Toll-like Receptors

Antipodal Enhancers or Suppressors of Tumorigenesis New Targets for Cancer Therapeutics

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Toll-like Receptors
Antipodal Enhancers or Suppressors of Tumorigenesis New Targets for Cancer Therapeutics
he broad expression of Toll-like Receptors (TLRs) in tumors and increasing amounts of information indicate that TLRs play an important albeit unclaried function in cancer biology. TLR recognize and respond to exogenous and endogenous ligands through signaling pathways leading to inammatory cascade mediator production which direct the innate and adaptive immune response. it is increasingly recognized that inammatory processes play a key role in tumorigenesis. TLRs, as in other human diseases, appear to act as double edged swords in tumorigenesis (reviewed in Conroy et al, 2008 and Huang et al, 2008). Overall, research studies suggest that TLRs as a family are involved in both inhibiting and promoting cancer (Rakoff-Nahoum and Medzhitov, 2009). Parallel to the recognition of the importance of TLRs as sensors and shapers of the overall anti-tumor response, TLRs have emerged as an important application area and focus of basic research and applied to development of cancer therapeutic and vaccine research. Table 1 below illustrates specic TLR targets and Pharma activities involved in development of Cancer Therapeutics pertaining to clinical trial staging. Research focusing on TLRs in tumor biology is growing rapidly. TLRs various TLR polymorphisms have now been identied in a number of tumor types as summarized in Table 2.
Table 1. Examples of Clinical Trials of TLR Realted Targets for Cancer Therapeutics TLR Target TLR3 TLR7 TLR7 TLR9 TLR9 TLR9 TLR9 TLR9 Cancer Breast Cancer Basal Cell Carcinoma Melanoma Non-Hodgkins Lymphoma Colorectal Cancer Metastatic colorectal cancer Renal cell carcinoma NSCLC Candidate Undisclosed Imiquimod (Aldara) Imidazoquinoline (852A) 1018ISS+ Rituximab 1018ISS+ chemotherapy DSLIM HYB2055, IMO2055 (IMOxine) MAGE-A3 + Vaximmune Company Innate Pharma 3M Pharma 3M Pharma Dynavax Dynavax Mologen Idera Pharm Coley/GSK Clinical Status Preclinical Approved Phase II Phase II Phase II Phase Ib/IIa Phase II Phase III

Source: Makkouk and Abdelnoor, 2009

Table 2. TLR Expression and Polymorphisms in Tumors

TLR
TLR1

TYPE OF CANCER
Colon cancer (RNA) Lymphoma (DNA) Prostate cancer (DNA)

NOTES
Low Expression Single-nucleotide polymorphism Single-nucleotide polymorphism Slightly Increased Expression Moderate Expression Increased Expression Increased Expression Weak Expression Single-nucleotide polymorphism Increased Expression Moderate Expression Moderate Expression Increased Expression Increased Expression Moderate Expression Moderate Expression Increased Expression Increased Expression Increased Expression Increased Expression Moderate Expression Increased Expression Moderate Expression Variable Expression in benign tumors, epithelial tumors, and ovarian cancer cell lines Increased Expression

APPLICATION
RT-PCR Functional Assay Functional Assay RT-PCR, Functional Assay, IHC IHC RT-PCR IHC RT-PCR Functional Assay IHC Functional Assay RT-PCR RT-PCR RT-PCR IHC, IF RT-PCR IP, WB, Functional Assay IHC RT-PCR RT-PCR Functional Assay RT-PCR, IHC RT-PCR, WB RT-PCR, IHC

