Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer.

Platinum (PT)-resistant Epithelial Ovarian Cancer (EOC) grows as a metastatic disease, disseminating in the abdomen and pelvis. Very few options are available for PT-resistant EOC patients, and little is known about how the acquisition of PT-resistance mediates the increased spreading capabilities of EOC. Here, using isogenic PT-resistant cells, genetic and pharmacological approaches, and patient-derived models, we report that Integrin α6 (ITGA6) is overexpressed by PT-resistant cells and is necessary to sustain EOC metastatic ability and adhesion-dependent PT-resistance. Using in vitro approaches, we showed that PT induces a positive loop that, by stimulating ITGA6 transcription and secretion, contributes to the formation of a pre-metastatic niche enabling EOC cells to disseminate. At molecular level, ITGA6 engagement regulates the production and availability of insulin-like growth factors (IGFs), over-stimulating the IGF1R pathway and upregulating Snail expression. In vitro data were recapitulated using in vivo models in which the targeting of ITGA6 prevents PT-resistant EOC dissemination and improves PT-activity, supporting ITGA6 as a promising druggable target for EOC patients.

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome-positive acute lymphoblastic leukemia through upregulation of integrin α6.

BACKGROUND: Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin ß1 (CD29) or ß4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. AIMS: Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. METHODS AND RESULTS: Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline. CONCLUSION: BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.

Targeted m6A demethylation of ITGA6 mRNA by a multisite dCasRx-m6A editor inhibits bladder cancer development.

INTRODUCTION: N6-methyladenosine (m6A) modification contributes to the pathogenesis and development of various cancers, including bladder cancer (BCa). In particular, integrin α6 (ITGA6) promotes BCa progression by cooperatively regulating multisite m6A modification. However, the therapeutic effect of targeting ITGA6 multisite m6A modifications in BCa remains unknown. OBJECTIVES: We aim to develop a multisite dCasRx- m6A editor for assessing the effects of the multisite dCasRx-m6A editor targeted m6A demethylation of ITGA6 mRNA in BC growth and progression. METHODS: The multisite dCasRx- m6A editor was generated by cloning. m6A-methylated RNA immunoprecipitation (meRIP), luciferase reporter, a single-base T3 ligase-based qPCR-amplification, Polysome profiling and meRIP-seq experiments were performed to determine the targeting specificity of the multisite dCasRx-m6A editor. We performed cell phenotype analysis and used in vivo mouse xenograft models to assess the effects of the multisite dCasRx-m6A editor in BC growth and progression. RESULTS: We designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based m6A-editing platform, comprising a nucleus-localized dCasRx fused with the catalytic domains of methyltransferase-like 3 (METTL3-CD) or α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5-CD), to simultaneously manipulate the methylation of ITGA6 mRNA at four m6A sites. The results confirmed the dCasRx-m6A editor modified m6A at multiple sites in ITGA6 mRNA, with low off-target effects. Moreover, targeted m6A demethylation of ITGA6 mRNA by the multisite dCasRx-m6A editor significantly reduced BCa cell proliferation and migration in vitro and in vivo. Furthermore, the dCasRx-ALKBH5-CD and ITGA6 multi-site gRNA delivered to 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice via adeno-associated viral vectors significantly inhibited BCa cell growth. CONCLUSION: Our study proposes a novel therapeutic tool for the treatment of BC by applying the multisite dCasRx-m6A editor while highlighting its potential efficacy for treating other diseases associated with abnormal m6A modifications.

Biophysical phenotype mixtures reveal advantages for tumor muscle invasion in vivo.

Bladder, colon, gastric, prostate, and uterine cancers originate in organs surrounded by laminin-coated smooth muscle. In human prostate cancer, tumors that are organ confined, without extracapsular extension through muscle, have an overall cancer survival rate of up to 97% compared with 32% for metastatic disease. Our previous work modeling extracapsular extension reported the blocking of tumor invasion by mutation of a laminin-binding integrin called α6ß1. Expression of the α6AA mutant resulted in a biophysical switch from cell-ECM (extracellular matrix) to cell-cell adhesion with drug sensitivity properties and an inability to invade muscle. Here we used different admixtures of α6AA and α6WT cells to test the cell heterogeneity requirements for muscle invasion. Time-lapse video microscopy revealed that tumor mixtures self-assembled into invasive networks in vitro, whereas α6AA cells assembled only as cohesive clusters. Invasion of α6AA cells into and through live muscle occurred using a 1:1 mixture of α6AA and α6WT cells. Electric cell-substrate impedance sensing measurements revealed that compared with α6AA cells, invasion-competent α6WT cells were 2.5-fold faster at closing a cell-ECM or cell-cell wound, respectively. Cell-ECM rebuilding kinetics show that an increased response occurred in mixtures since the response was eightfold greater compared with populations containing only one cell type. A synthetic cell adhesion cyclic peptide called MTI-101 completely blocked electric cell-substrate impedance sensing cell-ECM wound recovery that persisted in vitro up to 20 h after the wound. Treatment of tumor-bearing animals with 10 mg/kg MTI-101 weekly resulted in a fourfold decrease of muscle invasion by tumor and a decrease of the depth of invasion into muscle comparable to the α6AA cells. Taken together, these data suggest that mixed biophysical phenotypes of tumor cells within a population can provide functional advantages for tumor invasion into and through muscle that can be potentially inhibited by a synthetic cell adhesion molecule.

