Integrin Alpha Subunits Research Articles

Integrated bioinformatics analysis shows integrin alpha 3 is a prognostic biomarker for pancreatic cancer.

Integrin subunit alpha 3 (ITGA3) expression correlates with the development and prognosis of human cancers. This study aimed to investigate the association of ITGA3 expression with pancreatic cancer (PCa) prognosis. The ITGA3 gene expression data were extracted from The Cancer Genome Atlas (TCGA) pancreatic adenocarcinoma (PAAD) cohort and 14 Gene Expression Omnibus microarray datasets. The differences in ITGA3 expression levels between tumor and non-tumor tissues were compared using the Mann-Whitney U test. Cox regression analysis and meta-analysis were performed to detect the association of ITGA3 expression with PCa prognosis. ITGA3 expression was higher in tumors than in controls. Tumors with advanced grades (3/4) had higher ITGA3 levels compared with early-grade tumors (1/2). The meta-analysis of the TCGA PAAD cohort and seven microarray datasets (GSE28735, GSE62452, GSE79668, GSE71729, GSE57495, GSE78229, and GSE21501) showed that ITGA3 was a prognostic biomarker in PCa (hazard ratio (HR) = 1.38, 95% confidence interval (CI) 1.26-1.51, p < 0.00001). Five ITGA3-related genes, including ITGB1 (HR = 1.6), ITGB5 (HR = 1.6), ITGB6 (HR = 1.6), LAMA3 (HR = 2.1), and CD9 (HR = 2.3), correlated with PCa prognosis significantly (p < 0.05). Functional enrichment analysis showed that ITGA3 was related to "hsa04151: PI3K-Akt signaling pathway" and "hsa04510: Focal adhesion." We concluded that high ITGA3 expression was a potential prognostic biomarker in PCa.

ITGA9 Inhibits Proliferation and Migration of Dermal Microvascular Endothelial Cells in Psoriasis.

Cell proliferation, migration, and angiogenesis are aberrant in psoriatic human dermal microvascular endothelial cells (HDMECs), resulting in abnormal endothelial function and microvascular dilation in psoriasis. To explore the role of Integrin subunit alpha 9 (ITGA9) in proliferation and migration of dermal microvascular endothelial cells. HDMECs were isolated from the skin of 6 psoriatic patients and 6 healthy controls. Expression levels of ITGA9 mRNA and protein were assessed with qRT-PCR and Western blot, respectively, while miqRT-PCR was used to determine expression levels of miR-146a-3p. Cell proliferation and migration were assessed in human microvascular endothelial cell line (HMEC-1), following overexpression of either ITGA9 or miR-146a-3p, or co-transfection with miR-146a-3p-mimic and pLVX - ITGA9. Cell viability was detected by Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay. Cell apoptosis was assessed, using annexin V-FITC/PI apoptosis detection kit, while cell migration was detected by wound healing and transwell assay. Expression levels of ITGA9 were significantly decreased in psoriatic HDMECs compared to normal controls. Moreover, expression levels of miR-146a-3p were higher in psoriatic HDMECs than in normal controls. Overexpression of miR-146a-3p lowered expression levels of ITGA9, accompanied by increased proliferation and migration of HMEC-1 in vitro. In contrast, overexpression of ITGA9 inhibited proliferation and migration of HMEC-1, while increasing expression levels of cdc42, ki67, focal adhesion kinase (FAK), c-Src tyrosine kinase (Src), RAC1 and RhoA. ITGA9 can repress the proliferation and migration of HMEC-1, suggesting utility of ITGA9 as a potential therapeutic intervention for psoriasis.

Depletion of circPDSS1 inhibits ITGA11 production to confer cisplatin sensitivity through miR-515-5p in gastric cancer

