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Wnt and Frizzled Receptors As Potential Targets for Immunotherapy in Head and Neck Squamous Cell Carcinomas

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Wnt and Frizzled Receptors As Potential Targets for Immunotherapy in Head and Neck Squamous Cell Carcinomas Oncogene (2002) 21, 6598 – 6605 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Wnt and frizzled receptors as potential targets for immunotherapy in head and neck squamous cell carcinomas Chae-Seo Rhee1,2, Malini Sen1, Desheng Lu1, Christina Wu1, Lorenzo Leoni1, Jeffrey Rubin3, Maripat Corr1 and Dennis A Carson*,1 1Department of Medicine and The Sam and Rose Stein Institute for Research on Aging, University of California San Diego, La Jolla, California, CA 92093-0663, USA; 2Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University College of Medicine, Seoul, Korea; 3National Cancer Institute, Bethesda, Maryland, USA The diverse receptor-ligand pairs of the Wnt and frizzled Although metastatic HNSCC can respond to (Fz) families play important roles during embryonic chemotherapy and radiotherapy, it is seldom development, and thus may be overexpressed in cancers adequately controlled. Therefore, it is important to that arise from immature cells. Hence, we investigated identify new molecular determinants on HNSCC that the expression and function of five Wnt (Wnt-1, 5a, 7a, may be potential targets for chemotherapy or immu- 10b, 13) and two Fz (Fz-2, 5) genes in 10 head and neck notherapy. squamous carcinoma cell lines (HNSCC). In comparison In the past decade there has been tremendous to normal bronchial or oral epithelial cells, all the progress in identifying genetic and molecular changes HNSCC had markedly increased mRNA levels of Wnt- that occur during the transformation of malignant 1, 7a, 10b, and 13, as well as Fz-2. Moreover, the levels cells. Many malignant cells have a less differentiated of Wnt-1, 10b, and Fz-2 proteins were also markedly phenotype, and a higher growth fraction than normal increased in HNSCC, relative to normal epithelial cells. adult tissues. These basic characteristics are similar to Treatment of one HNSCC cell line (SNU 1076) with immature or embryonic cells. During the development anti-Wnt-1 antibodies reduced the activity of the Wnt/Fz of the embryo, various cell surface receptors and dependent transcription factor LEF/TCF, and diminished ligands direct tissue pattern formation, and cellular the expression of cyclin D1 and b-catenin proteins. differentiation (Hunter, 1997; Ng et al., 1999; Ramsdell Blocking Wnt-1 signaling also inhibited proliferation and and Yost, 1998). The expression of these receptors and induced apoptosis in these cells. These results show that ligands is often no longer required in fully matured HNSCC cell lines often overexpress one or more Wnt adult tissues. Because they are expressed on the cell and Fz genes, and suggest that the growth and survival of surface, the receptors and ligands important for a subset of HNSCC may depend on the Wnt/Fz morphologic patterning and tissue differentiation could pathway. Hence, the Wnt and Fz receptors may be be targets for the immunotherapy of tumors that have possible targets for immunotherapy therapy of this arisen from residual immature cells, or that have common cancer. undergone de-differentiation. Oncogene (2002) 21, 6598 – 6605. doi:10.1038/sj.onc. Genes of the wingless (Wnt) and frizzled (Fz) class 1205920 have an established role in cell morphogenesis and cellular differentiation (Parr et al., 1993; Riddle et al., Keywords: head and neck squamous cell carcinomas; 1995; Vogel et al., 1995). The Wnt genes encode a frizzled; beta-catenin; apoptosis family of 38 – 45 kDa secreted cysteine-rich proteins that lack transmembrane domains and are modified by N-linked glycosylation. The secreted Wnts associate Introduction with extracellular matrix proteins on or near the cell surface, and thus can exert autocrine or paracrine Head and neck squamous cell carcinoma (HNSCC) is effects. The Wnt proteins are ligands for the Fz the sixth most common cancer in developed countries, receptors, which resemble typical G protein coupled and of the 44 000 annual cases reported in the United receptors. The first member of the 19 known human States approximately 11 000 will result in an unfavor- Wnt genes, Wnt-1, was initially discovered because of able outcome (Landis et al., 1999; Parkin et al., 1999). its oncogenic properties (Nusse and Varmus, 1982). Signaling downstream of some Fz homologs, in response to Wnt-1 or other transforming Wnts, leads *Correspondence: DA Carson, Department of Medicine and The to activation of the phosphoprotein Dishevelled (Dsh), Sam and Rose Stein Institute for Research on Aging, University of which inhibits the ability of glycogen synthase kinase- California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0663, USA; E-mail: [email protected] 3b to phosphorylate b-catenin. Whereas phosphory- Received 21 January 2002; revised 30 July 2002; accepted 31 July lated b-catenin is unstable and degraded rapidly, the 2002 unphosphorylated protein accumulates in the cytosol, Wnt and Fz genes in head and neck cancers C-S Rhee et al 6599 and nucleus, where it binds to members of the TCF/ LEF transcription factor family (Cadigan and Nusse, 1997; Miller et al., 1999). These transcription factor complexes control the activities of specific Wnt target genes, including developmental regulators and other genes involved in co-ordinating cell proliferation, such as cyclin D1, c-myc and fibronectin. The overexpres- sion of b-catenin and LEF-1 has been demonstrated to result in the oncogenic transformation of chicken fibroblasts (Aoki et al., 1999). A recent survey using microarray techniques showed that most HNSCC overexpress mRNAs of the Wnt family (Leethanakul et al., 2000). However, the various Wnt mRNAs are very homologous, and hybridization in microarrays often cannot distinguish between closely related templates. Also, it is not known if Wnt and Fz proteins are expressed in HNSCC cells, and if they play any role in cell growth and survival. The hypothesis that prompted this study was that different clonal populations of HNSCC might over- express various receptors of the Wnt and Fz family, because of their immature cell of origin, and the growth and survival advantage provided by autocrine or paracrine Wnt/Fz signaling. We examined HNSCC and normal human epithelial cell lines for the expression of five Wnt and two Fz genes. The results showed that most HNSCCs did indeed overexpress one or more Wnt and Fz mRNAs. Moreover, the Wnt/Fz pathway was functional in some of the HNSCC cells, Figure 1 (a) RT – PCR amplification for Wnt/Fz family members as indicated by the constitutive expression of a LEF/ in cancer cell lines. Lane 1, DNA standard; lane 2, H2O; lane 3, TCF reporter gene. In the SNU 1076 cell line, anti- U87MG; lane 4, U373MG; lane 5, SCC25; lane 6, AMC4; lane 7, Wnt-1 or anti-Wnt-10b antibodies decreased the SNU1066; lane 8, SNU1076; lane 9, PCI 1; lane 10, PCI 13; lane 11, PCI 50; lane 12, KB; lane 13, Detroit 562; lane 14, RPMI expression of b-catenin and cyclin D1, inhibited cell 2650; lane 15, Lesch; lane 16, Ramos. (b) RT – PCR amplification growth, and induced apoptosis. Thus, the Wnt and Fz for Wnt/Fz families in normal cells. Lane 1: DNA standard; lane genes are frequently overexpressed in HNSCC, and 2: H2O; lanes 7 and 14: normal human bronchial epithelial cells; might be attractive targets for both immunotherapy other lanes: normal primary oral squamous epithelial cells and drug therapy. Results Table 1 Summary of Wnt and Fz gene expression detected by RT – PCR in normal and cancer cells Expression of Wnt and Fz mRNAs in HNSCC Normal (11) Cancer (14) Ten different HNSCC cell lines, two normal human mRNA Oral SC NHBE Glioblastoma HNSCC B cell tumor amplified (10) (2) (2) (10) (2) broncho-epithelial (NHBE) cell lines, and normal oral squamous epithelial cells were tested by RT – PCR for Wnt-1 0 0 2 10 2 the expression of five Wnts (Wnt-1, Wnt-5a, Wnt-7a, Wnt-5a 0 0 1 4 1 Wnt-10b, Wnt-13), and two Fzs (Fz-2 and 5). Wnt-7a 0 1 0 5 0 Wnt-10b 0 0 0 8 1 Representative results are illustrated in Figure 1 and Wnt-13 0 1 2 10 2 are summarized in Table 1. When compared to the Fz-2 0 1 2 10 2 housekeeping gene G3PDH, all the Wnts, as well as Fz-5 4 1 0 9 1 Fz-2, were expressed more frequently in HNSCC than The numbers indicate the number of cell lines yielding a detectable in normal cells, while there was no difference in Fz-5 PCR product after amplification with primers specific for the gene expression. Of the Wnt genes, Wnt-1, 5a, and 10b indicated Wnt and Fz genes. The top row shows the number of were most strongly expressed by the malignant cells, different squamous cell (SC), normal human bronchial epithelium but were barely detectable in the normal tissues tested. (NHBE), and head and neck squamous cell cancers (HNSCC) tested Real time PCR assays, confirmed that the gene expression levels of Wnt-1 were 17-fold higher, and Wnt-10b was threefold higher in the SNU1076 HNSCC cells than in the normal Detroit 551 cells. catenin and LEF/TCF pathway, and because affinity Subsequently, we focused on Wnt-1 and Wnt-10b, purified antibodies to the extracellular domains were since these Wnts signal through the canonical b- available. Oncogene Wnt and Fz genes in head and neck cancers C-S Rhee et al 6600 by RT – PCR. Beta-catenin was detected in all the Expression of Wnt/Fz proteins in HNSCC samples, including both HNSCC and NHBE lines. Cell lines were lysed and analysed for Wnt-1, Wnt-10b, Fz-2, and b-catenin protein expression by immunoblot- Effects of anti-Wnt antibodies and SFRP1 ting (Figure 2).
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