Poly I:C-Induced Tumor Cell Apoptosis Mediated by Pattern-Recognition Receptors

Abstract

Poly I:C is a synthetic dsRNA that can imitate a viral infection and elicit host immune responses by triggering specific pattern-recognition receptors (PRRs) such as toll-like receptor 3 and retinoic acid inducible gene I(RIG-I)-like receptors, including RIG-I and melanoma differentiation-associated gene 5. Activation of these PRRs by poly I:C triggers a signal transduction cascade that results in the activation of NF-κB and production of type I interferon. Poly I:C has been used as a vaccine adjuvant for cancer immunotherapy for several decades. Evidence from recent studies indicates that poly I:C can directly induce apoptosis in several types of tumor cells, thus providing a new therapeutic approach for cancer treatment. However, the molecular mechanism underlying the induction of apoptosis by poly I:C is still unclear. In this review, we summarize the current knowledge of poly I:C-induced tumor cell apoptosis, focusing on the key molecules and pathways involved in this process.

Introduction

Poly I:C is a synthetic dsRNA analogue that is recognized by pattern-recognition receptors (PRRs), triggering innate immune responses, and subsequently adaptive immunity against double-stranded viruses.^1,2 Poly I:C has been used as a vaccine adjuvant to treat cancer for the past decades^3
–5 based on its ability to enhance innate immunity and adapt immune responses and alter the tumor microenvironment.^6
–8 In recent years, evidence has accumulated that poly I:C can directly trigger apoptosis in many types of human malignant cells including breast cancer, melanoma, and hepatoma cells by activating specific PRRs such as toll-like receptor 3 (TLR3), retinoic acid inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5).^9

–12 Further, the proapoptotic effect of poly I:C is dose-dependent in human melanoma cells and in head and neck cancer cells.^13,14 Poly I:C can induce apoptosis in certain cancer cells even when applied at very low concentrations. In nasopharyngeal carcinoma cells, a poly I:C concentration of 100 ng/mL is sufficient to induce apoptosis when combined with the IAP inhibitor RMT 5265.¹⁵ Further, poly I:C enhanced the sensitivity of Hela cells (human cervical cancer) and MCA38 cells (murine colon cancer) to chemotherapeutics such as the protein synthesis inhibitor cycloheximide (CHX).^16,17 In athymic mice, poly I:C significantly reduced the metastatic capacity of cancer cells by activating TLR3.¹⁸ Evidence accumulated to date has indicated that poly I:C-induced apoptosis is a complex process involving a large number of factors.^12,19 Here, we summarize the key molecules involved in this process and outline a potential mechanism of poly I:C-induced apoptosis.

Overview of Poly I:C-Related PRR Signaling Pathways

TLR3 signaling pathway

TLRs are a family of PRRs that, together with the interleukin 1 receptor (IL-1R) family, form a superfamily that shares a homologous cytoplasmic domain, the toll/IL-1 receptor (TIR) domain.²⁰ TLR signaling pathways arise from this TIR domain, which is conserved among all TLRs.²⁰ The specificity of TLR signaling is dependent on the recruitment of different TIR domain-containing adaptor molecules such as MyD88 and TIRAP (Mal), TRIF and TRAM, which play an essential role in modulating TLR signaling pathways.^20,21

The TLR3 is primarily expressed in intracellular vesicles such as the endoplasmic reticulum, endosomes, and lysosomes,^22,23 and it has been detected on the surface of some cells such as fibroblasts.²⁴ TLR3 signaling, which is induced by dsRNA or its analog poly I:C, is mediated by the TRIF (TIR domain-containing adaptor-inducing interferon-β [IFN-β]) adaptor molecule through the TRIF-dependent pathway. TRIF is responsible for the activation of IRF3 (IFN regulatory factor 3) and NF-κB and the consequent induction of type I IFN and the production of inflammatory cytokines.^22,23 The TRIF-dependent pathway is essential for both TLR3 and TLR4 signaling. MyD88 is another important adaptor molecule for TLR signaling pathways, and the MyD88- dependent pathway is common to all TLRs except TLR3. TLR3-mediated signaling involves the recruitment of the adaptor protein TRIF, which recruits TNF receptor-associated factor 6 (TRAF6) and activates the transforming growth factor (TGF)-β-activated kinase 1 (TAK1)-mediated activation of NF-κB and mitogen-activated protein kinases (MAPKs).^21,23 This signaling pathway regulates the production of inflammatory cytokines. On the other hand, TRIF recruits TRAF3 to activate the noncanonical IκB kinases (IKKs) TANK-binding kinase 1 (TBK1) and IKKi, which catalyze the phosphorylation of IRF3 and induce its nuclear translocation to regulate the production of type I IFN.^23,25,26

