Targeting Integrin α3 Blocks ß1 Maturation, Triggers Endoplasmic Reticulum Stress, and Sensitizes Glioblastoma Cells to TRAIL-Mediated Apoptosis.

Glioblastoma (GBM) is a devastating brain cancer for which new effective therapies are urgently needed. GBM, after an initial response to current treatment regimens, develops therapeutic resistance, leading to rapid patient demise. Cancer cells exhibit an inherent elevation of endoplasmic reticulum (ER) stress due to uncontrolled growth and an unfavorable microenvironment, including hypoxia and nutrient deprivation. Cancer cells utilize the unfolded protein response (UPR) to maintain ER homeostasis, and failure of this response promotes cell death. In this study, as integrins are upregulated in cancer, we have evaluated the therapeutic potential of individually targeting all αß1 integrin subunits using RNA interference. We found that GBM cells are uniquely susceptible to silencing of integrin α3. Knockdown of α3-induced proapoptotic markers such as PARP cleavage and caspase 3 and 8 activation. Remarkably, we discovered a non-canonical function for α3 in mediating the maturation of integrin ß1. In its absence, generation of full length ß1 was reduced, immature ß1 accumulated, and the cells underwent elevated ER stress with upregulation of death receptor 5 (DR5) expression. Targeting α3 sensitized TRAIL-resistant GBM cancer cells to TRAIL-mediated apoptosis and led to growth inhibition. Our findings offer key new insights into integrin α3's role in GBM survival via the regulation of ER homeostasis and its value as a therapeutic target.

MUC1 promotes cervical squamous cell carcinoma through ERK phosphorylation-mediated regulation of ITGA2/ITGA3.

In contrast to the decreasing trends in developed countries, the incidence and mortality rates of cervical squamous cell carcinoma in China have increased significantly. The screening and identification of reliable biomarkers and candidate drug targets for cervical squamous cell carcinoma are urgently needed to improve the survival rate and quality of life of patients. In this study, we demonstrated that the expression of MUC1 was greater in neoplastic tissues than in non-neoplastic tissues of the cervix, and cervical squamous cell carcinoma patients with high MUC1 expression had significantly worse overall survival than did those with low MUC1 expression, indicating its potential for early diagnosis of cervical squamous cell carcinoma. Next, we explored the regulatory mechanism of MUC1 in cervical squamous cell carcinoma. MUC1 could upregulate ITGA2 and ITGA3 expression via ERK phosphorylation, promoting the proliferation and metastasis of cervical cancer cells. Further knockdown of ITGA2 and ITGA3 significantly inhibited the tumorigenesis of cervical cancer cells. Moreover, we designed a combination drug regimen comprising MUC1-siRNA and a novel ERK inhibitor in vivo and found that the combination of these drugs achieved better results in animals with xenografts than did MUC1 alone. Overall, we discovered a novel regulatory pathway, MUC1/ERK/ITGA2/3, in cervical squamous cell carcinoma that may serve as a potential biomarker and therapeutic target in the future.

MUC1 is overexpressed in cervical squamous cell carcinoma. MUC1 regulates ERK phosphorylation, and subsequently upregulates ITGA2 and ITGA3 expression to promote tumorigenesis in cervical squamous cell carcinoma. A combination drug regimen targeting MUC1 and ERK achieved better results compared than MUC1 alone.

Integrin α3 is required for high-frequency repetitive transcranial magnetic stimulation-induced glutamatergic synaptic transmission in mice with ischemia.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is an effective therapy in post-stroke motor recovery. However, the underlying mechanisms of rTMS regulates long-lasting changes with synaptic transmission and glutamate receptors function (including AMPARs or NMDARs) remains unclear. METHODS: Mice were received 10-Hz rTMS treatment once daily on the third day after photothrombotic (PT) stroke for 18 days. Motor behaviors and the Western blot were used to evaluate the therapeutic efficacy of 10-Hz rTMS in the mice with PT model. Moreover, we used wild-type (WT) and NEX-α3-/- mice to further explore the 10-Hz rTMS effect. RESULTS: We found that 10-Hz rTMS improved the post-stroke motor performance in the PT mice. Moreover, the levels of AMPAR, vGlut1, and integrin α3 in the peri-infarct were significantly increased in the rTMS group. In contrast, 10-Hz rTMS did not induce these aforementioned effects in NEX-α3-/- mice. The amplitude of AMPAR-mediated miniature excitatory postsynaptic currents (EPSCs) and evoked EPSCs was increased in the WT + rTMS group, but did not change in NEX-α3-/- mice with rTMS. CONCLUSIONS: In this study, 10-Hz rTMS improved the glutamatergic synaptic transmission in the peri-infract cortex through effects on integrin α3 and AMPARs, which resulted in motor function recovery after stroke.

