Breast tumors interfere with endothelial TRAIL at the premetastatic niche to promote cancer cell seeding.

Endothelial cells (ECs) grant access of disseminated cancer cells to distant organs. However, the molecular players regulating the activation of quiescent ECs at the premetastatic niche (PMN) remain elusive. Here, we find that ECs at the PMN coexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate death receptor 5 (DR5). Unexpectedly, endothelial TRAIL interacts intracellularly with DR5 to prevent its signaling and preserve a quiescent vascular phenotype. In absence of endothelial TRAIL, DR5 activation induces EC death and nuclear factor κB/p38-dependent EC stickiness, compromising vascular integrity and promoting myeloid cell infiltration, breast cancer cell adhesion, and metastasis. Consistently, both down-regulation of endothelial TRAIL at the PMN by proangiogenic tumor-secreted factors and the presence of the endogenous TRAIL inhibitors decoy receptor 1 (DcR1) and DcR2 favor metastasis. This study discloses an intracrine mechanism whereby TRAIL blocks DR5 signaling in quiescent endothelia, acting as gatekeeper of the vascular barrier that is corrupted by the tumor during cancer cell dissemination.

Isolation and separation of murine tumor-associated macrophages (TAMs) subpopulations from orthotopic 4T1 breast tumors.

Tumor-associated macrophages (TAMs) are highly heterogenous regarding their intratumoral localization, surface marker expression, and molecular properties. This protocol describes the complete procedure for isolation and digestion of murine breast cancer samples and fluorescence-activated cell sorting (FACS) of TAMs from murine orthotopic 4T1 breast tumors. This includes steps to separate PoEMs (podoplanin-expressing macrophages) and non-PoEMs (podoplanin-negative macrophages). Our FACS separation approach could also be used for other tumor types with TAM infiltration. For complete details on the use and execution of this protocol, please refer to Bieniasz-Krzywiec et al. (2019).

An Apoptotic Caspase Network Safeguards Cell Death Induction in Pyroptotic Macrophages.

Pyroptosis has emerged as a key mechanism by which inflammasomes promote host defense against microbial pathogens and sterile inflammation. Gasdermin D (GSDMD)-mediated cell lysis is a hallmark of pyroptosis, but our understanding of cell death signaling during pyroptosis is fragmented. Here, we show that independently of GSDMD-mediated plasma membrane permeabilization, inflammasome receptors engage caspase-1 and caspase-8, both of which redundantly promote activation of apoptotic executioner caspase-3 and caspase-7 in pyroptotic macrophages. Impaired GSDMD pore formation downstream of caspase-1 and caspase-8 activation suffices to unmask the apoptotic phenotype of pyroptotic macrophages. Combined inactivation of initiator caspase-1 and caspase-8, or executioner caspase-3 and caspase-7, is required to abolish inflammasome-induced DEVDase activity during pyroptosis and in apoptotic Gsdmd-/- cells. Collectively, these results unveil a robust apoptotic caspase network that is activated in parallel to GSDMD-mediated plasma membrane permeabilization and safeguards cell death induction in pyroptotic macrophages.

B55α/PP2A Limits Endothelial Cell Apoptosis During Vascular Remodeling: A Complementary Approach To Disrupt Pathological Vessels?

RATIONALE: How endothelial cells (ECs) migrate and form an immature vascular plexus has been extensively studied. Yet, mechanisms underlying vascular remodeling remain poorly established. A better understanding of these processes may lead to the design of novel therapeutic strategies complementary to current angiogenesis inhibitors. OBJECTIVE: Starting from our previous observations that PP2A (protein phosphatase 2) regulates the HIF (hypoxia-inducible factor)/PHD-2 (prolyl hydroxylase 2)-constituted oxygen machinery, we hypothesized that this axis could play an important role during blood vessel formation, tissue perfusion, and oxygen restoration. METHODS AND RESULTS: We show that the PP2A regulatory subunit B55α is at the crossroad between vessel pruning and vessel maturation. Blood vessels with high B55α counter cell stress conditions and thrive for stabilization and maturation. When B55α is inhibited, ECs cannot cope with cell stress and undergo apoptosis, leading to massive pruning of nascent blood vessels. Mechanistically, we found that the B55α/PP2A complex restrains PHD-2 activity, promoting EC survival in a HIF-dependent manner, and furthermore dephosphorylates p38, altogether protecting ECs against cell stress occurring, for example, during the onset of blood flow. In tumors, EC-specific B55α deficiency induces pruning of immature-like tumor blood vessels resulting in delayed tumor growth and metastasis, without affecting nonpathological vessels. Consistently, systemic administration of a pan-PP2A inhibitor disrupts vascular network formation and tumor progression in vivo without additional effects on B55α-deficient vessels. CONCLUSIONS: Our data underline a unique role of the B55α/PP2A phosphatase complex in vessel remodeling and suggest the use of PP2A-inhibitors as potent antiangiogenic drugs targeting specifically nascent blood vessels with a mode-of-action complementary to VEGF-R (vascular endothelial growth factor receptor)-targeted therapies. Graphical Abstract: A graphical abstract is available for this article.

