Expression of EphB4 in head and neck squamous cell carcinoma.

Abstract

EphB4 is a receptor tyrosine kinase that is expressed on epithelial cells during fetal life. It is also expressed on some venous endothelial cells. We conducted a study of six men with primary squamous cell carcinoma of the head and neck (HNSCC) that had metastasized to the cervical lymph nodes. Our goal was to determine if EphB4 is aberrantly expressed in cases of HNSCC and to determine if there is a qualitative difference between the expression of EphB4 on primary and metastatic tumors and its expression on normal mucosa adjacent to primary tumors. From each patient, we obtained specimens of the primary tumor, the nodal metastasis, and the adjacent normal mucosa, and we performed immunocytochemistry on each. We observed EphB4 expression in all primary and metastatic tumors and no expression in the normal tissue. In each of the six patients, expression was greater in the metastatic tumor than in the primary tumor. We conclude that EphB4 is a novel target in the treatment of HNSCC.

Introduction

Squamous cell carcinoma, the most common cancer of the head and neck, is caused primarily by tobacco-derived carcinogens. (1) Head and neck squamous cell carcinoma (HNSCC) has been shown to progress through a series of dysplastic histopathologic changes before it becomes an invasive cancer. The differences seen in normal epithelium in the upper aerodigestive tract and in cancer cells that arise from that tissue are the result of mutations in specific genes and alterations of their expression. (2) These genes control a variety of cellular processes, including proliferation and immortalization. Both the activation of oncogenes and the inactivation of tumor suppressor proteins are involved in these pathways.

Receptor tyrosine kinases have been widely implicated in the generation and progression of common human tumors, including HNSCC. (3) One of these kinases is the epidermal growth factor (EGF) receptor (EGFR) gene, which encodes a transmembrane receptor for EGF-family ligands. In HNSCC, EGFR and/or EGF ligand are over-expressed. (4,5)

The largest subfamily of receptor tyrosine kinases is the Eph family, which has 14 fully sequenced members. (6) The prototype of this gene family (EphA1) was isolated from an erythropoietin-producing hepatoma cell line. Initially, the ligands for these receptors were unknown, but they have since been identified. The Eph-family ligands are called ephrins (for Eph receptor interacting proteins). These ligands, like the receptors, are anchored on the plasma membrane via a glycosyl phosphatidylinositol linkage (ephrin-A) or a transmembrane domain (ephrin-B). (7) The binding of ephrins to Ephs requires cell-to-cell contact; soluble forms of ephrins cannot activate their receptors. To date, eight ephrins have been cloned, and each is capable of binding to several receptors. (8)

Both Ephs and ephrins are divided into two subclasses--A and B--on the basis of their sequence homology, structure, and binding affinity. Although initially characterized in the nervous system, (9) recent knock-out studies have suggested that ephrin-B2 and its EphB4 receptor play key roles in vascular development. (10) For example, mouse embryos that lack ephrin-B2 and EphB4 exhibit fatal defects in early angiogenic remodeling. Moreover, ephrin-B2 and EphB4 display remarkably reciprocal distribution patterns during vascular development, with ephrin-B2 marking the endothelium of primordial arterial vessels while EphB4 marks the endothelium of primordial venous vessels. These distributions suggest that ephrin-B2 and EphB4 are involved in establishing arterial vs venous identity, perhaps by fusing arterial and venous vessels at their junctions. Defects in these processes might account for the early lethality observed in mouse embryos that lack these proteins. (9)

EphB4 is also expressed on epithelial cells. Ectopic expression of EphB4 in the breast tissue of transgenic mice shows cooperative tumor induction and metastasis with neuT antigen. Preliminary studies have identified expression of the EphB4 gene in a number of human tumors, including hematologic malignancies, melanomas, glioblastomas, and lung, breast, and colon carcinomas. (10-14) However, data on EphB4 protein levels are limited, and there is a complete lack of data on the significance of this protein in tumor biology. Also, little is known about the expression and role of the ephrin-Eph system in HNSCC.

In this article, we describe our investigation of the pattern of EphB4 expression in a series of specimens obtained from human primary HNSCC tumors, cervical lymph node metastases, and normal mucosal tissue.

Patients and methods

Our study was conducted at the Keck School of Medicine at the University of Southern California in Los Angeles following approval from the university's institutional review hoard. Our study population was made up of six men, aged 38 to 77 years (mean: 59), who had stage IV HNSCC; there were three cases of tonsillar fossa squamous cell carcinoma and one each of laryngeal, tongue, and buccal squamous cell carcinoma. All six patients had cervical lymph node metastases.

Tissue harvest and preservation. Tissue specimens were harvested from three sites in each patient--from the primary tumor, from the clinically involved regional metastatic lymph node, and from normal tissue adjacent to the primary tumor. Each specimen was cut into two parts. One part was placed in a sterile container in dry ice, snap-frozen in liquid nitrogen, and stored at -80[degrees] C. The other part was left unfrozen, fixed in formalin, and analyzed on hematoxylin and eosin staining (H&E) to establish the diagnosis of HNSCC. All tissues were processed immediately following harvest.

