ERP57 membrane translocation dictates the immunogenicity of tumor cell death by controlling the membrane translocation of calreticulin

Several pieces of experimental evidence indicate the following: 1) the most efficient antitumor treatments (this principle applies on both chemotherapy and radiotherapy) are those that induce immunogenic cell death and are able to trigger a specific antitumor immune response; and 2) the immunogenicity of cell death depends very closely on the plasma membrane quantity of calreticulin (CRT), an endoplasmic reticulum (ER) stress protein exposed to the cell membrane after immunogenic treatment. Nevertheless, the mechanisms implicated in CRT translocation are unknown. CRT is known to interact in the ER with ERP57, another ER stress protein. I sought to determine whether ERP57 would have any role in tumor immunogenicity. In this article I report that CRT exposure is controlled by ERP57 exposure. CRT and ERP57 are translocated together in the same molecular complex. ERP57 knockdown suppressed CRT exposure as well as phagocytosis by dendritic cells and abolished the immunogenicity in vivo. Knockdown or the absence of CRT abolishes ERP57 exposure. Administration of recombinant ERP57, unlike the administration of recombinant CRT, did not restore the immunogenicity of CRT or ERP57 small interfering RNA-transfected tumor cells. Together, these studies identify ERP57 as a key protein that controls immunogenicity by controlling CRT exposure and illustrate the ability of ERP57 to serve as a new molecular marker of immunogenicity.     

Disruption of the PP1/GADD34 complex induces calreticulin exposure

In response to some chemotherapeutic agents, tumor cells can translocate calreticulin (CRT), which is usually contained in the lumen of the endoplasmic reticulum, to the surface of the plasma membrane. This effect requires the phosphorylation of the eukaryotic initiation factor 2a (eIF2a) by the eIF2a kinase PERK, yet may also be triggered by inhibition of the eIF2a phosphatase, which is composed by a catalytic subunit (PP1) and a regulatory subunit (GADD34). Here, we addressed the question whether the dissociation of the PP1/GADD34 complex would be sufficient to trigger CRT exposure. Molecular modeling led to the design of a GADD34-derived peptide that competitively disrupts the PP1/GADD34 complex. When added to intact cells, the GADD34-derived peptide fused to a plasma membrane translocation domain abolished the interaction between PP1 and GADD34, stimulated the phosphorylation of eIF2a, and triggered CRT exposure. However, the resolution of the PP1/GADD34 complex did not evoke apoptosis, allowing for the dissociation of CRT exposure and cell death. Anthracyclins, which are highly efficient in inducing CRT translocation to the cell surface also stimulated the dissociation of the PP1/GADD34 complex. These results suggest that the PP1/GADD34 complex plays a major role in the regulation of CRT exposure.     

Many faces of DAMPs in cancer therapy

A new concept of immunogenic cell death (ICD) has recently been proposed. The immunogenic characteristics of this cell death mode are mediated mainly by molecules called ‘damage-associated molecular patterns'' (DAMPs), most of which are recognized by pattern recognition receptors. Some DAMPs are actively emitted by cells undergoing ICD (e.g. calreticulin (CRT) and adenosine triphosphate (ATP)), whereas others are emitted passively (e.g. high-mobility group box 1 protein (HMGB1)). Recent studies have demonstrated that these DAMPs play a beneficial role in anti-cancer therapy by interacting with the immune system. The molecular pathways involved in translocation of CRT to the cell surface and secretion of ATP from tumor cells undergoing ICD are being elucidated. However, it has also been shown that the same DAMPs could contribute to progression of cancer and promote resistance to anticancer treatments. In this review, we will critically evaluate the beneficial and detrimental roles of DAMPs in cancer therapy, focusing mainly on CRT, ATP and HMGB1.     

