorylated proteins. The control condition was set to 1. Antibodies and dilutions used are as follows: p-eIF2α (monoclonal rabbit anti-human, #9721, 1:500; Cell Signaling Technology, Danvers, MA, USA), t-eIF2α (mouse monoclonal anti-human #2103, 1:1,000; Cell Signaling Technology), p-PERK (rabbit polyclonal anti-human, sc-32577, 1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), t-PERK (rabbit monoclonal anti-human, #3192, 1:1,000; Cell Signaling Technology), CRT (mouse monoclonal anti-human, 1:1,000; BD Biosciences, San Jose, CA, USA), β-actin (monoclonal mouse anti-human, 1:10,000; Sigma-Aldrich).
Flow cytometry
CRT expression on the cell surface was determined by flow cytometry. Cells were tripsinyzed and suspended in medium with 10% FBS. After washing with 1X PBS, cells were blocked with 1X PBS containing 0.5% BSA and then stained with phycoerytrin (PE)-conjugated anti-CRT (monoclonal anti-human, ab83220, 1:200; Abcam) in 1X PBS with 0.5% BSA at 4°C for 30 min, away from light. After washing with 1X PBS, stained cells were analyzed in a flow cytometer (LSRFortessa™ X-20 cell analyzer; BD Biosciences) and the data were processed with FlowJo software (Tree Star, Inc., Ashland, OR, USA). Results were expressed as mean fluorescence intensity (MFI); the control condition was set to 1.
Immunofluorescence
Cells were grown and treated in Lab-Tek chambers (Thermo Scientific™ Nunc™ Lab-Tek™ II Chamber Slide™ System). Next, treated cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were then permeabilized with triton 0.3% for 10 min, blocked with 5% BSA in PBS for 10 min and then incubated with 1:10,000 rabbit anti-human calreticulin (CRT) antibody and anti-KDEL antibody (mouse monoclonal anti-human, #12223, 1:500; Abcam) overnight. Another group of cells was incubated for 1 h with wheat germ agglutinin (WGA, W11261, 1:1,000; Thermo Fisher Scientific) for membrane staining immediately after fixation, and then blocked and incubated with 1:10,000 rabbit anti-human CRT, with no previous permeabilization step. Afterwards, all cells were washed and incubated with Alexa Fluor 594 conjugated anti-rabbit secondary antibody (#8889; Cell Signaling Technology) and Alexa Fluor 488 conjugated with anti-mouse secondary antibody (#4408; Cell Signaling Technology) at 1:500 for 1 h. Cells were mounted in mounting medium with DAPI (ProLong® Gold Antifade reagent with DAPI; Cell Signaling Technology) and observed under a confocal microscope (Zeiss LSM 700).
Image analysis
Immunofluorescence pictures were deconvoluted with the Huygens software (Scientific Volume Imaging, Hilversum, The Netherlands). Mean fluorescence intensity (MFI) was determined with ImageJ software. In order to evaluate fluorescence intensity across one cell, a line was traced and green and red fluorescence intensity was determined in that line.
Statistical analysis
Results are expressed as mean ± SEM. Normality was determined by the Kolmogorov-Smirnov test. Significant differences between groups were assessed by one-way analysis of variance (ANOVA) followed by a Bonferroni post test in case of parametric data, and by Kruskal Wallis followed by Dunn's post test in case of non-parametric distribution. P<0.05 were considered statistically significant. The statistical analysis was done with GhaphPad Prism 5 software (Graphpad Software, Inc., La Jolla, CA, USA).