Integrin α3 promotes TH17 cell polarization and extravasation during autoimmune neuroinflammation.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by CNS-infiltrating leukocytes, including TH17 cells that are critical mediators of disease pathogenesis. Although targeting leukocyte trafficking is effective in treating autoimmunity, there are currently no therapeutic interventions that specifically block encephalitogenic TH17 cell migration. Here, we report integrin α3 as a TH17 cell-selective determinant of pathogenicity in experimental autoimmune encephalomyelitis. CNS-infiltrating TH17 cells express high integrin α3, and its deletion in CD4+ T cells or Il17a fate-mapped cells attenuated disease severity. Mechanistically, integrin α3 enhanced the immunological synapse formation to promote the polarization and proliferation of TH17 cells. Moreover, the transmigration of TH17 cells into the CNS was dependent on integrin α3, and integrin α3 deficiency enhanced the retention of CD4+ T cells in the perivascular space of the blood-brain barrier. Integrin α3-dependent interactions continuously maintain TH17 cell identity and effector function. The requirement of integrin α3 in TH17 cell pathogenicity suggests integrin α3 as a therapeutic target for MS treatment.

ITGA3 acts as a purity-independent biomarker of both immunotherapy and chemotherapy resistance in pancreatic cancer: bioinformatics and experimental analysis.

Contribution of integrin superfamily genes to treatment resistance remains uncertain. Genome patterns of thirty integrin superfamily genes were analyzed of using bulk and single-cell RNA sequencing, mutation, copy number, methylation, clinical information, immune cell infiltration, and drug sensitivity data. To select the integrins that are most strongly associated with treatment resistance in pancreatic cancer, a purity-independent RNA regulation network including integrins were constructed using machine learning. The integrin superfamily genes exhibit extensive dysregulated expression, genome alterations, epigenetic modifications, immune cell infiltration, and drug sensitivity, as evidenced by multi-omics data. However, their heterogeneity varies among different cancers. After constructing a three-gene (TMEM80, EIF4EBP1, and ITGA3) purity-independent Cox regression model using machine learning, ITGA3 was identified as a critical integrin subunit gene in pancreatic cancer. ITGA3 is involved in the molecular transformation from the classical to the basal subtype in pancreatic cancer. Elevated ITGA3 expression correlated with a malignant phenotype characterized by higher PD-L1 expression and reduced CD8+ T cell infiltration, resulting in unfavorable outcomes in patients receiving either chemotherapy or immunotherapy. Our findings suggest that ITGA3 is an important integrin in pancreatic cancer, contributing to chemotherapy resistance and immune checkpoint blockade therapy resistance.

Aberrant glycosylation of α3 integrins as diagnostic markers in epithelial ovarian cancer.