Expression of type VI collagen α3 chain in canine mammary carcinomas.

This study aimed to investigate the expression of type VI collagen α3 chain (COL6a3) in neoplastic cells of canine mammary gland carcinomas (CMGCs) using immunohistochemistry (IHC) and to evaluate the association between COL6a3 expression and tumor histological features, histological grades, and the differentiation status of neoplastic epithelial cells. COL6a3 expression in carcinoma cells was significantly associated with histologically low malignancy and low mitotic indices. In addition, COL6a3+ carcinoma cells were more frequently detected in simple carcinomas (tubular and tubulopapillary types) than in solid carcinomas. These findings indicate that reduced expression of COL6a3 in carcinoma cells contributes to the malignant phenotype in CMGCs. We also showed that COL6a3 expression in the carcinoma cells was more frequently detected in CK19+/CD49f + and/or CK19+/CK5+ tumors. In addition, COL6a3+/CK19+/CD49f + and COL6a3+/CK19+/CK5+ tumors consisted of CK19+/CD49f + and CK19+/CD49f- cells, and CK19+/CK5+ and CK19+/CK5- cells, respectively. Most of these tumors more frequently expressed GATA3, but not Notch1. These results indicate that COL6a3 is expressed in CMGCs containing both luminal progenitor-like and mature luminal-like cells and showing differentiation ability into mature luminal cells. It is possible that COL6 may be involved in the differentiation of luminal progenitor-like carcinoma cells into mature luminal-like carcinoma cells in CMGCs, which may suppresses the development of malignant phenotypes in CMGCs.

Poor Prognosis of Oral Squamous Cell Carcinoma Correlates With ITGA6.

OBJECTIVES: Oral cancer is the ninth most common cancer worldwide and a leading cause of cancer-related death. Oral squamous cell carcinoma (OSCC) accounts for 90% of all oral cancers. Autophagy is a conserved essential catabolic process related to OSCC. The aim of this study was to elucidate diagnostic and prognostic autophagy-related biomarkers in OSCC. METHODS: The OSCC gene expression data set was obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between the OSCC samples and adjacent healthy tissues were identified by R software. The Human Autophagy Database was screened, which revealed 222 autophagy-related genes. The autophagy-related DEGs were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were applied. Protein-protein interaction network analysis was performed in the STRING database. cytoHubba in the Cytoscape software was applied to determine the top 10 hub genes. The data set of patients with OSCC from The Cancer Genome Atlas (TCGA) was used to evaluate the prognostic value of the 10 hub genes. The association between prognosis-related hub genes and immune infiltrates was explored. RESULTS: Twenty-seven autophagy-related DEGs were identified. The top 10 hub genes were CCL2, CDKN2A, CTSB, CTSD, CXCR4, ITGA6, MAP1LC3A, MAPK3, PARP1, and RAB11A. ITGA6 was identified as the most efficient biomarker. Receiver operating characteristic curve analysis indicated that ITGA6 had the highest diagnostic accuracy for OSCC (area under the curve = 0.925). ITGA6 expression was significantly related to immune infiltrates. CONCLUSIONS: The autophagy-related gene ITGA6 might be an efficient diagnostic and prognostic biomarker in OSCC.

TM4SF1 promotes esophageal squamous cell carcinoma metastasis by interacting with integrin α6.

