Blockade of integrin α3 attenuates human pancreatic cancer via inhibition of EGFR signalling.

The prognosis of pancreatic cancer remains dismal despite continuous and considerable efforts. Integrins (ITGs) are highly expressed in various malignant cancers. However, very few studies investigated the role of integrin α3 (ITGα3) in malignant cancers. Here, we determined the functional role of ITGα3 in pancreatic cancer. Analysis of public microarray databases and Western blot analysis indicated a unique expression of ITGα3 in human pancreatic cancer. Silencing ITGα3 expression significantly inhibited the viability and migration of human pancreatic cancer cells. Notably, ablation of ITGα3 expression resulted in a significant decrease of epidermal growth factor receptor (EGFR) expression compared with transfection of control-siRNA through an increased number of leucine-rich repeats and immunoglobulin-like domain protein 1 (LRIG1) expression. In addition, ablating ITGα3 inhibited tumour growth via blockade of EGFR signalling in vivo. Furthermore, the highly expressed ITGα3 led to a poor prognosis of pancreatic cancer patients. Our results provide novel insights into ITGα3-induced aggressive pancreatic cancer.

Regulation of Protein Activity and Cellular Functions Mediated by Molecularly Evolved Nucleic Acids.

Regulation of protein activity is essential for revealing the molecular mechanisms of biological processes. DNA and RNA achieve many uniquely efficient functions, such as genetic expression and regulation. The chemical capability to synthesize artificial nucleotides can expand the chemical space of nucleic acid libraries and further increase the functional diversity of nucleic acids. Herein, a versatile method has been developed for modular expansion of the chemical space of nucleic acid libraries, thus enabling the generation of aptamers able to regulate protein activity. Specifically, an aptamer that targets integrin alpha3 was identified and this aptamer can inhibit cell adhesion and migration. Overall, this chemical-design-assisted in vitro selection approach enables the generation of functional nucleic acids for elucidating the molecular basis of biological activities and uncovering a novel basis for the rational design of new protein-inhibitor pharmaceuticals.

Disease-associated single amino acid mutation in the calf-1 domain of integrin α3 leads to defects in its processing and cell surface expression.

Integrin α3ß1, a receptor for laminins, is involved in the structural and functional organization of epithelial organs, including the lung, kidney, and skin. Recently, a missense mutation that causes substitution of Arg628 with Pro (R628P) in the calf-1 domain of human α3 was shown to be associated with disorders of the lung, kidney, and skin. Here, we found that the R628P mutation leads to aberrations in the posttranslational processing of α3. Specifically, α3 with the R628P mutation showed hardly any cleavage at the calf-2 domain, which usually occurs in the Golgi apparatus during the delivery of de novo-synthesized α3. The mutant α3 retained the ability to associate with integrin ß1, but not with the tetraspanin CD151, and the bound ß1 was a partially glycosylated immature form, the maturation of which also takes place in the Golgi apparatus. Furthermore, the cell surface expression of the mutant protein was markedly reduced. These results suggest that the R628P mutation leads to a deficit in the transport of α3ß1 from the ER to the Golgi apparatus. When Arg628 was mutated to Gln or Glu, instead of Pro, the resulting mutants did not display aberrations in processing or CD151 binding, indicating that the presence of Pro, rather than the absence of Arg, at amino acid residue 628 of α3 is important for the abnormalities in the R628P mutant. In support of this notion, a homology modeling analysis of the calf-1 domain of α3 showed that replacement with Pro, but not with Gln or Glu, caused partial disruption of the ß-sheet structures. Furthermore, the ER-associated degradation of the R628P mutant was not enhanced compared with that of the wild-type protein, suggesting that the deficits in the posttranslational processing and cell surface expression of the R628P mutant are independent of the ER-associated degradation, but arise from the defect in its export from the ER. We conclude that the calf-1 domain is required for the transport of α3 from the ER to the Golgi apparatus to maintain the integrity of epithelial tissues, and hence the impairment of the calf-1 domain by the R628P mutation leads to severe diseases of the kidneys, lungs, and skin.

Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system.

The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.

Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1.

Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the alpha3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the alpha2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-alpha2beta1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-alpha3beta1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors.

Differential regulation of cell adhesive functions by integrin alpha subunit cytoplasmic tails in vivo.

Cell adhesion to fibronectin (FN) is crucial for early vertebrate morphogenesis. In Xenopus gastrulae, several distinct integrin-dependent adhesive behaviors can be identified: adhesion of cells to FN, assembly of FN fibrils, and initiation of cell spreading and migration in response to mesoderm inducing signals. We have taken a chimeric integrin approach to investigate the role of the integrin alpha cytoplasmic tail in the specification of these developmentally significant adhesive functions. Cytoplasmic tail-deleted alpha4 constructs and alpha4-ectodomain/alpha-cytoplasmic tail chimeras were generated and expressed in whole embryos. Normal gastrula cells lack integrin alpha4 and, correspondingly, are unable to adhere to the alpha4 ligand, the V-region of FN. The ability of alpha4 constructs to promote adhesive behaviors was established by placing tissue explants or dissociated cells on an FN V-region fusion protein that lacks the RGD (Arg-Gly-Asp)/synergy sites or treating whole embryos with antibodies that block endogenous integrin-FN interactions. We found that each alpha4 cytoplasmic domain deletion mutant and alpha-tail chimera examined could support cell attachment; however, activin induction-dependent cell spreading, mesoderm cell and explant motility, and the ability to assemble FN matrix on the blastocoel roof varied with specific alpha subunit tail sequences. These data suggest that alpha cytoplasmic tail signaling and changes in integrin activation state can regulate a variety of developmentally significant adhesive behaviors in both space and time.

Tetraspanin CD9 is a "proteolipid," and its interaction with alpha 3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of laminin-5-dependent cell motility.

GM3 ganglioside inhibits tetraspanin CD9-facilitated cell motility in various cell lines (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414-6421). We now report the following: (i) CD9 has the novel feature of being soluble in chloroform/methanol, and classifiable as "proteolipid"; (ii) CD9 and alpha(3) integrin were concentrated together in the low-density glycolipid-enriched microdomain (GEM) of ldlD/CD9 cells, and the alpha(3) expression ratio (value for cells grown under +Gal condition divided by the value for cells grown under -Gal condition) in GEM of ldlD/CD9 cells was higher than that in control ldlD/moc cells, suggesting that CD9 recruits alpha(3) in GEM under +Gal condition, whereby GM3 is present. (iii) Chemical levels of alpha(3) and CD9 in the total extract or membrane fractions from cells grown under +Gal versus -Gal condition were nearly identical, whereas alpha(3) expressed at the cell surface, probed by antibody binding in flow cytometry, was higher under -Gal than +Gal condition. These results suggest that GM3 synthesized under +Gal condition promotes interaction of alpha(3) with CD9, which restricts alpha(3) binding to its antibody. A concept of the alpha(3)/CD9 interaction promoted by GM3 was further supported by (i) co-immunoprecipitation of CD9 and alpha(3) under +Gal but not -Gal condition, (ii) enhanced co-immunoprecipitation of CD9 and alpha(3) when GM3 was added exogenously to cells under -Gal condition, and (iii) the co-localization images of CD9 with alpha(3) and of GM3 with CD9 in fluorescence laser scanning confocal microscopy. Based on the promotion of alpha(3)/CD9 interaction by GM3 and the status of laminin-5 as a true ligand for alpha(3), the laminin-5/alpha(3)-dependent motility of ldlD/CD9 cells was found to be greatly enhanced under -Gal condition, but strongly inhibited under +Gal condition. Such a motility difference under +Gal versus -Gal condition was not observed for ldlD/moc cells. The inhibitory effect observed in ldlD/CD9 cells under +Gal condition was reversed upon addition of anti-alpha(3) antibody and is therefore based on interaction between alpha(3), CD9, and GM3 in GEM.