Colorectal Cancer: From the Genetic Model to Posttranscriptional Regulation by Noncoding RNAs.

Colorectal cancer is the third most common form of cancer in developed countries and, despite the improvements achieved in its treatment options, remains as one of the main causes of cancer-related death. In this review, we first focus on colorectal carcinogenesis and on the genetic and epigenetic alterations involved. In addition, noncoding RNAs have been shown to be important regulators of gene expression. We present a general overview of what is known about these molecules and their role and dysregulation in cancer, with a special focus on the biogenesis, characteristics, and function of microRNAs. These molecules are important regulators of carcinogenesis, progression, invasion, angiogenesis, and metastases in cancer, including colorectal cancer. For this reason, miRNAs can be used as potential biomarkers for diagnosis, prognosis, and efficacy of chemotherapeutic treatments, or even as therapeutic agents, or as targets by themselves. Thus, this review highlights the importance of miRNAs in the development, progression, diagnosis, and therapy of colorectal cancer and summarizes current therapeutic approaches for the treatment of colorectal cancer.

Structural and lipid-binding characterization of human annexin A13a reveals strong differences with its long A13b isoform.

Annexin A13 is the founder member of the vertebrate family of annexins, which are comprised of a tetrad of unique conserved domains responsible for calcium-dependent binding to membranes. Its expression is restricted to epithelial intestinal and kidney cells. Alternative splicing in the N-terminal region generates two isoforms, A13a and A13b, differing in a deletion of 41 residues in the former. We have confirmed the expression of both isoforms in human colon adenocarcinoma cells at the mRNA and protein levels. We have cloned, expressed, and purified human annexin A13a for the first time to analyze its structural characteristics. Its secondary structure and thermal stability differs greatly from the A13b isoform. The only tryptophan residue (Trp186) is buried in the protein core in the absence of calcium but is exposed to the solvent after calcium binding even though circular dichroism spectra are quite similar. Non-myristoylated annexin A13a binds in a calcium-dependent manner to acidic phospholipids but not to neutral or raft-like liposomes. Calcium requirements for binding to phosphatidylserine are around 6-fold lower than those required by the A13b isoform. This fact could account for the different subcellular localization of both annexins as binding to basolateral membranes seems to be calcium-dependent and myristoylation-independent.

Annexin-phospholipid interactions. Functional implications.

Annexins constitute an evolutionary conserved multigene protein superfamily characterized by their ability to interact with biological membranes in a calcium dependent manner. They are expressed by all living organisms with the exception of certain unicellular organisms. The vertebrate annexin core is composed of four (eight in annexin A6) homologous domains of around 70 amino acids, with the overall shape of a slightly bent ring surrounding a central hydrophilic pore. Calcium- and phospholipid-binding sites are located on the convex side while the N-terminus links domains I and IV on the concave side. The N-terminus region shows great variability in length and amino acid sequence and it greatly influences protein stability and specific functions of annexins. These proteins interact mainly with acidic phospholipids, such as phosphatidylserine, but differences are found regarding their affinity for lipids and calcium requirements for the interaction. Annexins are involved in a wide range of intra- and extracellular biological processes in vitro, most of them directly related with the conserved ability to bind to phospholipid bilayers: membrane trafficking, membrane-cytoskeleton anchorage, ion channel activity and regulation, as well as antiinflammatory and anticoagulant activities. However, the in vivo physiological functions of annexins are just beginning to be established.

Resistance to butyrate impairs bile acid-induced apoptosis in human colon adenocarcinoma cells via up-regulation of Bcl-2 and inactivation of Bax.

