Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors.

BACKGROUND: The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established. METHODS: Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells. RESULTS: We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect. CONCLUSION: MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.

Basement membrane protein and matrix metalloproteinase deregulation in engineered human oral mucosa following infection with Candida albicans.

A variety of morphological changes in the basement membrane (BM) are known to occur in inflammatory diseases. Modifications of the BM can be associated with significant changes in protein content. Candida albicans (C. albicans) is normally a commensal organism and is a member of the natural flora of a large number of healthy individuals. However, under certain conditions, C. albicans can invade host tissues, causing inflammation and tissue damage. The aim of this study was to investigate the effect of C. albicans on the expression and production of structural (laminin-5 and type IV collagen) and inflammatory [matrix metalloproteinases (MMPs) and their inhibitors] proteins by human oral epithelial cells. Using engineered normal human oral mucosa infected with 10(5) C. albicans/cm2 for different periods of time, we were able to demonstrate that this yeast promotes significant laminin-5 and type IV collagen gene activation and protein secretion. These effects were accompanied by MMP-2 and MMP-9 gene activation. Interestingly, only the levels of active MMP-9 rose. The increase in MMP levels was paralleled by a decrease in the secretion of type 2 matrix metalloproteinase tissue inhibitors (TIMP-2). Our results demonstrated that C. albicans has a significant effect on tissue structure through BM protein and MMP modulation. This might help C. albicans overcome the mechanical and biological defenses of the tissue and allow it to disseminate, causing severe infections. If C. albicans uses MMPs (mainly MMP-9) to disseminate, inhibition of this protease could be of interest in treating a variety of inflammatory disorders, including oral candidiasis.

In vitro analyses of tissue structure and interleukin-1beta expression and production by human oral mucosa in response to Candida albicans infections.

Clinical and experimental observations suggest that oral epithelial cells play a key role in host defenses against candidal infections through cytokines and chemokines. We thus attempted to determine whether oral epithelial cells convey IL-1beta as a pro-inflammatory cytokine in response to Candida albicans infections. We created engineered human oral mucosa (EHOM), put them in contact with live and heat-inactivated C. albicans (10(5) yeast/cm2), and measured the expression of IL-1beta mRNA and protein. Tissue structure and C. albicans morphology were also evaluated. Only live C. albicans modulated IL-1beta expression and secretion. IL-1beta mRNA expression significantly increased during the early stages of infection and decreased during the later stages. The modulatory effect of C. albicans on IL-1beta expression was confirmed by the fact that increased amounts of inactive IL-1beta (33 kDa) were detected early during the infection which then dropped dramatically. There was a significant and time-dependent increase in the amount of the active form of IL-1beta (17 kDa) secreted into the supernatant by epithelial cells infected with live C. albicans. Histological features revealed damage to infected tissues (separation of epithelial cells, edema, vacuolization, reduction in thickness) compared to uninfected ones. Morphological analyses showed that C. albicans changed from a blastospore to a hyphal form at later infection periods. This transformation was very pronounced at 8 and 24 h post-infection. These results provide additional evidence for the contribution of oral epithelial cells to local defenses against exogenous stimulations such as C. albicans infections.

Tissue structure, and IL-1beta, IL-8, and TNF-alpha secretions after contact by engineered human oral mucosa with dentifrices.

The use of dentifrice is part of an oral prophylaxis that aims at keeping bacteria in check within the dental plaque. When introduced into the oral cavity, dentifrice also comes in close contact with the oral epithelium. Our goal was to evaluate the effects of dentifrices on tissue structure and pro-inflammatory mediator release by epithelial cells. For this purpose, tri-dimensional engineered human oral mucosa (EHOM) was produced using normal human palatal fibroblasts and epithelial cells. EHOMs were either treated with Aquafresh(R) or Crest(R) for 1, 4, 8, and 24 h, or untreated, then used for cell viability assessment and structural analyses. Cultured supernatants were used to evaluate cytokine (interleukin (IL)-1beta, IL-8 and tumor necrosis factor (TNF)-alpha) secretion, and metalloproteinase (MMP)-2 and -9 activities. The present in vitro study using engineered oral mucosa confirms that dentifrices (Aquafresh and Crest) contribute to tissue desquamation. The desquamation was substantial at 24 h of contact but was limited to the upper layers of the treated tissues. Cell death in these tissues was not increased, suggesting that the dentifrice had accelerated desquamation of the layers containing differentiated cells. Measurement of cytokines revealed that dentifrices up-regulated IL-1beta while down-regulating IL-8 and TNF-alpha secretion, thus indicating an impaired cascade of inflammatory responses. These dentifrices may also impair normal repair mechanisms as suggested by an up-regulation of gelatinase activities. In conclusion, this study suggested that, via cytokines, dentifrice contributes to the modulation of the inflammatory (pro-inflammatory/anti-inflammatory responses) process.