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Open Forum on Cell Signaling

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Highlights from Toll-Like Receptor Function in the Cancer Microenvironment

13 May 2008

Nancy R. Gough

This session took place Sunday 6 April 2008. The function of the immune system in cancer suppression and promotion is complex with important differences between rodents and humans. There were four speakers: Michael Karin (University of California, San Diego), Xiaoxia Li (Lerner Research Institute, Cleveland Clinic), Rong-Fu Wang (Baylor College of Medicine), and Arthur M. Krieg (Coley Pharmaceutical Group). This series of talks presented intriguing insights into how the immune system both contributes to cancer and how it may be manipulated to treat or possibly prevent cancer.

IKK-Dependent NF-κB Signaling in Inflammation and Cancer- Michael Karin

Although the title of his talk suggested that he was going to discuss NF-κB signaling and cancer, instead for much of his talk Dr. Karin discussed a relationship between metastasis and Toll-like receptor (TLR) signaling. He wanted to address the question of how the immune system may be involved in early stages of metastatic growth, prior to any necrosis that may trigger an inflammatory response.

Cancer cells can release factors [for example, interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α)] that activate macrophages, which in turn may release factors that trigger metastases of the cancer cells. Versican, a chondroitin-sulfate proteoglycan that binds hyaluronic acid, was secreted by a metastatic lung carcinoma cell line (LLC), enhanced metastases, activated TLR2 signaling, and stimulated the production of inflammatory cytokines from macrophages.

The importance of TLR2 signaling in cancer metastases of the LLC in mice was confirmed with TLR2-knockout mice. When injected with LLC, the TLR2-knockout mice had fewer or undetectable lung tumors at early stages compared to the wild-type mice and had a longer lifespan than did the wild-type mice.

The importance of versican was verified by silencing versican in the cancer cells and injecting these into the mice. The versican-deficient cells were less metastatic (fewer lung tumors following tail vein injection). Versican may act by binding large fragments of hyaluronic acid produced by tumor cells and then by presenting the hyaluronic acid fragments to macrophages, which stimulates TLR2 signaling. A low metastatic derivative cell line derived from the LLC line was deficient in versican production and did not stimulate macrophage IL-6 production. Thus, the model is that cancer cells with a high metastatic potential release versican, which binds to hyaluronic acid, and this versican-hyaluronic acid complex stimulates TLR2 signaling in macrophages, which respond by releasing factors, such as TNF-α, that feed back onto the cancer cells to stimulate metastasis. It may be that different types of cancers produce different proteoglycans and these have been reported in the literature to be inflammatory.

Selected References

W. W. Lin, M. Karin, A cytokine-mediated link between innate immunity, inflammation, and cancer. J. Clin. Invest. 117, 1175-1183 (2207). [PubMed]

The Toll-Like Interleukin-1 Receptor Member SIGIRR Regulates Colonic Epithelial Homeostasis, Inflammation, and Tumorigenesis- XiaoXia Li

SIGIRR is a founding member of a new subgroup of the TLR family, notable for having a single Ig extracellular domain. Characterization of SIGIRR-knockout mice by Wald et al. suggested that SIGIRR inhibits TLR2, TLR9, ST2, IL-1R, IL-18R, and TLR4 signaling by forming complexes with these receptors. The extracellular Ig domain of SIGIRR interferes with receptor dimerization and the intracellular domain of SIGIRR inhibits recruitment of downstream signaling components.

The mRNA expression pattern suggested that SIGIRR is important for commensal microflora survival and may contribute to microbial tolerance, for example, in the intestinal epithelial layer (Xiao et al.). The SIGIRR-knockout mice exhibit altered colon crypt structure (elongated relative to wildtype), which is consistent with the increased cell proliferation and constitutive TLR7 signaling in these animals. Consistent with a role in tolerance for commensal microflora, SIGIRR-knockout mice showed an increased inflammatory response and tissue damage and ultimately death in a chemical-induced intestinal inflammation model (wild-type mice do not die in this model) and exhibited increased colon tumor development in a colitis-associated model of cancer.

Cancer formation in the colon is influenced largely by the Wnt pathway. In SIGIRR-knockout and wild-type mouse tumors, β-catenin is in the nucleus, which indicates that the Wnt pathway is activated. However, SIGIRR deficiency enhances the number of colonic polyps, thus Dr. Li proposes that SIGIRR may serve as the gatekeeper in the intestinal epithelia, allowing commensal microflora to trigger a survival response in the colonic epithelia. In the absence of SIGIRR, cells respond to commensal microflora with an inflammatory response.

However, SIGIRR is also present in two subsets of CD4+ T cells: Th17 and Th2 cells, which are both important in epithelial immunity. In Th2 cells, SIGIRR appears to negatively regulate ST2 signaling and SIGIRR-knockout animals exhibit an allergic pulmonary inflammation in a mouse model of asthma. The function of SIGIRR in Th17 cells appears to be to decrease the production of interleukin-17 (IL-17) and thus negatively regulate Th17 function. This latest work means that the interpretation that the cancer and inflammatory effects of loss of SIGIRR are due to altered signaling in the epithelial cells needs to be reassessed, because SIGIRR is also important in the mucosal T cell population. Thus, it will be interesting to determine how the loss of SIGIRR in the T-cell population is contributing to the phenotypes of the SIGIRR knockout mice.