Chemoresistance limits cisplatin (DDP)-mediated treatment for gastric cancer (GC). Circular RNA (circRNA) acts an important role in chemoresistance. However, the underlying mechanism of circPDSS1 regulating DDP sensitivity in GC remains unclear. The expression patterns of circPDSS1, miR-515-5p and integrin subunit alpha 11 (ITGA11) were analyzed by qRT-PCR. Protein expression was checked by Western blotting analysis. Cell viability was investigated by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation was evaluated by colony formation assay and 5-ethynyl-2′-deoxyuridine (EdU) assay. The analysis of cell apoptosis, migration and invasion was performed by flow cytometry analysis and transwell assays. Dual-luciferase reporter assay and RNA immunoprecipitation assay were conducted to identify the associations among circPDSS1, miR-515-5p and ITGA11. In vivo assay was implemented using a xenograft mouse model assay. CircPDSS1 and ITGA11 expression were significantly upregulated, whereas miR-515-5p was downregulated in DDP-resistant GC tissues and cells in comparison with controls. CircPDSS1 depletion reduced DDP resistance, cell proliferation, migration and invasion but induced cell apoptosis in DDP-resistant GC cells. CircPDSS1 directly bound to miR-515-5p. CircPDSS1-mediated actions were dependent on the regulation of miR-515-5p. Besides, miR-515-5p was associated with ITGA11, and circPDSS1 regulated ITGA11 expression by binding to miR-515-5p. Overexpression of miR-515-5p improved DDP sensitivity owing to the downregulation of ITGA11. Further, circPDSS1 mediated DDP sensitivity by regulating miR-515-5p and ITGA11 in vivo. CircPDSS1 conferred DDP resistance through the miR-515-5p/ITGA11 axis in GC cells.

The Role of Integrin Beta-1 in the Chemoresistance of Acute Myeloid Leukemia

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive disease that has a poor prognosis with a survival rate of 30-40%. The bone marrow microenvironment provides protection to AML cells from chemotherapy and thus confers cell adhesion-mediated drug resistance (CAM-DR) and promotes relapse. Integrins are key anchor molecules in CAM-DR of leukemia cells; they are heterodimeric adhesion molecules on the cell surface consisting of an alpha and a beta unit. Upregulated integrin alpha 4 and 6 expression is associated with poor survival in leukemias. We hypothesize that the targeting of integrin beta 1 (ITGB1, β1, or CD29) will also affect the integrin alpha units as they are heterodimer partners. This could be a superior approach to overcome CAM-DR in AML than just targeting the individual alpha units. METHODS: ITGB1 gene was knocked out via CRISPR-Cas9 using 2 guide RNAs in AML cell lines and expression of integrin subunits was profiled by flow cytometry. AML cell lines were treated in vitro with the anti-human ITGB1 monoclonal antibody OS2966 in combination with Ara-C chemotherapy and we determined effects on cell viability by flow cytometry and on cell signaling using RNA-sequencing. Using human patient-derived AML xenograft mouse models, we tested if blockade of ITGB1 combined with chemotherapy can overcome CAM-DR in vivo. Mice were treated with control total Ig (10 mg/kg), OS2966 (10 mg/kg), total Ig + Ara-C (600 mg/kg), or OS2966 + Ara-C twice a week for two weeks. RESULTS: Analysis of patient-derived AML cells at diagnosis and relapse, and of AML cell lines, showed high expression of ITGB1 and varying levels of other alpha and beta units. Loss-of-function studies of ITGB1 using CRISPR/Cas9-mediated knockout of ITGB1 in MLL-rearranged and FLT3-ITD+ AML cell lines MV4-11 and MOLM-13, and in AML cell line U937, revealed that surface expression of integrin alphas 3, 4, 5 and 6 were markedly decreased as determined by flow cytometry analysis. ITGB2 expression was high in all 3 AML cell lines and not affected by ITGB1 deletion. Remarkably, ITGB3 was downregulated upon ITGB1 deletion in 2 out of 3 AML cell lines, whereas the 3rd AML cell line did not express any ITGB3. As a result of the ITGB1 deletion, AML cells showed less adhesion to murine bone marrow stroma (OP9) cells and to extracellular matrix proteins. We found that inhibition of surface ITGB1 on human AML using OS2966 de-adhered the AML cells from OP9 cells. Moreover, ITGB1 blockade using OS2966 synergized with Ara-C in vitro and significantly decreased cell viability compared to single agent controls in human AML cell lines U937 and MV4-11. RNA sequencing data suggest that the NFkB pathway is significantly dysregulated in the combination treatment Ara-C and OS2966 compared to Ara-C alone. To test ITGB1 inhibition in vivo, a patient-derived AML sample, HEM279 (Fig. 1A), and AML cell line, U937 (Fig. 1B), were engrafted into NSG-SGM3 mice. Leukemic mice were treated with single agents including control total Ig, OS2966, or Ara-C; and with total Ig in combination with Ara-C or OS2966. In both xenograft models, the combination treatment of OS2966 with Ara-C significantly prolonged survival of leukemic mice compared to Ara-C only: HEM279: MST 115 +/- 15.4 days vs 73.1 +/- 5.2 days post-AML injection; p=0.0005; U937: MST 170 days vs 32 days post-AML injection; p=0.0004; 50% of the mice engrafted with U937 and treated with OS2966 with Ara-C survived until day 290 post-AML injection, which is when the experiment was terminated. CONCLUSIONS: Integrin alpha subunit expression is dependent on ITGB1 expression, supporting our hypothesis that targeting integrin beta 1 affects alpha integrin units. ITGB1 inhibition with OS2966 combined with chemotherapy significantly decreases cell viability of AML cell lines in vitro and prolongs survival of xenograft mice in vivo. Our findings will allow for better understanding of the role of ITGB1 in drug resistance of AML and of ITGB1 as a preclinical target in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An artificial LAMA2-GelMA hydrogel microenvironment for the development of pancreatic endocrine progenitors

