SUMMARY The activation of Mixed Lineage Kinase-Like (MLKL) by Receptor Interacting Protein Kinase-3 (RIPK3) results in plasma membrane (PM) disruption and a form of regulated necrosis, called necroptosis. Here we show that during necroptosis, MLKL-dependent calcium (Ca++) influx and phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane preceded loss of PM integrity. Activation of MLKL results in the generation of broken, PM “bubbles” with exposed PS that are released from the surface of the otherwise intact cell. The ESCRT-III machinery is required for formation of these bubbles, and acts to sustain survival of the cell when MLKL activation is limited or reversed. Under conditions of necroptotic cell death, ESCRT-III controls the duration of plasma membrane integrity. As a consequence of the action of ESCRT-III, cells undergoing necroptosis can express chemokines and other regulatory molecules, and promote antigenic cross-priming of CD8+ T cells.

BackgroundTumor-associated macrophages (TAMs) are alternatively activated cells induced by interleukin-4 (IL-4)-releasing CD4+ T cells. TAMs promote breast cancer invasion and metastasis; however, the mechanisms underlying these interactions between macrophages and tumor cells that lead to cancer metastasis remain elusive. Previous studies have found microRNAs (miRNAs) circulating in the peripheral blood and have identified microvesicles, or exosomes, as mediators of cell-cell communication. Therefore, one alternative mechanism for the promotion of breast cancer cell invasion by TAMs may be through macrophage-secreted exosomes, which would deliver invasion-potentiating miRNAs to breast cancer cells.ResultsWe utilized a co-culture system with IL-4-activated macrophages and breast cancer cells to verify that miRNAs are transported from macrophages to breast cancer cells. The shuttling of fluorescently-labeled exogenous miRNAs from IL-4-activated macrophages to co-cultivated breast cancer cells without direct cell-cell contact was observed. miR-223, a miRNA specific for IL-4-activated macrophages, was detected within the exosomes released by macrophages and was significantly elevated in the co-cultivated SKBR3 and MDA-MB-231 cells. The invasiveness of the co-cultivated breast cancer cells decreased when the IL-4-activated macrophages were treated with a miR-223 antisense oligonucleotide (ASO) that would inhibit miR-223 expression. Furthermore, results from a functional assay revealed that miR-223 promoted the invasion of breast cancer cells via the Mef2c-β-catenin pathway.ConclusionsWe conclude that macrophages regulate the invasiveness of breast cancer cells through exosome-mediated delivery of oncogenic miRNAs. Our data provide insight into the mechanisms underlying the metastasis-promoting interactions between macrophages and breast cancer cells.

Defects in dying cell clearance are postulated to underlie the pathogenesis of systemic lupus erythematosus (SLE)1. Mice lacking molecules associated with dying cell clearance develop SLE-like disease2, and phagocytes from SLE patients often display defective clearance and increased inflammatory cytokine production when exposed to dying cells in vitro. Previously, we3–6 and others7 described a form of noncanonical autophagy called “LC3-associated phagocytosis” (LAP), wherein phagosomes containing engulfed particles, including dying cells3,4,7, recruit elements of the autophagy pathway to facilitate phagosome maturation and digestion of cargo. Genome-wide association studies have identified polymorphisms in atg58 and possibly atg79, involved in both canonical autophagy and LAP3–7, as predisposition markers for SLE. Here, we describe the consequences of defective LAP in vivo. Mice lacking any of several components of the LAP pathway display elevated serum inflammatory cytokines, autoantibodies, glomerular immune complex deposition, and evidence of kidney damage. Dying cells, injected into LAP-deficient animals, are engulfed but not efficiently degraded, and trigger acute elevation of pro-inflammatory cytokines but not the anti-inflammatory interleukin (IL)-10. Repeated injection of dying cells into LAP-deficient, but not LAP-sufficient animals accelerated SLE-like disease, including increased serum levels of autoantibodies. In contrast, animals deficient for genes required for canonical autophagy but not LAP do not display defective dead cell clearance, inflammatory cytokine production, or SLE-like disease, and like wild-type animals, produce IL-10 in response to dying cells. Therefore, defects in LAP, rather than canonical autophagy, can cause SLE-like phenomena, and may contribute to the pathogenesis of SLE.

Purpose: We aim to examine miR-21 expression in tongue squamous cell carcinomas (TSCC) and correlate it with patient clinical status, and to investigate its contribution to TSCC cell growth, apoptosis, and tumorigenesis. Experimental Design: MicroRNA profiling was done in 10 cases of TSCC with microarray. MiR-21 overexpression was quantitated with quantitative reverse transcription-PCR in 103 patients, and correlated to the pathoclinical status of the patients. Immunohistochemistry was used to examine the expression of TPM1 and PTEN, and terminal deoxynucleotidyl transferase-mediated dUTP labeling to evaluate apoptosis. Moreover, miR-21 antisense oligonucleotide (ASO) was transfected in SCC-15 and CAL27 cell lines, and tumor cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adherent colony formation, and soft agar assay, whereas apoptosis was determined by Annexin V assay, cytochrome c release, and caspase 3 assay. Tumorigenesis was evaluated by xenografting SCC-15 cells in nude mice. Results: MiR-21 is overexpressed in TSCC relative to adjacent normal tissues. The level of miR-21 is reversely correlated with TPM1 and PTEN expression and apoptosis of cancer cells. Multivariate analysis showed that miR-21 expression is an independent prognostic factor indicating poor survival. Inhibiting miR-21 with ASO in TSCC cell lines reduces survival and anchorage-independent growth, and induces apoptosis in TSCC cell lines. Simultaneous silencing of TPM1 with siRNA only partially recapitulates the effect of miR-21 ASO. Furthermore, repeated injection of miR-21 ASO suppresses tumor formation in nude mice by reducing cell proliferation and inducing apoptosis. Conclusions: miR-21 is an independent prognostic indicator for TSCC, and may play a role in TSCC development by inhibiting cancer cell apoptosis partly via TPM1 silencing.