Abnormal accumulation and activation of receptor tyrosine kinase Ron (recepteur d'origine nantais) has been demonstrated in a variety of primary human cancers. We show that RNA interference-mediated knockdown of Ron kinase in a highly tumorigenic colon cancer cell line led to reduced proliferation as compared with the control cells. Decreased Ron expression sensitized HCT116 cells to growth factor deprivation stress-induced apoptosis as reflected by increased DNA fragmentation and caspase 3 activation. In addition, cell motility was decreased in Ron knockdown cells as measured by wound healing assays and transwell assays. HCT116 cells are heterozygous for gain of function mutant PIK3CA H1047R. Analysis of signaling proteins that are affected by Ron knockdown revealed that phosphatidylinositol 3-kinase (PI3K) activity of the mutant PI3K as well as AKT phosphorylation was substantially reduced in the Ron knockdown cells compared with the control cells. Moreover, we demonstrated in vivo that knockdown of Ron expression significantly reduced lung metastasis as compared with the control cells in the orthotopic models. In summary, our results demonstrate that Ron plays an essential role in maintaining malignant phenotypes of colon cancer cells through regulating mutant PI3K activity. Therefore, targeting Ron kinase could be a potential strategy for colon cancer treatment, especially in patients bearing gain of function mutant PI3K activity.The receptor tyrosine kinase Ron (recepteur d'origine nantais) belongs to the Met proto-oncogene family (1, 2). Mature Ron is a 180-kDa heterodimer composed of a 40-kDa extracellular ␣-chain and a 150-kDa transmembrane ␤-chain with tyrosine kinase activity (2). Macrophage-stimulating protein is the only ligand that has been identified for Ron (3, 4). Upon ligand binding, Ron dimerizes, becomes autophosphorylated, and transduces a variety of signals that regulate different downstream pathways including Ras/mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), 3 c-Jun N-terminal kinase (JNK), ␤-catenin, and nuclear factor-B (5-12). Ron can be activated through ligand-dependent or -independent mechanisms (3,13,14), which lead to responses important for tumorigenesis and metastasis, including cell scattering, proliferation, motility, and survival (15,16).Ron is normally expressed at relatively low levels in cells of epithelial origin (4). Recent studies have shown that Ron is overexpressed in 47% of breast tumor tissues as compared with benign epithelium and that elevated Ron expression was strongly associated with invasive activity by tumors (17). In addition, Ron is moderately expressed in normal colorectal mucosa, but is significantly increased in the majority of primary human colorectal adenocarcinoma samples (18). Ron overexpression has also been demonstrated in head and neck tumors (19). Furthermore, splice variants of Ron have been identified in human colon cancer. These variants were found to confer constitutive Ron activity, transformation, and tumorige...

Fibrillar amyloid-␤ protein (A␤)1 deposition in senile plaques in the neuropil and in the walls of cerebral blood vessels is a common pathologic feature of patients with Alzheimer's disease (AD) and certain related disorders including Down's syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (1). A␤ is a 39 -43-amino acid peptide that has the propensity to self-assemble into insoluble, ␤-sheet-containing fibrils (1, 2). A␤ is proteolytically derived from a large type I integral membrane precursor protein, termed the amyloid ␤-protein precursor (A␤PP), encoded by a gene located on chromosome 21 (3-6). In this regard, full-length A␤PP is proteolytically cleaved by an enzyme, termed ␤-secretase, at the amino terminus of the A␤ domain. A novel aspartyl proteinase named BACE (for ␤-site A␤PP-Cleaving Enzyme) has been identified as the ␤-secretase enzyme (7-10). Subsequent cleavage of the remaining amyloidogenic membrane spanning A␤PP carboxyl-terminal fragment by an enzyme termed ␥-secretase liberates the 40-or 42-amino acid residue A␤ peptide. Although the exact identity of ␥-secretase remains unclear studies suggest that the presenilin proteins may function as this enzyme or as a required cofactor for ␥-secretase function (11-13). Alternatively, full-length A␤PP can be proteolytically processed by an enzyme termed ␣-secretase through the A␤ domain. This cleavage event generates a non-amyloidogenic membrane spanning carboxyl-terminal fragment and truncated secretory forms of A␤PP␣ (sA␤PP␣) that are released into the extracellular environment (14,15).Cerebrovascular A␤ deposition, known as cerebral amyloid angiopathy, is accompanied by smooth muscle cell degeneration suggesting a toxic effect of A␤ to these cells in vivo (16 -18). These degenerating smooth muscle cells have been implicated in the overproduction of A␤PP and A␤ in the cerebral vessel wall further suggesting the active involvement of these cells in the progression of this cerebrovascular pathology (17-19). Similar to these in vivo observations, we have reported that A␤-(1-42), the more pathogenic form of the wild-type peptide, causes severe cellular degeneration accompanied by a marked increase in the level of cell-associated A␤PP in cultured human cerebrovascular smooth muscle (HCSM) cells (20 -22). In more recent studies we demonstrated that mutations associated with familial forms of cerebral amyloid angiopathy (E22Q Dutch, E22K Italian, and D23N Iowa) markedly enhance both the fibrillogenic and cerebrovascular pathogenic properties of A␤ toward cultured HCSM cells (23)(24)(25)(26). These experiments showed that these pathogenic forms of A␤ assemble into an