The biomimetic pancreatic microenvironment improves the differentiation efficiency and function of human embryonic stem cell-derived β-cells (SC-β cells). Thus, a laminin subunit alpha 2-gelatin methacrylate (LAMA2-GelMA) hybrid hydrogel as a bionics carrier for the formation and maturation of endocrine lineage was developed in our research, based on pancreas proteomics analysis of postnatal mice. Pancreatic endocrine cells cultured on the hybrid hydrogel in vitro, which was composed of 0.5 μg/mL LAMA2 protein and 4% GelMA, the expression of transcription factors (TFs), including NKX6.1, NKX6.2, and NEUROD1 were upregulated. Single-cell transcriptomics was performed after LAMA2 knockdown during the early differentiation of pancreatic progenitor (PP) cells, a marked decrease in the forkhead box protein A2 (FOXA2+)/GATA-binding factor 6 (GATA6+) cluster was detected. Also, we clarified that as a receptor of LAMA2, integrin subunit alpha 7 (ITGA7) participated in Integrin-AKT signaling transduction and influenced the protein levels of FOXA2 and PDX1. In vivo experiments showed that, PP cells encapsulated in the LAMA2-GelMA hydrogel exhibited higher serum C-peptide levels compared to the GelMA and Matrigel groups in nude mice and reversed hyperglycemia more quickly in STZ-induced diabetic nude mice. Taken together, our findings highlighted the feasibility of constructing a pancreas-specific microenvironment based on proteomics and tissue engineering for the treatment of diabetes.

Integrin subunit alpha V is a potent prognostic biomarker associated with immune infiltration in lower-grade glioma

As a member of integrin receptor family, ITGAV (integrin subunit α V) is involved in a variety of cell biological processes and overexpressed in various cancers, which may be a potential prognostic factor. However, its prognostic value and potential function in lower-grade glioma (LGG) are still unclear, and in terms of immune infiltration, it has not been fully elucidated. Here, the expression preference, prognostic value, and clinical traits of ITGAV were investigated using The Cancer Genome Atlas database (n = 528) and the Chinese Glioma Genome Atlas dataset (n = 458). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and gene set enrichment analysis (GSEA) were used to explore the biological function of ITGAV. Using R package "ssGSEA" analysis, it was found thatthe ITGAV mRNA expression level showed intense correlation with tumor immunity, such as tumor-infiltrating immune cells and multiple immune-related genes. In addition, ITGAV is associated with some immune checkpoints and immune checkpoint blockade (ICB) and response to chemotherapy. and the expression of ITGAV protein in LGG patients was verified via immunohistochemistry (IHC). ITGAV expression was higher in LGG tissues than in normal tissues (P < 0.001) and multifactor analysis showed that ITGAV mRNA expression was an independent prognostic factor for LGG overall survival (OS; hazard ratio = 2.113, 95% confidence interval = 1.393-3.204, P < 0.001). GSEA showed that ITGAV expression was correlated with Inflammatory response, complement response, KRAS signal, and interferon response. ssGSEA results showed a positive correlation between ITGAV expression and Th2 cell infiltration level. ITGAV mRNA was overexpressed in LGG, and high ITGAV mRNA levels were found to be associated with poor protein expression and poor OS. ITGAV is therefore a potential biomarker for the diagnosis and prognosis of LGG and may be a potential immunotherapy target.

Lymph Node Metastasis-Related Gene ITGA4 Promotes the Proliferation, Migration, and Invasion of Gastric Cancer Cells by Regulating Tumor Immune Microenvironment.

