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Headpin: a Serpin with Endogenous and Exogenous Suppression of Angiogenesis

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Headpin: a Serpin with Endogenous and Exogenous Suppression of Angiogenesis Research Article Headpin: A Serpin with Endogenous and Exogenous Suppression of Angiogenesis Thomas D. Shellenberger,1 Abhijit Mazumdar,1 Ying Henderson,1 Katrina Briggs,1 Mary Wang,1 Chandrani Chattopadhyay,1 Arumugam Jayakumar,1 Mitchell Frederick,1 and Gary L. Clayman1,2 Department of 1Head and Neck Surgery and 2Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas Abstract range of cancers. Several highly conserved serpin genes, including Headpin is a novel serine proteinase inhibitor (serpin) with plasminogen activator inhibitor 1 and 2 (PAI-1 and PAI-2), constitutive mRNA expression in histologically normal oral squamous cell carcinoma antigen 1 and 2 (SCCA-1 and SCCA-2), mucosa but with lost or down-regulated expression in head maspin, and headpin, cluster to chromosome 18q21.3 to q23 (1). and neck squamous cell carcinoma. Several serpin family This locus is a site of frequent loss in multiple cancer types (2–5). members are similarly lost in multiple cancer types and Moreover, the loss of serpin expression has been correlated with hold tumor suppressor functions including the inhibition advanced tumor progression (6–9). of angiogenesis. However, the functional significance for We initially identified headpin as a head and neck serpin that the loss of headpin expression in cancer is not known. Using shows strong constitutive expression in histologically normal oral immunohistochemical analysis of invasive squamous cell mucosa but profoundly reduced mRNA expression in head and carcinoma and matched normal squamous mucosa of neck squamous cell carcinoma (HNSCC; ref. 10). The gene was patient specimens, headpin expression was lost or down- independently discovered by Abts et al. (11) on the basis of regulated in the vast majority of tumor specimens. We differential expression in UV-modulated keratinocytes. We then investigated the functions of exogenous recombinant head- showed the inactivity of the functional promoter region of headpin pin and endogenously expressed headpin related to angio- in head and neck carcinoma lines, consistent with dysregulation of genesis. In a rat corneal assay of neovascularization, transcription as an explanation for the differential expression of recombinant headpin protein blocked in vivo angiogenesis headpin (12). Subsequently, we andothersinvestigatedthe mediated by interleukin 8 (IL-8) and vascular endothelial interaction of recombinant headpin (r-headpin) protein with target growth factor (VEGF). In assays of cellular events in enzymes and found that r-headpin inhibits the cysteine proteinases angiogenesis, headpin blocked the invasion, migration, and cathepsin K and L (13, 14). tube formation of endothelial cells. In light of our findings of A paradigm for serpin function in cancer is based on the nuclear subcellular localization of headpin, we investigated inhibition of proteinases in the cascade of extracellular matrix the expression and secretion of angiogenic factors and found degradation, a critical pathway in angiogenesis, tumor invasion, reduced mRNA, protein, and promoter activities of IL-8 and and metastasis. For instance, by reacting to form a stable complex VEGF. Finally, using a murine flank tumor model, headpin with urokinase plasminogen activator, the extracellular forms of expression reduced growth and microvessel density in PAIs employ a suicide substrate-like inhibition mechanism (15). tumors derived from headpin-expressing UMSCC1 cells However, the role of serpins in cancer has proved more complex on relative to those from vector control cells. These findings the basis of the findings of several studies investigating mecha- of nuclear regulatory functions of a serpin in the inhibition nisms of serpin function. For example, Darnell et al. (16) have of angiogenesis bring new understanding to the cellular and shown a novel intracellular function of PAI-2 in the inhibition of molecular mechanisms of serpins. Therefore, this novel retinoblastoma protein degradation. Additionally, the tumor serpin targets diverse mechanisms against tumor angiogen- suppressor functions of maspin seem to involve complex non- esis on which to base therapeutic strategies. (Cancer Res 2005; inhibitory mechanisms associated with the cell surface and 65(24): 11501-9) cytoplasm in addition to extracellular secretion (17–19). Moreover, recent studies in which immunohistochemical analysis was done Introduction have shown the nuclear and cytoplasmic localization of maspin in specimens from patients with breast, lung, and ovarian cancers The serine proteinase inhibitor (serpin) family includes mem- (20–22). The