Transmembrane-4 L-six family member-1 (TM4SF1) is a member of the L6 family and functions as a signal transducer to regulate tumor cell behaviors. However, the function and mechanism of TM4SF1 in esophageal squamous cell carcinoma (ESCC) metastasis remains unclear. Here, we find that TM4SF1 expression is increased and positively correlated with clinical TNM stage, N classification, differentiation, tumor size, and poor prognosis in ESCC patients. Interestingly, we demonstrate that TM4SF1 promotes ESCC cell adhesion, spreading, migration, and invasion, but not cell proliferation, in a laminin-dependent manner by interacting with integrin α6. Mechanistically, the TM4SF1/integrin α6/FAK axis signal pathway mediates cell migration under laminin-coating condition. Inhibiting FAK or knocking down TM4SF1 can attenuate TM4SF1-mediated cell migration and lung metastasis. Clinically, the TM4SF1/integrin α6/FAK axis positively correlates with ESCC. Altogether, these findings reveal a new mechanism of TM4SF1 in promoting ESCC metastasis via binding to integrin α6 and suggest that the cross-talk between TM4SF1 and integrin α6 may serve as a therapeutic target for ESCC.

Integrin alpha 6 is upregulated and drives hepatocellular carcinoma progression through integrin α6ß4 complex.

Integrin α6 (ITGA6) forms integrin receptors with either integrin ß1 (ITGB1) or integrin ß4 (ITGB4). How it functions to regulate hepatocellular carcinoma (HCC) progression is not well-elucidated. We found that ITGA6 RNA and protein expression levels are significantly elevated in human HCC tissues in comparison with paired adjacent nontumor tissues by RNA sequencing, RT-qPCR, Western blotting and immunofluorescence staining. Stable knockdown of ITGA6 with different ITGA6 shRNA expression lentivectors significantly inhibited proliferation, migration and anchorage-independent growth of HCC cell lines in vitro, and xenograft tumor growth in vivo. The inhibition of anchorage-dependent and -independent growth of HCC cell lines was also confirmed with anti-ITGA6 antibody. ITGA6 knockdown was shown to induce cell-cycle arrest at G0/G1 phase. Immunoprecipitation assay revealed apparent interaction of ITGA6 with ITGB4, but not ITGB1. Expression studies showed that ITGA6 positively regulates the expression of ITGB4 with no or negative regulation of ITGB1 expression. Finally, while high levels of ITGA6 and ITGB4 together were associated with significantly worse survival of HCC patients in TCGA data set, the association was not significant for high levels of ITGA6 and ITGB1. In conclusion, ITGA6 is upregulated in HCC tumors and has a malignant promoting role in HCC cells through integrin α6ß4 complex. Thus, integrin α6ß4 may be a therapeutic target for treating patients with HCC.

The miR-410-5p /ITGA6 axis participates in the pathogenesis of recurrent abortion by regulating the biological function of trophoblast.

The purpose of this study was to determine the regulation of the miR-410-5p /ITGA6 axis on the biological functions of trophoblast cells and the mechanism involved in recurrent spontaneous abortion（RSA）. We used qRT-PCR and Western blotting to quantify the expression levels of Mir-410-5p and ITGA6 in placenta of RSA and normal, and found that compared with normal placenta, the placenta of RSA patients showed higher miR-410-5p and lower ITGA6 expression. Dual luciferase reporter gene assay confirmed the binding of miR-410-5p to ITGA6. The expression of miR-410-5p and ITGA6, and proliferation, apoptosis, invasion and migration of trophoblast cells and the effect on the polarization of M2 macrophages were detected in the trophoblast derived cell lines HTR8/Svneo transfected with miR-410-5p mimic, sh-miR-410-5p and si-ITGA6 respectively. Meanwhile, the molecular mechanism of ITGA6 regulation on trophoblast cells was explored. Transfection with miR-410-5p mimic or si-ITGA6 attenuated the proliferation, migration and invasion and induced apoptosis of HTR-8/SVneo cells. Transfection of sh-miR-410-5p promoted proliferation, migration and invasion, and weakened apoptosis of HTR-8/SVneo cells. In addition, overexpression of miR-410-5p in trophoblast cells inhibited the polarization of M2 macrophages, while knockdown of miR-410-5p was beneficial to recruitment of trophoblast cell and promoted the polarization of M2 macrophages. ITGA6 may affect the biological functions of trophoblast cells by regulating PI3K/AKT and MAPK signaling pathways. In conclusion, miR-410-5p mediates trophoblast cell proliferation, apoptosis, invasion and migration through regulating ITGA6 expression.

Effect of different iodine levels on the DNA methylation of PRKAA2, ITGA6, THEM4 and PRL genes in PI3K-AKT signaling pathway and population-based validation from autoimmune thyroiditis patients.