Multimodal immunogenic cancer cell death as a consequence of anticancer cytotoxic treatments

Apoptotic cell death generally characterized by a morphologically homogenous entity has been considered to be essentially non-immunogenic. However, apoptotic cancer cell death, also known as type 1 programmed cell death (PCD), was recently found to be immunogenic after treatment with several chemotherapeutic agents and oncolytic viruses through the emission of various danger-associated molecular patterns (DAMPs). Extensive studies have revealed that two different types of immunogenic cell death (ICD) inducers, recently classified by their distinct actions in endoplasmic reticulum (ER) stress, can reinitiate immune responses suppressed by the tumor microenvironment. Indeed, recent clinical studies have shown that several immunotherapeutic modalities including therapeutic cancer vaccines and oncolytic viruses, but not conventional chemotherapies, culminate in beneficial outcomes, probably because of their different mechanisms of ICD induction. Furthermore, interests in PCD of cancer cells have shifted from its classical form to novel forms involving autophagic cell death (ACD), programmed necrotic cell death (necroptosis), and pyroptosis, some of which entail immunogenicity after anticancer treatments. In this review, we provide a brief outline of the well-characterized DAMPs such as calreticulin (CRT) exposure, high-mobility group protein B1 (HMGB1), and adenosine triphosphate (ATP) release, which are induced by the morphologically distinct types of cell death. In the latter part, our review focuses on how emerging oncolytic viruses induce different forms of cell death and the combinations of oncolytic virotherapies with further immunomodulation by cyclophosphamide and other immunotherapeutic modalities foster dendritic cell (DC)-mediated induction of antitumor immunity. Accordingly, it is increasingly important to fully understand how and which ICD inducers cause multimodal ICD, which should aid the design of reasonably multifaceted anticancer modalities to maximize ICD-triggered antitumor immunity and eliminate residual or metastasized tumors while sparing autoimmune diseases.     

Mechanisms of pre‐apoptotic calreticulin exposure in immunogenic cell death

Dying tumour cells can elicit a potent anticancer immune response by exposing the calreticulin (CRT)/ERp57 complex on the cell surface before the cells manifest any signs of apoptosis. Here, we enumerate elements of the pathway that mediates pre‐apoptotic CRT/ERp57 exposure in response to several immunogenic anticancer agents. Early activation of the endoplasmic reticulum (ER)‐sessile kinase PERK leads to phosphorylation of the translation initiation factor eIF2α, followed by partial activation of caspase‐8 (but not caspase‐3), caspase‐8‐mediated cleavage of the ER protein BAP31 and conformational activation of Bax and Bak. Finally, a pool of CRT that has transited the Golgi apparatus is secreted by SNARE‐dependent exocytosis. Knock‐in mutation of eIF2α (to make it non‐phosphorylatable) or BAP31 (to render it uncleavable), depletion of PERK, caspase‐8, BAP31, Bax, Bak or SNAREs abolished CRT/ERp57 exposure induced by anthracyclines, oxaliplatin and ultraviolet C light. Depletion of PERK, caspase‐8 or SNAREs had no effect on cell death induced by anthracyclines, yet abolished the immunogenicity of cell death, which could be restored by absorbing recombinant CRT to the cell surface.     

Capsaicin as an inducer of damage-associated molecular patterns (DAMPs) of immunogenic cell death (ICD) in human bladder cancer cells

Few conventional cytotoxic anticancer therapeutics induce immunogenic cell death (ICD). This means that they induce tumor cells to undergo apoptosis while eliciting the emission of a spatiotemporal-defined combination of damage-associated molecular patterns (DAMPs) decoded by the immune system to activate antitumor immunity effective for long-term therapeutic success. The neurotoxin capsaicin (CPS) can induce both cancer cell apoptosis and immune-mediated tumor regression. In the present study, we investigated whether CPS is capable of eliciting the emission of ICD hallmarks in human bladder cancer cell lines undergoing apoptosis. We demonstrated that CPS induces pre- and early apoptotic cell surface exposure of calreticulin (CRT), HSP90, and HSP70 as well as ATP release. Moreover, CRT exposure was prevented by inhibition of endoplasmic reticulum–Golgi traffic by brefeldin A. Furthermore, high-mobility group box 1, HSP90, and HSP70 were passively released at late apoptotic stages. We provide the first evidence that CPS is an inducer of ICD hallmarks, suggesting CPS as a novel potential immunogenic cytotoxic agent.     

