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Purpose: Epithelial-to-mesenchymal transition (EMT) is a process whereby cells acquire molecular alterations that facilitate cell motility and invasion. In preliminary studies, we observed that oxaliplatin-resistant (OxR) colorectal cancer (CRC) cells underwent morphologic changes suggestive of a migratory phenotype, leading us to hypothesize that OxR CRC cells undergo EMT. Experimental Design: The human CRC cell lines KM12L4 and HT29 were exposed to increasing doses of oxaliplatin to establish stable cell lines resistant to oxaliplatin. Migration and invasion were assessed by modified Boyden chamber assays. Morphologic and molecular changes characteristic of EMT were determined by immunofluorescence staining and Western blot analyses. Results: The OxR cells showed phenotypic changes consistent with EMT: spindle-cell shape, loss of polarity, intercellular separation, and pseudopodia formation. KM12L4 and HT29 OxR cells exhibited an f8-to 15-fold increase in migrating and invading cells, respectively (P < 0.005 for both). Immunofluorescence staining of OxR cells revealed translocation of E-cadherin and h-catenin from their usual membrane-bound complex to the cytoplasm and nucleus, respectively. The OxR cells also had decreased expression of the epithelial adhesion molecules E-cadherin and plakoglobin and an increase in the mesenchymal marker vimentin. The KM12L4 OxR cells exhibited increased nuclear expression of Snail, an EMT-regulatory transcription factor, whereas the HT29 OxR cells exhibited an increase in nuclear expression of the EMT-associated transcription factor nuclear factor nB. Conclusion: We hypothesize that induction of EMT may contribute to the decreased efficacy of therapy in chemoresistant CRC, as the tumor cells switch from a proliferative to invasive phenotype. Further understanding of the mechanisms of chemoresistance in CRC will enable improvements in chemotherapy for metastatic disease.Oxaliplatin is a third-generation platinum compound and is the first platinum-based compound to show efficacy in the treatment of colorectal cancer (CRC; ref. 1). Its use in combination with 5-fluorouracil and leucovorin (FOLFOX) for metastatic CRC has led to response rates >50% and median survival approaching 2 years (2, 3). FOLFOX has also been found to be very effective in the adjuvant setting, leading to an increase in the number of patients who are cured after surgical resection when compared with the use of 5-fluorouracil and leucovorin alone (4). Despite these impressive accomplishments, virtually all metastatic CRC eventually become resistant to oxaliplatin, with a median time to progression of f8 months (5). Hypotheses on the mechanisms of oxaliplatin resistance include defects in oxaliplatin uptake, impaired DNA adduct formation, and increased expression of a copper efflux transporter (6 -9).Epithelial-to-mesenchymal transition (EMT) is a process initially observed in embryonic development in which cells lose epithelial characteristics and gain mesenchymal properties to increase motility and...

5-fluorouracil (5FU) and oxaliplatin are standard therapy for metastatic colorectal cancer (CRC), but the development of chemoresistance is inevitable. Since cancer stem cells (CSCs) are hypothesized to be chemoresistant, we investigated CSC properties in newly developed chemoresistant CRC cell lines and sought to identify targets for therapy. The human CRC cell line HT29 was exposed to increasing doses of 5FU (HT29/5FU-R) or oxaliplatin (HT29/Ox) to achieve resistance at clinically relevant doses. Western blotting and flow cytometry were done to determine molecular alterations. The insulin-like growth factor 1 receptor (IGF-1R) monoclonal antibody (MoAb) AVE-1642 was used to inhibit signaling in vitro and in vivo using murine xenograft models. HT29/5FU-R and HT29/OxR demonstrated 16- to 30-fold enrichment of CD133+ cells and 2-fold enrichment of CD44+ cells (putative CRC CSC markers). Resistant cells were enriched 5- to 22-fold for double-positive (CD133+/CD44+) cells. Consistent with the CSC phenotype, resistant cells exhibited a decrease in cellular proliferation in vitro (47–59%; p<0.05). Phosphorylated and total IGF-1R levels were increased in resistant cell lines. HT29/5FU-R and HT29/OxR cells were ~5-fold more responsive to IGF-1R inhibition relative to parental cells (p<0.01) in vitro. Tumors derived from HT29/OxR cells demonstrated significantly greater growth inhibition in response to an IGF-1R MoAB than did parental cells (p<0.05). Chemoresistant CRC cells are enriched for CSC markers and the CSC phenotype. Chemotherapy-induced IGF-1R activation provided for enhanced sensitivity to IGF-1R targeted therapy. Identification of CSC targets presents a novel therapeutic approach in this disease.

