Cellular and molecular features related to exceptional therapy response and extreme long-term survival in glioblastoma.

Glioblastoma Multiforme (GBM) remains the most common malignant primary brain tumor with a dismal prognosis that rarely exceeds beyond 2 years despite extensive therapy, which consists of maximal safe surgical resection, radiotherapy, and/or chemotherapy. Recently, it has become clear that GBM is not one homogeneous entity and that both intra-and intertumoral heterogeneity contributes significantly to differences in tumoral behavior which may consequently be responsible for differences in survival. Strikingly and in spite of its dismal prognosis, small fractions of GBM patients seem to display extremely long survival, defined as surviving over 10 years after diagnosis, compared to the large majority of patients. Although the underlying mechanisms for this peculiarity remain largely unknown, emerging data suggest that still poorly characterized both cellular and molecular factors of the tumor microenvironment and their interplay probably play an important role. We hereby give an extensive overview of what is yet known about these cellular and molecular features shaping extreme long survival in GBM.

ATP13A2/PARK9 regulates endo-/lysosomal cargo sorting and proteostasis through a novel PI(3, 5)P2-mediated scaffolding function.

ATP13A2 (also called PARK9), is a transmembrane endo-/lysosomal-associated P5 type transport ATPase. Loss-of-function mutations in ATP13A2 result in the Kufor-Rakeb Syndrome (KRS), a form of autosomal Parkinson's disease (PD). In spite of a growing interest in ATP13A2, very little is known about its physiological role in stressed cells. Recent studies suggest that the N-terminal domain of ATP13A2 may hold key regulatory functions, but their nature remains incompletely understood. To this end, we generated a set of melanoma and neuroblastoma cell lines stably overexpressing wild-type (WT), catalytically inactive (D508N) and N-terminal mutants, or shRNA against ATP13A2. We found that under proteotoxic stress conditions, evoked by the proteasome inhibitor Bortezomib, endo-/lysosomal associated full-length ATP13A2 WT, catalytically-inactive or N-terminal fragment mutants, reduced the intracellular accumulation of ubiquitin-conjugated (Ub) proteins, independent of autophagic degradation. In contrast, ATP13A2 silencing increased the intracellular accumulation of Ub-proteins, a pattern also observed in patient-derived fibroblasts harbouring ATP13A2 loss-of function mutations. In treated cells, ATP13A2 evoked endocytic vesicle relocation and increased cargo export through nanovesicles. Expression of an ATP13A2 mutant abrogating PI(3,5)P2 binding or chemical inhibition of the PI(3,5)P2-generating enzyme PIKfyve, compromised vesicular trafficking/nanovesicles export and rescued intracellular accumulation of Ub-proteins in response to proteasomal inhibition. Hence, our study unravels a novel activity-independent scaffolding role of ATP13A2 in trafficking/export of intracellular cargo in response to proteotoxic stress.

DAMP-Induced Allograft and Tumor Rejection: The Circle Is Closing.

The pathophysiological importance of the immunogenicity of damage-associated molecular patterns (DAMPs) has been pinpointed by their identification as triggers of allograft rejection following release from dying cells, such as after ischemia-reperfusion injury. In cancers, however, this strong trigger of a specific immune response gives rise to the success of cancer immunotherapy. Here, we review the recently literature on the pathophysiological importance of DAMP release and discuss the implications of these processes for allograft rejection and cancer immunotherapy, revealing a striking mechanistic overlap. We conclude that these two fields share a common mechanistic basis of regulated necrosis and inflammation, the molecular characterization of which may be helpful for both oncologists and the transplant community.

Transplantation and Damage-Associated Molecular Patterns (DAMPs).

Upon solid organ transplantation and during cancer immunotherapy, cellular stress responses result in the release of damage-associated molecular patterns (DAMPs). The various cellular stresses have been characterized in detail over the last decades, but a unifying classification based on clinically important aspects is lacking. Here, we provide an in-depth review of the most recent literature along with a unifying concept of the danger/injury model, suggest a classification of DAMPs, and review the recently elaborated mechanisms that result in the emission of such factors. We further point out the differences in DAMP responses including the release following a heat shock pattern, endoplasmic reticulum stress, DNA damage-mediated DAMP release, and discuss the diverse pathways of regulated necrosis in this respect. The understanding of various forms of DAMPs and the consequences of their different release patterns are prerequisite to associate serum markers of cellular stresses with clinical outcomes.