The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death

The exposure of calreticulin (CRT) on the plasma membrane can precede anthracycline-induced apoptosis and is required for cell death to be perceived as immunogenic. Mass spectroscopy, immunofluorescence and immunoprecipitation experiments revealed that CRT co-translocates to the surface with another endoplasmic reticulum-sessile protein, the disulfide isomerase ERp57. The knockout and knockdown of CRT or ERp57 inhibited the anthracycline-induced translocation of ERp57 or CRT, respectively. CRT point mutants that fail to interact with ERp57 were unable to restore ERp57 translocation upon transfection into crt(-/-) cells, underscoring that a direct interaction between CRT and ERp57 is strictly required for their co-translocation to the surface. ERp57(low) tumor cells generated by retroviral introduction of an ERp57-specific shRNA exhibited a normal apoptotic response to anthracyclines in vitro, yet were resistant to anthracycline treatment in vivo. Moreover, ERp57(low) cancer cells (which failed to expose CRT) treated with anthracyclines were unable to elicit an anti-tumor response in conditions in which control cells were highly immunogenic. The failure of ERp57(low) cells to elicit immune responses and to respond to chemotherapy could be overcome by exogenous supply of recombinant CRT protein. These results indicate that tumors that possess an intrinsic defect in the CRT-translocating machinery become resistant to anthracycline chemotherapy due to their incapacity to elicit an anti-cancer immune response.     

Ischemia and reperfusion injury combined with cisplatin induces immunogenic cell death in lung cancer cells

A first-line chemotherapeutic drug for non-small cell lung cancer (NSCLC), cisplatin (CDDP), fails to induce immunogenic cell death (ICD) because it fails to induce calreticulin (CRT) exposure on the cell surface. We investigated the potential of ischemia and reperfusion injury (I/R) combined with CDDP to induce ICD in lung cancer cells. The in vitro model of I/R, oxygen-glucose deprivation and reperfusion (OGD/R), effectively induced CRT exposure, ATP secretion, high mobility group box 1 (HMGB1) release and eIF2α phosphorylation in both Lewis lung carcinoma (LLC) and A549 cells when combined with CDDP. By using a vaccine assay and coculture with bone marrow-derived dendritic cells (BMDCs), we showed that OGD/R restored the immunogenicity of CDDP by phosphorylating eIF2α and demonstrated that OGD/R + CDDP (O + C) is an ICD inducer. Using the inguinal tumor model, we found that I/R significantly enhanced the tumor-killing effect of CDDP and Mitomycin C, and this effect relied on adaptive antitumor immunity. Consistently, I + C altered the ratio of interferon-gamma-secreting T lymphocytes, thus overcoming the immunosuppressive effect induced by CDDP. In conclusion, our research presents a new combination strategy and indicates that I/R is a potential anticancer immunogenic modality when combined with nonimmunogenic chemotherapy.Subject terms: Cancer immunotherapy, Chemotherapy     

Wogonin Induced Calreticulin/Annexin A1 Exposure Dictates the Immunogenicity of Cancer Cells in a PERK/AKT Dependent Manner

In response to ionizing irradiation and certain chemotherapeutic agents, dying tumor cells elicit a potent anticancer immune response. However, the potential effect of wogonin (5,7-dihydroxy-8-methoxyflavone) on cancer immunogenicity has not been studied. Here we demonstrated for the first time that wogonin elicits a potent antitumor immunity effect by inducing the translocation of calreticulin (CRT) and Annexin A1 to cell plasma membrane as well as the release of high-mobility group protein 1 (HMGB1) and ATP. Signal pathways involved in this process were studied. We found that wogonin-induced reactive oxygen species (ROS) production causes an endoplasmic reticulum (ER) stress response, including the phosphorylation of PERK (PKR-like endoplasmic reticulum kinase)/PKR (protein kinase R) and eIF2α (eukaryotic initiation factor 2α), which served as upstream signal for the activation of phosphoinositide 3-kinase (PI3K)/AKT, inducing calreticulin (CRT)/Annexin A1 cell membrane translocation. P22/CHP, a Ca²⁺-binding protein, was associated with CRT and was required for CRT translocation to cell membrane. The releases of HMGB1 and ATP from wogonin treated MFC cells, alone or together with other possible factors, activated dendritic cells and induced cytokine releases. In vivo study confirmed that immunization with wogonin-pretreated tumor cells vaccination significantly inhibited homoplastic grafted gastric tumor growth in mice and a possible inflammatory response was involved. In conclusion, the activation of PI3K pathway elicited by ER stress induced CRT/Annexin A1 translocation (“eat me” signal) and HMGB1 release, mediating wogonin-induced immunity of tumor cell vaccine. This indicated that wogonin is a novel effective candidate of immunotherapy against gastric tumor.     