Epithelial to mesenchymal transition (EMT) plays a dual role in tumor progression. It enhances metastasis of tumor cells by increasing invasive capacity and promoting survival, and it decreases tumor cell sensitivity to epithelial cell-targeting agents such as epithelial growth factor receptor kinase inhibitors. In order to study EMT in tumor cells, we have characterized 3 new models of ligand-driven EMT: the CFPAC1 pancreatic tumor model and the H358 and H1650 lung tumor models. We identified a diverse set of ligands that drives EMT in these models. Hepatocyte growth factor and oncostatin M induced EMT in all models, while transforming growth factor-β induced EMT in both lung models. We observed morphologic, marker and phenotypic changes in response to chronic ligand treatment. Interestingly, stimulation with 2 ligands resulted in more pronounced EMT compared with single-ligand treatment, demonstrating a spectrum of EMT states induced by parallel signaling, such as the JAK and PI3K pathways. The EMT changes observed in response to the ligand were reversed upon ligand withdrawal, demonstrating the ‘metastable’ nature of these models. To study the impact of EMT on cell morphology and invasion in a 3D setting, we cultured cells in a semisolid basement membrane extract. Upon stimulation with EMT ligands, the colonies exhibited changes to EMT markers and showed phenotypes ranging from modest differences in colony architecture (CFPAC1) to complex branching structures (H358, H1650). Collectively, these 3 models offer robust cell systems with which to study the roles that EMT plays in cancer progression.

This trial was registered with Clinicaltrials.gov as study NCT01759056 and with EudraCT as study 2012-004859-27.

Background Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the GI tract that is currently treated with injected monoclonal antibodies specific for tumor necrosis factor (TNF). We developed and characterized AVX-470, a novel polyclonal antibody specific for human TNF. We evaluated the oral activity of AVX-470m, a surrogate antibody specific murine TNF, in several well-accepted mouse models of IBD. Methods AVX-470 and AVX-470m were isolated from the colostrum of dairy cows that had been immunized with TNF. The potency, specificity and affinity of both AVX-470 and AVX-470m were evaluated in vitro and compared with infliximab. AVX-470m was orally administered to mice either before or after induction of colitis and activity was measured by endoscopy, histopathology, immunohistochemistry and quantitative measurement of mRNA levels. Colitis was induced using either 2,4,6-trinitrobenzene sulfonate (TNBS) or dextran sodium sulfate (DSS). Results AVX-470 and AVX-470m were shown to be functionally comparable in vitro. Moreover, the specificity, neutralizing potency and affinity of AVX-470 were comparable to infliximab. Orally administered AVX-470m effectively reduced disease severity in several mouse models of IBD. Activity was comparable to that of oral prednisolone or parenteral etanercept. The antibody penetrated the colonic mucosa and inhibited TNF-driven mucosal inflammation with minimal systemic exposure. Conclusions AVX-470 is a novel polyclonal anti-TNF antibody with an in vitro activity profile comparable to that of infliximab. Oral administration of a surrogate antibody specific for mouse TNF is effective in treating mouse models of IBD, delivering the anti-TNF to the site of inflammation with minimal systemic exposure.