Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.

When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here, we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In Escherichia coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating reactive oxygen species (ROS) that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear Listeria monocytogenes. Thus killer cells play an unexpected role in bacterial defense.

Hypoxia as a potential inducer of immune tolerance, tumor plasticity and a driver of tumor mutational burden: Impact on cancer immunotherapy.

In cancer patients, immune cells are often functionally compromised due to the immunosuppressive features of the tumor microenvironment (TME) which contribute to the failures in cancer therapies. Clinical and experimental evidence indicates that developing tumors adapt to the immunological environment and create a local microenvironment that impairs immune function by inducing immune tolerance and invasion. In this context, microenvironmental hypoxia, which is an established hallmark of solid tumors, significantly contributes to tumor aggressiveness and therapy resistance through the induction of tumor plasticity/heterogeneity and, more importantly, through the differentiation and expansion of immune-suppressive stromal cells. We and others have provided evidence indicating that hypoxia also drives genomic instability in cancer cells and interferes with DNA damage response and repair suggesting that hypoxia could be a potential driver of tumor mutational burden. Here, we reviewed the current knowledge on how hypoxic stress in the TME impacts tumor angiogenesis, heterogeneity, plasticity, and immune resistance, with a special interest in tumor immunogenicity and hypoxia targeting. An integrated understanding of the complexity of the effect of hypoxia on the immune and microenvironmental components could lead to the identification of better adapted and more effective combinational strategies in cancer immunotherapy. Clearly, the discovery and validation of therapeutic targets derived from the hypoxic tumor microenvironment is of major importance and the identification of critical hypoxia-associated pathways could generate targets that are undeniably attractive for combined cancer immunotherapy approaches.

Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.

How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

Role of Hypoxic Stress in Regulating Tumor Immunogenicity, Resistance and Plasticity.

Hypoxia, or gradients of hypoxia, occurs in most growing solid tumors and may result in pleotropic effects contributing significantly to tumor aggressiveness and therapy resistance. Indeed, the generated hypoxic stress has a strong impact on tumor cell biology. For example, it may contribute to increasing tumor heterogeneity, help cells gain new functional properties and/or select certain cell subpopulations, facilitating the emergence of therapeutic resistant cancer clones, including cancer stem cells coincident with tumor relapse and progression. It controls tumor immunogenicity, immune plasticity, and promotes the differentiation and expansion of immune-suppressive stromal cells. In this context, manipulation of the hypoxic microenvironment may be considered for preventing or reverting the malignant transformation. Here, we review the current knowledge on how hypoxic stress in tumor microenvironments impacts on tumor heterogeneity, plasticity and resistance, with a special interest in the impact on immune resistance and tumor immunogenicity.

Granzyme B-activated p53 interacts with Bcl-2 to promote cytotoxic lymphocyte-mediated apoptosis.

Granzyme B (GzmB) plays a major role in CTLs and NK cell-mediated elimination of virus-infected cells and tumors. Human GzmB preferentially induces target cell apoptosis by cleaving the proapoptotic Bcl-2 family member Bid, which, together with Bax, induces mitochondrial outer membrane permeabilization. We previously showed that GzmB also induces a rapid accumulation of the tumor-suppressor protein p53 within target cells, which seems to be involved in GzmB-induced apoptosis. In this article, we show that GzmB-activated p53 accumulates on target cell mitochondria and interacts with Bcl-2. This interaction prevents Bcl-2 inhibitory effect on both Bax and GzmB-truncated Bid, and promotes GzmB-induced mitochondrial outer membrane permeabilization. Consequently, blocking p53-Bcl-2 interaction decreases GzmB-induced Bax activation, cytochrome c release from mitochondria, and subsequent effector caspases activation leading to a decreased sensitivity of target cells to both GzmB and CTL/NK-mediated cell death. Together, our results define p53 as a new important player in the GzmB apoptotic signaling pathway and in CTL/NK-induced apoptosis.

Mechanisms of Cytotoxic Lymphocyte-Mediated Apoptosis and Relationship with the Tumor Suppressor p53.

Cytotoxic T lymphocytes and natural killer cells are key effector cells in the immune response against intracellular infection and transformed cells. These killer cells induce multiple programs of cell death to achieve their function of eliminating their targets. In this review, we summarize our current understanding of the signaling pathways involved in target cells apoptosis triggered by the cytotoxic effector cells. We also discuss the role of an important player in the field of apoptosis, the well-known p53 tumor suppressor, in the modulation of cytotoxic lymphocyte-mediated cell death.

Perforin: a key pore-forming protein for immune control of viruses and cancer.