The Integrin Subunit Alpha 4 (ITGA4) plays important roles in cancers pathogenesis. However, the expression and association with clinicopathological and survival probability have not been previously assessed in gastric cancer (GC). Protein expression of ITGA4 was assessed in TMA using immunohistochemistry and correlated with clinicopathological factors and survival. The mRNA expression of ITGA4 was also assessed in the HMU-GC cohort. Bioinformatics function analysis was conducted through GSEA. The “CIBERSORT” package was used for immune infiltration analysis. “SvyNom” package is used to construct prognosis model. ITGA4 knock down using shRNA. The evaluation of cell function was performed by CCK-8 and Transwell invasion and migration experiments. ITGA4 was significantly associated with N classification (P = 0.031), tumor location (P = 0.033), WHO classification (P = 0.007), and poor prognosis in mRNA level. GSEA analysis of the validation cohort suggested that ITGA4 was associated with macrophage infiltration. Immunohistochemistry showed that ITGA4 was associated with poor prognosis. Multivariate Cox regression analysis found that ITGA4 (P = 0.045) and lymph node metastasis rate (P = 0.026) were independent prognostic factors and could construct a prognosis model. ITGA4 knockdown cell line significantly reduced the ability of proliferation, invasion, and metastasis. ITGA4 is associated with patient survival in GC and may be an important prognostic biomarker.

The Abundance of Tumor-Infiltrating CD8+ Tissue Resident Memory T Lymphocytes Correlates with Patient Survival in Glioblastoma.

Glial tumors alone account for 40% of all CNS tumors and present a low survival rate. The tumor microenvironment is a critical regulator of tumor progression and therapeutic effectiveness in glioma. Growing evidence from numerous studies of human solid tumor-infiltrating CD8+ T cells indicates that tissue-resident memory T cells (TRM) represent a substantial subpopulation of tumor-infiltrating lymphocytes (TILs). Although it is reported that some types of cancer patients with high immune infiltration tend to have better outcomes than patients with low immune infiltration, it seems this does not happen in gliomas. This study aimed to characterize TRMs cells in the glioma tumor microenvironment to identify their potential predictive and prognostic role and the possible therapeutic applications. Fluorescence activated cell sorting (FACS) analysis and immunofluorescence staining highlighted a statistically significant increase in CD8+ TRM cells (CD103+ and CD69+ CD8+ T cells) in gliomas compared to control samples (meningioma). In-silico analysis of a dataset of n = 153 stage IV glioma patients confirmed our data. Moreover, the gene expression analysis showed an increase in the expression of TRM-related genes in tumor tissues compared to normal tissues. This analysis also highlighted the positive correlation between genes associated with CD8+ TRM and TILs, indicating that CD8+ TRMs cells are present among the infiltrating T cells. Finally, high expression of Integrin subunit alpha E (ITGAE), the gene coding for the integrin CD103, and high CD8+ TILs abundance were associated with more prolonged survival, whereas high ITGAE expression but low CD8+ TILs abundance were associated with lower survival.

Integrins are enriched on aberrantly fucosylated tumour‐derived urinary extracellular vesicles

AbstractUrinary 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.

Bi-allelic pathogenic variants in ITGA8 cause slowly progressive renal disease of unknown etiology.

Integrin Subunit Alpha 8 gene (ITGA8) encodes an integrin chain that is known to be critical in the early stage of the kidney development. Bi-allelic pathogenic variants in ITGA8 are associated with bilateral renal agenesis, as well as anomalies involving urogenital system. Here, we report two unrelated patients presenting with slowly progressing chronic kidney disease associated with bilateral renal hypodysplasia carrying homozygous loss of function variants in the ITGA8 gene. These results broaden the clinical and genotypic spectrum of ITGA8 defects, revealing the high and unexpected degree of phenotypic heterogeneity of this autosomal recessive disease. Our study emphasizes the usefulness of Next-Generation Sequencing in unraveling the genetic cause of chronic kidney disease of unknown etiology, and raises the question of genetic modifiers involved in the variation of the phenotypes associated with autosomal recessive ITGA8 pathogenic variants.

Transcriptomic analysis reveals genetic factors regulating early steroid-induced osteonecrosis of the femoral head.

