Brian M. Dolinski

Role of αvβ6 Integrin in Acute Biliary Fibrosis

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 αvβ6 integrin and that expression of this integrin is directly linked to fibrogenesis through activation of transforming growth factor beta (TGF‐β). Administration of blocking antibody to αvβ6 significantly reduces the extent of acute fibrosis after bile duct ligation. Moreover, in β6‐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 αvβ6, rather than inflammation, is linked to fibrogenesis. It is known that αvβ6 binds latent TGF‐β and that binding results in release of active TGFβ. Consistent with this, intracellular signaling from the TGFβ receptor is increased after bile duct ligation in wild‐type mice but not in β6−/− mice, and a competitive inhibitor of the TGFβ receptor type II blocks fibrosis to the same extent as antibody to αvβ6. In a survey of human liver disease, expression of αvβ6 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 αvβ6 integrin, which is closely linked to periductal fibrogenesis. The findings provide a rationale for the use of inhibitors of αvβ6 integrin or TGFβ for down‐regulating fibrosis in the setting of acute or ongoing biliary injury. (HEPATOLOGY 2007.)

Gene Profiling in Atherosclerosis Reveals a Key Role for Small Inducible Cytokines Validation Using a Novel Monocyte Chemoattractant Protein Monoclonal Antibody

Background—Pathological aspects of atherosclerosis are well described, but gene profiles during atherosclerotic plaque progression are largely unidentified. Methods and Results—Microarray analysis was performed on mRNA of aortic arches of ApoE−/− mice fed normal chow (NC group) or Western-type diet (WD group) for 3, 4.5, and 6 months. Of 10 176 reporters, 387 were differentially (>2×) expressed in at least 1 group compared with a common reference (ApoE−/−, 3- month NC group). The number of differentially expressed genes increased during plaque progression. Time-related expression clustering and functional grouping of differentially expressed genes suggested important functions for genes involved in inflammation (especially the small inducible cytokines monocyte chemoattractant protein [MCP]-1, MCP-5, macrophage inflammatory protein [MIP]-1&agr;, MIP-1&bgr;, MIP-2, and fractalkine) and matrix degradation (cathepsin-S, matrix metalloproteinase-2/12). Validation experiments focused on the gene cluster of small inducible cytokines. Real-time polymerase chain reaction revealed a plaque progression–dependent increase in mRNA levels of MCP-1, MCP-5, MIP-1&agr;, and MIP-1&bgr;. ELISA for MCP-1 and MCP-5 showed similar results. Immunohistochemistry for MCP-1, MCP-5, and MIP-1&agr; located their expression to plaque macrophages. An inhibiting antibody for MCP-1 and MCP-5 (11K2) was designed and administered to ApoE−/− mice for 12 weeks starting at the age of 5 or 17 weeks. 11K2 treatment reduced plaque area and macrophage and CD45+ cell content and increased collagen content, thereby inducing a stable plaque phenotype. Conclusions—Gene profiling of atherosclerotic plaque progression in ApoE−/− mice revealed upregulation of the gene cluster of small inducible cytokines. Further expression and in vivo validation studies showed that this gene cluster mediates plaque progression and stability.

Antibody-mediated blockade of integrin ?v?6 inhibits tumor progression in vivo by a transforming growth factor-?–regulated mechanism

The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(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 alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(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 alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-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 alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.

Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells CHARACTERIZATION OF A SELECTIVE ALLOSTERIC KINASE INHIBITOR

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1–5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

Overexpression of the αvβ6 Integrin in Endometrial Cancer

The alpha(v)beta(6) integrin (αvβ6) has been shown to be up-regulated in adenocarcinoma of the breast, colon, stomach, and ovary, generally reflecting a more aggressive phenotype. Expression in endometrial cancer has not been reported. We analyzed αvβ6 expression in the tissue from primary endometrial carcinomas (endometrioid type) using a mouse monoclonal antibody against human αvβ6, and correlated the findings with grade, stage, and nodal involvement. Normal cycling endometrium was studied for comparison. αvβ6 was only weakly expressed in normal epithelium and infrequently expressed in precancers, but up-regulated in the majority of endometrial carcinomas, especially with high grade. Nodal metastases strongly expressed αvβ6, even when the primary tumor showed only focal expression. No correlation was found between expression and depth of invasion or the presence of metastases. Overexpression of αvβ6 in endometrial carcinoma is common. Expression is high in metastatic lesions. The level of expression of the primary tumor was not indicative of the presence of nodal metastasis; however, the number of cases with nodal metastases was limited.

