Lena Seifert

The necrosome promotes pancreatic oncogenesis via CXCL1 and Mincle-induced immune suppression

Neoplastic pancreatic epithelial cells are believed to die through caspase 8-dependent apoptotic cell death, and chemotherapy is thought to promote tumour apoptosis. Conversely, cancer cells often disrupt apoptosis to survive. Another type of programmed cell death is necroptosis (programmed necrosis), but its role in pancreatic ductal adenocarcinoma (PDA) is unclear. There are many potential inducers of necroptosis in PDA, including ligation of tumour necrosis factor receptor 1 (TNFR1), CD95, TNF-related apoptosis-inducing ligand (TRAIL) receptors, Toll-like receptors, reactive oxygen species, and chemotherapeutic drugs. Here we report that the principal components of the necrosome, receptor-interacting protein (RIP)1 and RIP3, are highly expressed in PDA and are further upregulated by the chemotherapy drug gemcitabine. Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype. By contrast, in vivo deletion of RIP3 or inhibition of RIP1 protected against oncogenic progression in mice and was associated with the development of a highly immunogenic myeloid and T cell infiltrate. The immune-suppressive tumour microenvironment associated with intact RIP1/RIP3 signalling depended in part on necroptosis-induced expression of the chemokine attractant CXCL1, and CXCL1 blockade protected against PDA. Moreover, cytoplasmic SAP130 (a subunit of the histone deacetylase complex) was expressed in PDA in a RIP1/RIP3-dependent manner, and Mincle—its cognate receptor—was upregulated in tumour-infiltrating myeloid cells. Ligation of Mincle by SAP130 promoted oncogenesis, whereas deletion of Mincle protected against oncogenesis and phenocopied the immunogenic reprogramming of the tumour microenvironment that was induced by RIP3 deletion. Cellular depletion suggested that whereas inhibitory macrophages promote tumorigenesis in PDA, they lose their immune-suppressive effects when RIP3 or Mincle is deleted. Accordingly, T cells, which are not protective against PDA progression in mice with intact RIP3 or Mincle signalling, are reprogrammed into indispensable mediators of anti-tumour immunity in the absence of RIP3 or Mincle. Our work describes parallel networks of necroptosis-induced CXCL1 and Mincle signalling that promote macrophage-induced adaptive immune suppression and thereby enable PDA progression.

Dectin-1 Regulates Hepatic Fibrosis and Hepatocarcinogenesis by Suppressing TLR4 Signaling Pathways

Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1(-/-) mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS)-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF) expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis.

Radiation Therapy Induces Macrophages to Suppress T-Cell Responses Against Pancreatic Tumors in Mice.

BACKGROUND & AIMS The role of radiation therapy in the treatment of patients with pancreatic ductal adenocarcinoma (PDA) is controversial. Randomized controlled trials investigating the efficacy of radiation therapy in patients with locally advanced unresectable PDA have reported mixed results, with effects ranging from modest benefit to worse outcomes compared with control therapies. We investigated whether radiation causes inflammatory cells to acquire an immune-suppressive phenotype that limits the therapeutic effects of radiation on invasive PDAs and accelerates progression of preinvasive foci. METHODS We investigated the effects of radiation therapy in p48(Cre);LSL-Kras(G12D) (KC) and p48(Cre);LSLKras(G12D);LSL-Trp53(R172H) (KPC) mice, as well as in C57BL/6 mice with orthotopic tumors grown from FC1242 cells derived from KPC mice. Some mice were given neutralizing antibodies against macrophage colony-stimulating factor 1 (CSF1 or MCSF) or F4/80. Pancreata were exposed to doses of radiation ranging from 2 to 12 Gy and analyzed by flow cytometry. RESULTS Pancreata of KC mice exposed to radiation had a higher frequency of advanced pancreatic intraepithelial lesions and more foci of invasive cancer than pancreata of unexposed mice (controls); radiation reduced survival time by more than 6 months. A greater proportion of macrophages from radiation treated invasive and preinvasive pancreatic tumors had an immune-suppressive, M2-like phenotype compared with control mice. Pancreata from mice exposed to radiation had fewer CD8(+) T cells than controls, and greater numbers of CD4(+) T cells of T-helper 2 and T-regulatory cell phenotypes. Adoptive transfer of T cells from irradiated PDA to tumors of control mice accelerated tumor growth. Radiation induced production of MCSF by PDA cells. A neutralizing antibody against MCSF prevented radiation from altering the phenotype of macrophages in tumors, increasing the anti-tumor T-cell response and slowing tumor growth. CONCLUSIONS Radiation treatment causes macrophages murine PDA to acquire an immune-suppressive phenotype and disabled T-cell-mediated anti-tumor responses. MCSF blockade negates this effect, allowing radiation to have increased efficacy in slowing tumor growth.