Podoplanin-Expressing Macrophages Promote Lymphangiogenesis and Lymphoinvasion in Breast Cancer.

Among mammary tumor-infiltrating immune cells, the highest expression of podoplanin (PDPN) is found in a subset of tumor-associated macrophages (TAMs). We hereby demonstrate that PDPN is involved in the attachment of this TAM subset to lymphatic endothelial cells (LECs). Mechanistically, the binding of PDPN to LEC-derived galectin 8 (GAL8) in a glycosylation-dependent manner promotes the activation of pro-migratory integrin ß1. When proximal to lymphatics, PDPN-expressing macrophages (PoEMs) stimulate local matrix remodeling and promote vessel growth and lymphoinvasion. Anti-integrin ß1 blockade, macrophage-specific Pdpn knockout, or GAL8 inhibition impairs TAM adhesion to LECs, restraining lymphangiogenesis and reducing lymphatic cancer spread. In breast cancer patients, association of PoEMs with tumor lymphatic vessels correlates with incidences of lymph node and distant organ metastasis.

DPP8/DPP9 inhibition elicits canonical Nlrp1b inflammasome hallmarks in murine macrophages.

Activating germline mutations in the human inflammasome sensor NLRP1 causes palmoplantar dyskeratosis and susceptibility to Mendelian autoinflammatory diseases. Recent studies have shown that the cytosolic serine dipeptidyl peptidases DPP8 and DPP9 suppress inflammasome activation upstream of NLRP1 and CARD8 in human keratinocytes and peripheral blood mononuclear cells. Moreover, pharmacological inhibition of DPP8/DPP9 protease activity was shown to induce pyroptosis in murine C57BL/6 macrophages without eliciting other inflammasome hallmark responses. Here, we show that DPP8/DPP9 inhibition in macrophages that express a Bacillus anthracis lethal toxin (LeTx)-sensitive Nlrp1b allele triggered significantly accelerated pyroptosis concomitant with caspase-1 maturation, ASC speck assembly, and secretion of mature IL-1ß and IL-18. Genetic ablation of ASC prevented DPP8/DPP9 inhibition-induced caspase-1 maturation and partially hampered pyroptosis and inflammasome-dependent cytokine release, whereas deletion of caspase-1 or gasdermin D triggered apoptosis in the absence of IL-1ß and IL-18 secretion. In conclusion, blockade of DPP8/DPP9 protease activity triggers rapid pyroptosis and canonical inflammasome hallmarks in primary macrophages that express a LeTx-responsive Nlrp1b allele.

Oncogenic p95HER2/611CTF primes human breast epithelial cells for metabolic stress-induced down-regulation of FLIP and activation of TRAIL-R/Caspase-8-dependent apoptosis.

Oncogenic transformation triggers reprogramming of cell metabolism, as part of the tumorigenic process. However, metabolic reprogramming may also increase the sensitivity of transformed cells to microenvironmental stress, at the early stages of tumor development. Herein, we show that transformation of human breast epithelial cells by the p95HER2/611CTF oncogene markedly sensitizes these cells to metabolic stress induced by the simultaneous inhibition of glucose and glutamine metabolism. In p95HER2/611CTF-transformed cells, metabolic stress activates a TNF related apoptosis-inducing ligand (TRAIL)-R and caspase-8-dependent apoptotic process that requires prior down-regulation of cellular FLICE-like inhibitor protein (c-FLIP) levels. Importantly, sustained mTOR activation is involved in FLIP down-regulation and apoptosis induced by metabolic stress. In vivo experiments in immunodeficient mice demonstrate a requirement for caspase-8 in restraining primary tumor growth of xenografts with p95HER2/611CTF-transformed cells. Collectively, these data define a critical role of the extrinsic pathway of apoptosis in the control of tumor initiation by microenvironmental cues.