RIG-1 and MDA5 signaling pathways

RIG-I and MDA5 are cytosolic RNA sensors^6,27 that specifically recognize different RNA viruses and trigger host antiviral responses.^6,28 IFN-β promoter stimulator 1(IPS-1), also known as CARDIF, MAVS, or VISA, which is a key adaptor protein for RIG-I and MDA-5-mediated recognition of RNA ligands,²⁹ is located in the outer mitochondrial membrane.³⁰ RIG-I and MDA-5 recruit and bind with IPS-1 to activate downstream signaling pathways. IPS-1 induces the production of type I IFN by activating TBK1, which phosphorylates IRF-3 and IRF-7. On the other hand, IPS-1 can also activate NF-κB, which regulates the expression of inflammatory cytokines.²⁹ In vivo experiments have shown that IPS-1 deficiency impairs the activation of IRF-3 and NF-κB, resulting in severe defects in the induction of type I IFN and inflammatory cytokines by both RIG-I and MDA5.³⁰

The molecular mechanism of poly I:C-Induced apoptosis through the activation of PRRs

The molecular mechanism underlying the induction of apoptosis by poly I:C is not well understood. However, accumulating evidence indicates that it is a complex process involving multiple factors and signaling pathways. Moreover, certain PRRs signaling adaptor molecules have been shown to play important roles in poly I:C-mediated apoptosis.

TLR3 signaling-mediated apoptosis

Studies have shown that apoptosis triggered by poly I:C is partly mediated by TLR3 in certain types of tumor cells such as breast cancer Cam1 cells,¹⁹ human cervical cancer Hela cells,¹⁶ and colon carcinoma HCT116 cells.¹⁷ In the human hepatocellular carcinoma (HCC) cell line HepG2, the activation of TLR3 signaling is also skewed toward apoptosis.⁹ In addition, TLR3 directly inhibits cell proliferation and triggers apoptosis in human melanoma cells.³¹ Blocking TLR3 with an anti-TLR3 antibody greatly attenuated the proapoptotic effects of poly I:C on Hela cells.¹⁶ Further, in vivo experiments with mice carrying the TRAMP (transgenic adenocarcinoma of mouse prostate) transgene showed significant growth inhibition in TLR3 (+/+) mice compared with TLR3 (−/−) mice in response to stimulation with poly I:C.³² These results indicate that TLR3-induced apoptosis may be an important mechanism mediating the anticancer effects of poly I:C.

Recent studies have indicated that adaptor proteins involved in TLR3 signaling pathways play crucial roles in the process of apoptosis. In human breast cancer cells, synthetic dsRNA induced apoptosis in a TLR3-dependent manner,¹⁹ and the key adaptor protein of the TLR3 signaling pathway TRIF, and its downstream adaptor TRAF6 were indispensable for this process.²¹ A separate study demonstrated that TRIF is an essential regulator of TLR3-mediated apoptosis in Hela cells.³³ Moreover, siRNA-mediated silencing of TRIF in breast cancer cells significantly suppressed apoptosis.¹⁹ These data support the crucial role of TRIF in the TLR3-mediated apoptotic pathway.

RIG-I and MDA5 signaling-mediated apoptosis

MDA5 and RIG-I contain two N-terminal caspase recruitment domains, and have therefore been considered as potentially involved in proapoptotic pathways, which was confirmed in later studies.^34
–36 Because RIG-I and MDA5 are receptors that recognize poly I:C and mediate its signaling, they could be involved in the induction of apoptosis in tumor cells in response to poly I:C stimulation.^37,38 In human hepatoma cells, transfected poly I:C induced cell apoptosis by upregulating the expression of the cytoplasmic receptors RIG-I and MDA5 and activating the caspase pathway.³⁷ Transfection or electroporation of poly I:C into ovarian cancer cells also induced apoptosis through the activation of MDA5 and RIG-I.^38,39

In melanoma cells, RIG-I- and MDA5-mediated apoptotic pathways require the adaptor protein IPS-1, as demonstrated by siRNA-mediated silencing of IPS-1, which significantly decreased apoptosis.¹³ Although RIG-I, MDA5. and the adaptor protein IPS-1 are all required for the induction of apoptosis in melanoma cells, the downstream expression of IRF-3 and type I IFN signaling do not appear to be indispensable for this process.¹³ Moreover, the RNA-activated protein kinase PKR and TLR3 were not involved in apoptosis induction in melanoma cells, although PKR was reported to enhance the induction of IFN-β and apoptosis mediated by RIG-I and MDA5.⁴⁰ In colorectal carcinoma cells, the Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) signaling pathway was involved in MDA5-mediated growth inhibition and apoptosis.⁴¹ These studies suggest that apoptosis mediated by RIG-I and MDA5 may involve different downstream signaling pathways specific to each tumor cell type.

