Altered glycosylation is a common feature of cancer cells. Some subsets of glycans are found to be frequently enriched on the tumor cell surface and implicated in different tumor phenotypes. Among these, changes in sialylation have long been associated with metastatic cell behaviors such as invasion and enhanced cell survival. Sialylation typically exists in three prominent linkages: α2,3, α2,6, and α2,8, catalyzed by a group of sialyltransferases. The aberrant expression of all three linkages has been related to cancer progression. The increased α2,6 sialylation on N-glycans catalyzed by β-galactoside α2,6 sialyltransferase 1 (ST6Gal1) is frequently observed in many cancers. In contrast, functions of α2,3 sialylation on N-glycans catalyzed by at least three β-galactoside α2,3-sialyltransferases, ST3Gal3, ST3Gal4, and ST3Gal6 remain elusive due to a possibility of compensating for one another. In this minireview, we briefly describe functions of sialylation and recent findings that different α2,3 sialyltransferases specifically modify target proteins, as well as sialylation regulatory mechanisms vis a complex formation among integrin α3β1, Golgi phosphoprotein 3 (GOLPH3), phosphatidylinositol 4-kinase IIα (PI4KIIα), focal adhesion kinase (FAK) and sialyltransferase, which suggests a new concept for the regulation of glycosylation in cell biology.

Urinary extracellular vesicles (uEVs) are enriched with glycosylated proteins which have been extensively studied as putative biomarkers of urological cancers. Here, we characterized the glycosylation and integrin profile of EVs derived from urological cancer cell lines. We used fluorescent europium-doped nanoparticles coated with lectins and antibodies to identify a biomarker combination consisting of integrin subunit alpha 3 (ITGA3) and fucose. In addition, we used the same cancer cell line-derived EVs as analytical standards to assess the sensitivity of the ITGA3-UEA assay. The clinical performance of the ITGA3-UEA assay was analysed using urine samples of various urological pathologies including diagnostically challenging benign prostatic hyperplasia (BPH), prostate cancer (PCa) and bladder cancer (BlCa). The assay can significantly discriminate BlCa from all other patient groups: PCa (9.2-fold; p = 0.00038), BPH (5.5-fold; p = 0.004) and healthy individuals (and 23-fold; p = 0.0001). Our results demonstrate that aberrantly fucosylated uEVs and integrin ITGA3 can be detected with fucose-specific lectin UEA in a simple bioaffinity assay for the detection of BlCa directly from unprocessed urine.

Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin targeting has been used in the treatment of ocular disease and cancer, but little is known about its utility in the treatment of heart failure. Here we sought to determine whether the second generation orally available, αvβ3-specific RGD-mimetic, 29P , was cardioprotective. Male mice were subjected to transverse aortic constriction (TAC) and treated with 50 μg/kg 29P or volume-matched saline as Vehicle control. At 3 weeks post-TAC, echocardiography showed that 29P treatment significantly restored cardiac function and structure indicating the protective effect of 29P treatment in this model of heart failure. Importantly, 29P treatment improved cardiac function giving improved fractional shortening, ejection fraction, heart weight and lung weight to tibia length fractions, together with partial restoration of Ace and Mme levels, as markers of the TAC insult. At a tissue level, 29P reduced cardiomyocyte hypertrophy and interstitial fibrosis, both of which are major clinical features of heart failure. RNA sequencing identified that, mechanistically, this occurred with concomitant alterations to genes involved molecular pathways associated with these processes such as metabolism, hypertrophy and basement membrane formation. Overall, targeting αvβ3 with 29P provides a novel strategy to attenuate pressure-overload induced cardiac hypertrophy and fibrosis, providing a possible new approach to heart failure treatment.

Integrin αIIbβ3 mediates platelet aggregation by binding the Arginyl-Glycyl-Aspartic acid (RGD) sequence of fibrinogen. RGD binding occurs at a site topographically proximal to the αIIb and β3 subunits, promoting the conformational activation of the receptor from bent to extended states. While several experimental approaches have characterized RGD binding to αIIbβ3 integrin, applying computational methods has been significantly more challenging due to limited sampling and the need for a priori information regarding the interactions between the RGD peptide and integrin. In this study, we employed all-atom simulations using funnel metadynamics (FM) to evaluate the interactions of an RGD peptide with the αIIb and β3 subunits of integrin. FM incorporates an external history-dependent potential on selected degrees of freedom while applying a funnel-shaped restraint potential to limit RGD exploration of the unbound state. Furthermore, it does not require a priori information about the interactions, enhancing the sampling at a low computational cost. Our FM simulations reveal significant molecular changes in the β3 subunit of integrin upon RGD binding and provide a free-energy landscape with a low-energy binding mode surrounded by higher-energy prebinding states. The strong agreement between previous experimental and computational data and our results highlights the reliability of FM as a method for studying dynamic interactions of complex systems such as integrin.

