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.     

Premortem autophagy determines the immunogenicity of chemotherapy-induced cancer cell death

One particular strategy to render anticancer therapies efficient consists of converting the patient's own tumor cells into therapeutic vaccines, via the induction of immunogenic cell death (ICD). One of the hallmarks of ICD dwells in the active release of ATP by cells committed to undergo, but not yet having succumbed to, apoptosis. We observed that the knockdown of essential autophagy-related genes (ATG3, ATG5, ATG7 and BECN1) abolishes the pre-apoptotic secretion of ATP by several human and murine cancer cell lines undergoing ICD. Accordingly, autophagy-competent, but not autophagy-deficient, tumor cells treated with ICD inducers in vitro could induce a tumor-specific immune response in vivo. Cancer cell lines stably depleted of ATG5 or ATG7 normally generate tumors in vivo, and such autophagy-deficient neoplasms, upon systemic treatment with ICD inducers, exhibit the same levels of apoptosis (as monitored by nuclear shrinkage and caspase-3 activation) and necrosis (as determined by following the kinetics of HMGB1 release) as their autophagy-proficient counterparts. However, autophagy-incompetent cancers fail to release ATP, to recruit immune effectors into the tumor bed and to respond to chemotherapy in conditions in which autophagy-competent tumors do so. The intratumoral administration of ecto-ATPase inhibitors increases extracellular ATP concentrations, re-establishes the therapy-induced recruitment of dendritic cells and T cells into the tumor bed, and restores the chemotherapeutic response of autophagy-deficient cancers. Altogether, these results suggest that autophagy-incompetent tumor cells escape from chemotherapy-induced (and perhaps natural?) immunosurveillance because they are unable to release ATP.     

Premortem autophagy determines the immunogenicity of chemotherapy-induced cancer cell death

Multiple myeloma (MM) is a clonal plasma cell malignancy that accounts for 10–15% of newly diagnosed hematological cancers. Although significant advances have been made in the treatment of MM the disease still remains incurable. The oncolytic potential of reovirus has previously been demonstrated by others and us and is currently in phase III clinical trials for solid tumors. In addition a phase I clinical trial has recently been initiated for MM. Despite the clinical activity, the mechanism(s) of cell death caused by reovirus in MM is yet not well elucidated. A comprehensive understanding of reovirus-mediated histology-specific cell death mechanisms is imperative if this therapeutic is to become a standard of care for patients. Previously we have shown that reovirus-mediated cell death of breast and prostate cancer is orchestrated via apoptosis. The present study demonstrates for the first time that in addition to inducing apoptosis reovirus also upregulates autophagy during oncolysis of MM.     

An emerging role for nanomaterials in increasing immunogenicity of cancer cell death

In the last decade, it has become clear that anti-cancer therapy is more successful when it can also induce an immunogenic form of cancer cell death (ICD). ICD is an umbrella term covering several cell death modalities, including apoptosis and necroptosis. In general, ICD is characterized by the emission of damage-associated molecular patterns (DAMPs) and/or cytokines/chemokines, leading to the induction of strong anti-tumor immune responses. In experimental cancer therapy, new observations indicate that the immunogenicity of dying cancer cells can be improved by the use of biomaterials. In this review, after a brief overview of the basic principles of the concept of ICD and discussion of the potential use of DAMPs as biomarkers of therapy efficacy, we discuss an emerging role of nanomaterials as a promising strategy to modulate the immunogenicity of cancer cell death. We address how nanocarriers can be used to increase the immunogenicity of ICD and then turn our attention to their dual action. Nanocarriers can be used to increase the immunogenicity of dying cancer cells and to reduce the side effects of chemotherapy. Future studies will show whether biomaterials are truly an optimal strategy to modulate the immunogenicity of dying cancer cells and will provide the insights needed for the development of novel treatment strategies for cancer.     

Calreticulin and cancer

Calreticulin (CALR) is an endoplasmic reticulum (ER)-resident protein involved in a spectrum of cellular processes. In healthy cells, CALR operates as a chaperone and Ca²⁺ buffer to assist correct protein folding within the ER. Besides favoring the maintenance of cellular proteostasis, these cell-intrinsic CALR functions support Ca²⁺-dependent processes, such as adhesion and integrin signaling, and ensure normal antigen presentation on MHC Class I molecules. Moreover, cancer cells succumbing to immunogenic cell death (ICD) expose CALR on their surface, which promotes the uptake of cell corpses by professional phagocytes and ultimately supports the initiation of anticancer immunity. Thus, loss-of-function CALR mutations promote oncogenesis not only as they impair cellular homeostasis in healthy cells, but also as they compromise natural and therapy-driven immunosurveillance. However, the prognostic impact of total or membrane-exposed CALR levels appears to vary considerably with cancer type. For instance, while genetic CALR defects promote pre-neoplastic myeloproliferation, patients with myeloproliferative neoplasms bearing CALR mutations often experience improved overall survival as compared to patients bearing wild-type CALR. Here, we discuss the context-dependent impact of CALR on malignant transformation, tumor progression and response to cancer therapy.Subject terms: Oncogenes, Tumour immunology, Protein folding, Endoplasmic reticulum     

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.     