Pharmacologic or Genetic Targeting of Glutamine Synthetase Skews Macrophages toward an M1-like Phenotype and Inhibits Tumor Metastasis.

Glutamine-synthetase (GS), the glutamine-synthesizing enzyme from glutamate, controls important events, including the release of inflammatory mediators, mammalian target of rapamycin (mTOR) activation, and autophagy. However, its role in macrophages remains elusive. We report that pharmacologic inhibition of GS skews M2-polarized macrophages toward the M1-like phenotype, characterized by reduced intracellular glutamine and increased succinate with enhanced glucose flux through glycolysis, which could be partly related to HIF1α activation. As a result of these metabolic changes and HIF1α accumulation, GS-inhibited macrophages display an increased capacity to induce T cell recruitment, reduced T cell suppressive potential, and an impaired ability to foster endothelial cell branching or cancer cell motility. Genetic deletion of macrophagic GS in tumor-bearing mice promotes tumor vessel pruning, vascular normalization, accumulation of cytotoxic T cells, and metastasis inhibition. These data identify GS activity as mediator of the proangiogenic, immunosuppressive, and pro-metastatic function of M2-like macrophages and highlight the possibility of targeting this enzyme in the treatment of cancer metastasis.

Macrophage Metabolism Controls Tumor Blood Vessel Morphogenesis and Metastasis.

Hypoxic tumor-associated macrophages (TAMs) acquire angiogenic and immunosuppressive properties. Yet it remains unknown if metabolic changes influence these functions. Here, we argue that hypoxic TAMs strongly upregulate the expression of REDD1, a negative regulator of mTOR. REDD1-mediated mTOR inhibition hinders glycolysis in TAMs and curtails their excessive angiogenic response, with consequent formation of abnormal blood vessels. Accordingly, REDD1 deficiency in TAMs leads to the formation of smoothly aligned, pericyte-covered, functional vessels, which prevents vessel leakiness, hypoxia, and metastases. Mechanistically, highly glycolytic REDD1-deficient TAMs outcompete endothelial cells for glucose usage that thwarts vascular hyperactivation and promotes the formation of quiescent vascular junctions. Tuning down glycolysis in REDD1 knockout TAMs re-establishes abnormal angiogenesis and metastases. On this basis, we prove that the anti-tumor effect of mTOR inhibitors is partly countered by the deleterious outcome of these drugs on TAMs. Our data provide a functional link between TAM metabolism and tumor angiogenesis.

PHD1 regulates p53-mediated colorectal cancer chemoresistance.

Overcoming resistance to chemotherapy is a major challenge in colorectal cancer (CRC) treatment, especially since the underlying molecular mechanisms remain unclear. We show that silencing of the prolyl hydroxylase domain protein PHD1, but not PHD2 or PHD3, prevents p53 activation upon chemotherapy in different CRC cell lines, thereby inhibiting DNA repair and favoring cell death. Mechanistically, PHD1 activity reinforces p53 binding to p38α kinase in a hydroxylation-dependent manner. Following p53-p38α interaction and chemotherapeutic damage, p53 can be phosphorylated at serine 15 and thus activated. Active p53 allows nucleotide excision repair by interacting with the DNA helicase XPB, thereby protecting from chemotherapy-induced apoptosis. In accord with this observation, PHD1 knockdown greatly sensitizes CRC to 5-FU in mice. We propose that PHD1 is part of the resistance machinery in CRC, supporting rational drug design of PHD1-specific inhibitors and their use in combination with chemotherapy.

Activated ERBB2/HER2 licenses sensitivity to apoptosis upon endoplasmic reticulum stress through a PERK-dependent pathway.

HER2/Neu/ERBB2 is a receptor tyrosine kinase overexpressed in approximately 20% of human breast tumors. Truncated or mutant isoforms that show increased oncogenicity compared with the wild-type receptor are found in many breast tumors. Here, we report that constitutively active ERBB2 sensitizes human breast epithelial cells to agents that induce endoplasmic reticulum stress, altering the unfolded protein response (UPR) of these cells. Deregulation of the ERK, AKT, and mTOR activities elicited by mutant ERBB2 was involved in mediating this differential UPR response, elevating the response to endoplasmic reticulum stress, and apoptotic cell death. Mechanistic investigations revealed that the increased sensitivity of mutant ERBB2-expressing cells to endoplasmic reticulum stress relied upon a UPR effector signaling involving the PERK-ATF4-CHOP pathway, upregulation of the proapoptotic cell surface receptor TRAIL-R2, and activation of proapoptotic caspase-8. Collectively, our results offer a rationale for the therapeutic exploration of treatments inducing endoplasmic reticulum stress against mutant ERBB2-expressing breast tumor cells.

