PolyI:C–Induced, TLR3/RIP3-Dependent Necroptosis Backs Up Immune Effector–Mediated Tumor Elimination In Vivo

Abstract

Double-stranded RNA directly acts on fibroblast and myeloid lineages to induce necroptosis as in TNFa. Here, we investigated whether this type of cell death occurred in cancer cells in response to polyinosinic-polycytidylic acid (polyI:C) and the pan-caspase inhibitor z-Val-Ala-Asp fluromethyl ketone (zVAD). We found that the colon cancer cell line CT26 is highly susceptible to necroptosis, as revealed by staining with annexin V/propidium iodide. CT26 cells possess RNA sensors, TLR3 and MDA5, which are upregulated by interferon (IFN)-inducing pathways and linked to receptor-interacting protein kinase (RIP) 1/3 activation via TICAM-1 or MAVS adaptor, respectively. Although exogenously added polyI:C alone marginally induced necroptosis in CT26 cells, a combined regimen of polyI:C and zVAD induced approximately 50% CT26 necroptosis in vitro without secondary effects of TNFa or type I IFNs. CT26 necroptosis depended on the TLR3-TICAM-1-RIP3 axis in the tumor cells to produce reactive oxygen species, but not on MDA5, MAVS, or the caspases/inflammasome activation. However, the RNA-derived necroptosis was barely reproduced in vivo in a CT26 tumor-implanted Balb/c mouse model with administration of polyI:C þ zVAD. Significant shrinkage of CT26 tumors was revealed only when polyI:C (100 mg) was injected intraperitoneally and zVAD (1 mg) subcutaneously into tumor-bearing mice that were depleted of cytotoxic T lymphocytes and natural killer cells. The results were confirmed with immune-compromised mice with no lymphocytes. Although necroptosis-induced tumor growth retardation appears mechanistically complicated and dependent on the injection routes of polyI:C and zVAD, anti-caspase reagent directed to tumor cells will make RNA adjuvant immunotherapy more effective by modulating the formation of the tumoricidal microenvironment and dendritic cell-inducing antitumor immune system.

RIPK1 and RIPK3 - emerging targets in cancer?

“…On the one hand, there is little to suggest that oncogenic transformation per se renders cells significantly more sensitive to RIPK1/RIPK3 responses in a cell autonomous manner. On the other hand, data using combination of SMAC mimetics or TLR3 agonist poly(I:C) in combination with pan-caspase inhibitors suggest that such necroptosis-inducing stimuli may be well tolerated and efficacious (66,80). One possibility is that only a very limited degree of RIPK1/RIPK3 cell death may be needed to get the immune system fired up to target the cancer, as some of the positive responses were not observed in immune-deficient animals (35, 80).…”

“…Recent data suggest that necroptosis may be an immunogenic form of cancer cell death, associated with: the release of key immunogenic danger associated m o l e c u l a r p a t t e r n s ( D A M P s ) -AT P a n d H M G B 1 ; upregulation of expression of critical immune-stimulating cytokines, such as Type I interferon pathway components; maturation of dendritic cells exposed to dying/dead necroptotic cells; tumor-associated antigen crosspresentation to CD8 + cytotoxic lymphocytes (CTLs);and generation of long term adaptive anti-cancer immunity in mice exposed to necroptotic cancer cells (35,(78)(79)(80)(81) memory to orthotopic glioma was also recently proposed to be in part mediated by the induction of necroptosis by this virus (82).Further details of necroptosis-associated immune regulation can be found in several comprehensive recent reviews (83-86).…”

Toll-like receptor stimulation in cancer: A pro- and anti-tumor double-edged sword

“…Indeed, poly I:C, a specific agonist of TLR3, was used in vitro to stimulate human head and neck squamous-cell carcinoma cells (Nomi et al, 2010), pharyngeal carcinoma cell lines (Matijevic et al, 2009), human melanoma cell lines (Salaun et al, 2007), human hepatocellular carcinoma (HCC) cells (Yoneda et al, 2008) and colon carcinoma cells (Takemura et al, 2015). Such stimulation led to tumor cell apoptosis by decreased expression of survivin (an inhibitor of apoptosis) as well as downregulation of anti-apoptotic XIAP, FLIP and Bcl-xL (Nomi et al, 2010;Yoneda et al, 2008).…”

“…In colon carcinoma and HCC mouse models, poly I:C injection produced an anti-tumoral effect via the recruitment of NK and CD8 + T cells (Chew et al, 2012;Takemura et al, 2015) and activated CD4 + T cells (Ho et al, 2015). In a murine model of lung cancer, TLR3 stimulation by poly I:C decreased tumor progression by a mechanism implying the conversion of pro-tumoral myeloid cells into M1 macrophages with tumoricidal properties (Shime et al, 2012).…”

“…Gambara et al (2015),Matijevic et al (2009), Nomi et al (2010,Paone et al (2008),Salaun et al (2007),Takemura et al (2015),Xu et al (2013a),Yoneda et al (2008) HCC, colon cancer, lung cancer Cytotoxic immune cells (NK cells, M1, CD8+ T cells) Immunosuppressive immune cellsChew et al (2012), Ho et al (2015), Shime et al (2012), Takemura et al (2015), Xu et al (2013b), Yoneda et al cells (CTL, NK cells, ␥/␦ T cells) Shojaei et al (2009), Zhou et al (2015), Zhu et al (2015) Breast cancer, melanoma Activation of intratumoral pDC Aspord et al (2014), Le Mercier et al al. (2015), Huang et al (2007), Maruyama et al (2015), Shi et al (2014), Tye et al (2012) TLR4 Breast cancer, lung cancer, head and neck cancer, prostate cancer, HCC IL-6 + IL-8 + TGF-b + VEGF + Tumor cell proliferation Tumor cell apoptosis Dapito et al (2012), Gu et al (2015), He et al (2007), Hsiao et al (2015), Jain et al (2015), Kundu et al (2008), Szczepanski et al (2009), Yang et al…”