Tumor environment dictates medulloblastoma cancer stem cell expression and invasive phenotype

The neural precursor surface marker CD133 is thought to be enriched in brain cancer stem cells and in radioresistant DAOY medulloblastoma-derived tumor cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP) expression is a hallmark of highly invasive, radioresistant, and hypoxic brain tumor cells, we sought to determine whether MT1-MMP and other MMPs could regulate the invasive phenotype of CD133(+) DAOY cells. We found that when DAOY medulloblastoma or U87 glioblastoma cells were implanted in nude mice, only those cells specifically implanted in the brain environment generated CD133(+) brain tumors. Vascular endothelial growth factor and basic fibroblast growth factor gene expression increases in correlation with CD133 expression in those tumors. When DAOY cultures were induced to generate in vitro neurosphere-like cells, gene expression of CD133, MT1-MMP, MMP-9, and MDR-1 was induced and correlated with an increase in neurosphere invasiveness. Specific small interfering RNA gene silencing of either MT1-MMP or MMP-9 reduced the capacity of the DAOY monolayers to generate neurospheres and concomitantly abrogated their invasive capacity. On the other hand, overexpression of MT1-MMP in DAOY triggered neurosphere-like formation which was further amplified when cells were cultured in neurosphere medium. Collectively, we show that both MT1-MMP and MMP-9 contribute to the invasive phenotype during CD133(+) neurosphere-like formation in medulloblastoma cells. Increases in MMP-9 may contribute to the opening of the blood-brain barrier, whereas increased MT1-MMP would promote brain tumor infiltration. Our study suggests that MMP-9 or MT1-MMP targeting may reduce the formation of brain tumor stem cells.     

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80(high), CD83(high), CD86(high), MHC-II(high)) and functional stimulation (NO(high), IL-10(absent), IL-1β(high)) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress.     

Pten dose dictates cancer progression in the prostate

Complete inactivation of the PTEN tumor suppressor gene is extremely common in advanced cancer, including prostate cancer (CaP). However, one PTEN allele is already lost in the vast majority of CaPs at presentation. To determine the consequence of PTEN dose variations on cancer progression, we have generated by homologous recombination a hypomorphic Pten mouse mutant series with decreasing Pten activity: Pten^hy/+ > Pten^+/− > Pten^hy/− (mutants in which we have rescued the embryonic lethality due to complete Pten inactivation) > Pten prostate conditional knockout (Pten^pc) mutants. In addition, we have generated and comparatively analyzed two distinct Pten^pc mutants in which Pten is inactivated focally or throughout the entire prostatic epithelium. We find that the extent of Pten inactivation dictate in an exquisite dose-dependent fashion CaP progression, its incidence, latency, and biology. The dose of Pten affects key downstream targets such as Akt, p27^Kip1, mTOR, and FOXO3. Our results provide conclusive genetic support for the notion that PTEN is haploinsufficient in tumor suppression and that its dose is a key determinant in cancer progression.     

程序性细胞死亡与肿瘤

程序性细胞死亡（programmed cell death,PCD）是指由基因控制的细胞自主的有序性死亡方式,涉及一系列基因的激活、表达以及调控等。目前,经典细胞凋亡被称为Ⅰ型PCD,而自噬性细胞死亡称为Ⅱ型PCD,坏死样程序性细胞死亡则被称为Ⅲ型PCD,它们在肿瘤的发生、发展及治疗过程中起非常重要的作用。该文结合国内外最新研究进展主要针对不同细胞死亡模式及其相互作用、关键作用蛋白,细胞自噬与肿瘤发生,细胞自噬、凋亡与肿瘤治疗作一简要综述,并展望发展前景,提出在肿瘤治疗中如何利用不同死亡模式的协同作用最大限度地发挥其临床应用价值。     

How cell death shapes cancer

Apoptosis has been established as a mechanism of anti-cancer defense. Members of the BCL-2 family are critical mediators of apoptotic cell death in health and disease, often found to be deregulated in cancer and believed to lead to the survival of malignant clones. However, over the years, a number of studies pointed out that a model in which cell death resistance unambiguously acts as a barrier against malignant disease might be too simple. This is based on paradoxical observations made in tumor patients as well as mouse models indicating that apoptosis can indeed drive tumor formation, at least under certain circumstances. One possible explanation for this phenomenon is that apoptosis can promote proliferation critically needed to compensate for cell loss, for example, upon therapy, and to restore tissue homeostasis. However, this, at the same time, can promote tumor development by allowing expansion of selected clones. Usually, tissue resident stem/progenitor cells are a major source for repopulation, some of them potentially carrying (age-, injury- or therapy-induced) genetic aberrations deleterious for the host. Thereby, apoptosis might drive genomic instability by facilitating the emergence of pathologic clones during phases of proliferation and subsequent replication stress-associated DNA damage. Tumorigenesis initiated by repeated cell attrition and repopulation, as confirmed in different genetic models, has parallels in human cancers, exemplified in therapy-induced secondary malignancies and myelodysplastic syndromes in patients with congenital bone marrow failure syndromes. Here, we aim to review evidence in support of the oncogenic role of stress-induced apoptosis.     