PURPOSE: Autoimmune thyroiditis (AIT) is one of the most common autoimmune endocrine diseases. The currently recognized causes are genetic susceptibility, environmental factors and immune disorders. It is important to clarify the pathogenesis for the prevention, diagnosis, treatment of AIT and scientific iodine supplementation. This study analyzed the DNA methylation levels of PRKAA2, ITGA6, PRL and THEM4 genes related to PI3K-AKT signaling pathway, compared the DNA methylation levels between cases and controls from different water iodine levels in Shandong Province of China, and evaluated the contribution of PI3K-AKT signaling pathway-related genes in AIT. METHODS: A total of 176 adult AIT patients were included from three different water iodine areas, and 176 healthy controls were included according to gender, age and BMI. According to the results of the Illumina Methylation 850 K BeadChip in our previous research, the significant methylation differences of genes on the PI3K-AKT signaling pathway related to AIT were determined. The MethylTarget™ assay was used to detect the methylation levels of the target genes, and real-time PCR experiments were used to verify the mRNA expression levels. RESULTS: Compared with the control group, PRKAA2_3 and 15 CpG sites were hyper-methylated. ITGA6 gene and 2 CpG sites were hypo-methylated in AIT cases. The mRNA expression of ITGA6 gene was negatively correlated with the DNA methylation levels of ITGA6 gene and 2 CpG sites. Compared with cases and controls in areas with different water iodine levels, methylation differences were mainly in PRKAA2 and ITGA6 genes. The methylation levels of PRKAA2_1 and PRKAA2_3 were positively correlated with age. The methylation levels of PRL and THEM4 genes were negatively correlated with age. The methylation level of PRKAA2_3 was positively correlated with FT4. CONCLUSION: In summary, we identified aberrant DNA methylation levels of PRKAA2 and ITGA6 genes related to PI3K-AKT signaling pathway in the blood of AIT patients. Both iodine supplementation after long-term iodine deficiency and iodine excess can affect the DNA methylation levels of PRKAA2 and ITGA6 genes, and the former affects more obviously. In ITGA6 gene, this aberrant epigenetic modification is associated with the increased mRNA expression.

[MiR-4484 regulates the expression of integrin α 6 in gastric cancer tissues and its significance].

Objective: To investigate the relationship between the expression of integrin α 6 (ITGA6), miR-4484 and the pathologic stage of gastric cancer. Methods: Gastric cancer tissues and normal gastric mucosa tissues adjacent to cancer (>5 cm from tumor margin) of 30 patients with primary gastric cancer who underwent direct surgical resection without adjuvant therapy from June to September 2017 in West China Hospital of Sichuan University were selected. Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression levels of miR-4484 and ITGA6, western blot was used to detect the expression level of ITGA6 protein, dual luciferase reporter gene was used to verify the relationship between ITGA6 and miR-4484. Spearman's correlation analysis was used to determine the relationship between miR-4484 and ITGA6 expression levels in gastric cancer tissues. Results: The expression level of ITGΑ6 in gastric cancer (32.30±13.47) was higher than that in matched normal gastric tissues (24.55±10.25, P=0.015), the area under the receiver operating characteristic (ROC) curve was 0.660 and the diagnostic sensitivity and specificity were 43.3% and 96.7%, respectively. The expression level of miR-4484 in gastric cancer (4.11±2.87) was lower than that of matched normal gastric tissues (5.75±2.80, P=0.029), the area under the ROC curve was 0.690 and the diagnostic sensitivity and specificity were 30.0% and 86.7%, respectively. The expression level of miR-4484 was negatively correlated with ITGA6 in gastric cancer tissues (r=-0.621, P<0.001). The expression level of ITGA6 protein in gastric cancer tissues (0.65±0.19) was higher than that in normal adjacent tissues (0.26±0.12, P<0.001). Compared with ITGA6 3'UTR wild-type+ miR-NC group, ITGA6 3'UTR wild-type+ miRNA mimics group had lower luciferase activity (50.69±5.10, 34.00±1.19, P<0.001), while the luciferase activity of ITGA6 3'UTR wild-type+ ASO miR-4484 group was higher than that of ITGA6 3'UTR wild-type+ miR-NC group (82.44±6.37, 50.69±5.10, P<0.001), indicated that ITGA6 was the direct target gene of miR-4484. The expression levels of miR-4484 in T1, T2, T3 and T4 (4a and 4b) gastric cancer tissues were 9.98±2.24, 5.28±2.03, 2.92±2.04 and 4.11±2.87, respectively, with statistical significance (P<0.001). The expression levels of ITGA6 in N0, N1, N2 and N3 gastric cancer tissues were 29.55±8.32, 21.71±3.75, 24.60±8.79 and 40.69±15.83, respectively, with statistical significance (P=0.022). The expression levels of miR-4484 in N0, N1, N2 and N3 gastric cancer tissues were 5.01±3.52, 5.48±2.76, 5.88±1.83 and 2.30±1.56, respectively, with statistical significance (P=0.032). The expression levels of ITGA6 in M0 and M1 gastric cancer tissues were 26.28±7.66 and 52.08±8.12, respectively, with statistical significance (P<0.001). The expression levels of miR-4484 in M0 and M1 gastric cancer tissues were 4.95±2.74 and 1.34±0.80, respectively, with statistical significance (P<0.001). Conclusions: ITGA6 is upregulated in gastric cancer tissues, while miR-4484 is downregulated in the gastric cancer group, and its expression level is related to the clinicopathological features of gastric cancer. ITGA6 is the direct target gene of miR-4484, implicates that miR-4484 may inhibit the invasion and metastasis of gastric cancer by regulating the expression of ITGA6. Both miR-4484 and ITGA6 may be the new prognostic markers and potential therapeutic targets of gastric cancer.