Immunohistochemical analysis of frozen-tissue specimens. The tissue blocks of the frozen specimens were sectioned at 5 [micro]m of thickness in a cryostat, mounted on positively charged Superfrost slides (Fisher Scientific; Houston), and air-dried for 30 minutes. Tissues were fixed in cold acetone for 5 minutes and in 1:1 acetone/ chloroform for 5 minutes and then washed with phosphate-buffered saline (PBS) three times for 3 minutes each time. The slides were preincubated with a blocking buffer (0.2% Triton-X100 and 1% BSA in PBS) for 20 minutes. The tissues were then incubated with antibodies to EphB4 (1:100 dilution in PBS) in the blocking buffer at 4[degrees] C for 16 hours.

After three more washes, the slides were incubated with the appropriate fluorescein-conjugated secondary antibodies (Sigma-Aldrich; St. Louis). Nuclei were counter stained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), washed extensively with PBS, and mounted with Vectashield antifade mounting solution (Vector Laboratories; Burlingame, Calif.). Images were obtained with an Olympus AX70 fluorescence microscope and the Spot v2.2.2 digital imaging system (Diagnostic Instruments; Sterling Heights, Mich.).

For histopathologic study, the adjacent sections were stained with H&E. Both immunofluorescence and H&E staining were performed on each section of primary tumor, metastatic tumor, and normal mucosa.

Results

Expression of EphB4 as seen on immunofluorescence staining of the primary tumor (figure, A), metastatic tumor (figure, B), and normal tissue (figure, C) was compared with that seen on H&E staining of the adjacent sections (figure, D, E, and F, respectively).

Primary tumor specimens. EphB4 expression was seen on immunofluorescence staining of the primary tumor specimens obtained from all six patients. EphB4 expression was localized in the tumor and not in the stromal component (figure, A). The greatest signal intensity was seen at the leading edge of the tumor. On H&E staining, the primary tumor specimens exhibited areas of tumor adjacent to areas of the stromal component (figure, D).

Metastatic tumor specimens. Expression of EphB4 was detected in the metastatic nodes obtained from all six patients. On immunofluorescence staining, the EphB4 signal was limited exclusively to the tumor itself; normal areas of the lymph nodes exhibited no signal (figure, B). In each case, the intensity of immunofluorescence staining of EphB4 in the metastatic tumor was higher than that of the primary tumor. H&E staining confirmed tumor morphology in the metastatic specimens (figure, E).

No evidence of EphB4 expression on immunofluorescence staining (figure, C) or tumor on H&E staining (figure, F) was seen in the normal tissue specimens obtained from any patient.

Discussion

The observation that angiogenesis occurs around tumors was made nearly a century ago. (15) In the 1970s, an intense search for pro- and antiangiogenic molecules was spawned by the hypothesis that tumor growth and metastasis are angiogenesis-dependent. (16) It is now widely accepted that the "angiogenic switch" is "off" when the effect of proangiogenic molecules is balanced by that of antiangiogenic molecules, and the switch is "on" when the net balance is tipped in favor of angiogenesis. (17) Various factors that trigger this switch have been discovered. They include metabolic stress (e.g., low P[O.sub.2] or low pH), mechanical stress (e.g., pressure generated by proliferating cells), response to immune and/or inflammatory cells that have infiltrated tissue, and genetic mutations (e.g., activation of oncogenes or deletion of tumor-suppressor genes that control the production of angiogenesis regulators). (18) The means by which the interplay among environmental and genetic mechanisms influences tumor angiogenesis is a complex and largely unresolved matter.

Tumors and metastases may arise as small avascular masses that subsequently induce the angiogenic ingrowths that are required to allow for further growth of early tumors. (19) These tumors are initially separated from underlying vessels by a basement membrane that must be broken before tumor cells can gain access to the vasculature. However, recent studies indicate that tumor cells can initially "home in on" and grow by co-opting existing host vessels and thus start off as well-vascularized small tumors. (20) Angiopoietin 2 (Ang2) and vascular EGF (VEGF) inductions correlate remarkably well with this process. Soon after tumor co-option, host vessels begin expressing high autocrine levels of Ang2. Thus, Ang2 is one of the earliest tumor markers described; it is also one of the most general because it marks co-opted vessels and not the tumor cells themselves. Overexpression of Ang2 destabilizes vessels and enhances apoptotic processes. As vessels die, the tumor becomes avascular and hypoxic, resulting in a marked induction of tumor-derived VEGF. These high levels of VEGF prompt the onset of robust angiogenesis that sprouts from host vessels and allows for tumor survival and further growth.