Inducers of immunogenic cancer cell death

Recently, cytokine-based pro-tumourigenic signalling has been found to play a major role in the immune system's pro-tumourigenic activity. On the other hand, other recent findings have shown that immunogenic cancer cell death triggered by certain anticancer modalities might reset the dysfunctional immune system towards the activation of a long-lasting protective anti-tumour response. Therefore, using inducers of immunogenic cell death (ICD) that can prevent or impede tumour-promoting cytokine signalling is one of the best ways of instigating or restoring efficient anti-tumour immunity. In this review it is discussed, how the different ICD inducers interact with the immune system and influence cytokine-based pro-tumourigenic signalling. We believe that it is crucial to discover or develop new anti-cancer therapeutic modalities that can induce ICD and impede tumour-promoting cytokine signalling.     

Molecular characteristics of immunogenic cancer cell death

Apoptotic cell death is initiated by a morphologically homogenous entity that was considered to be non-immunogenic and non-inflammatory in nature. However, recent advances suggest that apoptosis, under certain circumstances, can be immunogenic. In particular, some characteristics of the plasma membrane, acquired at preapoptotic stage, can cause immune effectors to recognize and attack preapoptotic tumor cells. The signals that mediate the immunogenicity of tumor cells involve elements of the DNA damage response (such as ataxia telangiectasia mutated and p53 activation), elements of the endoplasmic reticulum stress response (such as eukaryotic initiation factor 2alpha phosphorylation), as well as elements of the apoptotic response (such as caspase activation). Depending on the signal-transduction pathway, tumor cells responding to chemotherapy or radiotherapy can express 'danger' and 'eat me' signals on the cell surface (such as NKG2D ligands, heat-shock proteins and calreticulin) or can secrete/release immunostimulatory factors (such as cytokines and high-mobility group box 1) to stimulate innate immune effectors. Likewise, the precise sequence of such events influences the 'decision' of the immune system to mount a cognate response or not. We therefore anticipate that the comprehension of the mechanisms governing the immunogenicity of cell death will have a profound impact on the design of anticancer therapies.     

Experience dictates stem cell fate in the adult hippocampus

Adult hippocampal neurogenesis has been implicated in cognitive and emotional processes, as well as in response to antidepressant treatment. However, little is known about how the adult stem cell lineage contributes to hippocampal structure and function and how this process is modulated by the animal's experience. Here we perform an indelible lineage analysis and report that neural stem cells can produce expanding and persisting populations of not only neurons, but also stem cells in the adult hippocampus. Furthermore, the ratio of stem cells to neurons depends on experiences of the animal or the location of the stem cell. Surprisingly, social isolation facilitated accumulation of stem cells, but not neurons. These results show that neural stem cells accumulate in the adult hippocampus and that the stem cell-lineage relationship is under control of anatomic and experiential niches. Our findings suggest that, in the hippocampus, fate specification may act as a form of cellular plasticity for adapting to environmental changes.     

Lysosomal involvement in cell death and cancer

Lysosomes, with their arsenal of degradative enzymes are increasingly becoming an area of interest in the field of oncology. The changes induced in this compartment upon transformation are numerous and whereas most are viewed as pro-oncogenic the same processes also render cancer cells susceptible to lysosomal death pathways. This review will provide an overview of the pro- and anti-oncogenic potential of this compartment and how these might be exploited for cancer therapy, with special focus on lysosomal death pathways.     

Triggering caspase-independent cell death to combat cancer

Caspase-mediated apoptosis is a major hindrance to tumour growth and metastasis. Accordingly, defects in signalling pathways leading to the activation of caspases are common in tumours. Moreover, many tumour cells can unexpectedly survive the activation of caspases. As a result, caspase-independent cell death programmes are gaining increasing interest among cancer researchers. The heterogeneity of cancer cells with respect to their sensitivity to various death stimuli further emphasizes the need for additional death pathways in the therapeutic control of cell death. An understanding of the molecular control of alternative death pathways is beginning to emerge, being comparable with that of the molecular anatomy of apoptosis at the time of the discovery of caspases less than a decade ago. Here, newly discovered triggers and molecular regulators of alternative cell death programmes are reviewed and their potential in future cancer therapy is discussed.