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Neoplasias da Mama , Fibroblastos Associados a Câncer , Resistencia a Medicamentos Antineoplásicos , Terapia Neoadjuvante , Neoplasias da Mama/tratamento farmacológico , Humanos , Feminino , Terapia Neoadjuvante/métodos , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacosRESUMO
Mouse lung branching morphogenesis creates epithelial tree structures required for respiration. Here, we present a protocol for studying mouse lung developmental branching using lung explant cultures. We describe steps for isolating lungs with a video at embryonic day 12.5 (E12.5) and culturing as an explant for 2 days. We also detail procedures for microscopic imaging on days 0-2 and analysis of peripheral lung buds. This technique has the potential to investigate lung development in various conditions. For complete details on the use and execution of this protocol, please refer to Talvi et al.1.
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Embigin (Gp70), a receptor for fibronectin and an ancillary protein for monocarboxylate transporters, is known to regulate stem cell niches in sebaceous gland and bone marrow. Here, we show that embigin expression is at high level during early mouse embryogenesis and that embigin is essential for lung development. Markedly increased neonatal mortality of Emb-/- mice can be explained by the compromised lung maturation: in Emb-/- mice (E17.5) the number and the size of the small airways and distal airspace are significantly smaller, there are fewer ATI and ATII cells, and the alkaline phosphatase activity in amniotic fluid is lower. Emb-/- lungs show less peripheral branching already at E12.5, and embigin is highly expressed in lung primordium. Thus, embigin function is essential at early pseudoglandular stage or even earlier. Furthermore, our RNA-seq analysis and Ki67 staining results support the idea that the development of Emb-/- lungs is rather delayed than defected.
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Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.
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Dipeptídeos , Isoenzimas , Prolil Hidroxilases , Prolil Hidroxilases/genética , Isoenzimas/genética , Colágeno Tipo I/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/química , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Colágeno/genética , Colágeno/metabolismo , Prolina/metabolismoRESUMO
Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. The prognosis of patients with metastatic cSCC is poor emphasizing the need for new therapies. We have previously reported that the activation of Ras/MEK/ERK1/2 and transforming growth factor ß (TGF-ß)/Smad2 signaling in transformed keratinocytes and cSCC cells leads to increased accumulation of laminin-332 and accelerated invasion. Here, we show that the next-generation B-Raf inhibitor PLX8394 blocks TGF-ß signaling in ras-transformed metastatic epidermal keratinocytes (RT3 cells) harboring wild-type B-Raf and hyperactive Ras. PLX8394 decreased phosphorylation of TGF-ß receptor II and Smad2, as well as p38 activity, MMP-1 and MMP-13 synthesis, and laminin-332 accumulation. PLX8394 significantly inhibited the growth of human cSCC tumors and in vivo collagen degradation in xenograft model. In conclusion, our data indicate that PLX8394 inhibits several serine-threonine kinases in malignantly transformed human keratinocytes and cSCC cells and inhibits cSCC invasion and tumor growth in vitro and in vivo. We identify PLX8394 as a potential therapeutic compound for advanced human cSCC.
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Carcinoma de Células Escamosas , Neoplasias Cutâneas , Humanos , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Laminina , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fator de Crescimento Transformador beta/metabolismo , AnimaisRESUMO
Introduction: Protein arginine deiminases (PADs) are intracellular enzymes that may, especially in pathological conditions, also citrullinate extracellular substrates, including matrisome proteins such as structural proteins in extracellular matrix (ECM). PADs are abundantly expressed in human cancer cells. Citrullination of matrisome proteins has been reported in colon cancer but the phenomenon has never been systematically studied. Methods: To gain a broader view of citrullination of matrisome proteins in cancer, we analyzed cancer proteomics data sets in 3 public databases for citrullinated matrisome proteins. In addition, we used three-dimensional cell cocultures of fibroblasts and cancer cells and analyzed citrullination of ECM. Results and discussion: Our new analysis indicate that citrullination of ECM occurs in human cancer, and there is a significant variation between tumors. Most frequently citrullinated proteins included fibrinogen and fibronectin, which are typically citrullinated in rheumatoid inflammation. We also detected correlation between immune cell marker proteins, matrix metalloproteinases and ECM citrullination, which suggests that in cancer, citrullination of matrisome proteins is predominantly an inflammation-related phenomenon. This was further supported by our analysis of three-dimensional spheroid co-cultures of nine human cancer cell lines and fibroblasts by mass spectrometry, which gave no evidence that cancer cells or fibroblasts could citrullinate matrisome proteins in tumor stroma. It also appears that in the spheroid cultures, matrisome proteins are protected from citrullination.