Several studies have shown that a high expression of ephrins may be associated with an increased potential for tumorigenesis, including growth and metastasis. (10-14) Up-regulation of ephrins during tumor progression is mediated by several mechanisms. The EphA2 receptor has been found in some tumor-associated endothelial cells. (19) Tumor necrosis factor alpha and interleukin 1-beta, which are produced by leukocytes, induce ephrin-A1 in melanomas as host inflammatory responses to tumor. (10) Members of the Eph receptor gene family and their ligands facilitate cell-cell repulsion (antiadhesive) and promote cell migration. Overexpression of ephrin-B2 and EphB4 has been observed in malignant colonic epithelium. (14)

Our study demonstrated overexpression of EphB4 in primary HNSCC and cervical lymph node metastases. Expression was greater in the metastatic lymph nodes than in the primary tumors, as evidenced by the greater intensity of immunofluorescence staining of EphB4 in the lymph nodes. Although our study did not demonstrate it, EphB4 may play a role in lymphatic metastasis of HNSCC. (14,21-23) We are currently investigating the quantitative differences in the expression of EphB4 with respect to tumor characteristics, induction of EGFRs, inactivation of tumor suppressor genes, and activation of oncogenes. We are also interested in defining the role that EphB4 plays in the development and metastasis of HNSCC. It is apparent that EphB4 can be used as a novel target for treatment delivery in refractory HNSCC. EphB4 inhibitors would be of therapeutic value if EphB4 can be proven to enhance tumor growth or spread.

References

(1.) Boyle JO, Hakim J, Koch W, et al. The incidence of p53 mutations increases with progression of head and neck cancer. Cancer Res 1993;53:4477-80.

(2.) Brachman DG. Molecular biology of head and neck cancer. Semin Oncol 1994;21:320-9.

(3.) van der Geer P, Hunter T, Lindberg RA. Receptor protein-tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol 1994;10:251-337.

(4.) Eisbruch A, Blick M, Lee JS, et al. Analysis of the epidermal growth factor receptor gene in fresh human head and neck tumors. Cancer Res 1987;47:3603-5.

(5.) Reiss M, Stash EB, Vellucci VF, Zhou ZL. Activation of the autocrine transforming growth factor alpha pathway in human squamous carcinoma cells. Cancer Res 1991;51(Pt 1):6254-62.

(6.) Drescher U. The Eph family in the patterning of neural development. Curr Biol 1997;7:R799-807.

(7.) Eph Nomenclature Committee. Unified nomenclature for Eph family receptors and their ligands, the ephrins. [letter]. Cell 1997; 90:403-4.

(8.) Pasquale EB. The Eph family of receptors. Curr Opin Cell Biol 1997;9:608-15.

(9.) Gale NW, Yancopoulos GD. Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development. Gene Dev 1999; 13:1055-66.

(10.) Gerety SS, Wang HU, Chen ZF, Anderson DJ. Symmetrical mutant phenotypes of the receptor EphB4 and its specific trans-membrane ligand ephrin-B2 in cardiovascular development. Mol Cell 1999;4:403-14.

(11.) Easty DJ, Hilt SP, Hsu MY, et al. Up-regulation of ephrin-A1 during melanoma progression. Int J Cancer 1999;84:494-501.

(12.) Kiyokawa E, Takai S, Tanaka M, et al. Overexpression of ERK, an EPH family receptor protein tyrosine kinase, in various human tumors. Cancer Res 1994;54:3645-50.

(13.) Hirai H, Maru Y, Hagiwara K, et al. A novel putative tyrosine kinase receptor encoded by the eph gene. Science 1987;238:1717-20.

(14.) Liu W, Ahmad SA, Jung YD, et al. Coexpression of ephrin-Bs and their receptors in colon carcinoma. Cancer 2002;94:934-9.

(15.) Goldman E. The growth of malignant disease and lower animal with special reference to the vascular system. Lancet 1907;2:1236-40.

(16.) Gullino PM. Angiogenesis and oncogenesis. J Natl Cancer Inst 1978;61:639-43.

(17.) Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57-70.

(18.) Carmeliet P. Developmental biology. Controlling the cellular brakes. Nature 1999;401:657-8.

(19.) Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996;86:353-64.

(20.) Holash J, Maisonpierre PC, Compton D, et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science 1999;284:1994-8.

(21.) Vogt T, Stolz W, Welsh J, et al. Overexpression of Lerk-5/Ep1g5 messenger RNA: A novel marker for increased tumorigenicity and metastatic potential in human malignant melanomas. Clin Cancer Res 1998;4:791-7.

(22.) Tang XX, Brodeur GM, Campling BG, Ikegaki N. Coexpression of transcripts Encoding EPHB receptor protein tyrosine kinases and their ephrin-B ligands in human small cell lung carcinoma. Clin Cancer Res 1999;5:455-60.

(23.) Tang XX, Evans AE, Zhao H, et al. High-level expression of EPHB6, EFNB2, and EFNB3 is associated with low tumor stage and high TrkA expression in human neuroblastomas. Clin Cancer Res 1999;5:1491-6.

From the Department of Otolaryngology-Head and Neck Surgery (Dr. Sinha and Mr. Parsa), the Department of Medicine (Mr. Kundra. Dr. Smith. Dr. Masood, and Dr. Gill), and the Department of Pathology (Dr. Sealia), Keck School of Medicine, University of Southern California, Los Angeles.

Reprint requests: Parkash S. Gill, MD, USC/Norris Comprehensive Cancer Center, Room 6332, 1441 Eastlake Ave., Los Angeles, CA 90089-9172. Phone: [phone]; fax: [phone]; e-mail: [e-mail]