Inhibition of αvβ5 Integrin Attenuates Vascular Permeability and Protects against Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is a leading cause of AKI. This common clinical complication lacks effective therapies and can lead to the development of CKD. The αvβ5 integrin may have an important role in acute injury, including septic shock and acute lung injury. To examine its function in AKI, we utilized a specific function-blocking antibody to inhibit αvβ5 in a rat model of renal IRI. Pretreatment with this anti-αvβ5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the αvβ5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that αvβ5 inhibition affected established injury markers and induced pathway alterations previously shown to be protective. Intravital imaging of post-ischemic kidneys revealed reduced vascular leak with αvβ5 antibody treatment. Immunostaining for αvβ5 in the kidney detected evident expression in perivascular cells, with negligible expression in the endothelium. Studies in a three-dimensional microfluidics system identified a pericyte-dependent role for αvβ5 in modulating vascular leak. Additional studies showed αvβ5 functions in the adhesion and migration of kidney pericytes in vitro Initial studies monitoring renal blood flow after IRI did not find significant effects with αvβ5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for αvβ5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal αvβ5 inhibition as a promising therapeutic strategy for AKI.

Abstract 685: PARP inhibitor MK-4827 is synthetic lethal for tumors with homologous recombination defects associated with ATM-deficiency, PTEN-deletion and microsatellite instability (MSI)

Poly(ADP-ribose) polymerase (PARP) −1 and −2 enzymes play a critical role in the base excision DNA repair (BER) pathway. PARP inhibition in oncology has the potential to target pre-defined sub-populations with higher sensitivity and to widen the therapeutic index of chemotherapy and radiotherapy. Pre-clinical and clinical evidence demonstrated that PARP inhibitors are synthetic lethal for tumors with defects in the homologous recombination (HR) pathway, such as tumors with mutations in the BRCA1 or BRCA2 genes. MK-4827 is a potent and selective orally available PARP-1/2 inhibitor currently in Phase 1 development. In pre-clinical models, MK-4827 displays excellent monotherapy activity in a large panel of BRCA mutant cell lines (10 nM ≤ CC50 ≤ 90nM) with at least 10-fold selectivity over BRCA wild-type celllines (CC50 ≥ 1.5 uM). We have explored the potential of MK-4827 for the therapy of tumors with defects in the HR pathway in addition to mutation in the BRCA genes. In this study we have evaluated the activity of MK-4827 on tumor cell lines with defects in the homologous recombination pathway as a consequence of 1) inactivation of ATM gene in melanoma and mantle cell lymphoma cell lines; 2) PTEN gene deletion in prostate cancer cells; 3) MRE11 expression level defects associated with the Microsatellite Instability (MSI) phenotype in colorectal cancer cells (CRCs). We demonstrated that cancer cells with an inactive ATM pathway as a consequence of homozygous ATM gene inactivation are very sensitive to MK-4827 inhibition (CC50 ≤ 50nM). MK-4827 also displays strong activity in ATM-deficient xenograft models. Heterozygous ATM inactivation is not sufficient to confer sensitivity to MK-4827. Our studies also showed that prostate cancer cells with homozygous-deletion of PTEN gene are sensitive to MK-4827 inhibition (100nM ≤ CC50 ≤ 600nM) as measured by long-term cell growth and proliferation assays. Homozygous, but not heterozygous, PTEN-deletion is required for MK-4827 sensitivity in agreement with data available for sensitivity of BRCA- and ATM-deficient cells to MK-4827. Finally, we have screened a panel of CRC cells with known Microsatellite Instability and have observed that MSI+ cells are sensitive to MK-4827 in long term proliferation assays, in contrast to Microsatellite stable (MSS) CRC cells which are resistant. In agreement with previous reports, we observed that MSI+ CRC cells show low expression levels of MRE11, a key protein for homologous recombination. Conclusion: Our pre-clinical data demonstrate that MK-4827 is synthetic lethal for tumors with ATM-deficiency, PTEN-deletion or MSI+ instability and suggest that patients with tumors carrying these genetic defects might benefit from treatment with PARP inhibitors Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 685.