NF-kB is an important nuclear transcription factor that regulates the expression of genes involved in immune responses, cell growth, and apoptosis.⁴² It is also an important downstream molecule of TLR3, RIG-I, and MDA5 signaling pathways. In certain tumor cells such as HCC, NF-kB was involved in poly I:C-mediated apoptosis,¹¹ whereas in multiple myeloma (MM) cells such as KMM1 cells or in other hematologic neoplasms, activated NF-kB promoted cell proliferation.^43,44 This suggests a dual function of NF-kB in the regulation of apoptosis and cell proliferation dependent on the signals received and transduced. The activation of NF-kB may therefore not be a necessary event for poly I:C-induced apoptosis in tumor cells.

Although the apoptotic pathway mediated by TLR3 and retinoic acid inducible gene I-like receptors (RLRs) (RIG-I and MDA5) remains largely unexplained, current data on these three PRR-mediated apoptotic pathways have confirmed their crucial roles in the response to poly I:C stimulation. However, the relationship between the PRR signaling pathway and its induced apoptotic pathway remains unclear. In addition, it is speculated that a dominant signaling pathway may exist for specific tumor cells in response to poly I:C stimulation. For example, certain hepatoma cells show a sensitive RLR-dependent pathway, while the TLR3-mediated apoptotic pathway is insensitive to poly I:C stimulation.³⁷ These results indicate that the existence of cell type-specific signaling pathways cannot be excluded.^23,45 Further, both synergistic and complementary roles of TLR3 and RLR signaling in poly I:C-induced apoptosis are possible, similar to their complementary correlation in poly I:C-mediated natural killer (NK) cell activation.⁴⁶ Further investigation is necessary to clarify these issues.

IFN signaling may be an important regulator of poly I:C-mediated apoptosis

Type I IFN, including IFN-α and IFN-β, plays an important role in the immune defense against tumors by enhancing the infiltration of T lymphocytes and NK cells into the tumor microenvironment to suppress the growth of tumor cells.³² As an effective pathway mediating TLR3, RIG-1, and MDA5 signaling,⁴⁷ the involvement of type I IFN in poly I:C-induced apoptosis has been demonstrated in several types of tumor cells such as colon carcinoma HCT116 cells,¹⁷ breast cancer cells,¹⁹ and MM cells.⁴³ In breast cancer cells, the induction of apoptosis involved the production of IFN-β and type I IFN autocrine signaling,¹⁹ while in MM cell lines such as NCI-H929 and RPMI 8226, cell death was mediated by IFN-α through TLR3 activation.⁴³ Pretreatment with IFN-α had a synergistic effect on enhancing poly I:C-mediated suppression of cell proliferation and inducing cell death in melanoma cells.³² Type I IFN was also reported to upregulate TLR3 expression¹⁷ and induce the expression of RIG-I and MDA-5 in certain cells in response to poly I:C.^34
–36 Therefore, type I IFN is a key mediator or an important regulator of poly I:C-induced apoptotic pathways in specific tumor cells.

In contrast, IFN-independent apoptosis has also been identified in melanoma cells¹³ and prostate cancer LNCaP cells¹² mediated by RIG-I, MDA-5. and TLR3. Moreover, certain hepatocellular tumor cells showed a slight response to IFN-β, indicating that it is not necessary for poly I:C-induced apoptosis.^16,48 A neutralization antibody test indicated that IFN-β is also not necessary for poly I:C/CHX-induced apoptosis in Hela cells.¹⁶

Type I IFN binding to its receptor activates the JAK-STAT signaling pathway, which induces the phosphorylation and translocation of the transcription factor STAT to the nucleus to regulate the expression of specific genes.⁴⁹ The p53 gene, an important tumor suppressor and proapoptotic molecule, is the downstream transcriptional target of type I IFN, and p53 gene transcription is induced by IFN-α/β.⁵⁰ The involvement of p53 may partially account for the mechanism of apoptosis mediated by IFN-α/β. In HCC, on the other hand, promyelocytic leukemia protein (PML) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have been identified as the central molecules responsible for IFN-α-induced apoptosis.⁴⁸