Results
Reticulum and cytotoxic stress induce CRT exposure to the cell surface in A2780 ovarian cancer cells
A2780 cells were treated for 4 h with thapsigargin (Tg), a reticulum stress inducer, or mitoxantrone (Mtx), a cytotoxic chemotherapeutic agent. Both compounds modified the CRT subcellular localization, as determined with confocal microscopy. In basal conditions CRT is found with a perinuclear distribution in A2780 cells, as shown in Fig. 1A–a, concordant with ER localization. CRT is shown in red, the nucleus is shown in blue and the cell membranes were stained in green. These cells were not permeabilized, however, paraformaldehyde fixation causes lost of cell membrane integrity, inducing partial permeabilization (36). Therefore, it is possible to detect intracellular CRT. Cell membranes were stained with WGA, which marks both the cell surface membrane and intracellular membranes. After Tg and Mtx stimuli, CRT cell distribution changes, and CRT can be detected in the cell periphery (Fig. 1A–b and A-c). These experiments were also done in triton-permeabilized cells. In this case, cells were also stained with an anti-KDEL antibody. KDEL is a peptide sequence found in ER proteins. CRT also has a KDEL sequence. CRT was marked with red fluorescence and KDEL was visualized in green (Fig. 1B). Since cells were permeabilized, the red and green marks correspond exclusively to intracellular staining. Fluorescence intensity for CRT (red line) and KDEL (green line) was measured along the line drawn in white. Under basal conditions (Fig. 1B–a2), the KDEL and CRT intensity signals are similar, indicating that CRT is mostly found in the ER, along with other KDEL proteins. After Tg or Mtx incubation, KDEL intensity is higher than CRT intensity near the nucleus, indicating that CRT has left the ER, leaving behind the other KDEL-bearing proteins (Fig. 1B–b2 and c2). All the above results suggest that Tg and Mtx are inducing CRT translocation; however, given the loss of membrane integrity due to paraformaldehyde fixing and to give more strength to the 2D plots, flow cytometry experiments were carried out to verify the CRT expression on the cell surface. Since flow cytometry is done on live cells that have intact membrane integrity, this technique allows for CRT staining exclusively on the cell surface. As seen in Fig. 1C–a, after Tg or Mtx treatments, CRT expression increased on the cell surface, expressed by a shift to the right: blue line for Tg, green line for Mtx, compared to the red basal line. CRT levels in the cell surface were also expressed as mean fluorescence intensity (MFI). As seen en Fig. 1C–b, MFI increases after Tg or Mtx treatment, which represents an increase in CRT translocation to the cell surface.
NGF increases CRT levels with no sub-localization change
Next, we determined the effect of the pro-angiogenic and pro-carcinogenic NGF molecule on CRT levels and subcellular localization in A2780 cells. After 4 h of incubation with NGF, we found an increase of CRT protein levels, measured by western blot analysis (Fig. 2A). A semi-quantitative analysis of CRT fluorescence intensity after NGF treatment, seen in Fig. 2C, shows a similar result, treatment of A2780 cells with NGF induces an increase of CRT levels compared to the basal condition. However, NGF does not alter CRT subcellular localization, as seen in Fig. 2B. This agrees with flow cytometry experiments, as seen in the histogram in Fig. 2D–a: the line representing the basal condition (red) and the line denoting NGF treatment (yellow) are similar. These results were also expressed as MFI (mean fluorescence intensity) (Fig. 2D–b), and no change is detected between basal and NGF-treated cells. These flow cytometry results indicate that after NGF treatment CRT is not being transported to the cell periphery.
NGF pre-incubation inhibits Mtx-induced CRT exposure to the cell surface in A2780 ovarian cancer cells
The ER stress response is necessary for CRT exposure to the cell surface and NGF can alter the ER stress response. In order to investigate the effect of NGF on CRT translocation, A2780 cells were incubated with NGF for 2 h previous to Tg or Mtx treatment. In the histogram shown in Fig. 3A–a, Mtx treatment is expressed as a dotted line, and it is shifted to the right, as compared to the basal condition (solid line), indicating that CRT was transported to the cell surface after Mtx treatment. NGF inhibited this shift (dashed line), meaning that CRT remained inside the cell. In Fig. 3A–b these results are expressed as MFI. However, NGF had no effect in CRT translocation to the cell surface after Tg treatment, as seen in the histogram in Fig. 3B–a: the dashed line, representing NGF-treated cells before Tg incubation, did not shift compared to the basal condition (solid line). Fig. 3B–b shows the MFI analysis. In summary, NGF inhibited CRT translocation induced by Mtx (Fig. 3A); nevertheless, NGF had no effect on Tg-induced CRT translocation (Fig. 3B).