BACKGROUND: Glycans are strongly involved in stability and function of integrins (ITG) and tetraspanin protein CD63 and their respective interaction partners as they are dysregulated in the tumorigenic processes. Glycosylation changes is a universal phenomenon of cancer cells. In this study, glycosylation changes in epithelial ovarian cancer (EOC) are explored using tetraspanin and integrin molecules. METHODS: ITG and CD63 were immobilized from 10 EOC and 5 benign ovarian cyst fluid on microtiter wells and traced with 3 glycan binding proteins (STn, WGA, UEA) conjugated on europium nanoparticles. Total protein measurements (ITG & CD63 immunoassays) were also performed. The most promising glycovariant candidates identified were then clinically evaluated on the whole cohort of 77 ovarian cyst fluids. Additional testing was performed in ascites fluid samples of liver cirrhosis (n = 2) and EOC (n = 4). RESULTS: Sialylated Tn antibody based glycovariants of ITGα3 (ITGα3STn) and CD63 (CD63STn) performed better than corresponding protein epitope-based immunoassays, ITGα3IA and CD63IA respectively. Combined ITGα3 based assays (ITGα3IA + ITGα3STn) detected 49 out of 55 malignant & borderline cases without detecting any of the 22 benign and healthy cysts. CONCLUSION: Our findings indicate the potential diagnostic application of ITGα3STn along with total ITGα3IA, which could help reduce the unnecessary surgeries. The results encourage studying further the potential use of these novel assays to detect EOC at earlier clinical stages.

Integrin α3 Mediates Stemness and Invasion of Glioblastoma by Regulating POU3F2.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor. Integrins have been implicated in the malignancy of GBM. A recent study demonstrated that integrin α3 (ITGA3) promoted the invasion of breast cancer cells by regulating transcriptional factor POU3F2. However, whether this also happened in GBM remained unknown. METHODS: Therefore, we explored the relationship between ITGA3 and POU3F2 in GBM. We measured the expression of ITGA3 and POU3F2 in GBM tissues. We generated ITGA3 knockdown and POU3F2 knockdown GBM U87MG cells and the proliferation, migration and invasion, the expression of stemness markers and epithelial to mesenchymal transition (EMT) markers were measured. We transplanted ITGA3 knockdown and POU3F2 knockdown GBM U87MG cells into mice. The mice were treated with anti-ITGA3 antibody. The tumor sizes, the expression of stemness markers and epithelial-to-mesenchymal transition (EMT) markers were measured. RESULTS: Both ITGA3 and POU3F2 were upregulated in GBM tissues. Knocking down ITGA3 resulted in reduced expression of POU3F2. Knocking down ITGA3 and POU3F2 suppressed U87MG cells proliferation, migration and invasion, inhibited the expression of stemness markers and prevented epithelial- to-mesenchymal transition. The transplantation of ITGA3 knockdown and POU3F2 knockdown U87MG cells decreased tumor size. CONCLUSION: Anti-ITGA3 antibody treatment reduced the tumor size. ITGA3 regulates stemness and invasion of glioblastoma through POU3F2.

YY1/ITGA3 pathway may affect trophoblastic cells migration and invasion ability.

Recurrent spontaneous abortion (RSA) is a disturbing pregnancy disorder experienced by ~2.5% of women attempting to conceive. The pathogenesis of RSA is still unclear. Previous findings revealed that transcription factor YIN-YANG 1(YY1) was related to the pathogenesis of RSA by influence trophoblastic cell invasion ability. Present study aimed to investigate more specific molecular mechanism of YY1 playing in trophoblastic cells. In our research, RNA-seq and Chip-seq were used to find significant changed genes between si-YY1(Knock down of YY1) HTR-8/SVneo cells(n = 3) and HTR-8/SVneo cells(n = 3). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis results suggested that Integrins related pathway maybe necessary to biological functions of trophoblastic cells. Chip-seq dataset analysis results predict YY1 can regulate ITGA3/7 expression by binding to the promoter region of ITGA3/7. Furthermore, results from chip experiment, RT-PCR, Dual-luciferase reporter gene assay showed that YY1 was able to bind to the promoter region of ITGA3 and regulate ITGA3 mRNA and protein expression. However, ITGA7 could not be significant influenced by YY1. Besides, gene silencing experiment, Western blot and Immunofluorescence assay confirmed that both YY1 and ITGA3 can accelerate phosphorylation focal adhesion kinase and affect cytoskeleton formation in HTR-8/SVneo cells. In conclusion, YY1/ITGA3 play a critical role in trophoblast invasion ability by regulating cytoskeleton formation.

Characterization and Identification of Aptamers against CD49c for the Detection, Capture, and Release of Cancer Cells.

