Disruption of the cell-cell junction with concomitant changes in the expression of junctional proteins is a hallmark of cancer cell invasion and metastasis. The role of adherent junction proteins has been studied extensively in cancer, but the roles of tight junction (TJ) proteins are less well understood. Claudins are recently identified members of the tetraspanin family of proteins, which are integral to the structure and function of TJs. Recent studies show changes in expression/cellular localization of claudins during tumorigenesis; however, a causal relationship between claudin expression/localization and cancer has not been established. Here, we report an increased expression of claudin-1 in human primary colon carcinoma and metastasis and in cell lines derived from primary and metastatic tumors. We also report frequent nuclear localization of claudin-1 in these samples. Genetic manipulations of claudin-1 expression in colon cancer cell lines induced changes in cellular phenotype, with structural and functional changes in markers of epithelial-mesenchymal transition. Furthermore, we demonstrate that changes in claudin-1 expression have significant effects on growth of xenografted tumors and metastasis in athymic mice. We further provide data suggesting that the regulation of E-cadherin expression and beta-catenin/Tcf signaling is a possible mechanism underlying claudin-1-dependent changes.

Neonatal necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of preterm infants. Increased intestinal epithelium permeability is an early event in NEC pathogenesis. Autophagy and apoptosis are induced by multiple stress pathways which may impact the intestinal barrier, and they have been associated with pathogenesis of diverse gastrointestinal diseases including inflammatory bowel disease. Using both in vitro and in vivo models, this study investigates autophagy and apoptosis under experimental NEC stresses. Furthermore this study evaluates the effect of erythropoietin (Epo), a component of breast milk previously shown to decrease the incidence of NEC and to preserve intestinal barrier function, on intestinal autophagy and apoptosis. It was found that autophagy and apoptosis are both rapidly up regulated in NEC in vivo as indicated by increased expression of the autophagy markers Beclin 1 and LC3II, and by evidence of apoptosis by TUNEL and cleaved caspase-3 staining. In the rat NEC experimental model, autophagy preceded the onset of apoptosis in intestine. In vitro studies suggested that Epo supplementation significantly decreased both autophagy and apoptosis via the Akt/mTOR signaling pathway and the MAPK/ERK pathway respectively. These results suggest that Epo protects intestinal epithelium from excessive autophagy and apoptosis in experimental NEC.

We have recently reported that the expression of a tight junction protein, claudin-1, is increased during colon carcinogenesis and particularly metastatic colorectal cancer. Manipulation of claudin-1 levels in colon cancer cells showed a positive correlation between claudin-1 expression and tumor growth and metastasis. However, the mechanisms underlying the increased claudin-1 expression in colorectal cancer remains unknown. The tumor suppressor Smad4 is a central intracellular signal transduction component of the transforming growth factor-B (TGF-B) family of cytokines. Loss of Smad4 protein expression is correlated with poor prognosis and is frequently observed in invasive and metastatic colorectal carcinoma. In the present study, we report an inverse relationship between Smad4 and claudin-1 expression in human colorectal carcinoma tumor samples and in human colon cancer cell lines. We found that the expression of Smad4 in Smad4-deficient but claudin-1-positive SW480 or HT29 colon cancer cell lines down-regulates claudin-1 expression through transcriptional repression by modulating B-catenin/ T-cell factor/lymphocyte enhancer factor activity. Furthermore, this Smad4-dependent inhibition of claudin-1 expression is independent of TGF-B signaling because Smad4 expression alone is insufficient to restore TGF-B signaling in the SW480 cells, and the selective TGF-B receptor kinase inhibitor LY364947 did not prevent the Smad4 suppression of claudin-1 protein expression in either SW480 or HT29 cells. Taken together, these findings suggest a novel mechanism underlying Smad4 tumor-suppressive function through regulation of a potential metastatic modulator, claudin-1, in a TGF-B-independent manner. [Cancer Res 2007;67(4):1571-9]