NGF alters the UPR induced by cytotoxic stress
CRT translocation requires the activation of the PERK/eIF2α branch from the UPR pathway, which is activated after ER stress. Activation of PERK/eIF2α was evaluated as p-PERK and p-eIF2α by western blot analysis. Representative gels of PERK and p-PERK are shown in Fig. 4A and B. When A2780 cells were treated with Tg, an increase was found in p-PERK. However, in cells previously treated with NGF, a significant inhibition of this activation was found, as shown in the semi-quantitative analysis of the western blot analysis (Fig. 4A). Results similar to those obtained with p-PERK were found in A2780 cells treated with Mtx alone or with Mtx after treatment with NGF, as shown in Fig. 4B.
Representative gel images of eIF2α and p-eIF2α are shown in Fig. 4C and D. In a semi-quantitative analysis of eIF2α activation in A2780 cells treated with Tg, we found a significant increase of p-eIF2α, however, pre-incubation with NGF did not change the activation of p-eIF2α after Tg treatment (Fig. 4C). In A2780 treated with Mtx we found a significant increase of p-eIF2α, and this activation was diminished by previous NGF treatment (Fig. 4D). These results agree with the flow cytometry experiments, since NGF pre-incubation could only inhibit Mtx-induced CRT exposure to the cell surface, having no effect on CRT expression on the cell surface after Tg stimulation. Moreover, these results suggest that NGF could interfere in the Mtx actions when this compound is used as a therapeutic drug.
Tg and Mtx did not change CRT total levels (Fig. 4E and F). CRT is a 50-kDa protein that has been shown to migrate at 60 kDa in SDS-PAGE gels (37,38). NGF, on the other hand, did not change the active levels of either PERK or eIF2α (Fig. 4A and B, respectively), but it did induce an increase in CRT protein levels (Fig. 4E and F).
NGF prevents cell death induced by a chemotherapeutic drug
Mtx is a drug used against cancer given its capacity to induce cell death. Because of the NGF ability to inhibit the UPR induced by Mtx (Fig. 4B and D), we decided to evaluate whether NGF has also an effect on cell death after Mtx treatment. Cells were incubated for 48 h with either Mtx or with NGF and Mtx and then cell viability was measured. As seen in Fig. 5, cells that were treated with both NGF and Mtx had a higher survival rate compared to cells treated with Mtx alone.
CRT levels and the UPR are altered in human ovarian samples
In order to better understand how these processes work in human ovarian cancer, we evaluated whether protein levels of CRT, PERK and/or eIF2α were altered in human ovarian cancer samples. As shown in Fig. 6, we found an increase in CRT levels in EOC II and EOC III as compared to normal inactive ovaries. p-PERK levels were also elevated in EOC as compared to normal ovaries. Notably, p-eIF2α levels were higher in EOC II than in less differentiated samples.
Discussion
CRT is a multifunctional, buffering and ubiquitous protein mainly involved in protein folding and the maintenance of calcium homeostasis (39). CRT is also involved in the pathogenesis of several diseases, including different types of cancer (40). In cancer cells exposed to reticulum and/or cytotoxic stress, CRT is translocated from the ER to the cell membrane, where it induces an anti-immune response against cancer cells. This immune response reduces or even destroy the tumor (28).
In human ovarian cancer cells, we found that thapsigargin (Tg) and mitoxantrone (Mtx) induce CRT translocation from the ER to the cell surface (Fig. 1). Tg, a compound that inhibits ER calcium pumps (41), induces a direct ER stress response that we found to be mediated by the activation of the UPR. Mtx, on the other hand, is an anticancer drug with DNA binding properties that induces cell death, while generating ER stress in an indirect manner (42). We found that both of these compounds stimulate CRT translocation from the ER to the cell surface (Fig. 1), which could indicate that ovarian cancer cells have the potential to respond to immunotherapy.
