Estructura del ectodominio de 4F2hc e implicación de 4F2hc en tumorigénesis: efectos del butirato y los ácidos biliares sobre células de adenocarcinoma de colon

Abstract

4F2hc is a type-II glycoprotein that form covalently-bound heterodimers with several described light chains and whose main function is the transport of amino acids. Likewise, the heavy chain interacts with β-integrins mediating integrin-dependent events such as survival, proliferation, migration and even transformation. Its large C-terminal domain resembles α-amylases structure, but lacking catalytic residues and thus enzymatic activity. In fact, this ectodomain contains the N-terminal domain A, a TIM barrel, connected by 6 residues in α-helix to the C-terminal domain C, comprised of a β-sandwich. Spectroscopic/structural characterization of recombinant 4F2hc-ED shows that its structure in solution is quite similar to that of the crystal, being compact and thermally stable. Moreover, this ectodomain is unable to homodimerize by itself, remaining monomeric in solution. According to the data obtained, the folding/unfolding mechanism of 4F2hc-ED may occur following a 4-state model with 2 intermediate forms. 4F2hc is a ubiquitous protein whose overexpression is related to tumor development and progression. Stable silencing of 4F2hc in HeLa cells shows impaired in vivo tumorigenicity and an ineffective proliferative response to mitogens. Moreover, silenced cells present defects in integrin- (FAK, Akt and ERK1/2) and hypoxia-dependent signaling, reduced expression/activity of MMP-2 and increased degradation of β-catenin. Furthermore, cell motility is impaired after protein silencing, showing for the first time the involvement of 4F2hc in tumor invasion via facilitating cell motiliy. Therefore, 4F2hc participates in tumor progression favoring the first steps of epithelial-mesenchymal transition, by inhibiting β-catenin proteasomal degradation through Akt/GSK-3β signaling and enabling cell motility and neovascularization. In addition, 4F2hc overexpression is irrespective of tumor tissue origin. Its association with β1-integrin does not occur in lipid rafts, but may be in tetraspaninenriched membrane domains. There is no clear correlation between 4F2hc protein levels and tumorigenicity activity or differentiation degree, although cells showing a more malignant phenotype present more complex glycosylation. 4F2hc silencing in tumorigenic BCS-TC2.BR2 and BCS-TC2.2 cells (the former more differentiated than the latter) gives rise to a delayed tumor growth rate. Colonic epithelium is exposed to a wide range of agents which may affect its homeostasis. Among them, butyrate, a product of anaerobic bacterial fermentation of dietary fiber, regulates it controlling proliferation, differentiation and apoptosis of intestinal cells. Other cytotoxic agents usually present in colonic lumen are bile acids. Acquisition of resistance to these apoptotic/cytotoxic effects is crucial for the development of colorectal carcinogenesis. Studies were carried out using a nontumorigenic human colon adenocarcinoma cell line sensitive to butyrate (BCS-TC2) and a cell subline resistant to its apoptotic effect (BCS-TC2.BR2). The latter is tumorigenic and present also resistance to metabolic, thermal and osmotic stress, and to other luminal components such as bile acids or LPS. Bile acids exert cytotoxic effects on BCS-TC2 cells triggering apoptosis by disruption of mitochondrial function. First, DCA and CDCA increase ROS generation by activating plasma membrane enzymes; this promotes mitochondrial transition permeability and subsequently caspase activation via apotosome. Caspase-3 induces amplification of apoptosis by degradation of Bcl-2 with consequent activation of Bax, which generates additional mitochondrial permeabilization and misfunction. In both cell lines butyrate uptake is carried out by two different mechanisms: at high concentrations a butyrate/bicarbonate antiporter and at low concentrations, a H+/monocarboxilate cotransporter. The latter is energy dependent, activated by PKCδ and mediated by MCT1. Butyrate mainly exerts its effects through inhibition of HDACs, regulating gene expression by controlling the acetylation status of histones and non-histone proteins, such as Smad2 in the regulation of butyrate-induced MMP11 gene. Moreover, many signaling pathways are altered by HDAC inhibitors. Butyrate triggers a pro-survival early activation of Akt and ERK1/2 in both cell lines, which later on falls off as a consequence of apoptosis in BCS-TC2 cells, whereas BCSTC2. BR2 maintain basal activation levels of these kinases. Moreover, HDAC inhibitors affect other signaling pathways such as JNK, HIF-1 dependent signaling and NFκB, among others. In addition, butyrate induces cell cycle arrest by transcriptional activation of p21 (which is independent of JNK and p38 signaling). Furthermore, butyrate provokes double strand breaks as confirmed by γH2AX expression in both cell lines, being delayed in BCS-TC2.BR2.