Extracellular vesicle-mediated delivery of miR-127-3p inhibits the proliferation and invasion of choriocarcinoma cells by targeting ITGA6.

BACKGROUND: Choriocarcinoma (CC) is a highly aggressive malignant tumor that mostly occurs in women of childbearing age. Chemotherapy is the main treatment for CC, but it has side effects and causes drug resistance, which can lead to treatment failure. Extracellular vesicles (EVs) that deliver microRNAs (miRNAs) have emerged as a novel and promising therapeutic tool for inhibiting tumor progression and metastasis. This research aimed to study the effects of miR-127-3p-enriched EVs (EV-miR-127-3p) on CC and underlying mechanisms. METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting were performed to determine the miR-127-3p and integrin subunit alpha-6 (ITGA6) expression levels. The interaction between miR-127-3p and ITGA6 was confirmed by a dual-luciferase reporter assay. Human umbilical cord mesenchymal stem cells (hUCMSCs) were identified using flow cytometry and multilineage differentiation. Uptake of labeled EVs was demonstrated using immunofluorescence staining and flow cytometry assays. EV-miR-127-3p were isolated from the culture medium of hUCMSCs and co-cultured with JEG-3 or JAR cells to evaluate their effects on cell proliferation, invasion, migration, and apoptosis, using the cell counting kit-8, Transwell, and flow cytometry assays. Epithelial-mesenchymal transition (EMT) and the transforming growth factor (TGF)-ß1/Smad pathway were investigated using qRT-PCR and western blotting. RESULTS: The expression of miR-127-3p was downregulated, while that of ITGA6 was upregulated in CC cell lines. ITGA6 was identified as a target gene of miR-127-3p. EV-miR-127-3p could inhibit the proliferation, invasion, migration, and promote the apoptosis of CC cells. We observed that EV-miR-127-3p suppressed EMT of CC cells by targeting ITGA6. In addition, the knockdown of ITGA6 inhibited the TGF-ß1/Smad pathway and reversed the EMT-promoting effect. CONCLUSION: These results indicate that EV-miR-127-3p from hUCMSCs exhibits anti-tumor effects by targeting ITGA6, which may be used as a novel therapeutic strategy for CC treatment.

Integrin α6A (ITGA6A)-type Splice Variant in Extracellular Vesicles Has a Potential as a Novel Marker of the Early Recurrence of Pancreatic Cancer.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) is one of the most common cancers worldwide, with a poor prognosis. Owing to the difficulty of early diagnosis, the aim of this study was to isolate biomarkers from extracellular vesicles (EVs) that can lead to early diagnosis. MATERIALS AND METHODS: EVs in the culture supernatant were isolated from a pancreatic cancer cell line (PK-1) and expanded by using two-dimensional gel electrophoresis, and protein identification from each spot was performed by using matrix-assisted laser desorption ionization mass spectrometry. The identified proteins were classified and compared with previously reported results for EVs from murine pancreatic cancer PAN02 cells, and their expression specificity was examined using PDAC cell lines and patient-derived PDAC tissues. In addition, the significance of selected biomarker(s) was examined based on the changes in biomarkers in the blood EVs of PDAC patients after surgery. RESULTS: We found that the ITGA6A splice variant was predominantly expressed in several pancreatic cancer cell lines and blood EVs from patients with PDAC, whereas the ITGA6B splice variant was predominantly expressed in EVs from the blood of normal volunteers. In the expression pattern of ITGA6 in EVs from blood samples of two PDAC patients before and after resection surgery, the expression of ITGA6A in EVs significantly decreased after surgery and increased several months before clinical recurrence. Furthermore, the increased expression of ITGA6A in EVs occurred much earlier than that of CA19-9. CONCLUSION: Determination of ITGA6A expression in blood EVs in PDAC patients could be a useful blood marker for the early diagnosis of PDAC recurrence.

miR-186-5p inhibits the progression of oral squamous cell carcinoma by targeting ITGA6 to impair the activity of the PI3K/AKT pathway.