Despite the ability of CRT to induce an anticancer immune response, it has also been associated with pro-carcinogenic properties (40). CRT has been found to be elevated in several types of cancer (39), although no evidence of efficient trans-location to the tumor cell surface has been presented. One of CRT properties is related to angiogenesis, a necessary process for the survival of solid tumors (25). In ovarian cancer, characterized by high levels of angiogenesis, this process is mostly driven by VEGF, a pro-angiogenic molecule overexpressed in most solid tumors (43). Another pro-angiogenic molecule overexpressed in ovarian cancer is NGF, which acts directly on endothelial cells and also on cancer cells, inducing them to increase their VEGF expression and secretion (12). In the present study, we found that NGF increases CRT levels (Fig. 2), which could be associated with pro-carcinogenic properties; however, CRT effects in cancer seem to be dependent on its subcellular localization. We found that NGF induces an increase of CRT protein levels; however, this increase is not accompanied by a change on its subcellular localization: CRT is maintained inside the cell (Fig. 2). As a consequence, CRT would be promoting angiogenesis and other pro-carcinogenic processes instead of inducing an anticancer immune response.
CRT translocation is mediated by the activation of the reticulum stress response, specifically the UPR branch involving the phosphorylation of PERK and its subtract, eIF2α (28). NGF has been found to inhibit the effects of reticulum stress, including reducing apoptosis after UPR activation (29,30). In this study, we determined that NGF also reduced Mtx-induced CRT exposure to the cell membrane (Fig. 3). This is an interesting result, given the high levels of NGF normally found in ovarian cancer. If NGF inhibits CRT transport from the ER to the cell membrane in ovarian cancer patients, this could mean that an anti-immune therapy based on chemotherapeutic drugs that induce this CRT translocation would be less efficient in these women than for patients suffering from other types of cancer.
NGF, on the other hand, did not affect the ability of Tg to induce a change in CRT sub-cellular localization (Fig. 3). This is consistent with the results obtained in the western blot analysis, as NGF was unable to prevent eIF2α phosphorylation induced by Tg. Both Mtx and Tg induced the UPR, a necessary step for CRT translocation; NGF, however, inhibited this response when induced by Mtx, while it only partially inhibited Tg effect, as eIF2α was still phosphorylated (Fig. 4). This could be explained by the existence of other pathways induced by Tg, including the activation of oxidative stress, which also induces eIF2α phosphorylation and CRT translocation to the cell periphery. In a therapeutic setting, this result could point to the advantage of accompanying chemotherapeutic treatment with a direct induction of the ER stress response, given its effect on CRT translocation, despite the participation of NGF.
Because of the NGF effect on the Mtx-induced UPR, we sought to determine if NGF had an effect on cell death induced by Mtx. Indeed, NGF diminished cell death after treatment with this cytotoxic agent (Fig. 5). Mtx is used in epithelial ovarian cancer; therefore, we sought to evaluate the effect of NGF on Mtx cytotoxicity.
In order to gain a better understanding of these processes in human ovarian cancer, we measured the UPR and CRT protein levels in six groups of frozen human ovary samples: normal inactive ovaries, benign tumors, borderline tumors and EOC I, EOC II and EOC III (respectively corresponding to well, moderately and poorly differentiated epithelial ovarian cancers). We found that CRT is increased in advanced stages of epithelial ovarian cancer, matching other reports in both ovarian cancer and other types of cancer (23). We also found an increase in p-PERK and p-eIF2α levels, consistent with the characteristic ER stress inducing microenvironment that surrounds most tumors. However, eIF2α is reduced in advanced cancer samples, which could be an impediment to CRT translocation. It is possible that cancer cells adapt to ER stress, which in turn could lead to the inhibition of CRT transport to the cell surface.
The induction of CRT translocation from the ER to the cell surface through the use of cytotoxic drugs is a novel strategy for the treatment of cancer (28). However, in this study we found that NGF inhibits CRT movement. Further studies should determine whether NGF can alter immune recognition and destruction of cancer cells, and the role of ER stress inducers in increasing the efficiency of immunotherapy against ovarian cancer.
Abbreviations:
CRT |
calreticulin |
NGF |
nerve growth factor |
Tg |
thapsigargin |
Mtx |
mitoxantrone |
ER |
endoplasmic reticulum |
Acknowledgments
The present study was supported by grants #1110372 and #1160139 from the Fondo Nacional de Desarrollo Científico y Tecnológico, Chile (to C.R.), the #1130099 grant from the Fondo Nacional de Desarrollo Científico y Tecnológico, Chile (to A.F.) and the #21120252 grant from the Comisión Nacional de Ciencia y Tecnología, Chile (to C.V.).
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