REFERENCES
Immunol 115:565574 (2005) Genes Immun 7:615624 (2006) J Natl Cancer Inst 97:525532 (2005) J Int Med Res 29:409420 (2001) Immunol 115:565574 (2005) Mol Pharmacol 66:14651477 (2004) S Biol Pharm Bull 28:886892 (2005) Eur Arch Otorhinolaryngol 264:525530 (2007) Mol Immunol 44:28502859 (2007) Genes Immun 7:615624 (2006) Cancer Immunol Immunother 58(9):1375-1385 (2009) Br J Cancer 97:598-604 (2007) JI 176:48944901 (2006) Immunol 115:565574 (2005) S Biol Pharm Bull 28:886892 (2005) Eur Arch Otorhinolaryngol 264:525530 (2007) Mol Immunol 44:28502859 (2007) Clin Cancer Res 13:45654574 (2007) Cancer Immunol Immunother 58(9):1375-1385 (2009) Mol Immunol 44:28502859 (2007) Immunol 115:565574 (2005) Clin Cancer Res 14:856-864 (2008) Cancer Immunol Immunother 58(9):1375-1385 (2009) Leuk Lymphoma 46:935-939 (2005) Cancer Immunol Immunother 58(9):1375-1385 (2009)

TLR2

Colon cancer (RNA, protein) Gastric cancer (protein) Hepatocellular carcinoma (RNA) Laryngeal carcinoma (protein) Lung cancer (RNA) Lymphoma (DNA) Ovarian tumor (protein) Pancreatic carcinoma (Incompletely resectable)

TLR3

Breast cancer (RNA) Colon cancer (RNA) Hepatocellular carcinoma (RNA) Laryngeal carcinoma (protein) Lung cancer (RNA) Melanoma (protein) Ovarian tumor (protein)

TLR4

Breast cancer (RNA) Colon cancer (RNA) Melanoma (IV stage) (protein) Ovarian tumor (protein)

TLR8

Chronic lymphocytic leukemia (RNA, protein) Ovarian tumor (protein)

TLR9

Breast cancer (RNA, protein)

RT-PCR, WB, IHC, FACS, Functional Assay IHC, Functional Assay IHC Functional Assay RT-PCR RT-PCR, IHC, Functional Assay Functional Assay RT-PCR IHC WB, IHC, Functional Assay RT-PCR RT-PCR RT-PCR Functional Assay

Mol Cancer Res 4:437447 (2006)

Cervical squamous cell carcinoma (protein) Gastric carcinoma (protein) Glioma (protein) Hepatocellular carcinoma (RNA) Lung cancer (RNA, protein) Lymphoma (DNA) Melanoma (IIIb/c or IV stage) (RNA) Ovarian tumor (protein) Prostate cancer (protein) Recurrent glioblastoma (RNA) Recurrent non-Hodgkin lymphoma (RNA) TLR10 Nasopharyngeal carcinoma (RNA) Prostate cancer (DNA)

Increased Expression Increased Expression Increased Expression Increased Expression Increased Expression Single-nucleotide polymorphism Moderate Expression Increased Expression Increased Expression Moderate Expression Moderate Expression Increased Expression Single-nucleotide polymorphism

Mol Carcinog 46:941947 (2007) Int J Med Microbiol 295:179185 (2005) Glia 54:526535 (2006) S Biol Pharm Bull 28:886892 (2005) Respir Res 6:1 (2005) Genes Immun 7:615624 (2006) J Clin Oncol 24:5716-5724 (2006) Cancer Immunol Immunother 58(9):1375-1385 (2009) Prostate 67:774781 (2007) Neuro- Oncol 8:60-66 (2006) J Immunother 29: 558-568 (2006) Cancer Epidemiol Biomarkers Prev 15:862866 (2006) J Natl Cancer Inst 97:525532 (2005)

TLRs in Tumor Cells and Tumor Cell Lines

Tumor cell lines are also widely used to study TLRs (Reviewed in Toll-like Receptors IMGENEX & Innate Immunity : The Story Tolld TLR Handbook 2009). One example is illustrated in Table 3. IMGENEX antibodies are widely used to analyze TLR expression in both tumor tissue and tumor cell lines. For example, Droemann et al (2005) analyzed the expression of TLR9 in both human lung carcinoma and in the A549 human lung carcinoma cell line (Fig 1 Top). The results showed that there was high TLR9 expression in the cytoplasm of tumors in most lung cancer specimens. TLR9 was also expressed in the cytoplasm of A549 and other tested tumor cell lines. In contrast, there was only sporadically weak TLR9
A