Mincle suppresses Toll-like receptor 4 activation

Regulation of Toll‐like receptor responses is critical for limiting tissue injury and autoimmunity in both sepsis and sterile inflammation. We found that Mincle, a C‐type lectin receptor, regulates proinflammatory Toll‐like receptor 4 signaling. Specifically, Mincle ligation diminishes Toll‐like receptor 4–mediated inflammation, whereas Mincle deletion or knockdown results in marked hyperresponsiveness to lipopolysaccharide in vitro, as well as overwhelming lipopolysaccharide‐mediated inflammation in vivo. Mechanistically, Mincle deletion does not up‐regulate Toll‐like receptor 4 expression or reduce interleukin 10 production after Toll‐like receptor 4 ligation; however, Mincle deletion decreases production of the p38 mitogen‐activated protein kinase‐dependent inhibitory intermediate suppressor of cytokine signaling 1, A20, and ABIN3 and increases expression of the Toll‐like receptor 4 coreceptor CD14. Blockade of CD14 mitigates the increased sensitivity of Mincle−/− leukocytes to Toll‐like receptor 4 ligation. Collectively, we describe a major role for Mincle in suppressing Toll‐like receptor 4 responses and implicate its importance in nonmycobacterial models of inflammation.

Mincle Signaling Promotes Con A Hepatitis

Con A hepatitis is regarded as a T cell–mediated model of acute liver injury. Mincle is a C-type lectin receptor that is critical in the immune response to mycobacteria and fungi but does not have a well-defined role in preclinical models of non-pathogen–mediated inflammation. Because Mincle can ligate the cell death ligand SAP130, we postulated that Mincle signaling drives intrahepatic inflammation and liver injury in Con A hepatitis. Acute liver injury was assessed in the murine Con A hepatitis model using C57BL/6, Mincle−/−, and Dectin-1−/− mice. The role of C/EBPβ and hypoxia-inducible factor-1α (HIF-1α) signaling was assessed using selective inhibitors. We found that Mincle was highly expressed in hepatic innate inflammatory cells and endothelial cells in both mice and humans. Furthermore, sterile Mincle ligands and Mincle signaling intermediates were increased in the murine liver in Con A hepatitis. Most significantly, Mincle deletion or blockade protected against Con A hepatitis, whereas Mincle ligation exacerbated disease. Bone marrow chimeric and adoptive transfer experiments suggested that Mincle signaling in infiltrating myeloid cells dictates disease phenotype. Conversely, signaling via other C-type lectin receptors did not alter disease course. Mechanistically, we found that Mincle blockade decreased the NF-κβ–related signaling intermediates C/EBPβ and HIF-1α, both of which are necessary in macrophage-mediated inflammatory responses. Accordingly, Mincle deletion lowered production of nitrites in Con A hepatitis and inhibition of both C/EBPβ and HIF-1α reduced the severity of liver disease. Our work implicates a novel innate immune driver of Con A hepatitis and, more broadly, suggests a potential role for Mincle in diseases governed by sterile inflammation.

Molecular Pathways: The Necrosome - A Target for Cancer Therapy

Necroptosis is a caspase-8–independent cell death that requires coactivation of receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3) kinases. The necrosome is a complex consisting of RIP1, RIP3, and Fas-associated protein with death domain leading to activation of the pseudokinase mixed lineage kinase like followed by a rapid plasma membrane rupture and inflammatory response through the release of damage-associated molecular patterns and cytokines. The necrosome has been shown to be relevant in multiple tumor types, including pancreatic adenocarcinoma, melanoma, and several hematologic malignancies. Preclinical data suggest that targeting this complex can have differential impact on tumor progression and that the effect of necroptosis on oncogenesis is cell-type and context dependent. The emerging data suggest that targeting the necrosome may lead to immunogenic reprogramming in the tumor microenvironment in multiple tumors and that combining therapies targeting the necrosome with either conventional chemotherapy or immunotherapy may have beneficial effects. Thus, understanding the interplay of necroptotic cell death, transformed cells, and the immune system may enable the development of novel therapeutic approaches. Clin Cancer Res; 23(5); 1132–6. ©2016 AACR.