A critical risk factor in colorectal carcinogenesis and tumor therapy is the resistance to the apoptotic effects of different compounds from the intestinal lumen, among them butyrate (main regulator of colonic epithelium homeostasis). Insensitivity to butyrate-induced apoptosis yields resistance to other agents, as bile acids or chemotherapy drugs, allowing the selective growth of malignant cell subpopulations. Here we analyze bile acid-induced apoptosis in a butyrate-resistant human colon adenocarcinoma cell line (BCS-TC2.BR2) to determine the mechanisms that underlay the resistance to these agents in comparison with their parental butyrate-sensitive BCS-TC2 cells. This study demonstrates that DCA and CDCA still induce apoptosis in butyrate-resistant cells through increased ROS production by activation of membrane-associated enzymes and subsequent triggering of the intrinsic mitochondrial apoptotic pathway. Although this mechanism is similar to that described in butyrate-sensitive cells, cell viability is significantly higher in resistant cells. Moreover, butyrate-resistant cells show higher Bcl-2 levels that confer resistance to bile acid-induced apoptosis sequestering Bax and avoiding Bax-dependent pore formation in the mitochondria. We have confirmed that this resistance is reverted using the Bcl-2 inhibitor ABT-263, thus demonstrating that the lower sensitivity of butyrate-resistant cells to the apoptotic effects of bile acids is mainly due to increased Bcl-2 levels.

The structure of human 4F2hc ectodomain provides a model for homodimerization and electrostatic interaction with plasma membrane.

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in amino acid transport and cell fusion, adhesion, and transformation. The structure of the ectodomain of human 4F2hc has been solved using monoclinic (Protein Data Bank code 2DH2) and orthorhombic (Protein Data Bank code 2DH3) crystal forms at 2.1 and 2.8 A, respectively. It is composed of a (betaalpha)(8) barrel and an antiparallel beta(8) sandwich related to bacterial alpha-glycosidases, although lacking key catalytic residues and consequently catalytic activity. 2DH3 is a dimer with Zn(2+) coordination at the interface. Human 4F2hc expressed in several cell types resulted in cell surface and Cys(109) disulfide bridge-linked homodimers with major architectural features of the crystal dimer, as demonstrated by cross-linking experiments. 4F2hc has no significant hydrophobic patches at the surface. Monomer and homodimer have a polarized charged surface. The N terminus of the solved structure, including the position of Cys(109) residue located four residues apart from the transmembrane domain, is adjacent to the positive face of the ectodomain. This location of the N terminus and the Cys(109)-intervening disulfide bridge imposes space restrictions sufficient to support a model for electrostatic interaction of the 4F2hc ectodomain with membrane phospholipids. These results provide the first crystal structure of heteromeric amino acid transporters and suggest a dynamic interaction of the 4F2hc ectodomain with the plasma membrane.

The tetraspanin CD9 inhibits the proliferation and tumorigenicity of human colon carcinoma cells.

The implication of the tetraspanin CD9 in cancer has received much recent attention and an inverse correlation between CD9 expression and the metastatic potential and cancer survival rate has been established for different tumor types. In contrast to the well-established role of CD9 in metastasis, very little is known about the involvement of this tetraspanin in the process of development of primary tumors. In the present study, we present evidence on the implication of CD9 in colon carcinoma tumorigenesis. We report here that ectopic expression of CD9 in colon carcinoma cells results in enhanced integrin-dependent adhesion and inhibition of cell growth. Consistently with these effects, treatment of these cells with anti-CD9-specific antibodies resulted in (i) increased beta1 integrin-mediated cell adhesion through a mechanism involving clustering of integrin molecules rather than altered affinity; (ii) induction of morphological changes characterized by the acquisition of an elongated cell phenotype; (iii) inhibition of cell proliferation with no significant effect on cell survival; (iv) increased expression of membrane TNF-alpha, and finally (v) inhibition of the in vivo tumorigenic capacity in nude mice. In addition, through the use of selective blockers of TNF-alpha, we have demonstrated that this cytokine partly mediates the antiproliferative effects of CD9. These results clearly establish for the first time a role for CD9 in the tumorigenic process.

Structure-function relationship in annexin A13, the founder member of the vertebrate family of annexins.