As a kind of recognition molecule, aptamers can be inserted into some regulatory sequences for the smart response of their targets. However, the molecular engineering might lead to the change of the binding affinity. Here, we present a stable aptamer ZAJ-2c and an environmentally sensitive aptamer ZAJ-2d optimized from an original cell-binding aptamer ZAJ-2, and the molecular target was further identified as CD49c on the cell membrane. ZAJ-2c was characterized with high binding ability independent of the presence of divalent cations at a temperature range from 4 to 37 °C, showing promise for measuring the expression of CD49c on cancer cells. Moreover, ZAJ-2d had a nanomolar binding affinity in the binding buffer at 4 °C, the same as ZAJ-2c, but lost the binding ability in a PBS buffer supplemented with 5 mM EDTA at 37 °C. This aptamer variant proved to selectively capture and release the CD49c positive cells by simply adjusting the temperatures and divalent cations. This set of aptamers might provide a toolbox for monitoring and operating of a wide range of cancer cells with CD49c expression on the surface, which will be helpful for the studying the heterogeneity of rare cells.

Integrin α3/α6 and αV are implicated in ADAM15-activated FAK and EGFR signalling pathway individually and promote non-small-cell lung cancer progression.

Disintegrin-metalloproteinase 15(ADAM15), a member of disintegrin metalloproteinases (ADAMs), plays important roles in various cancer types. However, the underlying ADAM15 functioning in lung cancer is still unclear. In the present study, we find that ADAM15 regulates the epidermal growth factor receptor/focal adhesion kinase (EGFR/FAK) signalling pathway by interactions with integrins. Integrin αV is involved in ADAM15-mediated FAK signalling. Further, we find that ADAM15 and CD151 were co-expressed, and the presence of ADAM15 affected the integrin α3/α6-related EGFR signalling pathway by cooperating with CD151. In addition, we also prove the effect of ADAM15 on proliferation in nude mice. Finally, we show that ADAM15 is a direct target of miR-204-5p by luciferase reporter assays, qRT-PCR and western blot analyses. Our findings provide molecular and cellular evidence that ADAM15 promotes cell proliferation and metastasis in NSCLC, which might provide a potential target for NSCLC treatment.

Effectiveness of Nano Bioactive Glass Fiber Loaded with Platelet-Rich Plasma on Thermal Wound Healing Process in Rats.

In this study we evaluated the impact of topical application of bioactive glass fibers loaded PRP on a deep seconddegree thermal wound and its healing process sub-streaming molecular pathway of re-epithelialization. Wistar rats were randomly divided into four groups: normal control group, model group (deep second-degree thermal wound), PRP group, and PRP+nanobioactive glass fiber group. After treatment, the changes of wounds were observed daily. H&E staining was used to evaluate the pathological changes and also, qRT-PCR was used to detect the mRNA expression of KGF, IL-1, IL-6, IL-10, TGF-ß, EGF, VEGF, HIF-1α, integrin α3 and integrin ß1 in wound tissues. In the current study, we observed that PRP group and the PRP group basically re-epithelized on the 21st day. The wound healing rates of the PRP+nanobioactive glass fiber group and PRP group at each time point were higher than those in the model group, while there was no significant difference in wound healing rate between the PRP+nanobioactive glass fiber group and PRP group at each time point. H&E staining showed that the pathological scores of skin wound repairing in the PRP+nanobioactive glass fiber group on the 7th, 14th and 21st day were higher than that of in the model group. The qPCR results suggested the mRNA expression of IL-1, IL-6 and IL-10 in the PRP+nanobioactive glass fiber group and the PRP group were lower than those in the untreated group on the 14th day; the expression of VEGF and EGF mRNA were higher on the 3rd day; the mRNA expression of TGF-ß, HIF-1α showed a tendency of increasing first and decreasing then; integrin ß1 mRNA expression increased significantly, which was highest; integrin α3 mRNA expression was higher on day 3rd and 21th, respectively. The PRP+nanobioactive glass fibers and PRP can shorten the wound healing time and improve the healing quality mainly by promoting the wound epithelization through increasing the expression of EGF, VEGF, TGF-ß, HIF-1α, Integrin α3, and meanwhile increasing the release of Integrin ß1 and other mechanisms.

