Glycosylation of Integrins in Melanoma Progression

Each stage of melanoma development from transformed melanocytes to metastatic lesions requires the involvement of cell adhesion receptors, among which integrins are of particular importance. Strong N-glycosylation of αβ integrin heterodimers influences their processing, activation, and functions related to the modulation of cell adhesion to extracellular matrix proteins (ECM) and the basement membrane. A lack of N-glycans on integrin chains significantly reduces their interactions with the ECM. Melanoma progression is accompanied by changes in the composition of N-glycans on integrin subunits. The glycosylation profile of integrins depends on the stage of melanoma development and on the location of the metastasis. Enhanced expression of β1,6-branched complex-type oligosaccharides and altered sialylation are well-characterized changes in the N-glycosylation of integrins observed in melanoma progression. This chapter summarizes the current state of knowledge about α3β1, α5β1, and αvβ3 integrin glycosylation in melanoma and the functional consequences of changed glycosylation for the development of this cancer

Identification of tumour-associated gycoproteins recognised by the lectin from Helix pomatia in breast cancer

Helix pomatia agglutinin (HPA) is a carbohydrate binding protein isolated from the Roman snail. There has been considerable interest in understanding HPA binding partners in cancer, as the lectin has been shown to identify glycosylation changes in cancers arising from the epithelia, from patients with poor prognosis. Identifying the HPA binding epitopes associated with a malignant phenotype may be useful for prognostication and may also offer potential as targets for immunotherapy. Previous studies have shown that HPA recognises a multitude of proteins in colorectal cancer (CRC). This study aimed to establish whether HPA recognises the same glycoproteins in breast cancer. An in vitro model of human breast cancer cell lines was used, ranging from HPA negative, non metastatic, to HPA positive and metastatic. Four human breast cell lines were chosen to represent phenotypes ranging from ‘normal’/benign (HMT3522), primary cancer (BT474) to metastatic cancer (T47D, MCF-7). HPA binding was assessed using confocal microscopy. Membrane proteins were extracted by differential centrifugation and the proteins were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Western blotting. The cell surface glycoproteins recognised by HPA were characterised using 2-dimensional electrophoresis (2-DE), Western blotting and mass spectrometry. HPA binding correlated with integrin α6 levels, this concurred with previous findings in CRC. HPA also bound transcription factors HnRNP H1, HnRNP D-like, HnRNP A2/B1 as well as Hsp27, GFAP and ENO1. The recognition of HnRNPs, Hsp 27 and ENO1 by HPA correlated with O-GlcNAcylation of these proteins. Interestingly, these HPA-binding glycoproteins were either absent or showed decreased levels in the non-metastatic breast cancer cell line BT474 and in ‘normal’ HMT3522. A comprehensive analysis of the breast cells proteome showed a number of proteins with elevated level in the metastatic breast cancer cell lines T47D and MCF-7, but this is the first report to show elevated levels of elongation factor Tu, Enoyl Coenzyme A hydratase 1 peroxisomal and macropain subunits. This work was extended to analyse the gene expression for UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase (ppGalNAc T1,T2, T3 and T6) and alpha 2,6 sialyltransferases (ST6GalNAc I and II) in the breast cell lines, but no correlation between the expression levels of mRNA of these enzymes and HPA binding was found in this study. The results from the present study show that, as in CRC cell lines, integrin α6 was the most abundant HPA binding glycoprotein extracted from the breast cancer cells with a metastatic phenotype. This is the first report in which HPA has been shown to bind O-GlcNAcylated transcription factors. This class of proteins represent a new means by which HPA differentiates cancer cells with an aggressive metastatic phenotype. New approaches aimed at targeting these changes might have broad application for the treatment of breast, colorectal and possibly other epithelial cancers

Functional analysis of the threonine motif in the β1 integrin cytoplasmic tail in mice

Integrins are ubiquitously expressed adhesion receptors with important functions in cellular adhesion, proliferation, migration and signaling. These functions are determined by integrin trafficking through endosomal compartments and receptor affinity regulation. In this thesis, we identified the distal NxxY motif of the β1 integrin cytoplasmic tail as a molecular switch modulating a spatiotemporally controlled binding of two FERM-domain proteins in different cellular compartments. Kindlins mediate integrin activation at the plasma membrane and they dislodge upon internalization. In the endosomal compartment, the free cytoplasmic domain is subsequently bound by sorting nexin 17 (SNX17) to inhibit integrin degradation. We identified SNX17 as a new β1 integrin adaptor protein, which uses the kindlin-binding site in endosomal compartments to stabilize integrins and to promote their recycling back to the plasma membrane.Integrine sind ubiquitär exprimierte Adhäsionsmoleküle mit entscheidender Bedeutung für die zelluläre Adhäsion, Wachstum, Migration und Signalgebung. Diese Funktionen werden durch konstantes Integrintrafficking durch endosomale Kompartimente sowie durch Modulation der Rezeptoraffinität kontrolliert. Im Rahmen der vorliegenden Dissertation wurde das distale NxxY-Motiv im zytoplasmatischen β1 Integrinschwanz als molekularer Schalter identifiziert, der die Bindung zweier FERM-Domänen-Proteine in unterschiedlichen zellulären Kompartimenten vermittelt. Während Kindline an der Zellmembran die Integrinaktivität regulieren, dislozieren sie nach der Internalisierung und Sorting Nexin 17 (SNX17) wird an den nun freien β1 Integrinschwanz rekrutiert, um die Integrindegradation zu hemmen. Wir haben SNX17 als neuen β1 Integrin-Bindungspartner identifiziert, der die Kindlin-Bindungsstelle im endosomalen Kompartiment verwendet, um Integrine zu stabilisieren und ihr Recycling zurück an die Zelloberfläche zu fördern