HER2-positive breast cancer, characterised by overexpressed HER2 levels, is associated with aggressive tumour behaviour and poor prognosis. Trastuzumab is a standard treatment; however, approximately 50% of patients develop resistance within one year. This study investigates the role of ITGβ3 in promoting stemness and resistance in HER2-positive breast cancer cell lines (HCC1599 and SKBR3). The findings demonstrate that chronic exposure to trastuzumab upregulates stem cell markers (SOX2, OCT4, KLF4, NANOG, SALL4, ALDH, BMI1, Nestin, Musashi 1, TIM3, CXCR4). Given the documented role of RGD-binding integrins in drug resistance and stemness, we specifically investigated their impact on resistant cells. Overexpression of ITGβ3 enhances the expression of these stem cell markers, while silencing ITGβ3 reduces their expression, suggesting a major role for ITGβ3 in maintaining stemness and resistance. Further analysis reveals that ITGβ3 activates the Notch signalling pathway, known for regulating stem cell maintenance. The combination of trastuzumab and cilengitide, an integrin inhibitor, significantly decreases the expression of stem cell markers in resistant cells, indicating a potential therapeutic strategy to overcome resistance. These results identify the importance of ITGβ3 in mediating stemness and trastuzumab resistance through Notch signalling in HER2-positive breast cancer, offering new approaches for enhancing treatment efficacy.

Dendritic cells (DCs) are the main antigen presenting cells of the immune system and are essential for anti-tumor responses. DC-based immunotherapies are used in cancer treatment, but their functionality is not optimized and their clinical efficacy is currently limited. Approaches to improve DC functionality in anti-tumor immunity are therefore required. We have previously shown that the loss of β2-integrin-mediated adhesion leads to epigenetic reprogramming of bone marrow-derived DCs (BM-DCs), resulting in an increased expression of costimulatory markers (CD86, CD80, and CD40), cytokines (IL-12) and the chemokine receptor CCR7. We now show that the loss of β2-integrin-mediated adhesion of BM-DCs also leads to a generally suppressed metabolic profile, with reduced metabolic rate, decreased ROS production, and lowered glucose uptake in cells. The mRNA levels of glycolytic enzymes and glucose transporters were reduced, indicating transcriptional regulation of the metabolic phenotype. Surprisingly, although signaling through a central regulator of immune cell metabolisms, the mechanistic target of rapamycin (mTOR), was increased in BM-DCs with dysfunctional integrins, rapamycin treatment revealed that mTOR signaling was not involved in suppressing DC metabolism. Instead, bioinformatics and functional analyses showed that the Ikaros transcription factor may be involved in regulating the metabolic profile of non-adhesive DCs. Inversely, we found that induction of metabolic stress through treatment of cells with low levels of an inhibitor of glycolysis, 2-deoxyglucose (2DG), led to increased BM-DC activation. Specifically, 2DG treatment led to increased levels of Il-12 and Ccr7 mRNA, increased production of IL-12, increased levels of cell surface CCR7 and increased in vitro migration and T cell activation potential. Furthermore, 2DG treatment led to increased histone methylation in cells (H3K4me3, H3K27me3), indicating metabolic reprogramming. Finally, metabolic stress induced by 2DG treatment led to improved BM-DC-mediated anti-tumor responses in vivo in a melanoma cancer model, B16-OVA. In conclusion, our results indicate a role for β2-integrin-mediated adhesion in regulating a novel type of metabolic reprogramming of DCs and DC-mediated anti-tumor responses, which may be targeted to enhance DC-mediated anti-tumor responses in cancer immunotherapy.