Zebrafish integrin a3b is required for cardiac contractility and cardiomyocyte proliferation.

In cardiac muscle cells, heterodimeric integrin transmembrane receptors are known to serve as mechanotransducers, translating mechanical force to biochemical signaling. However, the roles of many individual integrins have still not been delineated. In this report, we demonstrate that Itga3b is localized to the sarcolemma of cardiomyocytes from 24 to 96 hpf. We further show that heterozygous and homozygous itga3b/bdf mutant embryos display a cardiomyopathy phenotype, with decreased cardiac contractility and reduced cardiomyocyte number. Correspondingly, proliferation of ventricular and atrial cardiomyoctyes and ventricular epicardial cells is decreased in itga3b mutant hearts. The contractile dysfunction of itga3b mutants can be attributed to cardiomyocyte sarcomeric disorganization, including thin myofilaments with blurred and shortened Z-discs. Together, our results reveal that Itga3b localizes to the myocardium sarcolemma, and it is required for cardiac contractility and cardiomyocyte proliferation.

Glomerular endothelial cell-podocyte stresses and crosstalk in structurally normal kidney transplants.

Increased podocyte detachment begins immediately after kidney transplantation and is associated with long-term allograft failure. We hypothesized that cell-specific transcriptional changes in podocytes and glomerular endothelial cells after transplantation would offer mechanistic insights into the podocyte detachment process. To test this, we evaluated cell-specific transcriptional profiles of glomerular endothelial cells and podocytes from 14 patients of their first-year surveillance biopsies with normal histology from low immune risk recipients with no post-transplant complications and compared these to biopsies of 20 healthy living donor controls. Glomerular endothelial cells from these surveillance biopsies were enriched for genes related to fluid shear stress, angiogenesis, and interferon signaling. In podocytes, pathways were enriched for genes in response to growth factor signaling and actin cytoskeletal reorganization but also showed evidence of podocyte stress as indicated by reduced nephrin (adhesion protein) gene expression. In parallel, transcripts coding for proteins required to maintain podocyte adherence to the underlying glomerular basement membrane were downregulated, including the major glomerular podocyte integrin α3 and the actin cytoskeleton-related gene synaptopodin. The reduction in integrin α3 protein expression in surveillance biopsies was confirmed by immunoperoxidase staining. The combined growth and stress response of patient allografts post-transplantation paralleled similar changes in a rodent model of nephrectomy-induced glomerular hypertrophic stress that progress to develop proteinuria and glomerulosclerosis with shortened kidney life span. Thus, even among patients with apparently healthy allografts with no detectable histologic abnormality including alloimmune injury, transcriptomic changes reflecting cell stresses are already set in motion that could drive hypertrophy-associated glomerular disease progression.

miRNA-144-5p/ITGA3 Suppressed the Tumor-Promoting Behaviors of Thyroid Cancer Cells by Downregulating ITGA3.

OBJECTIVE: To ascertain the mechanism of miRNA-144-5p and ITGA3 in thyroid cancer (TC). METHODS: From The Cancer Genome Atlas (TCGA), RNA expression profiles were obtained for the expression analysis of miRNAs and mRNAs in TC. qRT-PCR and western blot were utilized to measure the expression of miRNA-144-5p and ITGA3 at RNA and protein levels, respectively. The association between miRNA-144-5p and ITGA3 was validated by the dual-luciferase assay. CCK-8, scratch healing, transwell, and flow cytometry assays were employed to evaluate tumor-related cell behaviors. RESULTS: Low-expressed miRNA-144-5p and high-expressed ITGA3 were found in TC cells relative to normal cells. miRNA-144-5p expression was positively associated with suppressive effects on proliferative, invasive, and migratory ability of TC cells while negatively associated with cell apoptosis. miRNA-144-5p inhibited ITGA3 expression in TC, and its overexpression remarkably reversed the tumor-promoting effects of overexpressed ITGA3 on the biological functions of TC. CONCLUSION: It is verified in our study that cell growth of TC is inhibited by the miRNA-144-5p/ITGA3 axis, which represents an underlying target for TC. This research proposed a new insight into the strategy of TC treatment.

