Targeting Interleukin-4 Receptors in Head and Neck Cancer
Targeting Interleukin-4 Receptors in Head and Neck Cancer
By Koji Kawakami, MD and Raj K. Puri, MD, PhD
Squamous cell carcinoma of the head and neck (scchn) represents approximately 5% of all cancers; about 70,000 new cases were diagnosed in the United States in 1997.1 Although advances in surgical reconstruction and combined modality therapy have improved functional outcome, there has been no significant improvement in survival in the past 30 years.2 In an attempt to develop novel therapeutic agents, we have observed that a variety of murine and human carcinoma cells express high levels of high-affinity receptors for interleukin 4 (IL-4) in vitro and in vivo.3-6 IL-4 is a pleiotropic immune cytokine predominantly produced by Th2 and mast cells.4 Although the importance of IL-4 receptor (IL-4R) expression on solid tumor cells is not known, we and others have observed that solid human tumor cells derived from malignant melanoma, breast carcinoma, ovarian carcinoma, mesothelioma, neuroblastoma, renal cell carcinoma, non-small cell lung carcinoma, and AIDS-associated Kaposi’s sarcoma respond to IL-4.5-10
Characterization of IL-4
We have previously reported that the IL-4R system could exist in three different types. Type I receptors are shown to consist of a primary IL-4 binding 140 kDa protein (IL-4Ra, also known as IL-4Rb) and IL-2 receptor g chain (IL-2Rgc). Type II receptors are composed of IL-4Rb and IL-13Ra’ (also known as IL-13Ra1) chains. In a third type of IL-4R, all three chains may form an IL-4R complex.11,12
In radiolabeled binding studies we have found that 125I-IL-4 binds to SCCHN cell lines at varying degrees and excess of unlabeled IL-4 displaced the binding of 125I-IL-4. IL-13 also displaced 125I-IL-4 binding; however, IL-4 was slightly superior to IL-13. To further characterize the IL-4Rs in SCCHN cell lines, we performed Scatchard analysis on one cell line (YCUM862). Scatchard analysis of the binding data showed a single type of receptor with a Kd value of ~38 nM. The number of IL-4Rs was calculated as 476,000 ± 5,000 IL-4 molecules bound/cell.
We next examined the structure of IL-4R on SCCHN cell lines. In 17 SCCHN cell lines studied, we found that IL-4Rb and IL-13Ra’ chains were expressed but IL-2Rgc was not expressed as determined by Northern and RT-PCR analyses. These results suggested that SCCHN cells express type II IL-4R in which the IL-4Rb chain forms a productive complex with the IL-13Ra’ chain.18 Furthermore, IL-4R appeared to be related to IL-13R as IL-13 also displaced 125I-IL-4 binding. The structure of IL-4R on SCCHN cells appeared to be similar to that observed on a variety of human solid tumor cell types, e.g., colon cancer, renal cell carcinoma, AIDS-associated Kaposi’s sarcoma, glioblastoma multiforme, and breast carcinoma.6,7,14,15 Thus, it is reasonable to assume that all solid cancers that express IL-4R may express type II receptors.
To determine whether IL-4Rs expressed on cancer cells are biologically functional, we analyzed STAT6 activation in response to IL-4 in four SCCHN cell lines. It has been shown that for signaling, IL-4 can activate STAT6 protein in various cell types.11,12,16 In SCCHN cell lines, IL-4 also induced STAT6 activation in three of four cell lines examined, suggesting that IL-4Rs on SCCHN cells are functional. To further analyze the function of IL-4R, we tested the effect of IL-4 on the growth of SCCHN cell lines. Myers et al reported that IL-4 could stimulate the growth of SCCHN cell lines.17 In contrast, in our observations, IL-4 showed no effect on SCCHN cells.13,18 The reason for this differential effect is not known; however, it is possible that the IL-4 effect is tumor specific, representing the heterogeneous nature of this cancer.
To target IL-4Rs, we utilized a chimeric protein, (IL4(38-37)-PE38KDEL or cpIL4-PE) composed of circular permuted IL-4 and a truncated form of a powerful bacterial toxin called Pseudomonas exotoxin (PE). This protein has been shown to be highly cytotoxic to IL-4R-positive tumor cells in vitro and in vivo.7,14,15,19-21 cpIL4-PE was also found to be highly and specifically cytotoxic to IL-4R-positive cells in 17 SCCHN cell lines. The IC50 (the protein concentration required for the inhibition of protein synthesis by 50%) in 15 of 17 SCCHN cell lines was less than 10 ng/mL, indicating that SCCHN cells are highly sensitive to cpIL4-PE. The cytotoxic activity of cpIL4-PE was neutralized by excess IL-4 and IL-13. YCUM862 cells expressed extremely high numbers of IL-4Rs; consequently, these cells were extremely sensitive to cpIL4-PE (IC50 = 0.75 ng/mL). cpIL4-PE induced DNA fragmentation and condensation of nuclei indicative of apoptotic cell death in KB SCCHN cell line. However, IL-4 itself did not show apoptosis in this cell line. The cytotoxic activity of cpIL4-PE was confirmed by clonogenic assay. These results suggest that IL-4Rs expressed on head and neck cancer cells serve as an efficient target for cpIL4-PE similar to what has been observed in several solid tumor cells.14,15,19-21
Antitumor Activity
Our previous studies have demonstrated that in vitro sensitivity to IL-4 toxin correlates with in vivo antitumor activity in brain tumor, AIDS-associated Kaposi’s sarcoma, epidermoid carcinoma, and breast tumor models in nude mice.7,14,19 Based on these observations, it is reasonable to predict that cpIL4-PE will have significant in vivo antitumor activity in SCCHN in athymic nude mice with subcutaneously growing tumors. We are currently examining the antitumor activity by systemic administration of cpIL4-PE in a SCCHN xenograft model.
Various innovative approaches, including gene transfer, are being tested for SCCHN. Although these techniques seem to be promising, currently no approach appears to be more effective. In addition, these approaches are limited by vector-related toxicities and suboptimal gene transfer. Since cpIL4-PE is a small molecule and does not involve an indirect mechanism of tumor cell kill, we believe that it may have superior antitumor activity without producing unknown virus or plasmid-related toxicities.
Conclusion
Of the SCCHN tumor cell lines we examined, 100% expressed surface IL-4 receptors that appear to be biologically functional. Since IL-4 toxin, IL4(38-37)-PE38KDEL, has profound cytotoxic activity against most of the tested cell lines, we conclude that IL-4R may serve as a unique target for the delivery of a cytotoxic agent to SCCHN. Further studies must be performed to investigate the antitumor activity of cpIL4-PE in animal models and perhaps a Phase I clinical trial should be undertaken to study its antitumor activity. (Dr. Kawakami is a Postdoctoral Fellow in the laboratory of Dr. Puri in the Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD. )
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