Therapeutic targeting of the tumor vasculature that destroys preexisting blood vessels of the tumor and antiangiogenesis therapy capitalize on the requirement of tumor cells on an intact vascular supply for oxygen and nutrients for growth, expansion and metastasis to the distal organs. Whereas these classes of agents show promise in delaying tumor progression, they also create glucose and oxygen deprivation conditions within the tumor that could trigger unintended prosurvival responses. The glucose-regulated protein GRP78, a major endoplasmic reticulum chaperone, is inducible by severe glucose depletion, anoxia, and acidosis. Here we report that in a xenograft model of human breast cancer, treatment with the vascular targeting agent, combretastatin A4P, or the antiangiogenic agent, contortrostatin, promotes transcriptional activation of the Grp78 promoter and elevation of GRP78 protein in surviving tumor cells. We further show that GRP78 is overexpressed in a panel of human breast cancer cells that has developed resistance to a variety of drug treatment regimens. Suppression of GRP78 through the use of lentiviral vector expressing small interfering RNA sensitizes human breast cancer cells to etoposide-mediated cell death. Our studies imply that antivascular and antiangiogenesis therapy that results in severe glucose and oxygen deprivation will induce GRP78 expression that could lead to drug resistance. (Cancer Res 2005; 65(13): 5785-91)

Type VII collagen is a major component of anchoring fibrils, attachment structures that mediate dermal-epidermal adherence in human skin. Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder caused by mutations in the type VII collagen gene and perturbations in anchoring fibrils. In this study, we produced recombinant human type VII collagen in stably transfected human 293 cell clones and purified large quantities of the recombinant protein from culture media. The recombinant type VII collagen was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. Purified type VII collagen bound to fibronectin, laminin-5, type I collagen, and type IV collagen and also supported human dermal fibroblast adhesion. In an attempt to establish genotype-phenotype relationships, we generated two individual substitution mutations that have been associated with recessive DEB, R2008G and G2749R, and purified the recombinant mutant proteins. The G2749R mutation resulted in mutant type VII collagen with increased sensitivity to protease degradation and decreased ability to form trimers. The R2008G mutation caused the intracellular accumulation of type VII collagen. We conclude that structural and functional studies of in vitro generated type VII collagen mutant proteins will aid in correlating genetic mutations with the clinical phenotypes of DEB patients.Type VII collagen resides within the basement membrane zone (BMZ) 1 beneath stratified squamous epithelium (1, 2). Type VII collagen is a major component of anchoring fibrils, attachment structures within the basement membrane between the epidermis and dermis of human skin (3, 4). In inherited and autoimmune forms of dystrophic epidermolysis bullosa (DEB) and epidermolysis bullosa acquisita, respectively, anchoring fibrils are diminutive and/or reduced in number (5-10). A genetic linkage has been established between the inherited forms of DEB and type VII collagen (11)(12)(13)(14).Type VII collagen is composed of three identical ␣ chains, each consisting of a 145-kDa central collagenous triple-helical segment characterized by repeating Gly-X-Y amino acid sequences, flanked by a large 145-kDa amino-terminal, noncollagenous domain (NC1), and a small 34-kDa carboxyl-terminal noncollagenous domain (NC2) (4, 15). Within the extracellular space, type VII collagen molecules form antiparallel, tail-to-tail dimers stabilized by disulfide bonding through a small carboxyl-terminal NC2 overlap between two type VII collagen molecules. A portion of the NC2 domain is then proteolytically removed (16, 17). The antiparallel dimers then aggregate laterally to form anchoring fibrils with large globular NC1 domains at both ends of the structure. NC1 domains have been suggested to interact at one end with BMZ components and at the other end with type IV collagen in "anchoring plaques" (2).Our recent study using recombinant NC1 demonstrated that NC1 interacts with various extracellular matrix (ECM) components including fibronectin, lami...

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC2 domain has been implicated in catalyzing the antiparallel dimer formation of type VII procollagen. In this study, we produced the entire 161 amino acids of the NC2 domain plus 186 amino acids of adjacent collagenous domain (NC2/COL) and purified large quantities of the recombinant NC2/COL protein. Recombinant NC2/ COL readily formed disulfide-bonded hexamers, each representing one antiparallel dimer of collagen VII. Removal of the collagenous helical domain from NC2/COL by collagenase digestion abolished the antiparallel dimer formation. Using site-directed mutagenesis, we found that mutation of either cysteine 2802 or cysteine 2804 alone within the NC2 domain blocked antiparallel dimer formation. In contrast, a single cysteine mutation, 2634, within the collagenous helical domain had no effect. A generated methionine to lysine substitution, M2798K, that is associated with recessive dystrophic epidermolysis bullosa, was unable to form antiparallel dimers. Furthermore, autoantibodies from epidermolysis bullosa acquisita patients also reacted with NC2/COL. We conclude that NC2 and its adjacent collagenous segment mediate antiparallel dimer formation of collagen VII. Epidermolysis bullosa acquisita autoantibodies bound to this domain may destabilize anchoring fibrils by interfering with antiparallel dimer assembly leading to epidermal-dermal disadherence.Type VII collagen, a genetically distinct member of the collagen family, is found within the basement membrane zone beneath stratified squamous epithelium (1, 2). Type VII collagen is a major component of anchoring fibrils, attachment structures within the basement membrane between the epidermis and dermis of human skin (3, 4). In inherited forms of dystrophic epidermolysis bullosa (DEB), 1 anchoring fibrils are diminutive and/or reduced in number (5-7). In addition to inherited DEB, epidermolysis bullosa acquisita (EBA) is an acquired autoimmune form of epidermolysis bullosa. EBA is characterized by circulating and tissue-bound IgG autoantibodies to type VII collagen (8, 9). Like DEB, ultrastructural studies have demonstrated a dramatic paucity of anchoring fibrils in EBA skin (10). These observations suggest that type VII collagen plays an important role in maintaining epidermal-dermal adherence. Type VII collagen has been cloned, and a genetic linkage has been established between inherited DEB and mutations in the gene that encodes for type VII collagen, COL7A1 (11-14). There have been over 100 distinct COL7A1 gene mutations identified in patients with DEB, and these mutations have occurred within NC1, NC2, and the helical domain (15,16).Type VII collagen is composed of three identical ␣ chains, each consisting of a 145-kDa central collagenous triple-helical segment, flanked by a large 145-kDa amino-terminal non-collagenous domain (NC1), and a smaller 34-kDa carboxyl-terminal non-collagen...