Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis.

Cathepsin B is a cysteine proteinase that primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during tumoral expansion, the regulation of cathepsin B can be altered at multiple levels, thereby resulting in its overexpression and export outside of the cell. This may suggest a possible role of cathepsin B in alterations leading to cancer progression. The aim of this study was to determine the contribution of intracellular and extracellular cathepsin B in growth, tumorigenesis, and invasion of colorectal cancer (CRC) cells. Results show that mRNA and activated levels of cathepsin B were both increased in human adenomas and in CRCs of all stages. Treatment of CRC cells with the highly selective and non-permeant cathepsin B inhibitor Ca074 revealed that extracellular cathepsin B actively contributed to the invasiveness of human CRC cells while not essential for their growth in soft agar. Cathepsin B silencing by RNAi in human CRC cells inhibited their growth in soft agar, as well as their invasion capacity, tumoral expansion, and metastatic spread in immunodeficient mice. Higher levels of the cell cycle inhibitor p27(Kip1) were observed in cathepsin B-deficient tumors as well as an increase in cyclin B1. Finally, cathepsin B colocalized with p27(Kip1) within the lysosomes and efficiently degraded the inhibitor. In conclusion, the present data demonstrate that cathepsin B is a significant factor in colorectal tumor development, invasion, and metastatic spreading and may, therefore, represent a potential pharmacological target for colorectal tumor therapy.

Constitutive activation of the MEK/ERK pathway inhibits intestinal epithelial cell differentiation.

The Ras/Raf/MEK/ERK cascade regulates intestinal epithelial cell proliferation. Indeed, while barely detectable in differentiated cells of the villi, ERK1/2-activated forms are detected in the nucleus of undifferentiated human intestinal crypt cells. In addition, we and others have reported that ERKs are selectively inactivated during enterocyte differentiation. However, whether inactivation of the ERK pathway is necessary for inhibition of both proliferation and induction of differentiation of intestinal epithelial cells is unknown. Human Caco-2/15 cells, undifferentiated crypt IEC-6 cells, and differentiating Cdx3-expressing IEC-6 cells were infected with retroviruses encoding either a hemagglutinin (HA)-tagged MEK1 wild type (wtMEK) or a constitutively active S218D/S222D MEK1 mutant (caMEK). Protein and gene expression was assessed by Western blotting, semiquantitative RT-PCR, and real-time PCR. Morphology was analyzed by transmission electron microscopy. We found that 1) IEC-6/Cdx3 cells formed multicellular layers after confluence and differentiated after 30 days in culture, as assessed by increased polarization, microvilli formation, expression of differentiation markers, and ERK1/2 inhibition; 2) while activated MEK prevented neither the inhibition of ERK1/2 activities nor the differentiation process in postconfluent Caco-2/15 cells, caMEK expression prevented ERK inhibition in postconfluent IEC-6/Cdx3 cells, thus leading to maintenance of elevated ERK1/2 activities; 3) caMEK-expressing IEC-6/Cdx3 cells exhibited altered multicellular structure organization, poorly defined tight junctions, reduced number of microvilli on the apical surface, and decreased expression of the hepatocyte nuclear factor 1α transcription factor and differentiation markers, namely apolipoprotein A-4, fatty acid-binding protein, calbindin-3, mucin 2, alkaline phosphatase, and sucrase-isomaltase; and 4) increased Cdx3 phosphorylation on serine-60 (S60) in IEC-6/Cdx3 cells expressing caMEK led to decreased Cdx2 transactivation potential. These results indicate that inactivation of the ERK pathway is required to ensure the full Cdx2/3 transcriptional activity necessary for intestinal epithelial cell terminal differentiation.

Loss of cathepsin L activity promotes claudin-1 overexpression and intestinal neoplasia.

Intestinal epithelial integrity and polarity are maintained by cohesive interactions between cells via the formation of tight junctions. Irregularities in tight junctions have only recently been found to be associated with the initiation and progression of intestinal neoplasia. The claudin family of proteins is integral to the structure and function of the tight junction but little is known of the molecular events that regulate the expression of these components. The present report identifies cathepsin L, classically a lysosomal cysteine protease, as being induced during intestinal epithelial cell polarization and differentiation. Inhibition of intracellular cathepsin L activity results in the accumulation of disorganized cell layers and a decline in the expression of differentiation markers in cultured intestinal epithelial cells. This coincides with a rapid up-regulation of claudin-1 protein accumulation. Mutant mice defective in cathepsin L activity (furless) display an elevated level of intestinal claudin-1 and claudin-2 expression. Loss of cathepsin L activity leads to a marked increase in tumor multiplicity in the intestine of Apc(Min) mice. Given the traditionally viewed biological role of cathepsin L in the processing of lysosomal content as well as in pathological extracellular matrix remodeling, the results here demonstrate an as yet unsuspected intracellular role for this protease in normal intestinal epithelial polarization and initiation of neoplasia.

The CDX2 transcription factor regulates furin expression during intestinal epithelial cell differentiation.