Immunogenic versus tolerogenic phagocytosis during anticancer therapy: mechanisms and clinical translation.

Phagocytosis of dying cells is a major homeostatic process that represents the final stage of cell death in a tissue context. Under basal conditions, in a diseased tissue (such as cancer) or after treatment with cytotoxic therapies (such as anticancer therapies), phagocytosis has a major role in avoiding toxic accumulation of cellular corpses. Recognition and phagocytosis of dying cancer cells dictate the eventual immunological consequences (i.e., tolerogenic, inflammatory or immunogenic) depending on a series of factors, including the type of 'eat me' signals. Homeostatic clearance of dying cancer cells (i.e., tolerogenic phagocytosis) tends to facilitate pro-tumorigenic processes and actively suppress antitumour immunity. Conversely, cancer cells killed by immunogenic anticancer therapies may stimulate non-homeostatic clearance by antigen-presenting cells and drive cancer antigen-directed immunity. On the other hand, (a general) inflammatory clearance of dying cancer cells could have pro-tumorigenic or antitumorigenic consequences depending on the context. Interestingly, the immunosuppressive consequences that accompany tolerogenic phagocytosis can be reversed through immune-checkpoint therapies. In the present review, we discuss the pivotal role of phagocytosis in regulating responses to anticancer therapy. We give particular attention to the role of phagocytosis following treatment with immunogenic or immune-checkpoint therapies, the clinical prognostic and predictive significance of phagocytic signals for cancer patients and the therapeutic strategies that can be employed for direct targeting of phagocytic determinants.

The PERKs of damage-associated molecular patterns mediating cancer immunogenicity: From sensor to the plasma membrane and beyond.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are emerging as key adaptation mechanisms in response to loss of proteostasis, with major cell autonomous and non-autonomous functions impacting cancer progression and therapeutic responses. In recent years, vital physiological roles of the ER in maintenance of proteostasis, Ca(2+) signaling and trafficking through the secretory pathway have emerged. Some of these functions have been shown to be decisive for mobilizing certain signals from injured/dying cancer cells in response to certain anticancer treatments, toward the plasma membrane and ultimately emit them into the extracellular environment, where they may act as danger signals. The spatiotemporally defined emission of these signals, better known as damage-associated molecular patterns (DAMPs), distinguishes this type of cancer cell death from physiological apoptosis, which is tolerogenic in nature, thereby enabling these dying cancer cells to alert the immune system and "re-activate" antitumor immunity. The emission of DAMPs, decisive for immunogenic cell death (ICD) and which include the ER chaperone calreticulin and ATP, is reliant on a danger signaling module induced by certain assorted anticancer treatments through oxidative-ER stress. The main focus of this review is to discuss the emerging role of ER-stress regulated pathways and processes in danger signaling thereby regulating the cancer cell-immune cell interface by the extracellular emission of DAMPs. In particular, we discuss signaling contexts existing upstream and around PERK, a major ER-stress sensor in ICD context, which have not been emphatically discussed in the context of antitumor immunity and ICD up until now. Finally, we briefly discuss the pros and cons of targeting PERK in the context of ICD.

Concurrent MEK and autophagy inhibition is required to restore cell death associated danger-signalling in Vemurafenib-resistant melanoma cells.

Vemurafenib (PLX4032), an inhibitor of BRAF(V600E), has demonstrated significant clinical anti-melanoma effects. However, the majority of treated patients develop resistance, due to a variety of molecular mechanisms including MAPK reactivation through MEK. The induction of a cancer cell death modality associated with danger-signalling resulting in surface mobilization of crucial damage-associated-molecular-patterns (DAMPs), e.g. calreticulin (CRT) and heat shock protein-90 (HSP90), from dying cells, is emerging to be crucial for therapeutic success. Both cell death and danger-signalling are modulated by autophagy, a key adaptation mechanism stimulated during melanoma progression. However, whether melanoma cell death induced by MAPK inhibition is associated with danger-signalling, and the reliance of these mechanisms on autophagy, has not yet been scrutinized. Using a panel of isogenic PLX4032-sensitive and resistant melanoma cell lines we show that PLX4032-induced caspase-dependent cell death and DAMPs exposure in the drug-sensitive cells, but failed to do so in the drug-resistant cells, displaying heightened MEK activation. MEK inhibitor, U0126, treatment sensitized PLX4032-resistant cells to death and re-established their danger-signalling capacity. Only melanoma cells exposing death-induced danger-signals were phagocytosed and induced DC maturation. Although the PLX4032-resistant melanoma cells displayed higher basal and drug-induced autophagy, compromising autophagy, pharmacologically or by ATG5 knockdown, was insufficient to re-establish their PLX4032 sensitivity. Interestingly, autophagy abrogation was particularly efficacious in boosting cell death and ecto-CRT/ecto-HSP90 in PLX4032-resistant cells upon blockage of MEK hyper-activation by U0126. Thus combination of MEK inhibitors with autophagy blockers may represent a novel treatment regime to increase both cell death and danger-signalling in Vemurafenib-resistant metastatic melanoma.