The present study aimed to explore the signaling pathways involved in development of early steroid-induced osteonecrosis of the femoral head (SONFH) and identify diagnostic biomarkers regulating peripheral blood in SONFH patients. We downloaded transcriptome data and identified differentially expressed genes (DEGs) using the R software. We used ClusterProfiler to perform enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, and analyzed protein–protein interactions using the STRING database. Network X was used to visualize the networks in Python. A total of 584 DEGs were identified, of which 294 and 290 were upregulated and downregulated, respectively. Enrichment analysis showed that the DEGs were mainly involved in red blood cell differentiation, cell protein catabolism, gas transportation, activation of myeloid leukocytes, phagocytosis, and inflammatory response. Pathway analysis revealed that these DEGs were involved in regulation of mitophagy-animal, human T-cell leukemia virus-1 infection, Forkhead box O, phagocytosis, osteoclast differentiation, and cytokine–cytokine receptor interaction. Quantitative real-time polymerase chain reaction results were consistent with findings from protein–protein interaction network analysis. Several genes, including peroxiredoxin 2, haptoglobin, matrix metallopeptidase 8, formyl peptide receptor 2, and integrin subunit alpha X, promote SONFH occurrence by regulating the redox, inflammatory response, and osteoblast and osteoclast structure and function pathways. They may be important targets for designing approaches for early diagnosis and treatment of SONFH.

Circ_0001658 regulates PI3K/AKT signaling via the miR-671-5p/ITGA2 axis and affects the progress of papillary thyroid carcinoma.

BackgroundCircular RNAs (circRNAs) are a newly described class of non-coding RNAs that play essential roles in regulating gene expression. However, to date, few studies have examined the roles of circRNAs in papillary thyroid carcinoma (PTC). In this study, we analyzed the expression of circ_0001658 in PTC as well as its functions and associated mechanisms of action in PTC cells.MethodsReal-time quantitative polymerase chain reaction was used to determine the expression of circ_0001658, miR-671-5p, and integrin subunit alpha 2 (ITGA2), and western blotting was performed to determine the levels of ITGA2 and phosphatidylinositide 3-kinase/protein kinase B pathway-related proteins in PTC cell lines. Cell Counting Kit-8 and Transwell analyses were conducted to examine the effects of circ_0001658 on PTC cell function following the knockdown of circ_0001658 expression. In addition, the targeting of circ_0001658 and ITGA2 by miR-671-5p was verified using dual-luciferase reporter assays.ResultsWe demonstrated that circ_0001658 and ITGA2 were significantly up-regulated in PTC tissues and cell lines. Knockdown of circ_0001658 inhibited the growth and metastatic potential of PTC cells. MiR-671-5p targeted both circ_0001658 and ITGA2. Mechanistically, circ_0001658 promoted PTC progression by sponging miR-671-5p, up-regulating ITGA2 expression, and activating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. In summary, circ_0001658 is a carcinogenesis-associated circRNA that regulates PI3K/AKT signaling via the miR-671-5p/ITGA2 axis and plays a role in promoting the progression of PTC.ConclusionsOur findings provide a theoretical basis for further molecular biological studies on the treatment of PTC.

5-Fluorouracil Alters Skeletal Muscle Mitochondrial Content And Inflammation In Mice

5-fluorouracil (5FU) is one of the most widely used chemotherapy drugs for colon, breast, head & neck, and pancreatic cancers; however, 5FU’s non-selective cytotoxic effects lead to debilitating fatigue coupled with decrements in skeletal muscle health and function. PURPOSE: To investigate the impacts of 5FU on physical activity, indices of skeletal muscle function, and the underlying changes in skeletal muscle mitochondrial content and inflammation. METHODS: Adult male CD2F1 mice were subjected to 1 cycle of 5FU (cycle: 5d, 30 mg/kg Lean Mass, i.p.) or PBS control. Cage activity, grip strength and neuromuscular function were assessed and skeletal muscle protein was isolated 48 hours following the last dose of 5FU or PBS. Mitochondria biogenesis [transcription factor A (TFAM)] and total content [electron transport chain complexes I-V (CI-V), cytochrome c (CytoC), voltage-dependent anion channels (VDAC)] was analyzed via western blot analysis. Real-time polymerase chain reaction was performed to analyze skeletal muscle macrophage [CD68, EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1), integrin subunit alpha X (Itgax), mannose receptor C-type 1 (MRC1)], pro-inflammatory [interleukin (IL) -1ß, -6, -13), tumor necrosis factor (TNF), interferon-gamma (IFNγ)] and anti-inflammatory [IL-10] gene expression. RESULTS: 5FU did not significantly impact grip strength or neuromuscular function (p > 0.05); however, cage activity and body weight (Δ8%) appreciably decreased following 5FU compared to controls (p < 0.05). Moreover, we observed a significant reduction in TFAM, VDAC, CIV, and CV following 5FU administration (p < 0.05) while no differences in CytoC, CI, CII, and CIII were found (p > 0.05). Interestingly, EMR1 (Δ43%) and CD68 (Δ37%) expression significantly decreased along with reductions in IL-1ß (Δ85%), IL-6 (Δ37%), TNF (Δ52%), and IFNγ (Δ42%) expression following 5FU compared to controls (p < 0.05). CONCLUSION: Collectively, these results demonstrate 1 cycle of 5FU notably impacts mitochondrial content and inflammation concomitant with decreases in body weight and cage activity. While changes in skeletal muscle function were not found, the observed changes in mitochondria and the inflammatory micro-environment may precede the functional decrements in skeletal muscle.