Perforin (PFN) is the key pore-forming molecule in the cytotoxic granules of immune killer cells. Expressed only in killer cells, PFN is the rate-limiting molecule for cytotoxic function, delivering the death-inducing granule serine proteases (granzymes) into target cells marked for immune elimination. In this chapter we describe our current understanding of how PFN accomplishes this task. We discuss where PFN is expressed and how its expression is regulated, the biogenesis and storage of PFN in killer cells and how they are protected from potential damage, how it is released, how it delivers Granzymes into target cells and the consequences of PFN deficiency.

Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling.

A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ~90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a-regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division.

Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis.

Cytotoxic T lymphocytes and natural killer cells destroy target cells via the polarized exocytosis of lytic effector proteins, perforin and granzymes, into the immunologic synapse. How these molecules enter target cells is not fully understood. It is debated whether granzymes enter via perforin pores formed at the plasma membrane or whether perforin and granzymes are first endocytosed and granzymes are then released from endosomes into the cytoplasm. We previously showed that perforin disruption of the plasma membrane induces a transient Ca(2+) flux into the target cell that triggers a wounded membrane repair response in which lysosomes and endosomes donate their membranes to reseal the damaged membrane. Here we show that perforin activates clathrin- and dynamin-dependent endocytosis, which removes perforin and granzymes from the plasma membrane to early endosomes, preserving outer membrane integrity. Inhibiting clathrin- or dynamin-dependent endocytosis shifts death by perforin and granzyme B from apoptosis to necrosis. Thus by activating endocytosis to preserve membrane integrity, perforin facilitates granzyme uptake and avoids the proinflammatory necrotic death of a membrane-damaged cell.

Modulation of the antitumor immune response by cancer-associated fibroblasts: mechanisms and targeting strategies to hamper their immunosuppressive functions.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous players that shape the tumor microenvironment and influence tumor progression, metastasis formation, and response to conventional therapies. During the past years, some CAFs subsets have also been involved in the modulation of immune cell functions, affecting the efficacy of both innate and adaptive anti-tumor immune responses. Consequently, the implication of these stromal cells in the response to immunotherapeutic strategies raised major concerns. In this review, current knowledge of CAFs origins and heterogeneity in the tumor stroma, as well as their effects on several immune cell populations that explain their immunosuppressive capabilities are summarized. The current development of therapeutic strategies for targeting this population and their implication in the field of cancer immunotherapy is also highlighted.

The Effect of Hypoxia and Hypoxia-Associated Pathways in the Regulation of Antitumor Response: Friends or Foes?

Hypoxia is an environmental stressor that is instigated by low oxygen availability. It fuels the progression of solid tumors by driving tumor plasticity, heterogeneity, stemness and genomic instability. Hypoxia metabolically reprograms the tumor microenvironment (TME), adding insult to injury to the acidic, nutrient deprived and poorly vascularized conditions that act to dampen immune cell function. Through its impact on key cancer hallmarks and by creating a physical barrier conducive to tumor survival, hypoxia modulates tumor cell escape from the mounted immune response. The tumor cell-immune cell crosstalk in the context of a hypoxic TME tips the balance towards a cold and immunosuppressed microenvironment that is resistant to immune checkpoint inhibitors (ICI). Nonetheless, evidence is emerging that could make hypoxia an asset for improving response to ICI. Tackling the tumor immune contexture has taken on an in silico, digitalized approach with an increasing number of studies applying bioinformatics to deconvolute the cellular and non-cellular elements of the TME. Such approaches have additionally been combined with signature-based proxies of hypoxia to further dissect the turbulent hypoxia-immune relationship. In this review we will be highlighting the mechanisms by which hypoxia impacts immune cell functions and how that could translate to predicting response to immunotherapy in an era of machine learning and computational biology.

p53 reactivating small molecule PRIMA­1MET/APR­246 regulates genomic instability in MDA­MB­231 cells.

Pharmacological reactivation of tumor­suppressor protein p53 has acted as a promising strategy for more than 50% of human cancers that carry a non­functional mutant p53 (mutp53). p53 plays a critical role in preserving genomic integrity and DNA fidelity through numerous biological processes, including cell cycle arrest, DNA repair, senescence and apoptosis. By contrast, non­functional mutp53 compromises the aforementioned genome stabilizing mechanisms through gain of function, thereby increasing genomic instability in human cancers. Restoring the functional activity of p53 using both genetic and pharmacological approaches has gained prominence in targeting p53­mutated tumors. Thus, the present study aimed to investigate the reactivation of p53 in DNA repair mechanisms and the maintenance of genomic stability using PRIMA­1MET/APR­246 small molecules, in both MDA­MB­231 and MCF­7 breast cancer cell lines, which carry mutp53 and wild­type p53, respectively. Results of the present study revealed that reactivation of p53 through APR­246 led to an increase in the functional activity of DNA repair. Prolonged treatment of MDA­MB­231 cells with APR­246 in the presence of cisplatin led to a reduction in mutational accumulation, compared with cells treated with cisplatin alone. These findings demonstrated that APR­246 may act as a promising small molecule to control the genomic instability in p53­mutated tumors.