Bladder cancer is one of the most common cancers worldwide, with transitional cell carcinoma (TCC) being the predominant form. Here we report a genomic analysis of TCC by both whole-genome and whole-exome sequencing of 99 individuals with TCC. Beyond confirming recurrent mutations in genes previously identified as being mutated in TCC, we identified additional altered genes and pathways that were implicated in TCC. Notably, we discovered frequent alterations in STAG2 and ESPL1, two genes involved in the sister chromatid cohesion and segregation (SCCS) process. Furthermore, we also detected a recurrent fusion involving FGFR3 and TACC3, another component of SCCS, by transcriptome sequencing of 42 DNA-sequenced tumors. Overall, 32 of the 99 tumors (32%) harbored genetic alterations in the SCCS process. Our analysis provides evidence that genetic alterations affecting the SCCS process may be involved in bladder tumorigenesis and identifies a new therapeutic possibility for bladder cancer.

SUMMARY We report a paracrine effect whereby endothelial cells (ECs) promote the cancer stem cell (CSC) phenotype of human colorectal cancer (CRC) cells. We showed that, without direct cell-cell contact, ECs secrete factors that promoted the CSC phenotype in CRC cells via Notch activation. In human CRC specimens, CD133 and Notch intracellular domain-positive cells co-localized with CRC cells in perivascular regions. An EC-derived, soluble form of Jagged-1, via ADAM17 proteolytic activity, led to Notch activation in CRC cells in a paracrine manner; these effects were blocked by immunodepletion of Jagged-1 in EC conditioned medium or blockade of ADAM17 activity. ECs play an active role in promoting Notch signaling and the CSC phenotype by secreting soluble Jagged-1.

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with betaine aldehyde as an intermediate. In this study, primary deuterium and solvent kinetic isotope effects have been used to elucidate the mechanism for substrate oxidation by choline oxidase using both steady-state kinetics and rapid kinetics techniques. The D(kcat/Km) value with 1,2-[2H4]-choline at saturating oxygen concentration was independent of pH in the range between 6.5 and 10, with a value of approximately 10.6, indicating that CH bond cleavage is not masked by other titratable kinetic steps belonging to the reductive half-reaction. In agreement with this conclusion, a Dkred value of approximately 8.9 was determined at pH 10 for the anaerobic reduction of the flavin by choline, irrespective of whether aqueous or deuterated solvent was used. At pH 10, both the D2(O)(kcat/Km) and the D2(O)kred values were not different from unity with choline or 1,2-[2H4]-choline, while the Dkcat and D2(O)kcat values were 7.3 and 1.1, respectively. The kcat and kred values were 133 s(-1) and 135 s(-1) with betaine aldehyde and 60 s(-1) and 93 s(-1) with choline. These data are consistent with a chemical mechanism in which the choline hydroxyl proton is not in flight in the transition state for CH bond cleavage and with chemical steps of flavin reduction by choline and betaine aldehyde being rate limiting for the overall turnover of the enzyme.

Astrocytes and microglia, the two immune-regulatory cells of the central nervous system (CNS), are activated by a variety of pathogens and cytokines to elicit rapid transcriptional responses. This program of activation is initiated by a set of intracellular signaling cascades that includes mitogen-activated protein kinase (MAPK), nuclear factor (NF) kappaB, and Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways. This study defines the critical role that NADPH oxidase(Phox)-derived reactive oxygen species (ROS) play in lipopolysaccharide (LPS)- and interferon (IFN)gamma-induced signaling cascades leading to gene expression in glial cells. Treatment of rat microglia and astrocytes with LPS and IFNgamma resulted in a rapid activation of Phox and the release of ROS followed by an induction of inducible nitric oxide synthase (iNOS) expression. iNOS induction was blocked by inhibitors of Phox, i.e., diphenylene iodonium chloride (DPI) and 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), suggesting an involvement of ROS signaling in iNOS gene expression. Exogenous catalase but not superoxide dismutase suppressed the basal activity and completely blocked induced levels of NO/iNOS, suggesting that hydrogen peroxide is the ROS involved. Phox inhibitors and catalase also suppressed LPS/IFNgamma-induced expression of cytokines, i.e., interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)alpha and blocked LPS activation of MAP kinases (i.e., p38 MAPK, c-Jun N-terminal kinase and extracellular signal-regulated kinase), NFkappaB, and IFNgamma-induced STAT1 phosphorylation. A microglial cell line stably transfected with a mutant form of Phox subunit, i.e., p47(phox) W(193)R, and primary astrocytes derived from Phox-deficient mice showed attenuated ROS production and induction of iNOS in response to LPS/IFNgamma, further strengthening the notion that Phox-derived ROS are crucial for proinflammatory gene expression in glial cells.

Endoglin (CD105) is an accessory protein of the transforming growth factor-h receptor system expressed on vascular endothelial cells. Mutation of the endoglin gene is associated with hereditary hemorrhagic telangiectasias, or Osler-Weber-Rendu syndrome, and has been studied extensively in the context of this disease.The expression of endoglin is elevated on the endothelial cells of healing wounds, developing embryos, inflammatory tissues, and solid tumors. Endoglin is a marker of activated endothelium, and its vascular expression is limited to proliferating cells. Recent studies identified endoglin expression in several solid tumor types, with the level of expression correlating with various clinicopathologic factors including decreased survival and presence of metastases. Attempts to target endoglin and the cells that express this protein in tumor-bearing mice have yielded promising results.