Traditional Apoptotic Pathways are Involved in Poly I:C-Induced Apoptosis

Extrinsic apoptotic pathways dependent on caspase-8 and Fas-associated death domain (FADD) and intrinsic apoptotic pathways dependent on caspase-9 and Apaf-1 are the most important apoptotic pathways identified to date, and these pathways are involved in poly I:C-mediated apoptosis in many tumor cells. In melanoma cells, TLR3-mediated cell death was found to involve both extrinsic and intrinsic apoptotic pathways,³¹ while RIG-I and MDA-5 mediated apoptosis was associated mainly with the intrinsic apoptotic pathway.¹³ In breast cancer cells¹⁹ and Hela cells,³³ TLR3-mediated apoptosis occurred mainly through the activation of intrinsic apoptotic pathways. TRIF, the key adaptor molecule of the TLR3 signaling pathway, was reported to bind receptor-interacting protein kinase 1(RIP1) and activate the RIP/FADD/caspase-8-dependent pathway.⁵¹ This interaction, which is essential for apoptosis triggered by poly I:C, could be impaired by HSV R1 in Hela cells.³³ IPS-1, the key adaptor protein of RIG-I and MDA-5 signaling pathways was also reported to interact with FADD and RIP1 to trigger apoptosis.^52,53

During poly I:C-mediated apoptosis, proapoptotic proteins such as Noca, Puma, and Bim were upregulated and antiapoptotic proteins such as Bcl-2, Bcl-xL, and survivin were downregulated, indicating their participation in the activation of apoptosis pathways.^11,54 In hepatoma cells, poly I:C significantly downregulated anti-apoptotic proteins,^9,11 and in melanoma cells, poly I:C significantly upregulated proapoptotic proteins such as Noxa, Puma, Bim, and Bik.¹³

Taken together, these results demonstrated that extrinsic and intrinsic apoptotic pathways play an important role in poly I:C-mediated apoptosis. The potential mechanisms of poly I:C-mediated apoptosis are illustrated in Figure 1.

FIG. 1.

Overview of apoptotic signaling pathway mediated by poly I:C through triggering PRRs in cancer cell poly I:C binds with its recognition receptors TLR3, RIG-I, and MDA-5, and then through the adaptor molecules TRIF and ISP-1 to initiate the downstream signal pathway, including the activation of IRF3/7 and NF-κB, and the consequent induction of type I interferon and the production of inflammatory cytokines. TRIF and ISP-1 are also the key mediators of poly I:C-induced apoptosis, which could bind with RIP1 or FADD to activate extrinsic apoptotic pathway, or through regulating the apoptotic proteins such as Noxa and Puma to activate intrinsic apoptotic pathway to induce tumor cells apoptosis. Moreover, as an effective pathway mediating TLR3, RIG-1, and MDA5 signaling, IFN signal also plays a crucial role in mediating poly I:C-induced apoptosis. PRR, pattern-recognition receptor; TLR3, Toll-like receptor 3; RIG-I, retinoic acid inducible gene I; MDA-5, melanoma differentiation-associated gene 5; RIP1, receptor-interacting protein kinase 1; FADD, Fas-associated death domain; IFN, interferon.

Conclusion

In summary, as a promising therapeutic agent for cancer immunotherapy, poly I:C exhibits multiple anticancer functions. Further, the newly discovered function of poly I:C as a direct inducer of tumor cell apoptosis makes it even more attractive for development as an anticancer agent. Moreover, a recent study reported that the uptake of poly I:C-induced apoptotic tumor cells by dendritic cells led to an enhanced ability to activate T cells compared with dendritic cells loaded with gamma-irradiated apoptotic tumor cells.⁵⁵ This research further confirms the potential value of poly I:C in cancer treatment.

Although the effect of poly I:C in the induction of tumor cell apoptosis has been demonstrated, several questions remain unanswered. First, the expression levels of TLR3, RIG-I, and MDA-5 and the signaling pathways and the mechanisms mediating the response to poly I:C stimulation may be different in specific cells.⁴³ Therefore, the elucidation of the cell type-specific apoptotic mechanisms induced by poly I:C is essential to design effective immunotherapy strategies in each cell type.^43,56,57 Second, the current data on poly I:C-induced tumor cell apoptosis suggest the existence of cross-talk among these signaling pathways,¹⁹ indicating that the elucidation of the molecular mechanisms of poly I:C-induced apoptosis in different types of tumor cells will require a better understanding of these complex signaling networks.

Footnotes

Acknowledgments

This work was supported by the Shandong Province Science Foundation for Outstanding Young Scientists (BS2011SW045). This work also was supported in part by the National Natural Science Foundation of China (No. 30973807) and Shandong medical and health science and technology development project (2011QW024).

Disclosure Statement

No conflict of interest exists for any of the authors.

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