Hypoxia increases melanoma-associated fibroblasts immunosuppressive potential and inhibitory effect on T cell-mediated cytotoxicity.

Cancer-associated fibroblasts (CAFs) and hypoxia are central players in the complex process of tumor cell-stroma interaction and are involved in the alteration of the anti-tumor immune response by impacting both cancer and immune cell populations. However, even if their independent immunomodulatory properties are now well documented, whether the interaction between these two components of the tumor microenvironment can affect CAFs ability to alter the anti-tumor immune response is still poorly defined. In this study, we provide evidence that hypoxia increases melanoma-associated fibroblasts expression and/or secretion of several immunosuppressive factors (including TGF-ß, IL6, IL10, VEGF and PD-L1). Moreover, we demonstrate that hypoxic CAF secretome exerts a more profound effect on T cell-mediated cytotoxicity than its normoxic counterpart. Together, our data suggest that the crosstalk between hypoxia and CAFs is probably an important determinant in the complex immunosuppressive tumor microenvironment.

Selection of tumor­resistant variants following sustained natural killer cell­mediated immune stress.

Resistance of tumor cells to cell­mediated cytotoxicity remains an obstacle to the immunotherapy of cancer and its molecular basis is poorly understood. To investigate the acquisition of tumor resistance to cell­mediated cytotoxicity, resistant variants were selected following long­term natural killer (NK) cell selection pressure. It was observed that these variants were resistant to NK cell­mediated lysis, but were sensitive to autologous cytotoxic T lymphocytes or cytotoxic drugs. This resistance appeared to be dependent, at least partly, on an alteration of target cell recognition by NK effector cells, but did not appear to involve any alterations in the expression of KIR, DNAM1 or NKG2D ligands on resistant cells, nor the induction of protective autophagy. In the present study, in order to gain further insight into the molecular mechanisms underlying the acquired tumor resistance to NK cell­mediated cytotoxicity, a comprehensive analysis of the variant transcriptome was conducted. Comparative analysis identified an expression profile of genes that best distinguished resistant variants from parental sensitive cancer cells, with candidate genes putatively involved in NK cell­mediated lysis resistance, but also in adhesion, migration and invasiveness, including upregulated genes, such as POT1, L1CAM or ECM1, and downregulated genes, such as B7­H6 or UCHL1. Consequently, the selected variants were not only resistant to NK cell­mediated lysis, but also displayed more aggressive properties. The findings of the present study emphasized that the role of NK cells may span far beyond the mere killing of malignant cells, and NK cells may be important effectors during cancer immunoediting.

The Most Common VHL Point Mutation R167Q in Hereditary VHL Disease Interferes with Cell Plasticity Regulation.

Von Hippel-Lindau disease (VHL) is a rare hereditary syndrome due to mutations of the VHL tumor suppressor gene. Patients harboring the R167Q mutation of the VHL gene have a high risk of developing ccRCCs. We asked whether the R167Q mutation with critical aspects of pseudo-hypoxia interferes with tumor plasticity. For this purpose, we used wild-type VHL (WT-VHL) and VHL-R167Q reconstituted cells. We showed that WT-VHL and VHL-R167Q expression had a similar effect on cell morphology and colony formation. However, cells transfected with VHL-R167Q display an intermediate, HIF2-dependent, epithelial-mesenchymal phenotype. Using RNA sequencing, we showed that this mutation upregulates the expression of genes involved in the hypoxia pathway, indicating that such mutation is conferring an enhanced pseudo-hypoxic state. Importantly, this hypoxic state correlates with the induction of genes belonging to epithelial-mesenchymal transition (EMT) and stemness pathways, as revealed by GSEA TCGA analysis. Moreover, among these deregulated genes, we identified nine genes specifically associated with a poor patient survival in the TCGA KIRC dataset. Together, these observations support the hypothesis that a discrete VHL point mutation interferes with tumor plasticity and may impact cell behavior by exacerbating phenotypic switching. A better understanding of the role of this mutation might guide the search for more effective treatments to combat ccRCCs.