Table 3
Tumor Cells/Cell Line NB-1 HCT-15 SW620 MCF7 UACC-2 MDA-MB435 Prostate Cancer Cells Cervical Tumor Cells Gastric Carcinoma Laryngeal Carcinoma Source: B Huang et al 2008 Oncogene 27:218-224 Tissue Organ Neuroblastoma Colon Colon Breast Melanoma Breast TLRs Expressed TLR4 Multiple Multiple Multiple TLR4 Multiple TLR9 TLR9 TLR4, 5, 9 TLR2, 3, 4

TLR9 (IMG-305A)

TLR3 Expression

TLR1 Expression

Figure 2. Western blot analysis of TLR expression in clinical tissue tumor /normal adjacent tissue lysates. A, TLR3 mAb (clone 40C1285.6: IMG-315A). B, TLR1 pAb (IMG-5012). C = colon; O = ovary., T = Tumor, N = Normal adjacent. Six patient donor pairs are shown. The tissue specimens were prepared as illustrated in Figure 4. The clinical tissue lysates are packaged native and should be denatured prior to SDS-PAGE and western blot analysis. Denatured packaging is available upon request. The variable banding pattern of TLR1 may result from TLR cleavage products or alternate expression forms in the samples, however this has not been characterized. Lysate Cat nos: CT1/N1(C003-005-T1/N1, CT2/N2 (C003-012-T1/N1), CT3/N3 (C003-019-T1/N1), CT4/N4 (C003-026-T1/N1), CT5/N5 (C003-035-T1/N1), OT1/N1 (C005-012-T1/N1).

expression in non-malignant (normal adjacent) lung tissue. The specicity of the antibody was veried in GFP-TLR9 A549 cells where TLR9 mAb and GFP uorescence colocalized (Fig 1 Bottom). The signicance of TLR expression patterns in cancer and their variation from normal/normal adjacent tissue as well as tumor-tumor variation remains to be fully elucidated. However, there is evidence that TLRs expressed in tumor cell lines are functional. For example, Droemann et al (2005) showed that A549 expressed TLR9 (Fig 2), and that CpG-ODN induced secretion of the chemokine MCP-1 which inhibited apoptosis. These results are indicative of functional TLR9 receptors in A549 tumor cells. TLR expression in tumor cells has also been conducted using Western Blotting techiques. An example of TLR expression screening in clinical tissue lysates in shown in Figure 3. The data shows that TLR3 was detected in all of the samples (Fig 3A). This data may suggest that TLR3 expression is ubiquitous in colon tumors/normal adjacent tissue.However, screening additional patient donors and tumor types would be needed to determine the signicance of this nding.

Figure 1. (Top) Immunohistochemisty (formalin-xed, parafn embedded) analysis of TLR9 in lung tumor/normal adjacent tissue and in A549 lung carcinoma cells using TLR9 mAb, Clone 26C593.2 (IMG-305A). Lung tumor and normal adjacent tissues were obtained from the same lung. TLR9 expression is observed in the cytoplasm as shown by the brown stain. Much less TLR9 staining is seen in the norma adjacent than the tumor tissue. Source: Droemann et al, 2005. (Bottom) Specicity validation of TLR9 mAb, Clone 26C593.2 (IMG-305A) by confocal microscopy using GFP-TLR9 transfected cells. Researchers sometimes perform specicity verication of antibodies in their own hands to determine the suitability of antibodies for their model systems. A549 cells were transiently transfected with a human GFP-TLR9 vector. TOTO-3 is a nuclear dye. GFP and TLR9 signals were cytoplasmic, and the GFP signals coincided with TLR9 mAb signals (overlay) indicating the specicity of the antibody for TLR9. Source: Droemann et al, 2005.