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Stem cell renewal and differentiation are regulated by interactions with the niche. Although multiple cell populations have been identified in distinct anatomical compartments, little is known about niche-specific molecular factors. Using skin as a model system and combining single-cell RNA-seq data analysis, immunofluorescence, and transgenic mouse models, we show that the transmembrane protein embigin is specifically expressed in the sebaceous gland and that the number of embigin-expressing cells is negatively regulated by Wnt. The loss of embigin promotes exit from the progenitor compartment and progression toward differentiation, and also compromises lipid metabolism. Embigin modulates sebaceous niche architecture by affecting extracellular matrix organization and basolateral targeting of monocarboxylate transport. We discover through ligand screening that embigin is a direct fibronectin receptor, binding to the N-terminal fibronectin domain without impairing integrin function. Our results solve the long-standing question of how embigin regulates cell adhesion and demonstrate a mechanism that couples adhesion and metabolism.
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Integrina alfa5beta1 , Glândulas Sebáceas , Animais , Adesão Celular , Diferenciação Celular , Fibronectinas , Integrina beta1 , Integrinas/metabolismo , CamundongosRESUMO
Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. Previous studies have shown the role of the complement system in cSCC progression. In this study, we have investigated the mechanistic role of serine proteinase C1r, a component of the classical pathway of the complement system, in cSCC. Knockout of C1r in cSCC cells using CRISPR/Cas9 resulted in a significant decrease in their proliferation, migration, and invasion through collagen type I compared with that of wild-type cSCC cells. Knockout of C1r suppressed the growth and vascularization of cSCC xenograft tumors and promoted apoptosis of tumor cells in vivo. mRNA-sequencing analysis after C1r knockdown revealed significantly regulated Gene Ontology terms cell-matrix adhesion, extracellular matrix component, basement membrane, and metalloendopeptidase activity and Kyoto Encyclopedia of Genes and Genomes pathway extracellular matrixâreceptor interaction. Among the significantly regulated genes were invasion-associated matrix metalloproteinases (MMPs) MMP1, MMP13, MMP10, and MMP12. Knockout of C1r resulted in decreased production of MMP-1, MMP-13, MMP-10, and MMP-12 by cSCC cells in culture. Knockout of C1r inhibited the expression of MMP-13 by tumor cells, suppressed invasion, and reduced the amount of degraded collagen in vivo in xenografts. These results provide evidence for the role of C1r in promoting the invasion of cSCC cells by increasing MMP production.
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Carcinoma de Células Escamosas , Complemento C1r , Metaloproteinase 13 da Matriz , Neoplasias Cutâneas , Animais , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Complemento C1r/genética , Complemento C1r/metabolismo , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.
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Biomarcadores Tumorais/genética , Proliferação de Células , Bases de Conhecimento , Neoplasias/patologia , Software , Esferoides Celulares/patologia , Microambiente Tumoral , Técnicas de Cultura de Células/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/classificação , Neoplasias/metabolismo , RNA-Seq , Reprodutibilidade dos Testes , Esferoides Celulares/imunologia , Esferoides Celulares/metabolismo , Células Tumorais CultivadasRESUMO
Collagen XIII is a conserved transmembrane collagen mainly expressed in mesenchymal tissues. Previously, we have shown that collagen XIII modulates tissue development and homeostasis. Integrins are a family of receptors that mediate signals from the environment into the cells and vice versa. Integrin α11ß1 is a collagen receptor known to recognize the GFOGER (O=hydroxyproline) sequence in collagens. Interestingly, collagen XIII and integrin α11ß1 both have a role in the regulation of bone homeostasis. To study whether α11ß1 is a receptor for collagen XIII, we utilized C2C12 cells transfected to express α11ß1 as their only collagen receptor. The interaction between collagen XIII and integrin α11ß1 was also confirmed by surface plasmon resonance and pull-down assays. We discovered that integrin α11ß1 mediates cell adhesion to two collagenous motifs, namely GPKGER and GF(S)QGEK, that were shown to act as the recognition sites for the integrin α11-I domain. Furthermore, we studied the in vivo significance of the α11ß1-collagen XIII interaction by crossbreeding α11 null mice (Itga11-/-) with mice overexpressing Col13a1 (Col13a1oe). When we evaluated the bone morphology by microcomputed tomography, Col13a1oe mice had a drastic bone overgrowth followed by severe osteoporosis, whereas the double mutant mouse line showed a much milder bone phenotype. To conclude, our data identifies integrin α11ß1 as a new collagen XIII receptor and demonstrates that this ligand-receptor pair has a role in the maintenance of bone homeostasis.