BACKGROUND: The intermediate filament protein vimentin is widely recognized as a molecular marker of epithelial-to-mesenchymal transition. Although vimentin expression is strongly associated with cancer metastatic potential, the exact role of vimentin in cancer metastasis and the underlying mechanism of its pro-metastatic functions remain unclear.
RESULTS: This study revealed that vimentin can enhance integrin β1 surface expression and induce integrin-dependent clustering of cells, shielding them against anoikis cell death. The increased integrin β1 surface expression in suspended cells was caused by vimentin-mediated protection of the internal integrin β1 pool against lysosomal degradation. Additionally, cell detachment was found to induce vimentin Ser38 phosphorylation, allowing the translocation of internal integrin β1 to the plasma membrane. Furthermore, the use of an inhibitor of p21-activated kinase PAK1, one of the kinases responsible for vimentin Ser38 phosphorylation, significantly reduced cancer metastasis in animal models.
CONCLUSIONS: These findings suggest that vimentin can act as an integrin buffer, storing internalized integrin β1 and releasing it when needed. Overall, this study provides insights regarding the strong correlation between vimentin expression and cancer metastasis and a basis for blocking metastasis using this novel therapeutic mechanism.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA based device in which the transition between single-stranded and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

UNASSIGNED: Integrin-dependent cell adhesion and migration play important roles in systemic sclerosis (SSc). The roles of integrin activating molecules including talins and kindlins, however, are unclear in SSc.
UNASSIGNED: We aimed to explore the function of integrin activating molecules in SSc.
UNASSIGNED: Transcriptome analysis of skin datasets of SSc patients was performed to explore the function of integrin-activating molecules including talin1, talin2, kindlin1, kindlin2 and kindlin3 in SSc. Expression of talin1 in skin tissue was assessed by multiplex immunohistochemistry staining. Levels of talin1 in serum were determined by ELISA. The effects of talin1 inhibition were analyzed in human dermal fibroblasts by real-time PCR, western blot and flow cytometry.
UNASSIGNED: We identified that talin1 appeared to be the primary integrin activating molecule involved in skin fibrosis of SSc. Talin1 was significantly upregulated and positively correlates with the modified Rodnan skin thickness score (mRSS) and the expression of pro-fibrotic biomarkers in the skin lesions of SSc patients. Further analyses revealed that talin1 is predominantly expressed in the dermal fibroblasts of SSc skin and promotes fibroblast activation and collagen production. Additionally, talin1 primarily exerts its effects through integrin β1 and β5 in SSc.
UNASSIGNED: Overexpressed talin1 is participated in skin fibrosis of SSc, and talin1 appears to be a potential new therapeutic target for SSc.

OBJECTIVE: In gout, monosodium urate crystals are taken up by macrophages, triggering the activation of the NLRP3 inflammasome and the maturation of IL-1β. This study aimed to investigate the role of integrin CD11b in inflammasome activation in macrophages stimulated by MSU.
METHODS: BMDM from WT and CD11b KO mice were stimulated in vitro with MSU crystals. Cellular supernatants were collected to assess the expression of the inflammatory cytokines by enzyme-linked immunosorbent assay and western blot methods. The role of integrin CD11b in MSU-induced gouty arthritis in vivo was investigated by intra-articular injection of MSU crystals. Real-time extracellular acidification rate and oxygen consumption rate of BMDMs were measured by Seahorse Extracellular Flux Analyzer.
RESULTS: We demonstrate that CD11b-deficient mice developed exacerbated gouty arthritis with increased recruitment of leukocytes in the joint and higher IL-1β levels in the sera. In macrophages, genetic deletion of CD11b induced a shift of macrophage metabolism from oxidative phosphorylation to glycolysis, thus decreasing the overall generation of intracellular ATP. Upon MSU stimulation, CD11b-deficient macrophages showed an exacerbated secretion of IL-1β. Treating wild-type macrophages with a CD11b agonist, LA1, inhibited MSU-induced release of IL-1β in vitro and attenuated the severity of experimental gouty arthritis. Importantly, LA1, was also effective in human cells as it inhibited MSU-induced release of IL-1β by peripheral blood mononuclear cells from healthy donors.
CONCLUSIONS: Our data identified the CD11b integrin as a principal cell membrane receptor that modulates NLRP3 inflammasome activation by MSU crystal in macrophages, which could be a potential therapeutic target to treat gouty arthritis in human patients.