Attack on tumor cells diminished or blocked

Tumor Cell
TLR Ligand LPS
MyD88 TLR4 TIRAM TIRAP TRIF
NF- B

Perforin Granzymes

NK

Activation Factors IFN TNF

X
Fas FasL
Treg

CTL

Immunosuppressive factors secreted inhibiting effector cell activity

TGF IL-35 IL-10

Pro-inflammatory factors secreted Nitric Oxide IL-12 IL-6 IL-1 TNF PGE 2

c c c
NK/T CD8+ suppressor

T
IFN IL17 TNF IL4 IL10

IFN TGF IL4 IL10

IFN TGF IL4 IL10

Figure 3. TLRs, inammatory processes and cancer. Cancer cells can express functional TLRs. PAMP and DAMP activated TLR signals on cancer cells may promote cancer progression, inhibition of apoptosis or other cell death pathways, or resistance to host immune responses.

In contrast to TLR3, TLR1 was differentially expressed (Fig 3B). For example, TLR1 was detected in the colon tumor (CT5) but not the normal adjacent (CN5) sample of Donor #5, and not in either sample of Donor #3 (neither CT3 nor CN3). This example reects the IMGENEX ready-to-use western blots (INSTA-BlotsTM) and protocols.

depth elucidation of the TLR control and functional activities directing tumorigenesis or tumoricidal activities represents an entirely new area approach. TLR and Cancer Biology is a rich opportunity for discovery of paradigms and development of research, diagnostic or therapeutic products . Harnessing the TLR signaling pathway for cancer immunotherapy and vaccines may prove to be among the most promising cancer prevention and management strategies of the twenty-rst century. An updated overview of TLRs can be found in the TLR Handbook and a complete IMGENEX TLR product listing follow the link at www.imgenex.com
References
Conroy H, NA Marshall, KHG Mills. TLR ligand suppression or enhancement of Treg cells? A double-edged sword in immunity to tumours. Oncogene 27:168-180 (2008). Droemann D, D Albrecht, J Gerdes, AJ Ulmer, D Branscheid, E Vollmer, K Dalhoff, P Zabel, T Goldmann. Human lung cancer cells express functionally active Toll-like receptor 9. Respiratory Res 6:1 doi:10.1186/1465-9921-6-1 (2005). Huang B, J Zhao, JC Unkeless, ZH Feng, H Xiong. TLR signaling by tumor and immune cells: a double-edged sword. Oncogene 27:218-224 (2008). Makkouk A, AM Abdelnoor. The potential use of Toll-like receptor (TLR agonists) and antagonists as prophylactic and/or therapeutic agents. Immunopharmacol and Immunotoxicol 31:331-338 (2009). Phrma. Pharmaceutical industry prole 2009. http://www.phrma.org/les/PhRMA%20 2009%20Prole%20FINAL.pdf (2009). Rakoff-Nahoum and R Medzhitov. Toll-like receptors and cancer. Nat Revs Cancer 9:5763 (2009). Toll-Like Receptors IMGENEX & Innate Immunity Overview & Handbook www.imgenex. com (2010).

TLRs and Mechanisms of Tumorigenesis

Tumors are inltrated with various types of immune cells, and immune cells can be the major cell population in the tumor microenvironment. Although tumor cells are dependent on the microenvironment for growth and survival signals, tumor cells are also under immune surveillance. Immune cells in the microenvironment can recognize tumor cells as foreign, and mechanisms such as TLR signaling can be activated to inhibit or destroy them. However, tumors have multiple mechanisms that evade or overcome immune surveillance and avoid attack by the hosts immune system. Emerging evidence suggests that TLR signaling is among these mechanisms. Figure 1 shows an overview of how TLR signaling may participate in helping tumor cells evade attack by the host immune TLR system. The signicance of TLR expression patterns in cancer and their variation from normal/normal adjacent tissue as well as tumor-tumor variation remains to be fully elucidated. More in

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