ITGAL infers adverse prognosis and correlates with immunity in acute myeloid leukemia

Integrin subunit alpha L (ITGAL) was found aberrantly expressed in multiple cancer types, suggesting its essential role in tumorigenesis. Hence, we aimed to explore its definite role in acute myeloid leukemia and emphasize its associations with immunity. Here, we found ITGAL was highly expressed in AML patients and elevated expression was associated with poor prognosis. ITGAL was associated with age and cytogenetic risk classifications, but not relevant to AML driver gene mutations. Univariate and multivariate Cox regression analyses determined ITGAL as an independent prognostic factor. The nomogram integrating ITGAL and clinicopathologic variables was constructed to predict 1-, 3- and 5-year overall survival (OS). Functional analyses revealed that ITGAL was mainly responsible for the production and metabolic process of cytokine. As for immunity, ITGAL was positively associated with MDSCs including iDCs, and macrophages in the TCGA-LAML cohort. We also found that ITGAL was positively associated with most immune checkpoint genes and cytokines. In addition, we found that ITGAL knockdown caused substantial inhibition of cell growth and significant induction of early apoptosis in AML cells. The xenograft study indicated that ITGAL knockdown prolonged the survival of recipient mice. Overall, ITGAL is an independent prognostic factor and is closely related to the number of MDSCs and cytokine production.

Abstract P1018: Bone Marrow Progenitor Cell-Derived Exosomes Activate Cardiac Fibroblasts Via Mir-21a-5p Mediated Integrin Alpha V Stabilization

Abstract: Heart failure is leading cause of death worldwide, and cardiac fibrosis is the hallmark of heart failure. We previously showed that interleukin-10 (IL10) treatment significantly reduces pressure overload-induced cardiac fibrosis. We also have demonstrated that fibroblast progenitor cells (bone marrow-prominin positive cells; FPCs) play a prominent role in developing cardiac fibrosis, yet the mechanism is largely unknown. Here, we hypothesized that pro-fibrotic miRNAs enriched in exosomes derived from IL10 KO FPCs promote fibroblast activation and cardiac fibrosis in pressure-overloaded myocardium. Methods: Exosomes were isolated from WT and IL-10KO FPCs. Exosomal miRNAs profiling was performed using fibrosis-associated miRNA profiler kit from Qiagen. To determine the contribution of exosomal miR21 on TAC-induced fibrosis, miR21 level was reduced in FPCs-derived exosomes using miR21 silencer. Modified exosomes were injected into heart immediately after TAC surgery. Results: FPCs were identified as CD45+/prominin-1 cells using FACS. TGFβ treatment exacerbated fibrotic genes (Col1α, fibronectin, periostin, and αSMA) expression in IL10KO FPCs compared to WT-FPCs. Pathway-based miRNA array revealed that IL10KO FPCs exosomes are enriched with miR-21a-5p and facilitated fibroblasts activation as observed by qPCR (αSMA and Col1α), western (Col1α and TGFβ levels), wound healing, and immunostaining assay (Col1α/DAPI expression) as compared to WT control. Interestingly, miR21 inhibition in IL10KO FPCs exosome using antimiR 21, reduced TGFβ-induced cardiac fibroblast activation. Target prediction database analysis revealed that Integrin Subunit Alpha V (ITGAV) is a strong regulatory target for miRNA21. At molecular level, we found that exosomal miR-21a-5p stabilizes ITGAV, which ultimately enhances fibroblast activation and cardiac fibrosis both in vitro and in vivo. Conclusions: In conclusion, we report that FPCs derived miR21 packaged in exosomes activates cardiac fibroblasts in IL10-KO mice via miR-21a-5p/ITGAV/Col1α signaling axis and increased cardiac fibrosis. IL10 treatment significantly reduced miR21 exosomal packaging, inhibited fibroblasts activation, and reduced cardiac fibrosis following TAC.

