Tumor cytotoxicity and endothelial Rac inhibition induced by TNP-470 in anaplastic thyroid cancer.

Anaplastic thyroid carcinoma is an aggressive form of cancer with no treatment. Angiogenesis inhibitors, such as TNP-470, a synthetic derivative of fumagillin, have been shown to reduce tumor size and increase survival in heterotopic animal models of thyroid cancer. Our goals were to determine the effect of TNP-470 on anaplastic thyroid cancer using an orthotopic murine model, to identify the molecular pathways of TNP-470 actions on endothelial cells, and to determine the non-endothelial tumor effects of TNP-470. We injected human anaplastic thyroid carcinoma cells (DRO'90) into the thyroid glands of nude mice. Mice received TNP-470 (30 mg/kg) s.c. for 6 weeks. TNP-470 prolonged survival and reduced liver metastases. TNP-470 had direct cytotoxic effects on anaplastic thyroid carcinoma cells in vitro and in vivo. Paradoxically, TNP-470 increased vascular endothelial growth factor secretion from tumor cells in vitro and in vivo. However, there was no associated increase in tumor microvessel density. In endothelial cells, TNP-470 prevented vascular endothelial growth factor-induced endothelial permeability, intercellular gap formation, and ruffle formation by preventing Rac1 activation.

NADPH oxidase mediates vascular endothelial cadherin phosphorylation and endothelial dysfunction.

Vascular endothelial activation is an early step during leukocyte/endothelial adhesion and transendothelial leukocyte migration in inflammatory states. Leukocyte transmigration occurs through intercellular gaps between endothelial cells. Vascular endothelial cadherin (VE-cadherin) is a predominant component of endothelial adherens junctions that regulates intercellular gap formation. We found that tumor necrosis factor (TNF) caused tyrosine phosphorylation of VE-cadherin, separation of lateral cell-cell junctions, and intercellular gap formation in human umbilical vein endothelial cell (HUVEC) monolayers. These events appear to be regulated by intracellular oxidant production through endothelial NAD(P)H (nicotinamide adenine dinucleotide phosphate) oxidase because antioxidants and expression of a transdominant inhibitor of the NADPH oxidase, p67(V204A), effectively blocked the effects of TNF on all 3 parameters of junctional integrity. Antioxidants and p67(V204A) also decreased TNF-induced JNK activation. Dominant-negative JNK abrogated VE-cadherin phosphorylation and junctional separation, suggesting a downstream role for JNK. Finally, adenoviral delivery of the kinase dead PAK1(K298A) decreased TNF-induced JNK activation, VE-cadherin phosphorylation, and lateral junctional separation, consistent with the proposed involvement of PAK1 upstream of the NADPH oxidase. Thus, PAK-1 acts in concert with oxidase during TNF-induced oxidant production and loss of endothelial cell junctional integrity.

Mutations in the Trp53 gene of UV-irradiated Xpc mutant mice suggest a novel Xpc-dependent DNA repair process.

Mutational hot spots in the human p53 gene are well established in tumors in the human population and are frequently negative prognosticators of the clinical outcome. We previously developed a mouse model of skin cancer with mutations in the xeroderma pigmentosum group C gene (Xpc). UVB radiation-induced skin cancer is significantly enhanced in these mice when they also carry a mutation in one copy of the Trp53 gene (Xpc-/-Trp53+/-). Skin tumors in these mice often contain inactivating mutations in the remaining Trp53 allele and we have previously reported a novel mutational hot spot at a non-dipyrimidine site (ACG) in codon 122 of the Trp53 gene in the tumors. Here we show that this mutation is not a hot spot in Xpa or Csa mutant mice. Furthermore, the mutation in codon T122 can be identified in mouse skin DNA from (Xpc-/-Trp53+/-) mice as early as 2 weeks after exposure to UVB radiation, well before histological evidence of dysplastic or neoplastic changes. Since this mutational hot spot is not at a dipyrimidine site and is apparently Xpc-specific, we suggest that some form of non-dipyrimidine base damage is normally repaired in a manner that is distinct from conventional nucleotide excision repair, but that requires XPC protein.

A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc mutant mice alters transactivation functions.

A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122-->L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc(-/-)) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122-->L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122-->L allele in p53(-/-) mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122-->L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122-->L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122-->L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122-->L mutant allele in the Xpc(-/-) background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.