Annexin A13 is considered the original progenitor of the 11 other members of vertebrate annexins, a superfamily of calcium/phospholipid-binding proteins. It is highly tissue-specific, being expressed only in intestinal and kidney epithelial cells. Alternative splicing generates two isoforms, both of which bind to rafts. In view of the lack of structural information supporting the physiological role of this annexin subfamily, we have cloned, expressed and purified human annexin A13b to investigate its structural and functional properties. The N-terminus of annexin A13b: (i) destabilizes the conserved protein core, as deduced from the low melting temperature in the absence (44 degrees C) or presence of calcium (55 degrees C), and (ii) impairs calcium-dependent binding to acidic phospholipids, requiring calcium concentrations >400 microM. Truncation of the N-terminus restores thermal stability and decreases the calcium requirement for phospholipid binding, confirming its essential role in the structure-function relationship of this annexin. Non-myristoylated annexin A13b only binds to acidic phospholipids at high calcium concentrations. We show for the first time that myristoylation of annexin A13b enables the direct binding to phosphatidylcholine, raft-like liposomes and acidic phospholipids in a calcium-independent manner. The conformational switch induced by calcium binding, from a 'closed' to an 'open' conformation with exposure of Trp227, can be mimicked by a decrease in pH, a process that may be relevant for membrane interactions. Our studies confirm that the common structural and functional characteristics that are dependent on the protein core of vertebrate annexins are likely to be common conserved features, whereas their variable N-termini confer distinct functional properties on annexins, as we report for myristoylation of annexin A13b.

Effects of periodate and chondroitin 4-sulfate on proteoglycan stabilization of ostrich pericardium. Inhibition of calcification in subcutaneous implants in rats.

Chemical modification of biological materials used in the manufacture of cardiac valves tends to reduce the relatively high degree of biodegradation and calcification of the implanted bioprostheses. The most widely used treatment to reduce biodegradability of the valves is glutaraldehyde fixation. However, this treatment is potentially toxic and induces tissue calcification. In order to minimize these undesirable effects, we have analyzed the effect of a pre-fixation of endogenous proteoglycans and exogenous glycosaminoglycans, as well as the borohydride reduction influence on the different modified ostrich pericardium implants after subcutaneous implantation in rats. The presence of calcific deposits was detected in all implanted GA-fixed samples; however, calcification was highly reduced in both groups of periodate-prefixed materials, which showed also a very low Ca/P molar ratio. Borohydride post-treatment of these biomaterials resulted in a significant increase in calcium phosphate precipitation, with the appearance of calcium deposits mainly in an amorphous form even though X-ray diffraction allowed the detection of brushite- and apatite-like crystals. Regarding tissue stability, no significant differences were found among the borohydride-untreated implants but higher levels of matrix metalloproteinases were observed by gelatin zymography in the periodate pre-fixed materials. This increase was partially reduced by pre-fixation of exogenous chondroitin 4-sulfate. On the other hand, borohydride post-treatment not only increased calcification, but also reduced tissue stability and increased the presence of matrix-degrading activities.

A functionally relevant conformational epitope on the CD9 tetraspanin depends on the association with activated beta1 integrin.

Tetraspanins associate on the cell membrane with several transmembrane proteins, including members of the integrin superfamily. The tetraspanin CD9 has been implicated in cell motility, metastasis, and sperm-egg fusion. In this study we characterize the first CD9 conformation-dependent epitope (detected by monoclonal antibody (mAb) PAINS-13) whose expression depends on changes in the activation state of associated beta(1) integrins. MAb PAINS-13 precipitates CD9 under conditions that preserve the association of this tetraspanin with integrins, but not under conditions that disrupt these interactions. Induction of activation of beta(1) integrins by temperature, divalent cation Mn(2+), or mAb TS2/16 correlated with enhanced expression of the PAINS-13 epitope on a variety of cells. Through the use of different K562 myeloid leukemia transfectant cells expressing specific members of the beta(1) integrin subfamily we show that the expression of the PAINS-13 epitope depends on CD9 association with alpha(6)beta(1) integrin. The mAb PAINS-13 reactivity has been mapped to the CD9 region comprising residues 112-154 in the NH(2) half of the large extracellular loop. Also, we show that the CD9 conformation recognized by mAb PAINS-13 is functionally relevant in beta(1) integrin-mediated cellular processes including wound healing migration, tubular morphogenesis, cell adhesion and spreading and in signal transduction involving phosphatidylinositol 3-kinase activation.