We have shown previously that gamma-glutamyl transpeptidase (GGT) activity is essential for the nephrotoxicity of cisplatin. In this study we asked whether GGT activity was necessary for the antitumor activity of cisplatin. GGT was transfected into PC3 cells, a human prostate tumor cell line. Two independent GGT-positive cell lines were isolated and characterized. GGT cleaves extracellular glutathione providing the cells with access to additional cysteine. Expression of GGT had no effect on the growth rate of the cells in vitro where the culture medium contains high levels of cysteine. However, when the cells were injected into nude mice the GGT-positive tumors grew at more than twice the rate of the GGT-negative tumors. Weekly treatment with cisplatin was toxic to both GGT-positive and -negative tumors. The GGT-positive tumors were significantly more resistant to the toxicity of cisplatin than the GGT-negative tumors. Therefore, expression of GGT is required for the nephrotoxicity of cisplatin, but diminishes the tumor toxicity of the drug. These results indicate that the nephrotoxicity and the tumor toxicity of cisplatin are via two distinct pathways.

Integrin a6b4Ámediated adhesion interactions play key roles in keratinocyte and epithelial tumor cell biology. In order to evaluate how a6b4 adhesion interactions contribute to these important cellular processes, the authors generated soluble versions of the integrin by recombinant expression of the subunit ectodomains fused to a human immunoglobulin G (IgG) Fc constant domain. Coexpression of the appropriate subunits enabled dimerization, secretion and purification of stable Fc-containing a6b4 heterodimers. The soluble proteins exhibited the same metal ion and ligand dependency in their binding characteristics as intact a6b4. Using these reagents in combination with anti-b4 antibodies, the authors identified two distinct functional epitopes on the b4 subunit. They demonstrated the involvement of one epitope in adhesion interactions and the other in regulating adhesion-independent growth in a6b4-expressing tumor cell lines. The availability of these soluble integrin reagents and the data provided herein help to further delineate the structure-function relationships regulating a6b4 signaling biology.

Integrin alpha6beta4 signaling interactions have been implicated in tumor progression, and beta4 expression has been linked to poor prognosis in certain breast cancer subtypes. We generated human antibodies to alpha6beta4 to further evaluate its role in tumor cell signaling. Biochemical characterization indicated these antibodies are specific for alpha6beta4, recognize distinct epitopes and have low nanomolar affinities for both human and murine protein. The antibodies demonstrated differing effects on alpha6beta4-mediated cellular adhesion, highlighting the existence of different functional epitopes on alpha6beta4. Interestingly however both antibodies blocked adhesion-independent growth in a panel of breast cancer cell lines. Antibody induced apoptosis and inhibition of phosphoinositide 3-kinase (PI3K) signaling were also observed within the context of matrix adhesion. Enhanced inhibitory effects were observed when the alpha6beta4 antibodies were used in combination with antibodies to epidermal growth factor receptor (EGFR) or erythoblastic leukemia viral oncogene homolog 2 (ErbB2). These findings illustrate a role for both the adhesive and signaling functions of alpha6beta4 in breast cancer cell survival. The antibodies and data generated herein advance our understanding of alpha6beta4 in regulating tumorigenic processes, and suggest that combination therapies involving alpha6beta4 may be therapeutically effective in breast cancer.

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