Epithelial-mesenchymal transition during invasion of cutaneous squamous cell carcinoma is paralleled by AKT activation.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is required for tumour invasion and dissemination to occur. OBJECTIVES: To investigate EMT during invasion of cutaneous squamous cell carcinoma (cSCC) and the involvement of AKT. METHODS: Using a tissue microarray, we measured expression of EMT-markers and AKT activation in 140 samples from patients with skin cancer and matched samples of normal skin adjacent to cSCC in cSCC in situ (cSCCIS) and in invasive cSCC. We investigated EMT using functional assays and the expression of EMT markers in an isogenic skin cancer progression model using cell lines derived from dysplastic forehead skin (PM1), primary invasive cSCC (MET1) and its lymph node metastasis (MET4). This model was used to investigate AKT-specific inhibition of the EMT process. RESULTS: In comparison with normal skin, and normal skin plus cSCCIS, the invasive cSCCs show significantly increased vimentin expression, decreased E-cadherin expression and increased expression of the active form of AKT. In the cell culture model, the primary MET1 cells display the lowest adhesion potential, the highest migratory and invasive ability through a Matrigel-coated porous membrane, the highest expression of EMT markers vimentin and Slug and the lowest expression of the epithelial marker E-cadherin. Pharmacological AKT inhibition in this model suppressed EMT mechanisms. CONCLUSIONS: AKT may serve as a therapeutic target to avoid dissemination of cSCC cells.

BNIP3 supports melanoma cell migration and vasculogenic mimicry by orchestrating the actin cytoskeleton.

BNIP3 is an atypical BH3-only member of the BCL-2 family of proteins with reported pro-death as well as pro-autophagic and cytoprotective functions, depending on the type of stress and cellular context. In line with this, the role of BNIP3 in cancer is highly controversial and increased BNIP3 levels in cancer patients have been linked with both good as well as poor prognosis. In this study, using small hairpin RNA (shRNA) lentiviral transduction to stably knockdown BNIP3 (BNIP3-shRNA) expression levels in melanoma cells, we show that BNIP3 supports cancer cell survival and long-term clonogenic growth. Although BNIP3-shRNA increased mitochondrial mass and baseline levels of reactive oxygen species production, which are features associated with aggressive cancer cell behavior, it also prevented cell migration and completely abolished the ability to form a tubular-like network on matrigel, a hallmark of vasculogenic mimicry (VM). We found that this attenuated aggressive behavior of these melanoma cells was underscored by severe changes in cell morphology and remodeling of the actin cytoskeleton associated with loss of BNIP3. Indeed, BNIP3-silenced melanoma cells displayed enhanced formation of actin stress fibers and membrane ruffles, while lamellopodial protrusions and filopodia, tight junctions and adherens junctions were reduced. Moreover, loss of BNIP3 resulted in re-organization of focal adhesion sites associated with increased levels of phosphorylated focal adhesion kinase. Remarkably, BNIP3 silencing led to a drop of the protein levels of the integrin-associated protein CD47 and its downstream signaling effectors Rac1 and Cdc42. These observations underscore that BNIP3 is required to maintain steady-state levels of intracellular complexes orchestrating the plasticity of the actin cytoskeleton, which is integral to cell migration and other vital processes stimulating cancer progression. All together these results unveil an unprecedented pro-tumorigenic role of BNIP3 driving melanoma cell's aggressive features, like migration and VM.

Danger signalling during cancer cell death: origins, plasticity and regulation.