CDX2, a member of the caudal family of transcription factors, is involved in enterocyte lineage specification. CDX2 activates many intestine-specific genes, such as sucrase-isomaltase and lactase-phlorizin hydrolase (LPH), and adhesion proteins, namely, LI-cadherin and claudin-2. In this study, we show that the proprotein convertase furin, involved in proteolytic maturation of proprotein substrates including LPH and cell surface proteins, is a CDX2 target. Indeed, expression of the rat furin homolog was induced 1.5-fold, as determined by microarray experiments that compared control with CDX2-expressing intestinal epithelial cells (IEC-6). As determined by transient transfection assays in Caco-2/15 cells, the furin P1 promoter 1.3-kb fragment between SacI and NheI was essential for CDX2 transcriptional activation. Electrophoretic mobility shift/supershift assays followed by site-specific mutagenesis and chromatin immunoprecipitation identified the CDX DNA-binding site (CBS)2 sequence from nt -1827 to -1821 as the major CBS involved in furin P1 promoter activation. Increased furin mRNA and protein expression correlated with both CDX2 expression and intestinal epithelial cell differentiation. In addition, furin mRNAs were detected predominantly in differentiated epithelial cells of the villus, as determined by in situ hybridization. Treatment of Caco-2/15 cells with a furin inhibitor led to inhibition of LPH activity. Morphological differentiation of enterocyte-like features in Caco-2/15 such as epithelial cell polarity and brush-border formation were strongly attenuated by furin inhibition. These results suggest that CDX2 regulates furin expression in intestinal epithelial cells. Furin may be important in modulating the maturation and/or activation of key factors involved in enterocyte differentiation.

Cdk2-dependent phosphorylation of homeobox transcription factor CDX2 regulates its nuclear translocation and proteasome-mediated degradation in human intestinal epithelial cells.

By having demonstrated previously that p27(Kip1), a potent inhibitor of G(1) cyclin-cyclin-dependent kinases complexes, increases markedly during intestinal epithelial cell differentiation, we examined the effect of p27(Kip1) on the activity of the transcription factor CDX2. The present results revealed the following. 1) p27(Kip1) interacts with the CDX2 transcription factor. 2) In contrast to CDX2 mRNA levels, CDX2 protein expression levels significantly increased as soon as Caco-2/15 cells reached confluence, slowed their proliferation, and began their differentiation. The mechanism of CDX2 regulation is primarily related to protein stability, because inhibition of proteasome activity increased CDX2 levels. The half-life of CDX2 protein was significantly enhanced in differentiated versus undifferentiated proliferative intestinal epithelial cells. 3) Cdk2 interacted with CDX2 and phosphorylated CDX2, as determined by pull-down glutathione S-transferase and immunoprecipitation experiments with proliferating undifferentiated Caco-2/15 cell extracts. 4) Treatment of Caco-2/15 cells with MG132 (a proteasome inhibitor) and (R)-roscovitine (a specific Cdk2 inhibitor) induced an increase in CDX2 protein levels. 5) Conversely, ectopic expression of Cdk2 resulted in decreased expression of CDX2 protein. 6) Of note, treatment of proliferative Caco-2/15 cells with (R)-roscovitine or leptomycin (an inhibitor of nuclear export through CRM1) led to an accumulation of CDX2 into the nucleus. These data suggest that CDX2 undergoes CRM1-dependent nuclear export and cytoplasmic degradation in cells in which Cdk2 is activated, such as in proliferative intestinal epithelial cells. The targeted degradation of CDX2 following its phosphorylation by Cdk2 identifies a new mechanism through which CDX2 activity can be regulated in coordination with the cell cycle machinery.

Extracellular signal-regulated kinase mediates phosphorylation of tropomyosin-1 to promote cytoskeleton remodeling in response to oxidative stress: impact on membrane blebbing.

Oxidative stress induces in endothelial cells a quick and transient coactivation of both stress-activated protein kinase-2/p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. We found that inhibiting the ERK pathway resulted, within 5 min of oxidative stress, in a misassembly of focal adhesions characterized by mislocalization of key proteins such as paxillin. The focal adhesion misassembly that followed ERK inhibition with the mitogen-activated protein kinase kinase (MEK) inhibitor PD098059 (2'-amino-3'-methoxyflavone) or with a kinase negative mutant of ERK in the presence of H(2)O(2) resulted in a quick and intense membrane blebbing that was associated with important damage to the endothelium. We isolated by two-dimensional gel electrophoresis a PD098059-sensitive phosphoprotein of 38 kDa that we identified, by mass spectrometry, as tropomyosin-1. In fact, H(2)O(2) induced a time-dependent phosphorylation of tropomyosin that was sensitive to inhibition by PD098059 and UO126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butanediane). Tropomyosin phosphorylation was also induced by expression of a constitutively activated form of MEK1 (MEK(CA)), which confirms that its phosphorylation resulted from the activation of ERK. In unstimulated cells, tropomyosin-1 was found diffuse in the cells, whereas it quickly colocalized with actin and stress fibers upon stimulation of ERK by H(2)O(2) or by expression of MEK(CA). We propose that phosphorylation of tropomyosin-1 downstream of ERK by contributing to formation of actin filaments increases cellular contractility and promotes the formation of focal adhesions. Incidentally, ML-7 (1-[5iodonaphthalene-1-sulfonyl]homopiperazine, HCl), an inhibitor of cell contractility, inhibited phosphorylation of tropomyosin and blocked the formation of stress fibers and focal adhesions, which also led to membrane blebbing in the presence of oxidative stress. Our finding that tropomyosin-1 is phosphorylated downstream of ERK, an event that modulates its interaction with actin, may lead to further understanding of the role of this protein in regulating cellular functions associated with cytoskeletal remodeling.