correlation of nuclear localization of maspin with bers with tumor suppressor functions that oppose tumorigenesis, favorable clinicopathologic features in these cancers suggests that proliferation, angiogenesis, invasion, and metastasis in a broad serpins regulate nuclear functions. Therefore, although the role of serpins in cancer is known, the cellular and molecular events responsible for tumor suppression Note: Presented at the American Association of Cancer Research 96th Annual are not well understood. Because of the diverse involvement of Meeting, April 18, 2005, Anaheim, California. serpins in cancer progression, these proteins offer promising T.D. Shellenberger and A. Mazumdar contributed equally to this work. therapeutic potential by targeting multiple mechanisms against Requests for reprints: Gary L. Clayman, Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, tumor growth, invasion, and metastasis. However, an improved Unit 441, Houston, TX 77030-4009. Phone: 713-794-1032; Fax: 713-745-2234; E-mail: understanding of the cellular and molecular mechanism of serpins [email protected]. I2005 American Association for Cancer Research. in cancer is requisite to developing serpin-based strategies of doi:10.1158/0008-5472.CAN-05-2262 tumor suppression. www.aacrjournals.org 11501 Cancer Res 2005; 65: (24). December 15, 2005 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Cancer Research To gain a clearer understanding of the mechanisms whereby determine the area of neovascularization by calculating the total area of serpins function in tumor suppression, we sought to determine the new blood vessels within the cornea of each image as previously described biological and functional significance of the loss of headpin (24). The total vascularity was determined by summing the pixel values of expression in HNSCC. We investigated the tumor suppressor effects that area within each image containing new vessels. Experiments using at least six animals were done twice using VEGF and thrice using IL-8 as the of headpin on angiogenesis by studying the effects of r-headpin mediator of angiogenesis. protein on effector cells of the tumor microenvironment and by Endothelial cell assays. HUVECs were grown to 80% to 90% confluence returning the expression of headpin to head and neck cancer cell in DMEM supplemented with 5% fetal bovine serum (FBS). Before the lines. We found that headpin localizes to the nucleus and regulates assays, cells were resuspended in DMEM containing 0.1% bovine serum the transcription of mediators of cell proliferation. Furthermore, albumin. In assays of endothelial invasion, cells were seeded in the top headpin functions intracellularly to regulate the transcription of chamber (150,000 per 250 AL) of 24-well plates with a Matrigel-coated, key angiogenic factors and extracellularly to block several steps in fluorescence-blocking membrane insert (BD Bioscience). Culture medium endothelial cells vital for angiogenesis. (5% FCS) alone or with various concentrations of headpin (750 AL) was placed in the bottom chambers. In assays of endothelial migration, cells were starved in basal growth medium for 2 to 3 hours at 37jC and seeded in Materials and Methods the top chamber (150,000 in 250 AL) of 24-well plates with a fibronectin- Cell cultures, reagents, transfection, and cell extracts. Vector and coated, fluorescence-blocking membrane insert (BD Bioscience). Serum-free headpin stable clones in UMSCC1 and parental Tu138 cell lines were medium with VEGF at 20 ng/mL alone or with various concentrations of maintained in DMEM-F12 with 10% FCS. Human umbilical vein endothelial headpin (750 AL) was placed in the bottom chambers. In assays of cells (HUVEC) were maintained in Medium 131 with microvascular growth endothelial tube formation, cells were seeded (20,000 in 50 AL) into 96-well supplement from Cascade Biologics (Portland, OR). Primary normal oral plates coated with a Matrigel (BD Bioscience) and incubated with 50 ALof epithelial cells were explanted and cultivated in keratinocyte growth serum-free basal growth media alone, with VEGF or IL-8 at 20 ng/mL, or medium (Clonetics, Walkersville, MD) as previously described (23). SF9 with headpin. Cells were allowed to invade or migrate for 22 F 1 hours insect cells were maintained in Insect-XPRESS from Cambrex BioScience or form tubes for 18 F 1 hours at 37jC. Following incubation, cells were (Walkersville, MD). Actin antibody and propidium iodide were obtained labeled with 4 Ag/mL of calcein AM (Molecular Probes, Eugene, OR). from Sigma (St. Louis, MO); antibodies against headpin and CD31 and Migrating or invading cells and endothelial tube lengths were measured by endothelial assays of tube formation, migration, and invasion assay direct fluorescence of cells with a plate reader
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