Acute biliary obstruction leads to periductal myofibroblasts and fibrosis, the origin of which is uncertain. Our study provides new information on this question in mice and humans. We show that bile duct obstruction induces a striking increase in cholangiocyte ␣v␤6 integrin and that expression of this integrin is directly linked to fibrogenesis through activation of transforming growth factor beta (TGF-␤). Administration of blocking antibody to ␣v␤6 significantly reduces the extent of acute fibrosis after bile duct ligation. Moreover, in ␤6-null mice subjected to the injury, fibrosis is reduced by 50% relative to that seen in wild-type mice, whereas inflammation occurs to the same extent. The data indicate that ␣v␤6, rather than inflammation, is linked to fibrogenesis. It is known that ␣v␤6 binds latent TGF-␤ and that binding results in release of active TGF␤. Consistent with this, intracellular signaling from the TGF␤ receptor is increased after bile duct ligation in wild-type mice but not in ␤6 ؊/؊ mice, and a competitive inhibitor of the TGF␤ receptor type II blocks fibrosis to the same extent as antibody to ␣v␤6. In a survey of human liver disease, expression of ␣v␤6 is increased in acute, but not chronic, biliary injury and is localized to cholangiocyte-like cells. Conclusion: Cholangiocytes respond to acute bile duct obstruction with markedly increased expression of ␣v␤6 integrin, which is closely linked to periductal fibrogenesis. The findings provide a rationale for the use of inhibitors of ␣v␤6 integrin or TGF␤ for down-regulating fibrosis in the setting of acute or ongoing biliary injury. (HEPATOLOGY 2007;46:1404-1412

The transforming growth factor (TGF)-␤-inducible integrin ␣v␤6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-␤. Herein , we show that ␣v␤6 is overexpressed in human kidney epithelium in membranous glomerulonephritis , diabetes mellitus , IgA nephropathy , Goodpasture's syndrome , and Alport syndrome renal epithelium. To assess the potential regulatory role of ␣v␤6 in renal disease , we studied the effects of functionblocking ␣v␤6 monoclonal antibodies (mAbs) and genetic ablation of the ␤6 subunit on kidney fibrosis in Col4A3 ؊/؊ mice , a mouse model of Alport syndrome. Expression of ␣v␤6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with ␣v␤6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in ␤6-deficient Alport mice. Transcript profiling of kidney tissues showed that ␣v␤6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-␤ RII treatment , suggesting shared regulatory functions of ␣v␤6 and TGF-␤. These findings demonstrate that ␣v␤6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target.

Although we have made great progress in understanding the complex genetic alterations that underlie human cancer, it has proven difficult to identify which molecularly targeted therapeutics will benefit which patients. Drug-specific modulation of oncogenic signaling pathways in specific patient subpopulations can predict responsiveness to targeted therapy. Here, we report a pathway-based phosphoprofiling approach to identify and quantify clinically relevant, drug-specific biomarkers for phosphatidylinositol 3-kinase (PI3K) pathway inhibitors that target AKT, phosphoinositide-dependent kinase 1 (PDK1), and PI3K-mammalian target of rapamycin (mTOR). We quantified 375 nonredundant PI3K pathway-relevant phosphopeptides, all containing AKT, PDK1, or mitogen-activated protein kinase substrate recognition motifs. Of these phosphopeptides, 71 were drug-regulated, 11 of them by all three inhibitors. Drug-modulated phosphoproteins were enriched for involvement in cytoskeletal reorganization (filamin, stathmin, dynamin, PAK4, and PTPN14), vesicle transport (LARP1, VPS13D, and SLC20A1), and protein translation (S6RP and PRAS40). We then generated phosphospecific antibodies against selected, drug-regulated phosphorylation sites that would be suitable as biomarker tools for PI3K pathway inhibitors. As proof of concept, we show clinical translation feasibility for an antibody against phospho-PRAS40(Thr246). Evaluation of binding of this antibody in human cancer cell lines, a PTEN (phosphatase and tensin homolog deleted from chromosome 10)-deficient mouse prostate tumor model, and triple-negative breast tumor tissues showed that phospho-PRAS40(Thr246) positively correlates with PI3K pathway activation and predicts AKT inhibitor sensitivity. In contrast to phosphorylation of AKT(Thr308), the phospho-PRAS40(Thr246) epitope is highly stable in tissue samples and thus is ideal for immunohistochemistry. In summary, our study illustrates a rational approach for discovery of drug-specific biomarkers toward development of patient-tailored treatments.