The clinical significance of integrin subunit alpha V in cancers: from small cell lung carcinoma to pan-cancer

BackgroundLittle is known about the relationship between integrin subunit alpha V (ITGAV) and cancers, including small cell lung cancer (SCLC).MethodsUsing large sample size from multiple sources, the clinical roles of ITGAV expression in SCLC were explored using differential expression analysis, receiver operating characteristic curves, Kaplan–Meier curves, etc.ResultsDecreased mRNA (SMD = − 1.05) and increased protein levels of ITGAV were detected in SCLC (n = 865). Transcription factors—ZEB2, IK2F1, and EGR2—may regulate ITGAV expression in SCLC, as they had ChIP-Seq (chromatin immunoprecipitation followed by sequencing) peaks upstream of the transcription start site of ITGAV. ITGAV expression made it feasible to distinguish SCLC from non-SCLC (AUC = 0.88, sensitivity = 0.78, specificity = 0.84), and represented a risk role in the prognosis of SCLC (p < 0.05). ITGAV may play a role in cancers by influencing several immunity-related signaling pathways and immune cells. Further, the extensive pan-cancer analysis verified the differential expression of ITGAV and its clinical significance in multiple cancers.ConclusionITGAV served as a potential marker for prognosis and identification of cancers including SCLC.

FEV Maintains Homing and Expansion by Activating ITGA4 Transcription in Primary and Relapsed AML.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy that recurs in approximately 50% of cases. Elevated homing and uncontrolled expansion are characteristics of AML cells. Here, we identified that Fifth Ewing Variant (FEV) regulates the homing and expansion of AML cells. We found that FEV was re-expressed in 30% of primary AML samples and in almost all relapsed AML samples, and FEV expression levels were significantly higher in relapsed samples compared to primary samples. Interference of FEV expression in AML cell lines delayed leukemic progression and suppressed homing and proliferation. Moreover, FEV directly activated integrin subunit alpha 4 (ITGA4) transcription in a dose-dependent manner. Inhibition of integrin α4 activity with natalizumab (NZM) reduced the migration and colony-forming abilities of blasts and leukemic-initiating cells (LICs) in both primary and relapsed AML. Thus, our study suggested that FEV maintains the homing and expansion of AML cells by activating ITGA4 transcription and that targeting ITGA4 inhibits the colony-forming and migration capacities of blasts and LICs. Thus, these findings suggested that the FEV-ITGA4 axis may be a therapeutic target for both primary and relapsed AML.

Abstract 2920: Targeting integrin alpha V beta 5 heterodimer stability using a novel small molecular inhibitor for tumor suppression

Abstract The cell surface proteome provides a pivotal interphase that connects the intracellular signaling with external environments, which may harbor molecular targets for therapeutic development. To identify essential cell surface proteins in cancers, we established a CRISPR library that contains 2,910 sgRNAs targeting 582 genes encoding cell surface receptors (to cytokines, extracellular matrices [ECM], etc.), molecular channels, and signaling components. We then screened this library in 3 solid cancer (MDA-MB-231, SW620, PANC1) and 3 liquid cancer (MV4-11, HUT78, CCRF-CEM) cell models expressing the Cas9 endonuclease, and observed an essential role of ITGAV (Integrin Alpha V) specifically in the solid cancer. As integrins require the dimerization between an alpha and beta subunit to function as cell surface ECM receptors, we further developed a focused CRISPR library with 650 sgRNAs targeting all 26 integrin alpha and beta subunits. This validation screen revealed a strong correlation between ITGAV and ITGB5 (Integrin Beta 5) in solid tumors. CRISPR targeting either ITGAV or ITGB5 led to attenuated cell proliferation with increased cell cycle stalling and apoptosis. Knockout of ITGAV-ITGB5 also suppressed the capacity of tumor cells to migrate and invade through the ECM, highlighting the therapeutic benefit via blocking the ITGAV-ITGB5 heterodimer. To identify novel compounds that can disrupt ITGAV-ITGB5 dimerization, we first modeled a 3D crystal structure of ITGAV (PDB ID: 3IJE) using AutoSite (Scripps) to reveal the protein surface pockets amenable to the small molecular binding. This analysis revealed an AVB pocket at the N-terminal extracellular domain of ITGAV, which is in close contact with the beta subunit. CRISPR targeting this AVB pocket abolished the ITGAV-ITGB5 heterodimers (detected by an AV-B5-complex antibody) and induced rapid cell death, rationalizing this pocket for further therapeutic development. Based on these observations, we computationally docked &amp;gt;100,000 compounds from the NCI DTP Open Chemicals Repository using AutoDock Vina (Scripps), subsequently obtaining the top 500 binder compounds for in-lab validation, and identified a lead AVB pocket inhibitor “Cpd_2” (C32H40N4). Treatment of Cpd_2 killed tumor cells and diminished the AV-B5-complex antibody stain. Furthermore, we confirmed the capacity of Cpd_2 to dissociate ITGAV and ITGB5 on the cell surface by a NanoBRET energy transfer system. Of note, while the traditional ITGAV inhibitor cilengitide (an RGD peptide mimic) blocked the ECM interaction and detached the cells from TC dishes, they failed to dissociate the ITGAV-ITGB5 heterodimer nor induce rapid cell death. In summary, our study highlights a novel strategy of targeting the ITGAV-ITGB5 heterodimer stability by the small molecular inhibitor Cpd_2 for future anti-tumor treatment. Citation Format: Nicole Mattson, Kazuya Miyashita, Anthony Chan, Lu Yang, Sheela Pangeni Pokharel, Wei Lu, Mingli Li, Qiao Liu, Xiaobao Xu, Mingye Feng, Chun-Wei Chen. Targeting integrin alpha V beta 5 heterodimer stability using a novel small molecular inhibitor for tumor suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2920.