Role of Basement Membrane and Extracellular Matrix Proteins in the Adhesion and Spreading of Immortalized Human Corneal Epithelial Cells

The repair of corneal wounds requires both epithelial cell adhesion and migration. Basement membrane (BM) and extracellular matrix (ECM) proteins function in these processes via integrin and non-integrin receptors. We have studied the adhesion, spreading and migration of immortalized human corneal epithelial (HCE) cells and their interactions with the laminins (Lms), fibronectins and tenascins produced. Human corneal BM expresses Lms-332 and -511, while Lm-111 was not found in these experiments. HCE cells produced both processed and unprocessed Lm-332, whereas neither Lm-111 nor Lm-511 was produced. Because HCE cells did not produce Lm-511, although it was present in corneal BM, we suggest that Lm-511 is produced by stromal keratocytes. The adhesion of HCE cells to Lms-111, -332 and -511 was studied first by determining the receptor composition of HCE cells and then by using quantitative cell adhesion assays. Immunofluorescence studies revealed the presence of integrin α2, α3, α6, β1 and β4 subunits. Among the non-integrin receptors, Lutheran (Lu) was found on adhering HCE cells. The cells adhered via integrin α3β1 to both purified human Lms-332 and -511 as well as to endogenous Lm-332. However, only integrin β1 subunit functioned in HCE cell adhesion to mouse Lm-111. The adhesion of HCE cells to Lm-511 was also mediated by Lu. Since Lm-511 did not induce Lu into focal adhesions in HCE cells, we suggest that Lm-511 serves as an ECM ligand enabling cell motility. HCE cells produced extradomain-A fibronectin, oncofetal fibronectin and tenascin-C (Tn-C), which are also found during corneal wound healing. Monoclonal antibodies (MAbs) against integrins α5β1 and αvβ6 as well as the arginine-glycine-aspartic acid (RGD) peptide inhibited the adhesion of HCE cells to fibronectin. Although the cells did not adhere to Tn-C, they adhered to the fibronectin/Tn-C coat and were then more efficiently inhibited by the function-blocking MAbs and RGD peptide. During the early adhesion, HCE cells codeposited Lm-332 and the large subunit of tenascin-C (Tn-CL) beneath the cells via the Golgi apparatus and microtubules. Integrin β4 subunit, which is a hemidesmosomal component, did not mediate the early adhesion of HCE cells to Lm-332 or Lm-332/Tn-C. Based on these results, we suggest that the adhesion of HCE cells is initiated by Lm-332 and modulated by Tn-CL, as it has been reported to prevent the assembly of hemidesmosomes. Thereby, Tn-CL functions in the motility of HCE cells during wound healing. The different distribution of processed and unprocessed Lm-332 in adhering, spreading and migrating HCE cells suggests a distinct role for these isoforms. We conclude that the processed Lm-332 functions in cell adhesion, whereas the unprocessed Lm-332 participates in cell spreading and migration.Terveeseen sarveiskalvoon kohdistuva taittovirhekirurgia sekä vammat ja sarveiskalvon sairaudet asettavat suuret vaatimukset haavan paranemisen hallinnalle. Sarveiskalvon uloin kerros muodostuu tyvikalvoon kiinnittyvistä epiteelisoluista. Tyvikalvot ovat soluväliaineen tihentymiä ja toimivat epiteelisolujen kiinnittymisessä, liikkeessä, kasvussa ja erilaistumisessa. Nämä prosessit ovat tärkeitä haavanparanemisessa ja vaativat epiteelisolujen ja soluväliaineen välistä viestintää reseptoreiden välityksellä. Väitöskirjatyössäni selvitimme ne tyvikalvo- ja soluväliaineproteiinit joita ihmisen sarveiskalvon kuolemattomaksi tehdyt epiteelisolut (HCE-solut) tuottavat. Tuotettujen proteiinien merkitys HCE-solujen kiinnittymisessä, leviämisessä ja liikkeessä oli keskeinen tutkimuskohde. Laminiinit, fibronektiinit ja tenaskiini-C ovat glykoproteiineja, joiden on osoitettu välittävän epiteelisolujen kiinnittymistä ja liikettä haavanparanemisen aikana. Aikaisemmat tutkimukset osoittavat että fibronektiini ja tenaskiini-C lisääntyvät sarveiskalvohaavoissa ja tenaskiini-C estää hemidesmosomien muodostumista. Väitöskirjatyössäni osoitimme että ihmisen sarveiskalvon tyvikalvossa on laminiini-332:ta ja -511:tä. Aikaisemmista havainnoista poiketen näissä tyvikalvoissa ei ollut laminiini-111:tä. Tutkimuksemme osoittivat että HCE-solut tuottivat kuitenkin vain laminiini-332:ta, joten ehdotamme että sarveiskalvon strooman keratosyytit tuottavat laminiini-511:tä. Immunopresipitaatio ja Western blotting-menetelmällä selvitimme että HCE solut tuottivat myös EDA-fibronektiinia, onkofetaalista fibronektiiniä ja tenaskiini-C:tä. Tässä tutkimuksessa selvitimme myös ne integriinit ja muut reseptorit, joiden välityksellä HCE solut kiinnittyvät tuottamiinsa proteiineihin. HCE solujen kiinnittyessä alustaan ne tuottivat alleen prosessoimatonta ja prosessoitua laminiini-332:ta sekä tenaskiini-C:tä. Kiinnittymiskokeissa havaitsimme että HCE solut eivät kiinnittyneet tenaskiini-C:hen. Tuloksemme osoittivat myös että integriini β4 alayksikkö, joka toimii vahvaa eopiteelisolujen ja tyvikalvojen välistä kiinnittymistä välittävänä hemidesmosomin osana, ei toiminut HCE solujen kiinnittymisessä laminiini-332:teen tai laminiin-332/tenaskiini-C:hen. Näiden tulosten perusteella ehdotamme että tenaskiini-C estää hemidesmosomien muodostumista ja välittää HCE-solujen liikettä. Kiinnittymis- ja haavakokeisiimme perustuen ehdotamme myös, että prosessoimaton laminiini-332 toimii HCE solujen leviämisessä ja liikkeessä ja prosessoitu laminiini-332 toimii solujen varhaisessa kiinnittymisessä