Necroptotic cell death – An unexpected driver of pancreatic oncogenesis

Cell growth and death are tightly regulated mechanisms that, in concert, facilitate tissue formation and integrity. Disruption in either pathway is a well-described hallmark that cancer cells exploit to survive noxious stimuli such as DNA damage, Reactive Oxygen Species (ROS), Fas-Fas-Ligand interaction or loss of cell-cell adhesion. Thus, many chemotherapeutics aim to disrupt survival and drive cells down a Caspase 8 mediated apoptotic pathway. Necroptosis is a more recently described mechanism of cell death that denotes coordinated cellular necrosis, leading to pore formation in the cell membrane and the release of cellular components. Evolutionarily, necroptosis can be considered a second line of defense against viruses, which enter cells and inhibit cellular demise via Caspase 8mediated apoptosis. In the presence of dysfunctional Caspase 8, receptor interacting kinase (RIP) 1 and 3 associate and assemble the necrosome, which in turn recruits mixed lineage kinase domain-like protein (MLKL) leading to necroptosis and the release of danger signals. Our recent work shows that the components of the necrosome are highly expressed in human and murine pancreatic ductal adenocarcinoma (PDA). Expression was further inducible by the chemotherapeutic agent Gemcitabine. To investigate the role of necroptosis in PDA progression, we deleted RIP3 in pancreatic cancer cells. In accordance with our hypothesis, RIP3 deletion resulted in a more aggressive oncogenic phenotype in vitro. However, contrary to these observations RIP3 deletion in vivo resulted in marked protection against PDA including a significant survival benefit. RIP1 inhibition was similarly protective. We found that targeting the necrosome led to a more immunogenic inflammatory infiltrate as evidenced by an increase in tumor infiltrating CD8C T cells and Th1-polarized CD4C T cells, B cells, as well as a reduction in myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs). Furthermore, TAMs exhibited a shift toward an M1-like immunogenic phenotype. Interestingly, pro-tumorigenic effects of necroptosis seemed to be specific to the pancreatic tumor microenvironment (TME), as neither subcutaneously implanted PDA nor B16 melanoma cells exhibited altered tumor growth in the context of RIP3 deletion. We discovered that CXCL1, a potent chemoattractant for myeloid cells, was highly expressed in a RIP3 dependent manner. We postulated that CXCL1 could mediate the protumorigenic immune suppression associated with RIP3 signaling by mobilizing myeloid cells. CXCL1 blockade protected against PDA, reduced myeloid cell infiltration, and increased peri-tumoral T cells. However, CXCL1 overexpression alone could not account for the entire immunosuppressive phenotype associated with intact necroptosis signaling in PDA. Since necroptosis produces danger signals to the surrounding environment, we postulated that the release of damage associated molecular patterns (DAMPs) by necroptotic cells within the TME triggers immune-suppressive inflammation. Mincle is a C type lectin receptor involved in fungal immunity and has recently been implicated in promoting sterile inflammation by ligating SAP130, a subunit of the histone deacetylase complex, which is released by necrotic cells. We found that Mincle and its associated signaling intermediates were highly expressed on antigen presenting cells (APCs) and Mincle coassociated with SAP130 in the PDA TME. Further emphasizing the role of necroptosis, SAP130 levels and Mincle signaling were reduced in RIP3¡/¡ tumors. Moreover, Mincle deletion was protective against PDA, extended survival, and directly phenocopied the immunogenic infiltrate associated with RIP3 deletion. In contrast, Mincle ligation led to tumor progression, promoted MDSC infiltration and M2-polarization of TAMs and induced adaptive immune suppression. Cellular depletion experiments revealed that TAMs promote tumorigenesis in PDA; however, they lose their immune-suppressive effects when RIP3 or Mincle are deleted. As such, T cells, which do not protect against PDA progression in hosts with intact RIP3 or Mincle signaling, are reprogrammed into potent mediators of anti-tumor immunity in the absence of RIP3 or Mincle. Our work describes parallel axes of necroptosis-induced CXCL1 and Mincle signaling that promote macrophage-induced adaptive immune suppression and thereby enable pancreatic cancer progression (Fig. 1). Each of these axes represents a novel target for experimental therapeutics.