Role of Hypoxia-Mediated Autophagy in Tumor Cell Death and Survival.

Programmed cell death or type I apoptosis has been extensively studied and its contribution to the pathogenesis of disease is well established. However, autophagy functions together with apoptosis to determine the overall fate of the cell. The cross talk between this active self-destruction process and apoptosis is quite complex and contradictory as well, but it is unquestionably decisive for cell survival or cell death. Autophagy can promote tumor suppression but also tumor growth by inducing cancer-cell development and proliferation. In this review, we will discuss how autophagy reprograms tumor cells in the context of tumor hypoxic stress. We will illustrate how autophagy acts as both a suppressor and a driver of tumorigenesis through tuning survival in a context dependent manner. We also shed light on the relationship between autophagy and immune response in this complex regulation. A better understanding of the autophagy mechanisms and pathways will undoubtedly ameliorate the design of therapeutics aimed at targeting autophagy for future cancer immunotherapies.

Hypoxia increases mutational load of breast cancer cells through frameshift mutations.

Tumor hypoxia-induced downregulation of DNA repair pathways and enhanced replication stress are potential sources of genomic instability. A plethora of genetic changes such as point mutations, large deletions and duplications, microsatellite and chromosomal instability have been discovered in cells under hypoxic stress. However, the influence of hypoxia on the mutational burden of the genome is not fully understood. Here, we attempted to elucidate the DNA damage response and repair patterns under different types of hypoxic stress. In addition, we examined the pattern of mutations exclusively induced under chronic and intermittent hypoxic conditions in two breast cancer cell lines using exome sequencing. Our data indicated that hypoxic stress resulted in transcriptional downregulation of DNA repair genes which can impact the DNA repair induced during anoxic as well as reoxygenated conditions. In addition, our findings demonstrate that hypoxic conditions increased the mutational burden, characterized by an increase in frameshift insertions and deletions. The somatic mutations were random and non-recurring, as huge variations within the technical duplicates were recognized. Hypoxia also resulted in an increase in the formation of potential neoantigens in both cell lines. More importantly, these data indicate that hypoxic stress mitigates DNA damage repair pathways and causes an increase in the mutational burden of tumor cells, thereby interfering with hypoxic cancer cell immunogenicity.

Tumor resistance to specific lysis: a major hurdle for successful immunotherapy of cancer.

Research over the past decade in tumor immunology has shown that immune reactivity to tumor antigens can decrease tumor growth in experimental models. These observations have been translated into clinical studies involving both passive and active forms of immunotherapy. Immunotherapy, an alternative treatment for cancer, is confronted to a major hurdle: tumor escape of specific lysis. Cancer antigen-specific cytotoxic T lymphocytes (CTL) are the major effectors used in immunotherapy against cancer cells. However, large established tumors are usually not fully controlled by CTL. These effector cells could indeed have a dual activity, which allow cancer cells to escape destruction. In this review, we will focus on the essential role of the p53 tumor suppressor gene in the dynamic regulation of tumor cell death induced by cytotoxic T lymphocytes and the involving of structural changes of cytoskeleton in the acquisition of tumor resistance.

The pharmalogical reactivation of p53 function improves breast tumor cell lysis by granzyme B and NK cells through induction of autophagy.

Cytotoxic T lymphocytes (CTL) and natural killer cells (NK)-mediated elimination of tumor cells is mostly dependent on Granzyme B apoptotic pathway, which is regulated by the wild type (wt) p53 protein. Because TP53 inactivating mutations, frequently found in human tumors, could interfere with Granzyme B-mediated cell death, the use of small molecules developed to reactivate wtp53 function in p53-mutated tumor cells could optimize their lysis by CTL or NK cells. Here, we show that the pharmalogical reactivation of a wt-like p53 function in p53-mutated breast cancer cells using the small molecule CP-31398 increases their sensitivity to NK-mediated lysis. This potentiation is dependent on p53-mediated induction of autophagy via the sestrin-AMPK-mTOR pathway and the ULK axis. This CP31398-induced autophagy sequestrates in autophagosomes several anti-apoptotic proteins, including Bcl-XL and XIAP, facilitating Granzyme B-mediated mitochondrial outer membrane permeabilization, caspase-3 activation and Granzyme B- or NK cell-induced apoptosis. Together, our results define a new way to increase cytotoxic lymphocyte-mediated lysis of p53-mutated breast cancer cell, through a p53-dependent autophagy induction, with potential applications in combined immunotherapeutic approaches.