Cabozantinib Inhibits Growth of Androgen-Sensitive and Castration-Resistant Prostate Cancer and Affects Bone Remodeling

Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. In vivo, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease.     

Dynamic Changes in Mucus Thickness and Ion Secretion during Citrobacter rodentium Infection and Clearance

Citrobacter rodentium is an attaching and effacing pathogen used as a murine model for enteropathogenic Escherichia coli. The mucus layers are a complex matrix of molecules, and mucus swelling, hydration and permeability are affected by many factors, including ion composition. Here, we used the C. rodentium model to investigate mucus dynamics during infection. By measuring the mucus layer thickness in tissue explants during infection, we demonstrated that the thickness changes dynamically during the course of infection and that its thickest stage coincides with the start of a decrease of bacterial density at day 14 after infection. Although quantitative PCR analysis demonstrated that mucin mRNA increases during early infection, the increased mucus layer thickness late in infection was not explained by increased mRNA levels. Proteomic analysis of mucus did not demonstrate the appearance of additional mucins, but revealed an increased number of proteins involved in defense responses. Ussing chamber-based electrical measurements demonstrated that ion secretion was dynamically altered during the infection phases. Furthermore, the bicarbonate ion channel Bestrophin-2 mRNA nominally increased, whereas the Cftr mRNA decreased during the late infection clearance phase. Microscopy of Muc2 immunostained tissues suggested that the inner striated mucus layer present in the healthy colon was scarce during the time point of most severe infection (10 days post infection), but then expanded, albeit with a less structured appearance, during the expulsion phase. Together with previously published literature, the data implies a model for clearance where a change in secretion allows reformation of the mucus layer, displacing the pathogen to the outer mucus layer, where it is then outcompeted by the returning commensal flora. In conclusion, mucus and ion secretion are dynamically altered during the C. rodentium infection cycle.     

Cytokine gene polymorphism and graft-versus-host disease: a survey in Iranian bone marrow transplanted patients

Graft versus host disease (GVHD) is a major complication of bone marrow transplantation (BMT). Numerous studies have shown the potential role of cytokine genotypes in the occurrence of GVHD. In this retrospective, case–control study we aimed to investigate the association between 13 cytokine genes and acute GVHD (aGVHD) after HLA-identical sibling BMT in 91 Iranian subjects. Negative association was found between aGVHD and donor IL-10/GCC haplotype or donor IL-4Ra-A allele in the population study. When compared within the leukemia subgroup, we observed positive association between recipient IL-1α ?889/C allele and aGVHD. Also there were negative association between recipient IL-10/CAA haplotype and donor IL-4Ra/A allele and development of aGVHD. Among the different genotypes only donor IL-4Ra and donor IL-12 showed significant association. We conclude that several cytokine polymorphisms are positively and negatively associated with aGVHD in Iranian HLA matched siblings, of which IL-4Ra and IL-12 may play important roles.     

Gastrointestinal Cell Lines Form Polarized Epithelia with an Adherent Mucus Layer when Cultured in Semi-Wet Interfaces with Mechanical Stimulation

Mucin glycoproteins are secreted in large quantities by mucosal epithelia and cell surface mucins are a prominent feature of the glycocalyx of all mucosal epithelia. Currently, studies investigating the gastrointestinal mucosal barrier use either animal experiments or non-in vivo like cell cultures. Many pathogens cause different pathology in mice compared to humans and the in vitro cell cultures used are suboptimal because they are very different from an in vivo mucosal surface, are often not polarized, lack important components of the glycocalyx, and often lack the mucus layer. Although gastrointestinal cell lines exist that produce mucins or polarize, human cell line models that reproducibly create the combination of a polarized epithelial cell layer, functional tight junctions and an adherent mucus layer have been missing until now. We trialed a range of treatments to induce polarization, 3D-organization, tight junctions, mucin production, mucus secretion, and formation of an adherent mucus layer that can be carried out using standard equipment. These treatments were tested on cell lines of intestinal (Caco-2, LS513, HT29, T84, LS174T, HT29 MTX-P8 and HT29 MTX-E12) and gastric (MKN7, MKN45, AGS, NCI-N87 and its hTERT Clone5 and Clone6) origins using Ussing chamber methodology and (immuno)histology. Semi-wet interface culture in combination with mechanical stimulation and DAPT caused HT29 MTX-P8, HT29 MTX-E12 and LS513 cells to polarize, form functional tight junctions, a three-dimensional architecture resembling colonic crypts, and produce an adherent mucus layer. Caco-2 and T84 cells also polarized, formed functional tight junctions and produced a thin adherent mucus layer after this treatment, but with less consistency. In conclusion, culture methods affect cell lines differently, and testing a matrix of methods vs. cell lines may be important to develop better in vitro models. The methods developed herein create in vitro mucosal surfaces suitable for studies of host-pathogen interactions at the mucosal surface.     

FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.     

PKC-beta1 isoform activation is required for EGF-induced NF-kappaB inactivation and IkappaBalpha stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers

Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-kappaB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-beta1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-beta1 prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, F-actin, and barrier dysfunction. We used wild-type (WT) and transfected cells. The latter were transfected with varying levels of cDNA to overexpress or underexpress PKC-beta1. Cells were pretreated with EGF or PKC modulators +/- oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-beta1 activity, decreasing IkappaBalpha phosphorylation/degradation, suppressing NF-kappaB activation (p50/p65 subunit nuclear translocation/activity), enhancing stable actin (increased F-actin-to-G-actin ratio), increasing stability of actin cytoskeleton, and reducing barrier hyperpermeability. Cells stably overexpressing PKC-beta1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IkappaBalpha stabilization, NF-kappaB inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-beta1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-beta1 substantially prevented all measures of EGF's protection against NF-kappaB activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-beta1 activation and NF-kappaB suppression. The molecular event underlying this unique effect of PKC-beta1 involves inhibition of phosphorylation and increases in stabilization of IkappaBalpha. The ability to inhibit the dynamics of NF-kappaB/IkappaBalpha and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.     

Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the -isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC- is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC- (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC- showed monolayer injury as indicated by decreased native PKC- activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC- was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC- was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-/tubulin complex. Second, DN transfection to inhibit the endogenous PKC- led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC- led to a mostly cytosolic distribution of -isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1) PKC- isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2) the molecular event underlying this novel biological effect of PKC- involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-. microtubules; tubulin; occludin; epithelial barrier permeability; protein kinase C isoform