Accumulating data indicates that following anti-cancer treatments, cancer cell death can be perceived as immunogenic or tolerogenic by the immune system. The former is made possible due to the ability of certain anti-cancer modalities to induce immunogenic cell death (ICD) that is associated with the emission of damage-associated molecular patterns (DAMPs), which assist in unlocking a sequence of events leading to the development of anti-tumour immunity. In response to ICD inducers, activation of endoplasmic reticulum (ER) stress has been identified to be indispensable to confer the immunogenic character of cancer cell death, due to its ability to coordinate the danger signalling pathways responsible for the trafficking of vital DAMPs and subsequent anti-cancer immune responses. However, in recent times, certain processes apart from ER stress have emerged (e.g., autophagy and possibly viral response-like signature), which have the ability to influence danger signalling. In this review, we discuss the molecular nature, emerging plasticity in the danger signalling mechanisms and immunological impact of known DAMPs in the context of immunogenic cancer cell death. We also discuss key effector mechanisms modulating the interface between dying cancer cells and the immune cells, which we believe are crucial for the therapeutic relevance of ICD in the context of human cancers, and also discuss the influence of experimental conditions and animal models on these.

Perk-dependent repression of miR-106b-25 cluster is required for ER stress-induced apoptosis.

Activation of the unfolded protein response sensor PKR-like endoplasmic reticulum kinase (Perk) attenuates endoplasmic reticulum (ER) stress levels. Conversantly, if the damage is too severe and ER function cannot be restored, this signaling branch triggers apoptosis. Bcl-2 homology 3-only family member Bim is essential for ER stress-induced apoptosis. However, the regulatory mechanisms controlling Bim activation under ER stress conditions are not well understood. Here, we show that downregulation of the miR-106b-25 cluster contributes to ER stress-induced apoptosis and the upregulation of Bim. Hypericin-mediated photo-oxidative ER damage induced Perk-dependent cell death and led to a significant decrease in the levels of miRNAs belonging to miR-106b-25 cluster in wild-type (WT) but not in Perk⁻/⁻ MEFs. Further, we show that expression of miR-106b-25 and Mcm-7 (host gene of miR-106b-25) is co-regulated through the transcription factors Atf4 (activating transcription factor 4) and Nrf2 (nuclear factor-erythroid-2-related factor 2). ER stress increased the activity of WT Bim 3'UTR (untranslated region) construct but not the miR-106b-25 recognition site-mutated Bim 3'UTR construct. Overexpression of miR-106b-25 cluster inhibits ER stress-induced cell death in WT but did not confer any further protection in Bim-knockdown cells. Further, we show downregulation in the levels of miR-106b-25 cluster in the symptomatic SOD1(G86R) transgenic mice. Our results suggest a molecular mechanism whereby repression of miR-106b-25 cluster has an important role in ER stress-mediated increase in Bim and apoptosis.

PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress.

Endoplasmic reticulum stress is emerging as an important modulator of different pathologies and as a mechanism contributing to cancer cell death in response to therapeutic agents. In several instances, oxidative stress and the onset of endoplasmic reticulum (ER) stress occur together; yet, the molecular events linking reactive oxygen species (ROS) to ER stress-mediated apoptosis are currently unknown. Here, we show that PERK (RNA-dependent protein kinase (PKR)-like ER kinase), a key ER stress sensor of the unfolded protein response, is uniquely enriched at the mitochondria-associated ER membranes (MAMs). PERK(-/-) cells display disturbed ER morphology and Ca(2+) signaling as well as significantly weaker ER-mitochondria contact sites. Re-expression of a kinase-dead PERK mutant but not the cytoplasmic deletion mutant of PERK in PERK(-/-) cells re-establishes ER-mitochondria juxtapositions and mitochondrial sensitization to ROS-mediated stress. In contrast to the canonical ER stressor thapsigargin, during ROS-mediated ER stress, PERK contributes to apoptosis twofold by sustaining the levels of pro-apoptotic C/EBP homologous protein (CHOP) and by facilitating the propagation of ROS signals between the ER and mitochondria through its tethering function. Hence, this study reveals an unprecedented role of PERK as a MAMs component required to maintain the ER-mitochondria juxtapositions and propel ROS-mediated mitochondrial apoptosis. Furthermore, it suggests that loss of PERK may cause defects in cell death sensitivity in pathological conditions linked to ROS-mediated ER stress.

SHIP-1 inhibits CD95/APO-1/Fas-induced apoptosis in primary T lymphocytes and T leukemic cells by promoting CD95 glycosylation independently of its phosphatase activity.