BACKGROUND: microRNAs (miRNAs) are pivotal regulators of multiple biological processes. miR-186-5p functions as a tumor suppressor in a variety of cancers and promotes the malignant proliferation of oral squamous cell carcinoma (OSCC). This study aimed to clarify the role and regulatory mechanism of miR-186-5p in OSCC. METHODS: The levels of miR-186-5p and integrin subunit alpha 6 (ITGA6) were investigated in clinical specimens and OSCC cell lines by reverse transcription-quantitative polymerase chain reaction. The effects of miR-186-5p and ITGA6 on the cell migration, proliferation, and phosphatidylinositol 3-kinase (PI3K)/serine-threonine kinase (AKT) pathway activity were evaluated by transwell assay, cell counting kit 8 assay, and western blotting, respectively. A xenograft model was used to analyze the effect of miR-186-5p on tumor growth. Bioinformatic analyses were conducted to identify the putative targets of miR-186-5p in OSCC. RESULTS: Decreased miR-186-5p expression levels were observed in OSCC tumor tissues and cell lines. The overexpression of miR-186-5p suppressed the proliferation and migration of OSCC cells, and weakened the phosphorylation of PI3K and AKT. Moreover, the overexpression of miR-186-5p in xenograft tumor models impedes tumor growth. miR-186-5p is bound to ITGA6 and negatively related to ITGA6 expression in tumor tissues. The forced expression of ITGA6 promoted OSCC cell proliferation and migration and enhanced the phosphorylation levels of PI3K and AKT, while additional miR-186-5p enrichment partly abolished these effects. CONCLUSION: miR-186-5p binds to ITGA6 to impair the activity of the PI3K/AKT signaling pathway, thereby blocking the development of OSCC. This study provides insight to understand the pathogenesis of OSCC.

Long non-coding RNA OIP5-AS1 suppresses microRNA-92a to augment proliferation and metastasis of ovarian cancer cells through upregulating ITGA6.

OBJECTIVE: Recently, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been identified as essential biomarkers during development of malignancies. This study was performed to study the roles of lncRNA opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) and miR-92a in ovarian cancer (OC). METHODS: OIP5-AS1, miR-92a and integrin alpha 6 (ITGA6) expression in OC tissues and cells was assessed. The screened OC cells were respectively with OIP5-AS1-, miR-92a- and ITGA6-related vectors or oligonucleotides . The viability, migration, invasion and apoptosis of the cells were determined and the levels of epithelial-mesenchymal transition (EMT)-related proteins were also measured. The interactions between OIP5-AS1 and miR-92a, and between miR-92a and ITGA6 were confirmed. RESULTS: OIP5-AS1 and ITGA6 were upregulated while miR-92a was downregulated in OC. Inhibited OIP5-AS1 or downregulated ITGA6 or elevated miR-92a repressed EMT, viability, migration and invasion, and promoted apoptosis of OC cells. OIP5-AS1 as a competing endogenous RNA interacted with miR-92a to regulate ITGA6. These effects that induced by silenced OIP5-AS1 could be reversed by miR-92a inhibition while those that induced by up-regulated miR-92a were reduced by restored ITGA6. CONCLUSION: OIP5-AS1 silencing promoted miR-92a to repress proliferation and metastasis of OC cells through inhibiting ITGA6.

Silencing integrin α6 enhances the pluripotency-differentiation transition in human dental pulp stem cells.

