Autophagy Links Pattern Recognition Receptors to Tumor Cell Apoptosis
Authors: Jörg Vollmer
Journal: Molecular Therapy (2009)

Pathogen nucleic acids are detected by the infected body's immune cells via specific innate immune receptors, resulting in reactions such as secretion of T helper type 1 (Th1)-like and proinflammatory cytokines. Two studies have now reported the induction of cell death in melanoma cells, but not healthy skin cells, via stimulation of the cytoplasmic receptor MDA5 by the synthetic double-stranded RNA (dsRNA) agonist poly I:C, and one of the studies elucidated the mechanism for this response. Polyethyleneimine (PEI)-formulated poly I:C engages an intracellular process called autophagy via MDA5 to result in the induction of prodeath programs that ultimately lead to apoptosis of melanoma cells. Together with previous work, these reports demonstrate that immune detection of foreign nucleic acids involves diverse signaling pathways and thus yields different cellular outcomes that may overcome the inherent resistance of cancer cells to current cancer treatments. Pattern recognition receptors (PRRs) are specialized for sensing pathogen-associated molecular patterns (PAMPs) such as viral or bacterial nucleic acids to induce specific innate immune responses. These include the Toll-like receptors (TLRs) 3, 7, 8, and 9, which are expressed mainly in intracellular endolysosomal compartments of immune cells, where they respond to single- (TLR7, 8, 9) or double-stranded (TLR3) pathogen DNA or RNA.1Vollmer J Krieg A Mechanisms and therapeutic applications of immune modulatory oligodeoxynucleotide and oligoribonucleotide ligands for Toll-like receptors. Crooke, ST (ed). Antisense Drug Technology, Principles, Strategies, and Applications, 2nd edn. CRC Press Boca Raton, FL2008: 747-772Google Scholar Most nonimmune cells rely on cytoplasmic PRRs of the retinoic acid–inducible protein I (RIG-I)-like receptor family, such as MDA5 or RIG-I, to sense RNA accumulating in the cytoplasm during virus infection. MDA5 and TLR3 have previously been demonstrated to be involved in the response to long dsRNA species such as the synthetic long dsRNA poly I:C.2Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4936) Google Scholar,3Gitlin L Barchet W Gilfillan S Cella M Beutler B Flavell RA et al.Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus.Proc Natl Acad Sci USA. 2006; 103: 8459-8464Crossref PubMed Scopus (911) Google Scholar Signaling induced by poly I:C via MDA5 results predominantly in the production of type I interferons, whereas TLR3 mediates proinflammatory effects.3Gitlin L Barchet W Gilfillan S Cella M Beutler B Flavell RA et al.Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus.Proc Natl Acad Sci USA. 2006; 103: 8459-8464Crossref PubMed Scopus (911) Google Scholar It was also demonstrated that poly I:C can induce immune activation at least to some degree via RIG-I.4Kato H Takeuchi O Mikamo-Satoh E Hirai R Kawai T Matsushita K et al.Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.J Exp Med. 2008; 205: 1601-1610Crossref PubMed Scopus (1160) Google Scholar Differences in the length dependencies for the recognition of long dsRNA by MDA5 or RIG-I have been observed. Long dsRNAs above 1 kbp stimulate MDA5, dsRNAs with a length between ~70 bp and 1 kbp stimulate RIG-I,4Kato H Takeuchi O Mikamo-Satoh E Hirai R Kawai T Matsushita K et al.Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.J Exp Med. 2008; 205: 1601-1610Crossref PubMed Scopus (1160) Google Scholar and shorter single-stranded RNA (ssRNA) and dsRNA species activate RIG-1 only in the presence of a 5′-triphosphate moiety.5Schlee M Hartmann E Coch C Wimmenauer V Janke M Barchet W et al.Approaching the RNA ligand for RIG-I.Immunol Rev. 2009; 227: 66-74Crossref PubMed Scopus (67) Google Scholar PRRs were demonstrated not only to exert strong immune effects but also to be capable of inducing apoptosis. TLR9, which stimulates healthy B cells by recognizing so-called CpG motifs in pathogen or synthetic ssDNA, results in the induction of proliferation and protection from apoptosis involving upregulation of Bcl-Xl (6Yi AK Hornbeck P Lafrenz DE Krieg AM CpG DNA rescue of murine B lymphoma cells from anti-IgM-induced growth arrest and programmed cell death is associated with increased expression of c-myc and bcl-xL.J Immunol. 