光免疫治疗的抗肿瘤研究进展

光免疫治疗是一种新兴的肿瘤靶向光疗手段,它将单克隆抗体的肿瘤特异性与光吸收剂的光毒性相结合,可以快速且极具免疫原选择性地诱导靶肿瘤细胞的死亡。由于靶向性强,光免疫治疗的副作用小。而且因为该疗法诱导的免疫原性死亡会引起垂死肿瘤细胞周围未成熟树突状细胞的快速成熟,继而将肿瘤抗原提呈给CD8+T细胞,导致治疗后CD8+T细胞的激活和增殖,增强宿主抗肿瘤免疫反应。不仅如此,光免疫治疗还能通过增强纳米药物的肿瘤组织穿透性而提高疗效。鉴于光免疫治疗的优良应用前景,文中从其免疫激活机制、超级高渗透长滞留效应、新进展与联合治疗等方面进行综述,旨在为其深入研究和临床转化提供参考。     

Retracted: Apoptosis of CT26 colorectal cancer cells induced by Clostridium difficile toxin A stimulates potent anti-tumor immunity

Clostridium difficile toxin A (TcdA) is one of the main pathogenic factors released by C. difficile. Due to its potent cytotoxic and proinflammatory activities, we investigated the anti-tumor activity of TcdA. CT26 colorectal cancer cells were challenged with recombinant TcdA, and it was found that TcdA could induce apoptosis of CT26 cells. Calreticulin (CRT) exposure to the cell surface during TcdA-induced apoptosis suggested that this apoptosis may correlate with immunogenicity. Moreover, TcdA-treated apoptotic CT26 cells were highly immunogenic since they could stimulate DC activation, T-cell activation, and anti-tumor activity. Furthermore, the anti-tumor immune response generated was specific and long-term. In summary, these studies demonstrate that C. difficile toxin A can induce apoptotic death of CT26 colorectal cancer cells and stimulate potent anti-tumor immunity.     

Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin

Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.     

Tumor stress, cell death and the ensuing immune response

A cornucopia of physiological and pathological circumstances including anticancer chemotherapy and radiotherapy can induce cell death. However, the immunological consequences of tumor cell demise have remained largely elusive. The paradigm opposing 'apoptosis versus necrosis' as to their respective immunogenicity does not currently hold to predict long-term immunity. Moreover, the notion that tumor cells may be 'stressed' before death to be recognized by immune cells deserves to be underlined. 'Eat-me', 'danger' and 'killing' signals released by stressed tumor under the pressure of cytotoxic compounds may serve as links between the chemotherapy-elicited response of tumor cells and subsequent immune responses. This review will summarize the state-of-the-art of cancer immunity and describe how tumor cell death dictates the links between innate and acquired immunity.     

Influence of neuraminidase and mitomycin C on the immunogenicity of Novikoff tumor cells

Summary Novikoff rat ascites tumor cells were strongly immunogenic in the normal host (Sprague-Dawley strain rat), a single SC inoculum of 5×10⁴ cells rendering the host resistant to a subsequent IP challenge of 1.5×10⁶ tumor cells. Removal of cell-surface sialic acid did not abolish the tumorigenicity of the cells, nor did it modify their immunogenicity. Treatment of the cells with mitomycin C, an agent that blocks cell replication, caused a loss of immunogenicity, indicating that replicative capacity or other mitomycin C-sensitive cell processes are required for immunogenicity of Novikoff tumor cells.     