Preterm infants face many challenges in transitioning from the in utero to extrauterine environment while still immature. Failure of the preterm gut to successfully mature to accommodate bacteria and food substrate leads to significant morbidity such as neonatal necrotizing enterocolitis. The intestinal epithelial barrier plays a critical role in gut protection. Heat shock protein 70 (Hsp70) is an inducible cytoprotective molecule shown to protect the intestinal epithelium in adult models. To investigate the hypothesis that Hsp70 may be important for early protection of the immature intestine, Hsp70 expression was evaluated in intestine of immature rat pups. Data demonstrate that Hsp70 is induced by exposure to mother’s milk. Hsp70 is found in mother’s milk, and increased Hsp70 transcription is induced by mother’s milk. This Hsp70 colocalizes with the tight junction protein ZO-1. Mother’s milk-induced Hsp70 may contribute to maintenance of barrier function in the face of oxidant stress. Further understanding of the means by which mother’s milk increases Hsp70 in the ileum will allow potential means of strengthening the intestinal barrier in at-risk preterm infants.

Metastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-␤ (TGF-␤) signaling may play an important role in cancer cell invasion and metastasis; however, the underlying mechanisms remain unclear. In the present study, we report that autocrine TGF-␤ supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly, treatment of paracrine/exogenous TGF-␤ at higher concentrations than autocrine TGF-␤ further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-␤ is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-␤-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus, through the Smad pathway, autocrine TGF-␤ maintains uPA expression through facilitated protein secretion, thereby supporting tumor cell invasiveness, whereas exogenous TGF-␤ further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells.Malignant tumors are characterized by their ability to metastasize to distant organs. The initial steps of metastasis involve invasive growth of tumors across the basement membrane and migration through the extracellular matrix (ECM).2 Because the enzymatic degradation of both the basement membrane and ECM barriers requires a number of ECM-degrading proteases (1, 2) and is a critical early event in metastasis, invasiveness may be modulated by the expression of ECM-degrading proteases in tumor cells in response to autonomous and microenvironmental signals. Among the increasing number of ECMdegrading proteases implicated in metastasis, considerable attention has been focused on the family of matrix metalloproteinases (MMPs) and the plasminogen activator system. One of the regulators of these ECM-degrading proteases is transforming growth factor-␤ (TGF-␤).TGF-␤ is a multifunctional cytokine that regulates cell proliferation, differentiation, plasticity, and migration in a contextdependent manner (reviewed in Refs. 3 and 4). TGF-␤ transduces signaling through a transmembrane type II receptor (T␤RII), a constitutively active serine/threonine kinase receptor (5). Upon ligand binding, the T␤RII recruits and transphosphorylates intracellular TGF-␤ type I receptor (T␤RI), thereby stimulating T␤RI serine/threonine kinase activity (6). The T␤RI then activates the downstream effectors, Smad2 and Smad3, by phosphorylation. The activated Smad proteins form complexes with the common Smad mediator, Smad4, and then translocate to the nucleus, where the Smad complexes regulate transcription of TGF-␤ target genes in conjunction with vario...

No abstract

No abstract

To improve the efficacy of tumor cell-based and dendritic cell (DC)-based cancer vaccines, this study explored the potential of a new cancer vaccine strategy, that is, the use of CD40 ligand-transfected tumor (CD40L-tumor) cells to simultaneously deliver both tumor-derived antigens (Ag) and maturation stimuli to DCs. Materials from frozen/thawed or irradiated human tumor cells, with or without surface CD40L, were internalized efficiently by immature DCs after coincubation. However, during the internalization process, only coculturing with irradiated CD40L-tumor cells resulted in concurrent, optimal DC maturation and production of proinflammatory chemokines and pro-Th1 cytokines, such as IL-6, IL-8, IL-12, IFN-g, and TNF-a. These activated DCs were the most potent cells to support the growth of CD8 þ , IFN-g-producing T cells, and to process tumor Ag for the generation of specific cytotoxic T cells in vitro. Animals vaccinated with irradiated CD40L-tumor cell-pulsed DCs were better protected against subsequent challenge of a weakly immunogenic tumor cell line than animals vaccinated with irradiated CD40L-tumor cells alone. Thus, our results strongly support the future clinical application of using DCs pulsed with irradiated CD40L-tumor cells as a cancer vaccine.