A novel ITGA3 homozygous splice mutation in an ILNEB syndrome child with slow progression.

BACKGROUND AND AIMS: ILNEB (interstitial lung disease, nephrotic syndrome, epidermolysis bullosa) syndrome is caused by ITGA3 mutations. Demises usually happened at infancy. This study reports a complete ILNEB syndrome child with slow disease progression. MATERIALS AND METHODS: Clinical data and related specimens were collected. Genomic DNA was extracted for genetic sequencing. Integrin α3 expression was detected by western blotting and immunofluorescence staining. RESULTS: The patient was male. He experienced recurrent rashes shortly after birth. His sparse eyebrows and eyelashes gradually lost. The patient was vulnerable to respiratory infections and had recurrent fever after vaccine immunization after 4 years. He was found with nephrotic syndrome and polycystic renal dysplasia at 8 years and progressed to end-stage renal disease at 12 years. A chest Computed Tomography revealed intestinal lung disease at 8 years. Continuous oxygen supplementation was needed at 13 years. Counts of lymphocyte subsets revealed elevated percentage of double-negative T cells and activated T cells. Next-generation sequencing revealed a novel homozygous splice mutation c.2219 + 4A > Cin ITGA3 that was predicted to be deleterious. The mutation resulted in exon17 skipping with the loss of 80 bp in the mRNA. The aberrant integrin α3 mRNA level was lower compared to the healthy control. Integrin α3 protein was not detected in urine epithelial cells and skin of the patient. CONCLUSIONS: We report a patient harboring a novel ITGA3 homozygous splice mutation who presented with complete ILNEB syndrome but slow disease progression. Immune disorders were suspected.

First patient with ILNEB syndrome due to pathogenic variants in ITGA3 surviving to adulthood.

Interstitial Lung disease, Nephrotic syndrome and Epidermolysis Bullosa, also referred to as ILNEB syndrome is an extremely rare autosomal recessive condition, caused by pathogenic variants in ITGA3. 11 patients have previously been diagnosed with ILNEB syndrome of whom 7 died in infancy or early childhood. We report the only patient with ILNEB syndrome who survived past adolescence, partly due to a double lung transplant. Additionally, our patient showed oral, nasal and gynecological symptoms not previously reported in patients with ILNEB syndrome.

CD9 and ITGA3 are regulated during HIV-1 infection in macrophages to support viral replication.

Monocytes/macrophages are important target cells for HIV-1. Here, we investigated whether HIV-1 induces changes in the macrophage gene expression profile to support viral replication. We observed that the macrophage gene expression profiles dramatically changed upon HIV-1 infection. The majority of the HIV-1 regulated genes were also differentially expressed in M2a macrophages. The biological functions associated with the HIV-1 induced gene expression profile in macrophages were mainly related to inflammatory responses. CD9 and ITGA3 were among the top genes upregulated upon HIV-1 infection. We showed that these genes support viral replication and that downregulation of these genes decreased HIV-1 replication in macrophages. Here we showed that HIV-1 infection of macrophages induces a gene expression profile that may dampen inflammatory responses. CD9 and ITGA3 were among the top genes regulated by HIV-1 and were shown to support viral production most likely at the level of viral budding and release.

Detection of bladder cancer with aberrantly fucosylated ITGA3.

We describe a simple, non-invasive assay to identify fucosylated-glycoisoform of integrin alpha-3 (ITGA3) directly from unprocessed urine. ITGA3 was detected directly from the urine of bladder cancer (BlCa) (n = 13) and benign prostatic hyperplasia (BPH) (n = 9) patients with the use of lectins coated on europium-doped-nanoparticles (Eu3+-NPs). Lectin Ulex europaeus agglutinin-I (UEA) showed enhanced binding with BlCa-derived ITGA3. The evaluation with individual samples showed that a glycovariant ITGA3-UEA assay could significantly discriminate BlCa from BPH patients (p = 0.007). The detection of aberrantly fucosylated-isoform of ITGA3 from urine can be used to distinguish BlCa from age-matched benign controls in a simple sandwich assay.