The A v B 6 integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of A v B 6 expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of A v B 6 in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent A v B 6 expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate A v B 6 expression. Detroit 562 cells showed A v B 6 -dependent adhesion and activation of transforming growth factor-B (TGF-B) that was inhibited >90% with an A v B 6 blocking antibody, 6.3G9. Although both recombinant soluble TGF-B receptor type-II (rsTGF-BRII-Fc) and 6.3G9 inhibited TGF-B-mediated Smad2/ 3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-BRII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by A v B 6 antibodies, our findings support a role for A v B 6 in human cancer and underscore the therapeutic potential of function blocking A v B 6 antibodies. [Cancer Res 2008;68(2):561-70]

Integrin ␣ 4 ␤ 1 plays an important role in inflammatory processes by regulating the migration of lymphocytes into inflamed tissues. Here we evaluated the biochemical, pharmacological, and pharmacodynamic properties and efficacy in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, of two types of ␣ 4 ␤ 1 inhibitors, the anti-rat ␣ 4 monoclonal antibody TA-2 and the small molecule inhibitor. TA-2 has been extensively studied in rats and provides a benchmark for assessing function. BIO5192 is a highly selective and potent (K D of Ͻ10 pM) inhibitor of ␣ 4 ␤ 1 . Dosing regimens were identified for both inhibitors, which provided full receptor occupancy during the duration of the study.Both inhibitors induced leukocytosis, an effect that was used as a pharmacodynamic marker of activity, and both were efficacious in the EAE model. Treatment with TA-2 caused a decrease in ␣ 4 integrin expression on the cell surface, which resulted from internalization of ␣ 4 integrin/TA-2 complexes. In contrast, BIO5192 did not modulate cell surface ␣ 4 ␤ 1 . Our results with BIO5192 indicate that ␣ 4 ␤ 7 does not play a role in this model and that blockade of ␣ 4 ␤ 1 /ligand interactions without down-modulation is sufficient for efficacy in rat EAE. BIO5192 is highly selective and binds with high affinity to ␣ 4 ␤ 1 from four of four species tested. These studies demonstrate that BIO5192, a novel, potent, and selective inhibitor of ␣ 4 ␤ 1 integrin, will be a valuable reagent for assessing ␣ 4 ␤ 1 biology and may provide a new therapeutic for treatment of human inflammatory diseases.Integrins are a large family of cell surface receptors that mediate cell/cell and cell/matrix interactions and signal transduction. They exist as noncovalent ␣␤ heterodimers of different combinations of ␣ and ␤ chains and share extensive structural homology. The leukocyte integrin ␣ 4 ␤ 1 regulates normal lymphocyte trafficking (Lobb and Hemler, 1994) and provides a key costimulatory signal supporting cell activation (Clark and Brugge, 1995). During inflammatory responses, it regulates lymphocyte migration into the damaged tissues and thus has been recognized as an attractive therapeutic target. In vivo studies using blocking monoclonal antibodies (Lobb and Hemler, 1994) and inhibitory peptides (Molossi et al., 1995) have verified the critical role of ␣ 4 ␤ 1 integrins in leukocyte-mediated inflammation. Numerous EAE models of multiple sclerosis have been designed to recapitulate important aspects of the disease and are responsive to ␣ 4 inhibitors (Yednock et al., 1992). Recent positive phase II data using the anti-␣ 4 antibody 1 Current address: Wyeth, Cambridge, MA.