1996; 157: 4918-4925PubMed Google Scholar) The apoptotic process is tightly controlled by members of the Bcl-2 family of proapoptotic (NOXA, Bid, Bad, Bak, and others) and antiapoptotic (Bcl-2, Bcl-Xl) factors in the mitochondrial outer membrane. Upon initiation of the apoptotic process, cytochrome c released from mitochondria forms complexes with Apaf-1 and pro-caspase-9, resulting in activation of caspase-9 and terminal caspases such as caspase-3. This latter process is initiated upon TLR9 activation in malignant B-cell chronic lymphocytic leukemia cells but not in normal B cells, and it was speculated that upregulation of members of the tumor necrosis factor receptor family is involved.7Jahrsdorfer B Wooldridge JE Blackwell SE Taylor CM Griffith TS Link BK et al.Immunostimulatory oligodeoxynucleotides induce apoptosis of B cell chronic lymphocytic leukemia cells.J Leukoc Biol. 2005; 77: 378-387Crossref PubMed Scopus (88) Google Scholar Upregulation of Fas (CD95) is also involved in the apoptotic processes induced by imiquimod in tumor cells. Imiquimod is an immune-response modifier, a class of small molecules stimulating TLR7 and/or 8 (8Jurk M Heil F Vollmer J Schetter C Krieg AM Wagner H et al.Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848.Nat Immunol. 2002; 3: 499Crossref PubMed Scopus (817) Google Scholar) and was approved as imiquimod 5% cream (Aldara; 3M Pharmaceuticals) in 1997 for the treatment of external genital warts and in 2004 for the treatment of actinic keratoses. Since then, it has shown efficacy for a variety of skin cancers, including superficial basal cell carcinoma.9Gaspari A Tyring SK Rosen T Beyond a decade of 5% imiquimod topical therapy.J Drugs Dermatol. 2009; 8: 467-474PubMed Google Scholar,10Schon MP Schon M The small-molecule immune response modifier imiquimod—its mode of action and clinical use in the treatment of skin cancer.Expert Opin Ther Targets. 2006; 10: 69-76Crossref PubMed Scopus (26) Google Scholar Imiquimod not only mediates an efficient cellular immune response upon activating TLR7 but also develops a triple mode of action that appears to profoundly contribute to its clinical success. Its mode of action includes stimulating the production of Th1-like cytokines via TLR7, antagonizing adenosine receptor A2A to induce proinflammatory cytokines, and direct proapoptotic effects beyond CD95, via Bcl-2 and caspase pathways in nonmelanoma (e.g., basal cell carcinoma) and melanoma skin cancer cells, but not in healthy skin cells, including keratinocytes and melanocytes.10Schon MP Schon M The small-molecule immune response modifier imiquimod—its mode of action and clinical use in the treatment of skin cancer.Expert Opin Ther Targets. 2006; 10: 69-76Crossref PubMed Scopus (26) Google Scholar It is compelling that two independent research groups have now shown that two other PAMPs, poly I:C and 5′-triphosphate RNA, can induce apoptosis in melanoma cells, but not healthy skin cells, via stimulation of MDA5 and RIG-I, respectively.11Tormo D Checinska A Alonso-Curbelo D Perez-Guijarro E Canon E Riveiro-Falkenbach E et al.Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells.Cancer Cell. 2009; 16: 103-114Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar,12Besch R Poeck H Hohenauer T Senft D Hacker G Berking C et al.Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells.J Clin Invest. 2009; 119: 2399-2411Crossref PubMed Google Scholar Although both reports demonstrate a linkage between MDA5 and the induction of apoptotic processes, the group of Tormo et al.11Tormo D Checinska A Alonso-Curbelo D Perez-Guijarro E Canon E Riveiro-Falkenbach E et al.Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells.Cancer Cell. 2009; 16: 103-114Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar extended this by unraveling the mechanism that results in melanoma cell apoptosis: programmed cell death type II (Figure 1). Autophagy—an evolutionarily ancient cytoplasmic homeostasis process in eukaryotes13Delgado M Singh S De Haro S Master S Ponpuak M Dinkins C et al.Autophagy and pattern recognition receptors in innate immunity.Immunol Rev. 2009; 227: 189-202Crossref PubMed Scopus (147) Google Scholar—includes an initiation process whereby nascent autophagosomal structures in the cytoplasm are formed; an elongation stage, during which the autophagosomal structures (phagophores) envelop cytoplasmic targets to form an autophagosome; and maturation, in which autophagosomes fuse with late endosomal/lysosomal organelles to degrade the cytoplasmic targets. These processes, which normally serve as a mechanism for removing intracellular pathogens, in keeping with the primary role as a cytoplasmic cleanup process,13Delgado M Singh S De Haro S Master S Ponpuak M Dinkins C et al.Autophagy and pattern recognition receptors in innate immunity.Immunol Rev. 2009; 227: 189-202Crossref PubMed Scopus (147) Google Scholar were demonstrated to take place in melanoma cell lines upon MDA5-mediated cellular activation,11Tormo D Checinska A Alonso-Curbelo D Perez-Guijarro E Canon E Riveiro-Falkenbach E et al.Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells.Cancer Cell. 2009; 16: 103-114Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar and it appears feasible that a similar mode of action occurs with RIG-I, although this has yet to be proven. Importantly, the induction of autophagy precedes the later apoptotic program, and cellular death is mediated by caspases and proapoptotic factors such as Bak and NOXA.11Tormo D Checinska A Alonso-Curbelo D Perez-Guijarro E Canon E Riveiro-Falkenbach E et al.Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells.Cancer Cell. 2009; 16: 103-114Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar,12Besch R Poeck H Hohenauer T Senft D Hacker G Berking C et al.Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells.J Clin Invest. 2009; 119: 2399-2411Crossref PubMed Google Scholar However, normal melanocytes were insensitive to MDA5- or RIG-I-mediated apoptosis induction,11Tormo D Checinska A Alonso-Curbelo D Perez-Guijarro E Canon E Riveiro-Falkenbach E et al.Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells.Cancer Cell. 2009; 16: 103-114Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar,12Besch R Poeck H Hohenauer T Senft D Hacker G Berking C et al.Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells.J Clin Invest. 2009; 119: 2399-2411Crossref PubMed Google Scholar and upregulation of Bcl-Xl in normal cells seems to result in the tumor selectivity of apoptosis.12Besch R Poeck H Hohenauer T Senft D Hacker G Berking C et al.Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells.J Clin Invest. 2009; 119: 2399-2411Crossref PubMed Google Scholar Both reports exclude the involvement of other PRRs, TLR3, and PKR, as well as of indirect apoptotic effects via type I interferons that are induced upon treatment of melanoma cells with poly I:C or 5′-triphosphate RNA. Furthermore, a direct apoptotic activity on melanoma cells was readily demonstrated in vivo in animal cancer models using severe combined immunodeficiency (SCID)/beige mice11Tormo D Checinska A Alonso-Curbelo D Perez-Guijarro E Canon E Riveiro-Falkenbach E et al.Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells.Cancer Cell. 2009; 16: 103-114Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar or non-obese diabetic/SCID12Besch R Poeck H Hohenauer T Senft D Hacker G Berking C et al.Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells.J Clin Invest. 2009; 119: 2399-2411Crossref PubMed Google Scholar mice with strongly impaired cellular immune systems, arguing for a direct apoptotic effect on tumor cells in vivo. Antitumor activity was dependent on the use of a delivery agent, as was the induction of autophagy and apoptosis in vitro. Two delivery systems were used—PEI11Tormo D Checinska A Alonso-Curbelo D Perez-Guijarro E Canon E Riveiro-Falkenbach E et al.Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells.Cancer Cell. 2009; 16: 103-114Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar or a liposomal formulation12Besch R Poeck H Hohenauer T Senft D Hacker G Berking C et al.Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells.J Clin Invest. 2009; 119: 2399-2411Crossref PubMed Google Scholar—that had similar proapoptotic effects in vitro (only PEI was used in vivo). The need for a delivery agent may be explained by the requirement for an efficient transport of poly I:C into the cytoplasm where MDA5 resides. It is important to note that poly I:C has been used for decades as an immune modifier in preclinical and clinical studies, and the results have been discouraging—there were no detectable antitumor effects in melanoma patients.14Robinson RA DeVita VT Levy HB Baron S Hubbard SP Levine AS A phase I–II trial of multiple-dose polyriboinosic-polyribocytidylic acid in patients with leukemia or solid tumors.J Natl Cancer Inst. 1976; 57: 599-602PubMed Google Scholar However, in contrast to the present studies, most previous studies did not use a formulation allowing for enhanced stability as well as cytosolic delivery of poly I:C. Several other PRRs have been shown to induce autophagy, including TLR3, TLR7, TLR8, and TLR9 (13Delgado M Singh S De Haro S Master S Ponpuak M Dinkins C et al.Autophagy and pattern recognition receptors in innate immunity.Immunol Rev. 2009; 227: 189-202Crossref PubMed Scopus (147) Google Scholar). For example, the TLR7 agonists imiquimod and ssRNA were reported to result in autophagy, although this has yet to be linked to apoptosis. Given the amount of data on imiquimod and its ability to induce apoptosis in malignant cells,10Schon MP Schon M The small-molecule immune response modifier imiquimod—its mode of action and clinical use in the treatment of skin cancer.Expert Opin Ther Targets. 