OBJECTIVES: Although integrins have been shown to be associated with proliferation and differentiation in some stem cells, the regulatory effect of integrin α6 (ITGα6) on the human dental pulp stem cells (hDPSCs) has not been reported. Here, we detected the roles of ITGα6 in hDPSCs. MATERIALS AND METHODS: Attached to Cytodex 3 microcarriers, hDPSCs grown under stimulated microgravity (SMG) or conventional culture conditions were measured the proliferation and different gene expression. Further, ITGα6 was silenced in hDPSCs, and its effect on proliferation, differentiation, and cytoskeletal organization was analyzed. RESULTS: SMG conditions increased the number of Ki67-positive hDPSCs and progression into S phase of cell cycle. WB analysis showed the expression of ITGα6 was upregulated in hDPSCs under SMG conditions. Knockdown of ITGα6 decreased the expression of stemness markers, CD105 and STRO-1 in hDPSCs, but promoted the osteogenic and odontogenic differentiation by increased ALP expression and Alizarin Red nodules. Moreover, RNA-seq demonstrated that RHO/ROCK signaling pathway upregulated silencing ITGα6-hDPSCs. Treatment with Y-27632 inhibited the effect of ITGα6 depletion on hDPSCs stemness, rearranged the cytoskeleton, promoted the pluripotency, proliferation ability, and inhibited the differentiation. CONCLUSION: ITGα6 promotes hDPSCs stemness via inhibiting RHO/ROCK and restoring cytoskeleton.

IGF1R-α6 integrin-S100A4 network governs the organ-specific metastasis of chemoresistant epithelial ovarian cancer cells.

Recurrent metastatic epithelial ovarian cancer (EOC) is challenging and associated with treatment limitations, as the mechanisms governing the metastatic behavior of chemoresistant EOC cells remain elusive. Using orthotopic xenograft mouse models of sensitive and acquired platinum-taxol-resistant A2780 EOC cells, we studied the mechanistic role of insulin like growth factor 1 receptor (IGF1R) signaling in the regulation of organ-specific metastasis of EOC cells undergoing acquirement of chemoresistance. Biochemical assays and organ-specific fibroblast-EOC cell co-culture were used to study the differential metastatic characteristics of sensitive vs. chemoresistant EOC cells, and the key molecule/s underlying the organ-specific homing of chemoresistant EOC cells were identified through subtractive LC/MS profiling of the co-culture secretome. The role of the identified molecule was validated through genetic/pharmacologic perturbation experiments. Acquired chemoresistance augmented organ-specific metastasis of EOC cells and enhanced lung homing, particularly for the late-stage chemoresistant cells, which was abrogated after IGF1R silencing. Escalation of chemoresistance (intrinsic and acquired) conferred EOC cells with higher adhesion toward primary lung fibroblasts, largely governed by the α6 integrin-IGF1R dual signaling axes. Subtractive analysis of the co-culture secretome revealed that interaction with lung fibroblasts induced the secretion of S100A4 from highly resistant EOC cells, which reciprocally activated lung fibroblasts. Genetic and pharmacologic inhibition of S100A4 significantly lowered distant metastases and completely abrogated lung-tropic nature of late-stage chemoresistant EOC cells. These results indicate that chemoresistance exacerbates organ-specific metastasis of EOC cells via the IGF1R-α6 integrin-S100A4 molecular network, of which S100A4 may serve as a potential target for the treatment of recurrent metastatic EOC.

MiR-4484 regulates the expression of integrin α 6 in gastric cancer tissues and its significance / 中华肿瘤杂志