Identification of tumour-associated glycoproteins recognised by the lectin from Helix pomatia in breast cancer

Helix pomatia agglutinin (HPA) is a carbohydrate binding protein isolated from the Roman snail. There has been considerable interest in understanding HPA binding partners in cancer, as the lectin has been shown to identify glycosylation changes in cancers arising from the epithelia, from patients with poor prognosis. Identifying the HPA binding epitopes associated with a malignant phenotype may be useful for prognostication and may also offer potential as targets for immunotherapy. Previous studies have shown that HPA recognises a multitude of proteins in colorectal cancer (CRC). This study aimed to establish whether HPA recognises the same glycoproteins in breast cancer. An in vitro model of human breast cancer cell lines was used, ranging from HPA negative, non metastatic, to HPA positive and metastatic. Four human breast cell lines were chosen to represent phenotypes ranging from ‘normal’/benign (HMT3522), primary cancer (BT474) to metastatic cancer (T47D, MCF-7). HPA binding was assessed using confocal microscopy. Membrane proteins were extracted by differential centrifugation and the proteins were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Western blotting. The cell surface glycoproteins recognised by HPA were characterised using 2-dimensional electrophoresis (2-DE), Western blotting and mass spectrometry. HPA binding correlated with integrin α6 levels, this concurred with previous findings in CRC. HPA also bound transcription factors HnRNP H1, HnRNP D-like, HnRNP A2/B1 as well as Hsp27, GFAP and ENO1. The recognition of HnRNPs, Hsp 27 and ENO1 by HPA correlated with O-GlcNAcylation of these proteins. Interestingly, these HPA-binding glycoproteins were either absent or showed decreased levels in the non-metastatic breast cancer cell line BT474 and in ‘normal’ HMT3522. A comprehensive analysis of the breast cells proteome showed a number of proteins with elevated level in the metastatic breast cancer cell lines T47D and MCF-7, but this is the first report to show elevated levels of elongation factor Tu, Enoyl Coenzyme A hydratase 1 peroxisomal and macropain subunits. This work was extended to analyse the gene expression for UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase (ppGalNAc T1,T2, T3 and T6) and alpha 2,6 sialyltransferases (ST6GalNAc I and II) in the breast cell lines, but no correlation between the expression levels of mRNA of these enzymes and HPA binding was found in this study. The results from the present study show that, as in CRC cell lines, integrin α6 was the most abundant HPA binding glycoprotein extracted from the breast cancer cells with a metastatic phenotype. This is the first report in which HPA has been shown to bind O-GlcNAcylated transcription factors. This class of proteins represent a new means by which HPA differentiates cancer cells with an aggressive metastatic phenotype. New approaches aimed at targeting these changes might have broad application for the treatment of breast, colorectal and possibly other epithelial cancers.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Therapeutic Monoclonal Antibodies and Antibody Products, Their Optimization and Drug Design in Cancers

The book broadly deals with therapeutic monoclonal antibodies (mAbs) and various relevant topics, including different antibody formats such as Antibody–Drug Conjugates (ADC), bispecifics, nanoparticle-based mAbs and HER2+ cancers, immune checkpoint inhibitors and other closely related topics. Each paper was written by leading active research groups in their fields both from academia and industry. The book should be of interest to those scientists and researchers who develop or use biologics, biotherapeutics, biosimilars and biobetters in cancer treatment