It is commonly assumed that the only goal of anticancer chemotherapy, like antimicrobial antibiotic therapy, is to eradicate by direct cytotoxic effects all tumor cells. According to this mechanism, complete and permanent cure would be obtained by antineoplastic agents that succeed in killing all cancer cells including cancer stem cells and micrometastases. 1,2 In fact, cancer has long been conceived and treated as a cell-autonomous phenomenon, regardless of the immune system's contribution to the therapeutic response. Recently, we have challenged this idea by showing that, at least in the case of anthracyclin-mediated chemotherapy, the antitumor immune response plays a major role in therapeutic success. Thus, immunocompetent mice bearing CT26 colon carcinomas or MCA205 fibrosarcomas can be cured by intratumoral injection of anthracyclins, whereas immunodeficient mice lacking T cells only exhibit partial responses with a delay in tumor growth. [3][4][5] Detailed molecular studies revealed that anthracyclins have the peculiar capacity of inducing immunogenic cell death. In contrast, many other cytotoxic agents including agents that damage nuclear DNA (such as etoposide and mitomycin C), mitochondria, the endoplasmic reticulum or lysosomes fail to induce immunogenic cell Cells were transfected with a control siRNA or a CRT-specific siRNA heteroduplex (sense strand: 5 0 -rCrCrGrCUrGrGrGUrCrGrArAUrCrRrArATT-3 0 ). Thirty-six hours later, the cells were subjected to g-irradiation (g) or UVC light (h), cultured for 4 h, optionally treated with recombinant CRT protein (3 mg/10 6 cells in PBS on ice for 30 min, followed by three washes) and subjected to immunofluorescence staining of CRT as above. (i, j) Requirement of CRT exposure for the immunogenic effect of ionizing irradiation. CT26 colon cancer cells were transfected with the indicated siRNAs, g-irradiation (i), UV light (j) and/or recombinant CRT (as in g and h) and then injected subcutaneously (3 Â 10 6 cells) into the left flank of BALB/c mice. One week after this vaccination, the mice were challenged with live tumor cells in the opposite flank (day 0) and the frequency of tumor-free animals was monitored (mean7S.E.M.); n represents the absolute number of mice enrolled in each cohort. *Po0.001 (Student's t-test)

The conventional treatment of cancer relies upon radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Nonetheless, there are circumstances in which conventional anti-cancer therapy can induce a modality of cellular demise that elicits innate and cognate immune responses, which in turn mediate part of the anti-tumor effect. Although different chemotherapeutic agents may kill tumor cells through an apparently homogeneous apoptotic pathway, they differ in their capacity to stimulate immunogenic cell death. We discovered that the pre-apoptotic translocation of intracellular calreticulin (endo-CRT) to the plasma membrane surface (ecto-CRT) is critical for the recognition and engulfment of dying tumor cells by dendritic cells. Thus, anthracyclines and gamma-irradiation that induce ecto-CRT cause immunogenic cell death, while other pro-apoptotic agents (such as mitomycin C and etoposide) induce neither ecto-CRT nor immunogenic cell death. Depletion of CRT abolishes the immunogenicity of cell death elicited by anthracyclines, while exogenous supply of CRT or enforcement of CRT exposure by pharmacological agents that favor CRT translocation can enhance the immunogenicity of cell death. For optimal anti-tumor vaccination and immunogenic chemotherapy, the same cells have to expose ecto-CRT and to succumb to apoptosis; if these events affect different cells, no anti-tumor immune response is elicited. These results may have far reaching implications for tumor immunology because (i) ecto-CRT exposure by tumor cells allows for the prediction of therapeutic outcome and because (ii) the re-establishment of ecto-CRT may ameliorate the efficacy of chemotherapy.

Mutations in PINK1 cause early-onset Parkinson's disease (PD). Studies in Drosophila melanogaster have highlighted mitochondrial dysfunction on loss of Pink1 as a central mechanism of PD pathogenesis. Here we show that global analysis of transcriptional changes in Drosophila pink1 mutants reveals an upregulation of genes involved in nucleotide metabolism, critical for neuronal mitochondrial DNA synthesis. These key transcriptional changes were also detected in brains of PD patients harbouring PINK1 mutations. We demonstrate that genetic enhancement of the nucleotide salvage pathway in neurons of pink1 mutant flies rescues mitochondrial impairment. In addition, pharmacological approaches enhancing nucleotide pools reduce mitochondrial dysfunction caused by Pink1 deficiency. We conclude that loss of Pink1 evokes the activation of a previously unidentified metabolic reprogramming pathway to increase nucleotide pools and promote mitochondrial biogenesis. We propose that targeting strategies enhancing nucleotide synthesis pathways may reverse mitochondrial dysfunction and rescue neurodegeneration in PD and, potentially, other diseases linked to mitochondrial impairment.The role of mitochondrial impairment in PD has long been debated. Recently, the identification of causative mutations in PINK1, a gene encoding a mitochondrial kinase in PD patients has renewed interest in the role of mitochondrial damage in PD (ref. 1).