SHIP-1 (SH2 (Src homology 2)-containing inositol 5'-phosphatase-1) functions as a negative regulator of immune responses by hydrolyzing phosphatidylinositol-3,4,5-triphosphate generated by phosphoinositide-3 (PI 3)-kinase activity. As a result, SHIP-1 deficiency in mice results in myeloproliferation and B-cell lymphoma. On the other hand, SHIP-1-deficient mice have a reduced T-cell population, but the underlying mechanisms are unknown. In this work, we hypothesized that SHIP-1 plays anti-apoptotic functions in T cells upon stimulation of the death receptor CD95/APO-1/Fas. Using primary T cells from SHIP-1(-/-) mice and T leukemic cell lines, we report that SHIP-1 is a potent inhibitor of CD95-induced death. We observed that a small fraction of the SHIP-1 pool is localized to the endoplasmic reticulum (ER), in which it promotes CD95 glycosylation. This post-translational modification requires an intact SH2 domain of SHIP-1, but is independent of its phosphatase activity. The glycosylated CD95 fails to oligomerize upon stimulation, resulting in impaired death-inducing signaling complex (DISC) formation and downstream apoptotic cascade. These results uncover an unanticipated inhibitory function for SHIP-1 and emphasize the role of glycosylation in the regulation of CD95 signaling in T cells. This work may also provide a new basis for therapeutic strategies using compounds inducing apoptosis through the CD95 pathway on SHIP-1-negative leukemic T cells.

Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria.

Autophagy and apoptosis are two important and interconnected stress-response mechanisms. However, the molecular interplay between these two pathways is not fully understood. To study the fate and function of autophagic proteins at the onset of apoptosis, we used a cellular model system in which autophagy precedes apoptosis. IL-3 depletion of Ba/F3 cells caused caspase (casp)-mediated cleavage of Beclin-1 and PI3KC3, two crucial components of the autophagy-inducing complex. We identified two casp cleavage sites in Beclin-1, TDVD(133) and DQLD(149), cleavage at which yields fragments lacking the autophagy-inducing capacity. Noteworthy, the C-terminal fragment, Beclin-1-C, localized predominantly at the mitochondria and sensitized the cells to apoptosis. Moreover, on isolated mitochondria, recombinant Beclin-1-C was able to induce the release of proapoptotic factors. These findings point to a mechanism by which casp-dependent generation of Beclin-1-C creates an amplifying loop enhancing apoptosis upon growth factor withdrawal.

Deregulation of cell-death pathways as the cornerstone of skin diseases.

Deregulation of cell-death pathways plays a key role in the pathogenesis of various skin diseases. The different types of cell death are mainly defined by morphological criteria, and include apoptosis, autophagic cell death, and necrosis. The process of apoptosis is well characterized at the molecular level and involves the activation of two main pathways, the intrinsic and extrinsic pathways, converging into the execution of apoptosis by intracellular cysteine proteases, called caspases. The relevance and implication of these apoptotic pathways in the pathophysiology of skin diseases, such as toxic epidermal necrolysis, graft-versus-host disease and skin cancer, has been extensively studied. The role of autophagic cell death in progression of skin tumours and response to cytotoxic drugs is only beginning to be elucidated.

Molecular effectors and modulators of hypericin-mediated cell death in bladder cancer cells.

Photodynamic therapy (PDT) is an anticancer approach utilizing a light-absorbing molecule and visible light irradiation to generate, in the presence of O(2), cytotoxic reactive oxygen species, which cause tumor ablation. Given that the photosensitizer hypericin is under consideration for PDT treatment of bladder cancer we used oligonucleotide microarrays in the T24 bladder cancer cell line to identify differentially expressed genes with therapeutic potential. This study reveals that the expression of several genes involved in various metabolic processes, stress-induced cell death, autophagy, proliferation, inflammation and carcinogenesis is strongly affected by PDT and pinpoints the coordinated induction of a cluster of genes involved in the unfolded protein response pathway after endoplasmic reticulum stress and in antioxidant response. Analysis of PDT-treated cells after p38(MAPK) inhibition or silencing unraveled that the induction of an important subset of differentially expressed genes regulating growth and invasion, as well as adaptive mechanisms against oxidative stress, is governed by this stress-activated kinase. Moreover, p38(MAPK) inhibition blocked autonomous regrowth and migration of cancer cells escaping PDT-induced cell death. This analysis identifies new molecular effectors of the cancer cell response to PDT opening attractive avenues to improve the therapeutic efficacy of hypericin-based PDT of bladder cancer.