The long and winding road to cancer treatment: the TRAIL system.

Activation of cell surface death receptors of the tumor necrosis factor (TNF) receptor superfamily by the appropriate ligands represents an attractive therapeutic strategy to induce cell death by apoptosis in cancer cells. However, the toxic effects of TNF-alpha and CD95/Fas ligand (FasL) in normal tissues have significantly hampered the clinical application of these ligands in cancer treatment. TNF-related apoptosis-inducing ligand (TRAIL/APO-2L), another member of the TNF family, has been shown to induce apoptosis selectively in many tumor cell lines. Interestingly, TRAIL treatment also results in significant growth suppression of TRAIL-sensitive human cancer xenografts in mice and nonhuman primates. At the same time, recombinant TRAIL and agonistic TRAIL receptor antibodies show no significant cytotoxicity in these studies. Despite some adverse effects of certain TRAIL preparations, activation of proapoptotic TRAIL receptors represents a promising approach in cancer therapy. Herein we review what is known about proapoptotic TRAIL signaling, the role of intracellular survival pathways in the regulation of resistance to TRAIL and the activation of non-apoptotic signaling by TRAIL. We also discuss the role of the TRAIL system in tumorigenesis and the results of clinical trials with recombinant TRAIL and various TRAIL receptor agonistic antibodies, either as monotherapy or in combination with targeted or conventional chemotherapy.

ER stress sensitizes cells to TRAIL through down-regulation of FLIP and Mcl-1 and PERK-dependent up-regulation of TRAIL-R2.

Despite recent evidences suggesting that agents inducing endoplasmic reticulum (ER) stress could be exploited as potential antitumor drugs in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the mechanisms of this anticancer action are not fully understood. Moreover, the effects of ER stress and TRAIL in nontransformed cells remain to be investigated. In this study we report that ER stress-inducing agents sensitizes both transformed and nontransformed cells to TRAIL-induced apoptosis. In addition, glucose-regulated protein of 78 kDa (GRP78) knockdown by RNA interference induces ER stress and facilitates apoptosis by TRAIL. We demonstrate that TRAIL death-inducing signaling complex (DISC) formation and early signaling are enhanced in ER stressed cells. ER stress alters the cellular levels of different apoptosis-related proteins including a decline in the levels of FLIP and Mcl-1 and the up-regulation of TRAIL-R2. Up-regulation of TRAIL-R2 following ER stress is dependent on the expression of PKR-like ER kinase (PERK) and independent of CAAT/enhancer binding protein homologous protein (CHOP) and Ire1α. Silencing of TRAIL-R2 expression by siRNA blocks the ER stress-mediated sensitization to TRAIL-induced apoptosis. Furthermore, simultaneous silencing of cFLIP and Mcl-1 expression by RNA interference results in a marked sensitization to TRAIL-induced apoptosis. Finally, in FLIP-overexpressing cells ER stress-induced sensitization to TRAIL-activated apoptosis is markedly reduced. In summary, our data reveal a pleiotropic mechanism involving both apoptotic and anti-apoptotic proteins for the sensitizing effect of ER stress on the regulation of TRAIL receptor-mediated apoptosis in both transformed and nontransformed cells.

Autophagy inhibition sensitizes multiple myeloma cells to 17-dimethylaminoethylamino-17-demethoxygeldanamycin-induced apoptosis.

The Hsp90 inhibitor 17DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) is currently undergoing clinical trials as an antitumor drug. We show here that treatment of human multiple myeloma (MM) cells with 17DMAG induces mTOR inhibition and microtubule-associated protein light chain 3 (LC3) conversion (LC3-I to LC3-II), an indicator of autophagy. Interestingly, 17DMAG synergistically induces apoptosis through a mitochondria-operated pathway in the presence of the autophagy inhibitor 3-methyladenine (3-MA). Inhibition of autophagy by 3-MA facilitated caspase activation, cytochrome c release from mitochondria and poly (ADP-ribose) polymerase (PARP) cleavage in myeloma cells treated with 17DMAG. The potential use of Hsp90 and autophagy inhibitors combinations as a therapeutic tool in MM is further discussed in our work.