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Osso e Ossos , Colágeno Tipo XIII/metabolismo , Cadeias alfa de Integrinas/metabolismo , Integrinas/metabolismo , Receptores de Colágeno/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Adesão Celular , Linhagem Celular , Humanos , Camundongos , Camundongos KnockoutRESUMO
Biomimetic functionalization to confer stealth and targeting properties to nanoparticles is a field of intense study. Extracellular vesicles (EV), sub-micron delivery vehicles for intercellular communication, have unique characteristics for drug delivery. We investigated the top-down functionalization of gold nanoparticles with extracellular vesicle membranes, including both lipids and associated membrane proteins, through mechanical extrusion. EV surface-exposed membrane proteins were confirmed to help avoid unwanted elimination by macrophages, while improving autologous uptake. EV membrane morphology, protein composition and orientation were found to be unaffected by mechanical extrusion. We implemented complementary EV characterization methods, including transmission- and immune-electron microscopy, and nanoparticle tracking analysis, to verify membrane coating, size and zeta potential of the EV membrane-cloaked nanoparticles. While successful EV membrane coating of the gold nanoparticles resulted in lower macrophage uptake, low yield was found to be a significant downside of the extrusion approach. Our data incentivize more research to leverage EV membrane biomimicking as a unique drug delivery approach in the near future.
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Vesículas Extracelulares/metabolismo , Nanopartículas Metálicas/química , Animais , Humanos , Camundongos , RatosRESUMO
BACKGROUND: Tumor microenvironment or stroma has the potency to regulate the behavior of malignant cells. Fibroblast-like cells are abundant in tumor stroma and they are also responsible for the synthesis of many extracellular matrix components. Fibroblast-cancer cell interplay can modify the functions of both cell types. METHODS: We applied mass spectrometry and proteomics to unveil the matrisome in 3D spheroids formed by DU145 prostate cancer cells, PC3 prostate cancer cells, or prostate-derived fibroblasts. Similarly, DU145/fibroblast and PC3/fibroblast coculture spheroids were also analyzed. Western blot analysis and immunofluorescence were used to confirm the presence of specific proteins in spheroids. Cancer dissemination was studied by utilizing "out of spheroids" migration and invasion assays. RESULTS: In the spheroid model cancer cell-fibroblast interplay caused remarkable changes in the extracellular matrix and accelerated the invasion of DU145 cells. Fibroblasts produced structural matrix proteins, growth factors, and matrix metalloproteinases. In cancer cell/fibroblast cocultures basement membrane components, including laminins (α3, α5, ß2, and ß3), heparan sulfate proteoglycan (HSPG2 gene product), and collagen XVIII accumulated in a prominent manner when compared with spheroids that contained fibroblasts or cancer cells only. Furthermore, collagen XVIII was intensively processed to different endostatin-containing isoforms by cancer cell-derived cathepsin L. CONCLUSIONS: Fibroblasts can promote carcinoma cell dissemination by several different mechanisms. Extracellular matrix and basement membrane proteins provide attachment sites for cell locomotion promoting adhesion receptors. Growth factors and metalloproteinases are known to accelerate cell invasion. In addition, cancer cell-fibroblast interplay generates biologically active fragments of basement membrane proteins, such as endostatin.
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Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Proteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Autoantígenos/metabolismo , Membrana Basal/metabolismo , Catepsina L/metabolismo , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Masculino , Espectrometria de Massas , Invasividade Neoplásica , Colágenos não Fibrilares/metabolismo , Células PC-3 , Proteômica/métodos , Esferoides Celulares , Colágeno Tipo XVIIRESUMO
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers.
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Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. The molecular basis of cSCC progression to invasive and metastatic disease is still incompletely understood. Here, we show that fibroblasts and transforming growth factor-ß (TGF-ß) signaling promote laminin-332 synthesis in cancer cells in an activated H-Ras-dependent manner, which in turn promotes cancer cell invasion. Immunohistochemical analysis of sporadic UV-induced invasive human cSCCs (nâ¯=â¯208) revealed prominent cSCC cell specific immunostaining for laminin-332 γ2 chain, located in the majority of cases (90%, nâ¯=â¯173) in the invasive edge of the tumors. To mimic the progression of cSCC we established 3D spheroid cocultures using primary skin fibroblasts and HaCaT/ras-HaCaT human keratinocytes. Our results indicate that in 3D spheroids, unlike in monolayer cultures, TGF-ß upregulates laminin-332 production, but only in cells that harbour oncogenic H-Ras. Accumulation of laminin-332 was prevented by both H-Ras knock down and inhibition of TGF-ß signaling by SB431542 or RAdKD-ALK5 kinase-defective adenovirus. Furthermore, fibroblasts accelerated the invasion of ras-HaCaT cells through collagen I gels in a Ras/TGF-ß signaling dependent manner. In conclusion, we demonstrate the presence of laminin-332 in the invasive front of cSCC tumors and report a new Ras/TGF-ß-dependent mechanism that promotes laminin-332 accumulation and cancer cell invasion.