Selected References

D. Wald, J. Qin, Z. Zhao, Y. Qian, M. Naramura, L. Tian, J. Towne, J. E. Sims, G. R. Stark, X. Li, SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat. Immunol. 4, 920-927 (2007). [PubMed]

H. Xiao, M. F. Gulen, J. Qin, J. Yao, K. Bulek, D. Kish, C. Z. Altuntas, D. Wald, C. Ma, H. Zhou, V. K. Tuohy, R. L. Fairchild, C. de la Motte, D. Cua, B. A. Vallance, X. Li, The Toll-interleukin-1 receptor member SIGIRR regulates colonic epithelial homeostasis, inflammation, and tumorigenesis. Immunity 26, 461-475 (2007).[PubMed]

TLR Signaling and Regulatory T Cells in Cancer- Rong-Fu Wang

Dr. Wang is interested in how the connections between cancer and the immune system can be applied to the development of a "cancer vaccine." He focuses on human disease because there are some important differences between mouse and human in the function of TLR signaling that impact the use of mice as a model system for manipulating the immune system for the development of human cancer therapies.

TLRs may be present on tumor cells, T cells, and antigen-presenting cells (APCs); thus, there is a complex effect of TLR signaling in cancer progression and treatment. He divides the tumor environment into three immune components: (i) the CD8+ T cells and Th1 effector T cells, (ii) the pro-inflammatory Th17 cells, and (iii) the immunosuppressive Treg cells. The environment may be further complicated because the Treg cells are a diverse population, at least partially due to the activity of the transcription factor Foxp3. A subset of Treg cells, the CD4+ Tregs, recognize some tumor-associated antigens (LAGE1, ARTC1, EBNA1).

Different types of tumors are associated with different types of T cells. Analysis of cells associated with normal prostate tissue and prostate cancer samples indicated that CD8+, Foxp3+ Tregs are only associated with prostate cancer cells. γδ T cells, which inhibit naïve and effector T cell function and dendritic cell maturation through release of soluble factors, are commonly associated with breast and prostate cancers, but not with melanoma.

The tumor environment appears to promote the Treg arm of the immune system, which inhibits the CD8+ and Th1 arm of the immune system to promote tumorigenesis. Th17 cell differentiation also occurs in response to soluble factors in the tumor environment. Dr. Wang suggests that one approach to cancer treatment would be to interfere with the immune response and alter the environment of the tumor tissue. In humans, this might be achieved by stimulating TLR8 signaling, which would decrease the immunosuppressive response by inhibiting Treg function, or by stimulating TLR9 signaling, which would directly stimulate an immune response against the tumor.

It is worth noting that in mice, TLR8 does not appear to have the same functions as in humans. In mice, dendritic cells may be more important for regulating effector T cell function in response to TLR ligands; whereas in humans, TLR8 signaling on Tregs may be the critical connection to regulating effector T cells. Reconstitution of human TLR8 in mice restores similar function as that observed in a human tumor environment; the TLR8 ligand Poly-G becomes effective in the mouse for decreasing tumor size.

Selected References

R. F. Wang, Y. Miyhara, H. Y. Wang, Toll-like receptors and immune regulation: Implication for cancer therapy. Oncogene 27, 181-189 (2008). [PubMed]

Clinical Development of TLR Agonists for Cancer Therapy- Arthur M. Krieg

Dr. Krieg is from the commercial sector and focused on TLR agonists, oligoribonucleotides (ORNs) that are ligands for TLR7 and TLR8 or oligodeoxyribonucleotides (ODNs) that are ligands for TLR9, as adjuvants for cancer vaccines or as monotherapies for cancer. ORNs stimulate B cells, plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), monocytes, and natural killer (NK) cells; whereas DNA agonists, of which ODNs would be one type, only stimulate B cells and pDCs. Usually, ODNs are much more effective than ORNs for decreasing tumor load in mice.

TLR9 agonists have reached phase III clinical trials for allergy*, anti-idiotype (Id) vaccine, cancer vaccine, lung cancer therapy* (*terminated). CpG ODN are becoming the "gold standard" for vaccine adjuvants and have been used in greater than 35 human trials. These increase the antibody response and T cell response. There are many examples where addition of a CpG ODN with a tumor-specific antigenic peptide vaccine for cancer patients leads to higher Th1 responses and antibody titers than are produced with peptide alone. Studies by GlaxoSmithKline (poster only, not published) have shown decreased tumor load in response to vaccination. Seven to ten vaccinations were required, whereas many studies stop after four or five, which is an important detail for considering clinical applications.

There are several additional mechanisms by which TLR9 agonists may be useful in the treatment of cancer. Several studies with human cancers (metastatic melanoma, cutaneous T cell lymphoma, and non-Hodgkins lymphoma) suggest that TLR9 activation may be effective not only as an adjuvant, but as a stand-alone therapy (monotherapy). In mice, radiotherapy followed by a TLR9 agonist or combining chemotherapy with a TLR9 agonist improved survival. The additive effect required CD8+ T cells and did not occur in nude mice or in CD4-deficient mice. Unfortunately, in clinical trials, chemotherapy plus a TLR9 agonist did not improve survival of patients with a highly metastatic nonsmall cell lung cancer.

As the previous speaker, Dr. Wang, suggested, suppression or depletion of Tregs may also an effective cancer therapy. Some chemotherapy treatments alter the immune profile. For example, in mice treated with paclitaxel, there was a decreased abundance of Tregs in the blood but not the spleen, decreased abundance of Foxp3+ CD4+ T cells, and an increase in tumor-specific T cells.

Selected References

A. M. Krieg, Toll-like receptor 9 (TLR9) agonists in the treatment of cancer. Oncogene 27, 161-167 (2008). [PubMed]

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