In contrast to prior belief, tumor cell apoptosis is not necessarily silent but can be immunogenic. By tracing how anthracyclines and ;-irradiation trigger immunogenic cell deaths, we found that they were causally connected to the exposure of calreticulin on the tumor cell surface, before apoptosis in the tumor cell itself occurred. Furthermore, we showed that calreticulin exposure was necessary and sufficient to increase proimmunogenic killing by other chemotherapies. Our findings suggest that calreticulin could serve as a biomarker to predict therapy-associated immune responses, and that tactics to expose calreticulin might improve the clinical efficacy of many cancer therapies. [Cancer Res 2007;67(17):7941-4]

Protein misfolding has a key role in several neurological disorders including Parkinson's disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control.

Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal-lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF-1 and forskolin, two wellknown inhibitors of CGC apoptosis, as well as by adenovirusmediated overexpression of Bcl-2. 3-Methyladenine (3-MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3-MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase-3 activation which, in turn, appears to be mediated by cathepsin B, as CA074-Me, a selective inhibitor of this enzyme, fully blocks the processing of pro-caspase-3. Immunofluorescence analysis demonstratesd that cathepsin B, normally confined inside the lysosomal-endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death.

The endoplasmic reticulum (ER) is the cellular site of polypeptide folding and modification. When these processes are hampered, an unfolded protein response (UPR) is activated. If the damage is too broad, the mammalian UPR launches the apoptotic program. As a consequence, mobilization of ER calcium stores sensitizes mitochondria to direct proapoptotic stimuli. We make use of a mouse Apaf1-deficient cell system of proneural origin to understand the roles played in this context by the apoptosome, the most studied apoptotic machinery along the mitochondrial pathway of death. We show here that in the absence of the apoptosome ER stress induces cytochrome c release from the mitochondria but that apoptosis cannot occur. Under these circumstances, Grp78/BiP and GADD153/ CHOP, both hallmarks of UPR, are canonically up-regulated, and calcium is properly released from ER stores. We also demonstrate that caspase 12, a protease until now believed to play a central role in the initiation of ER stress-induced cell death in the mouse system, is dispensable for the mitochondrial pathway of death to take place.

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 anthracyclintreated tumor cells. Here, we investigated the role of cellular Ca 2 þ 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 þ concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca 2 þ depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca 2 þ , targeted expression of the ligand-binding domain of the IP 3 receptor and inhibition of the sarco-endoplasmic reticulum Ca 2 þ -ATPase pump reduced endoplasmic reticulum Ca 2 þ 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 þ levels control the exposure of CRT. In contrast to prior belief, apoptotic cell death can be immunogenic and hence elicits an active immune response against dying tumor cells. 1 This is therapeutically relevant because immune defects that compromise the response against apoptotic cells reduce the efficacy of anticancer chemotherapy, both in suitable animal models and in patients. 2 We found that anthracyclins and g-irradiation are particularly efficient in inducing immunogenic cell death, at least in mouse models. [3][4][5] The immunogenicity of cellular demise correlates with the exposure of calreticulin (CRT) on the plasma membrane (PM) surface of stressed tumor cells, a phenomenon that manifests before the cells acquire signs of apoptosis such as phosphatidylserine exposure. Inhibition of CRT exposure curtails the capacity of anthracyclin-treated or irradiated tumor cells to vaccinate against cancer and reduces the therapeutic efficacy of anthracyclins on tumors established in immunocompetent hosts. 3-5 Provision of recombinant CRT or drug-mediated enforcement of CRT exposure rendered per se nonimmunogenic chemotherapies (for instance with etoposide and mitomycin C) immunogenic and boosted their therapeutic efficacy. 5 These results suggest that CRT exposure is both necessary and sufficient to render conventional chemotherapies immunogenic.CRT is an abundant Ca 2 þ -binding chaperone that is mostly present in the endoplasmic reticulum (ER) lumen, although it can also be found in other subcellular localizations. 6,7 When present on the surface of damaged cells, it can serve as an 'eat-me' signal and hence facilitate the recognition and later engulfment of the dying cells by macrophages 8 or by dendrit...