Jerome Thiery

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.

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.

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity

By exploiting NK cell LROs (known as lytic granules) as a model, a new role is defined for Arl8b in regulating motility and exocytosis of lytic granules of NK cells. Not only lytic granules but also the MTOC is unable to polarize toward the immune synapse formed between the NK cell and its target in Arl8b-depleted NK cells.

Opposite effects of estrogen receptors alpha and beta on MCF-7 sensitivity to the cytotoxic action of TNF and p53 activity

We have investigated the effect of estrogen on p53 cellular location and its influence on tumor cell susceptibility to tumor necrosis factor (TNF)-mediated cytotoxic action. For this purpose, we have used the TNF-sensitive human breast adenocarcinoma MCF-7 and its derivative, the TNF-resistant 1001 clone. Our data indicate that although estrogen receptor (ER)α is present in both cell lines, estrogen treatment (1 × 10−8 M) has an influence only on the MCF-7 cells and protects these cells from the TNF cytotoxicity. This protective effect is associated with translocation of p53 from the nucleus to the cytoplasm in p53 wild-type MCF-7 and not in p53-mutated 1001 cells. The translocation of p53 in MCF-7 cells results in a decrease in its transcriptional activity, as revealed by diminished p21WAF1/CIP1 induction and an altered ratio of Bax and Bcl-2 proteins. The estrogen-induced effects are reversed by the selective estrogen inhibitor 182, 780 (1 × 10−6 M). Interestingly, transient transfection of MCF-7 cells with ERβ but not ERα cDNA encoding plasmid results in retention of p53 in the nucleus, a subsequent potentiation of its transcriptional activity, and in an increased MCF-7 sensitivity to TNF. The estrogen effects on p53 location and transcriptional activity may involve the mdm2 protein since both events were reversed following MCF-7 transfection with plasmid encoding the ARF cDNA. These studies suggest that estrogen-induced MCF-7 cell survival in the presence of TNF requires a transcriptionally active p53 and, more importantly, indicate that introduction of ERβ can attenuate the estrogen effects on the p53 protein location, its transcriptional activity and also results in a potentiation of cell sensitivity to TNF-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.

Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins

Killer lymphocytes induce apoptosis by the release of cytotoxic mediators from specialized secretory lysosomes, called cytotoxic granules, into the immunological synapse formed with a cell targeted for elimination. Methods are presented here for isolating CTL and NK cell cytotoxic granules using cell disruption by nitrogen cavitation followed by continuous Percoll density gradient fractionation. Protocols are also given for purifying the key cytolytic molecules (perforin, granzyme A, granzyme B, and granulysin) from isolated cytotoxic granules by fast protein liquid chromatography. Curr. Protoc. Cell Biol. 47:3.37.1‐3.37.29.

Granzyme B-induced Cell Death Involves Induction of p53 Tumor Suppressor Gene and Its Activation in Tumor Target Cells

In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-α-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-α resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications.

Granzymes and cell death.

Granzymes are cell death-inducing serine proteases released from cytotoxic granules of cytotoxic T lymphocytes and natural killer cells during granule exocytosis in response to viral infection or against transformed cells marked for elimination. A critical cofactor for the granule exocytosis pathway is perforin, which mediates the entry of granzymes into target cells, where they cleave specific substrates that initiate DNA fragmentation and apoptosis. One of the biggest challenges in studying the biology of granzymes has been the functional redundancy of granzymes in animal models making an in vitro experimental system essential. This chapter discusses methods to study granzyme function in vitro under physiologically relevant experimental conditions.

Melanoma-associated fibroblasts decrease tumor cell susceptibility to NK cell-mediated killing through matrix-metalloproteinases secretion

Cancer-associated fibroblasts (CAFs) play a central role in the complex process of tumor-stroma interaction and promote tumor growth. Emerging evidences also suggest that these fibroblasts are involved in the alteration of the anti-tumor immune response by impacting several immune cell populations, especially through their secretion of pro-inflammatory and immunosuppressive factors in the tumor microenvironment. However, the underlying immuno-modulating mechanisms triggered by these fibroblasts are still only partially defined. In this study, we provide evidence that melanoma-associated fibroblasts decrease the susceptibility of melanoma tumor cells to NK-mediated lysis through the secretion of active matrix metalloproteinases. This secretion reduces the expression of the two NKG2D ligands, MICA/B, at the surface of tumor cells and consequently decreases the NKG2D-dependent cytotoxic activity of NK cells against melanoma tumor cells. Together, our data demonstrate that the modification of tumor cell susceptibility to killer cells is an important determinant of the anti-tumor immune response alteration triggered by CAFs.