The <Emphasis Type="Italic">n</Emphasis>3-polyunsaturated fatty acid docosahexaenoic acid induces immunogenic cell death in human cancer cell lines via pre-apoptotic calreticulin exposure

Some anticancer chemotherapeutics, such as anthracyclines and oxaliplatin, elicit immunogenic apoptosis, meaning that dying cancer cells are engulfed by dendritic cells and tumor antigens are efficiently presented to CD8+ T cells, which control residual tumor cells. Immunogenic apoptosis is characterized by pre-apoptotic cell surface exposure of calreticulin (CRT), which usually resides into the endoplasmic reticulum. We investigated the ability of the n3-polyunsaturated fatty acid docosahexaenoic acid (22:6n3, DHA) to induce pre-apoptotic CRT exposure on the surface of the human PaCa-44 pancreatic and EJ bladder cancer cell lines. Cells were treated with 150 μM DHA for different time periods, and, by immunoblot and immunofluorescence, we showed that DHA induced CRT exposure, before the apoptosis-associated phosphatidylserine exposure. As for the known immunogenic compounds, CRT exposure was inhibited by the antioxidant GSH, the pan-caspase zVAD-FMK, and caspase-8 IETD-FMK inhibitor. We provide the first evidence that DHA induces CRT exposure, representing thus a novel potential anticancer immunogenic chemotherapeutic agent.     

Immunogenic cell death modalities and their impact on cancer treatment

It is still enigmatic under which circumstances cellular demise induces an immune response or rather remains immunologically silent. Moreover, the question remains open under which circumstances apoptotic, autophagic or necrotic cells are immunogenic or tolerogenic. Although apoptosis appears to be morphologically homogenous, recent evidence suggests that the pre-apoptotic surface-exposure of calreticulin may dictate the immune response to tumor cells that succumb to anticancer treatments. Moreover, the release of high-mobility group box 1 (HMGB1) during late apoptosis and secondary necrosis contributes to efficient antigen presentation and cytotoxic T-cell activation because HMGB1 can bind to Toll like receptor 4 on dendritic cells, thereby stimulating optimal antigen processing. Cell death accompanied by autophagy also may facilitate cross priming events. Apoptosis, necrosis and autophagy are closely intertwined processes. Often, cells manifest autophagy before they undergo apoptosis or necrosis, and apoptosis is generally followed by secondary necrosis. Whereas apoptosis and necrosis irreversibly lead to cell death, autophagy can clear cells from stress factors and thus facilitate cellular survival. We surmise that the response to cellular stress like chemotherapy or ionizing irradiation, dictates the immunological response to dying cells and that this immune response in turn determines the clinical outcome of anticancer therapies. The purpose of this review is to summarize recent insights into the immunogenicity of dying tumor cells as a function of the cell death modality.     

Immunogenic cell death,DAMPs and anticancer therapeutics: An emerging amalgamation

Immunogenic profile of certain cancer cell death mechanisms has been transmuted by research published over a period of last few years and this change has been so drastic that a new (sub)class of apoptotic cancer cell death, redefined as ‘immunogenic apoptosis’ has started taking shape. In fact, it has been shown that this chemotherapeutic agent-specific immunogenic cancer cell death modality has the capabilities to induce ‘anticancer vaccine effect’, in vivo. These new trends have given an opportunity to combine tumour cell kill and antitumour immunity within a single paradigm, a sort of ‘holy grail’ of anticancer therapeutics. At the molecular level, it has been shown that the immunological silhouette of these cell death pathways is defined by a set of molecules called ‘damage-associated molecular patterns (DAMPs)’. Various intracellular molecules like calreticulin (CRT), heat-shock proteins (HSPs), high-mobility group box-1 (HMGB1) protein, have been shown to be DAMPs exposed/secreted in a stress agent/factor-and cell death-specific manner. These discoveries have motivated further research into discovery of new DAMPs, new pathways for their exposure/secretion, search for new agents capable of inducing immunogenic cell death and urge to solve currently present problems with this paradigm. We anticipate that this emerging amalgamation of DAMPs, immunogenic cell death and anticancer therapeutics may be the key towards squelching cancer-related mortalities, in near future.