2006; 10: 69-76Crossref PubMed Scopus (26) Google Scholar it is tempting to speculate that TLR7-dependent autophagy mediates the apoptotic effect of imiquimod. However, the physiological roles of autophagy as a cytoplasmic cleanup process or its quintessential survival function—for example, during times of nutrient starvation—do not always result in apoptosis, and it will be important to investigate in more detail the control mechanisms that underlie the transition of autophagy as an initial mechanism of protection into a prodeath program. In addition, it will be interesting to see this work being extended to other tumor types such as B-cell chronic lymphocytic leukemia. On the basis of the studies described, it seems possible that some PRRs require a combinatorial stimulation to result in autophagy. In their reports, Tormo et al. and Besch et al. were unable to block apoptosis completely upon knockdown of MDA5 or RIG-I, suggesting additional factors that act in parallel or in concert.11Tormo D Checinska A Alonso-Curbelo D Perez-Guijarro E Canon E Riveiro-Falkenbach E et al.Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells.Cancer Cell. 2009; 16: 103-114Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar,12Besch R Poeck H Hohenauer T Senft D Hacker G Berking C et al.Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells.J Clin Invest. 2009; 119: 2399-2411Crossref PubMed Google Scholar Indeed, agonists with specificity only to the heterodimeric TLR1/2 or TLR2/6 were demonstrated to be unable to induce autophagy, whereas PAMPs that engage more than a single receptor—for example, yeast cell-wall particles stimulating both TLR2/6 and dectin-1—strongly induce autophagy.13Delgado M Singh S De Haro S Master S Ponpuak M Dinkins C et al.Autophagy and pattern recognition receptors in innate immunity.Immunol Rev. 2009; 227: 189-202Crossref PubMed Scopus (147) Google Scholar One candidate receptor contributing to the apoptotic effect of MDA5 may be TLR5, which was shown to respond normally to bacterial flagellin. However, PEI complexes encapsulating dsRNA such as silencing RNA (siRNA) were recently demonstrated to result in TLR3- and TLR5-dependent immune activation,15Cubillos-Ruiz JR Engle X Scarlett UK Martinez D Barber A Elgueta R et al.Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity.J Clin Invest. 2009; 119: 2231-2244Crossref PubMed Scopus (182) Google Scholar indicating that TLR5 may be involved in, but not required for, the apoptotic processes induced by PEI-formulated poly I:C. Before this technology can be applied in disease indications such as melanoma, potential hurdles must be overcome. The search for a better defined synthetic ligand for both of these receptors has not yet been successful; poly I:C is generated through annealing of enzymatically produced inosine and cytidine homopolymers of undefined length.16Pichlmair A Schulz O Tan CP Rehwinkel J Kato H Takeuchi O et al.Activation of MDA5 requires higher order RNA structures generated during virus infection.J Virol. 2009; Google Scholar This search may be futile, because recent work demonstrates that MDA5 responds to RNA aggregates consisting of ssRNA and dsRNA moieties, potentially including branched RNA.16Pichlmair A Schulz O Tan CP Rehwinkel J Kato H Takeuchi O et al.Activation of MDA5 requires higher order RNA structures generated during virus infection.J Virol. 2009; Google Scholar In contrast, the use of synthetic 5′-triphosphate RNA—such as in an siRNA as a therapeutic modality with different modes of action, including apoptosis induction—seems more promising.17Poeck H Besch R Maihoefer C Renn M Tormo D Morskaya SS et al.5′-triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma.Nat Med. 2008; 14 (e-pub ahead of print 5 August 2009): 1256-1263Crossref PubMed Scopus (319) Google Scholar In addition, the requirement for a delivery system makes the development of MDA5 and RIG-I ligands more complex, but this may benefit greatly from the current active search for delivery systems in the development of siRNAs. However, the induction of an array of antitumoral activities with single agents such as poly I:C or 5′-triphosphate RNA represents an exciting and promising therapeutic concept that may greatly benefit patients with different kind of tumors.