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Carcinoma de Células Escamosas/patologia , Moléculas de Adesão Celular/metabolismo , Fibroblastos/citologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Cutâneas/patologia , Adolescente , Adulto , Animais , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Adulto Jovem , CalininaRESUMO
Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-ß-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and ß-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance.
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Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Quinase 1 de Adesão Focal/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Platina/farmacologia , Animais , Cisplatino/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Células-TroncoRESUMO
Co- and post-translational hydroxylation of proline residues is critical for the stability of the triple helical collagen structure. In this review, we summarise the biology of collagen prolyl 4-hydroxylases and collagen prolyl 3-hydroxylases, the enzymes responsible for proline hydroxylation. Furthermore, we describe the potential roles of hydroxyproline residues in the complex interplay between collagens and other proteins, especially integrin and discoidin domain receptor type cell adhesion receptors. Qualitative and quantitative regulation of collagen hydroxylation may have remarkable effects on the properties of the extracellular matrix and consequently on the cell behaviour.
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Colágeno/metabolismo , Hidroxiprolina/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Colágeno/química , Humanos , Hidroxilação , Hidroxiprolina/química , Integrinas/metabolismo , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
Long non-coding RNAs (lncRNAs) regulate various cellular processes, and they have emerged as potential biomarkers and therapeutic targets in cancer. We have previously characterized the oncogenic role of lncRNA PICSAR (p38 inhibited cutaneous squamous cell carcinoma associated lincRNA) in cutaneous squamous cell carcinoma (cSCC), the most common metastatic skin cancer. In this study, we show that knockdown of PICSAR in cSCC cells upregulates expression of α2, α5 and ß1 integrins, resulting in increased cell adhesion and decreased cell migration on collagen I and fibronectin. In contrast, overexpression of PICSAR in cSCC cells downregulates expression of α2, α5 and ß1 integrins on cell surface, resulting in decreased cell adhesion on collagen I and fibronectin and increased cell migration. These results demonstrate a novel mechanism for regulation of the expression of collagen and fibronectin binding integrins by lncRNA PICSAR, leading to altered adhesion and migration of cSCC cells.This article has an associated First Person interview with the first author of the paper.
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High expression level of integrin α2ß1 is a hallmark of prostate cancer stem cell like cells. The role of this collagen receptor is controversial since it is down regulated in poorly differentiated carcinomas, but concomitantly proposed to promote metastasis. Here, we show that docetaxel resistant DU145 prostate cancer cells express high levels of α2ß1 and that α2ß1High subpopulation of DU145 cells proliferates slower than the cells representing α2ß1Low subpopulation. To further study this initial observation we used Crispr/Cas9 technology to create an α2ß1 negative DU145 cell line. Furthermore, we performed rescue experiment by transfecting α2 knockout cells with vector carrying α2 cDNA or with an empty vector for appropriate control. When these two cell lines were compared, α2ß1 positive cells proliferated slower, were more resistant to docetaxel and also migrated more effectively on collagen and invaded faster through matrigel or collagen. Integrin α2ß1 was demonstrated to be a positive regulator of p38 MAPK phosphorylation and a selective p38 inhibitor (SB203580) promoted proliferation and inhibited invasion. Effects of α2ß1 integrin on the global gene expression pattern of DU145 cells in spheroid cultures were studied by RNA sequencing. Integrin α2ß1 was shown to regulate several cancer progression related genes, most notably matrix metalloproteinase-1 (MMP-1), a recognized invasion promoting protein. To conclude, the fact that α2ß1 decelerates cell proliferation may explain the dominance of α2ß1 negative/low cells in primary sites of poorly differentiated carcinomas, while the critical role of α2ß1 integrin in invasion stresses the importance of this adhesion receptor in cancer dissemination.
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The enteric species F human adenovirus types 40 and 41 (HAdV-40 and -41) are the third most common cause of infantile gastroenteritis in the world. Knowledge about HAdV-40 and -41 cellular infection is assumed to be fundamentally different from that of other HAdVs since HAdV-40 and -41 penton bases lack the αV-integrin-interacting RGD motif. This motif is used by other HAdVs mainly for internalization and endosomal escape. We hypothesised that the penton bases of HAdV-40 and -41 interact with integrins independently of the RGD motif. HAdV-41 transduction of a library of rodent cells expressing specific human integrin subunits pointed to the use of laminin-binding α2-, α3- and α6-containing integrins as well as other integrins as candidate co-receptors. Specific laminins prevented internalisation and infection, and recombinant, soluble HAdV-41 penton base proteins prevented infection of human intestinal HT-29 cells. Surface plasmon resonance analysis demonstrated that HAdV-40 and -41 penton base proteins bind to α6-containing integrins with an affinity similar to that of previously characterised penton base:integrin interactions. With these results, we propose that laminin-binding integrins are co-receptors for HAdV-40 and -41.