Sugar Kelp Consumption Did Not Alter Circulating and Hepatic Lipids in Low-Density Lipoprotein Receptor Knockout Mice Fed a Diet High in Fat and Cholesterol

ObjectivesWe have previously shown that sugar kelp (Saccharina latissima), a brown seaweed, decreased serum triglycerides (TG) and total cholesterol (TC), and prevented adipose tissue inflammation and fibrosis in male diet-induced obesity mice. In this study, we investigated whether the consumption of sugar kelp can prevent metabolic and inflammatory features in low-density lipoprotein receptor knockout (Ldlr KO) mice fed a diet high in fat and cholesterol. MethodsMale Ldlr KO mice at the age of 7 weeks from the Jackson laboratory were randomly assigned to a high-fat/high-cholesterol control (HF/HC; 16% fat, 0.25% cholesterol, w/w, n = 12), or a HF/HC supplemented with sugar kelp (HF/HC-SK; 6.0% dried sugar kelp power, w/w, n = 12). Mice were fed the experimental diets for 16 weeks and then serum and tissue samples were collected to measure blood and liver lipids, serum alanine aminotransferase, and hepatic gene expression. ResultsMice fed HF/HC-SK diet showed a trend towards an increase in final body weight compared to HF/HC control. Serum TG and TC concentrations were not significantly different between control and sugar kelp-fed mice. In the liver, sugar kelp consumption did not alter weights, TG, TC, and lipogenic gene expression. However, the expression of macrophage markers, such as adhesion G protein-coupled receptor E1 and cluster of differentiation 68, and M1 macrophage markers, including tumor necrosis factor, and integrin subunit alpha X, were significantly higher in HF/HC-SK group than HF/HC control mice. Despite the increases in inflammatory markers in the liver of the HF/HC-SK group, serum ALT activity was not different between the two groups. ConclusionsSugar kelp consumption did not alter circulating and hepatic TC and TG, and serum liver injury marker in HF/HC-fed Ldlr KO mice. Further study is warranted to determine whether sugar kelp can impact the development of atherosclerosis in this mouse model. Funding SourcesUSDA Hatch CONS00993.

An Myh11 single lysine deletion causes aortic dissection by reducing aortic structural integrity and contractility

Pathogenic variants in myosin heavy chain (Myh11) cause familial thoracic aortic aneurysms and dissections (FTAAD). However, the underlying pathological mechanisms remain unclear because of a lack of animal models. In this study, we established a mouse model with Myh11 K1256del, the pathogenic variant we found previously in two FTAAD families. The Myh11∆K/∆K aorta showed increased wall thickness and ultrastructural abnormalities, including weakened cell adhesion. Notably, the Myh11∆K/+ mice developed aortic dissections and intramural haematomas when stimulated with angiotensin II. Mechanistically, integrin subunit alpha2 (Itga2) was downregulated in the Myh11∆K/∆K aortas, and the smooth muscle cell lineage cells that differentiated from Myh11∆K/∆K induced pluripotent stem cells. The contractility of the Myh11∆K/∆K aortas in response to phenylephrine was also reduced. These results imply that the suboptimal cell adhesion indicated by Itga2 downregulation causes a defect in the contraction of the aorta. Consequently, the defective contraction may increase the haemodynamic stress underlying the aortic dissections.