Objective: To investigate the relationship between the expression of integrin α 6 (ITGA6), miR-4484 and the pathologic stage of gastric cancer. Methods: Gastric cancer tissues and normal gastric mucosa tissues adjacent to cancer (>5 cm from tumor margin) of 30 patients with primary gastric cancer who underwent direct surgical resection without adjuvant therapy from June to September 2017 in West China Hospital of Sichuan University were selected. Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression levels of miR-4484 and ITGA6, western blot was used to detect the expression level of ITGA6 protein, dual luciferase reporter gene was used to verify the relationship between ITGA6 and miR-4484. Spearman's correlation analysis was used to determine the relationship between miR-4484 and ITGA6 expression levels in gastric cancer tissues. Results: The expression level of ITGΑ6 in gastric cancer (32.30±13.47) was higher than that in matched normal gastric tissues (24.55±10.25, P=0.015), the area under the receiver operating characteristic (ROC) curve was 0.660 and the diagnostic sensitivity and specificity were 43.3% and 96.7%, respectively. The expression level of miR-4484 in gastric cancer (4.11±2.87) was lower than that of matched normal gastric tissues (5.75±2.80, P=0.029), the area under the ROC curve was 0.690 and the diagnostic sensitivity and specificity were 30.0% and 86.7%, respectively. The expression level of miR-4484 was negatively correlated with ITGA6 in gastric cancer tissues (r=-0.621, P<0.001). The expression level of ITGA6 protein in gastric cancer tissues (0.65±0.19) was higher than that in normal adjacent tissues (0.26±0.12, P<0.001). Compared with ITGA6 3'UTR wild-type+ miR-NC group, ITGA6 3'UTR wild-type+ miRNA mimics group had lower luciferase activity (50.69±5.10, 34.00±1.19, P<0.001), while the luciferase activity of ITGA6 3'UTR wild-type+ ASO miR-4484 group was higher than that of ITGA6 3'UTR wild-type+ miR-NC group (82.44±6.37, 50.69±5.10, P<0.001), indicated that ITGA6 was the direct target gene of miR-4484. The expression levels of miR-4484 in T1, T2, T3 and T4 (4a and 4b) gastric cancer tissues were 9.98±2.24, 5.28±2.03, 2.92±2.04 and 4.11±2.87, respectively, with statistical significance (P<0.001). The expression levels of ITGA6 in N0, N1, N2 and N3 gastric cancer tissues were 29.55±8.32, 21.71±3.75, 24.60±8.79 and 40.69±15.83, respectively, with statistical significance (P=0.022). The expression levels of miR-4484 in N0, N1, N2 and N3 gastric cancer tissues were 5.01±3.52, 5.48±2.76, 5.88±1.83 and 2.30±1.56, respectively, with statistical significance (P=0.032). The expression levels of ITGA6 in M0 and M1 gastric cancer tissues were 26.28±7.66 and 52.08±8.12, respectively, with statistical significance (P<0.001). The expression levels of miR-4484 in M0 and M1 gastric cancer tissues were 4.95±2.74 and 1.34±0.80, respectively, with statistical significance (P<0.001). Conclusions: ITGA6 is upregulated in gastric cancer tissues, while miR-4484 is downregulated in the gastric cancer group, and its expression level is related to the clinicopathological features of gastric cancer. ITGA6 is the direct target gene of miR-4484, implicates that miR-4484 may inhibit the invasion and metastasis of gastric cancer by regulating the expression of ITGA6. Both miR-4484 and ITGA6 may be the new prognostic markers and potential therapeutic targets of gastric cancer.

Increased Expression of Integrin Alpha 6 in Nucleus Pulposus Cells in Response to High Oxygen Tension Protects against Intervertebral Disc Degeneration.

The destruction of the low oxygen microenvironment in nucleus pulposus (NP) cells played a critical role in the pathogenesis of intervertebral disc degeneration (IVDD). The purpose of this study was to determine the potential role of integrin alpha 6 (ITG α6) in NP cells in response to high oxygen tension (HOT) in IVDD. Immunofluorescence staining and western blot analysis showed that the levels of ITG α6 expression were increased in the NP tissue from IVDD patients and the IVDD rat model with mild degeneration, which were reduced as the degree of degeneration increases in severity. In NP cells, the treatment of HOT resulted in upregulation of ITG α6 expression, which could be alleviated by blocking the PI3K/AKT signaling pathway. Further studies found that ITG α6 could protect NP cells against HOT-induced apoptosis and oxidative stress and protect NP cells from HOT-inhibited ECM protein synthesis. Upregulation of ITG α6 expression by HOT contributed to maintaining NP tissue homeostasis through the interaction with hypoxia-inducible factor-1α (HIF-1α). Furthermore, silencing of ITG α6 in vivo could obviously accelerate puncture-induced IVDD. Taken together, these results revealed that the increase of ITG α6 expression by HOT in NP cells might be a protective factor in IVD degeneration as well as restore NP cell function.

Integrin alpha 6 as a stemness driver is a novel promising target for HPV (+) head and neck squamous cell carcinoma.

Although the incidence rates of head and neck squamous cell carcinoma (HNSCC) associated with human papilloma virus (HPV) infection have recently been on the rise, the underlying mechanism of its tumorigenesis remains largely unknown. Here, we investigated whether HNSCC cells with high expression of integrin alpha 6 (ITGα6), one of the HPV receptors, have a preference during HPV infection. In addition, we examined the gain or loss of function of the ITGα6 gene in HPV + ve HNSCC cells, as well as its prognostic value in patients with HNSCC. HPV pseudovirus was found to be more infective, with HNSCC cells featuring an overexpressed ITGα6 gene compared to the control cells. Overexpression and suppression of ITGα6 respectively increases and decreases stemness phenotypes of HPV + ve HNSCC cells. Furthermore, ITGα6 can regulate stemness by partially mediating AKT pathway in HPV + ve HNSCC cells. Finally, patients with HPV + ve HNSCC had a poor prognosis in cases of elevated ITGα6 expression; however, the expression levels of ITGα6 did not influence the survival rates of HPV-negative HNSCC patients. In conclusion, ITGα6 can serve as a potential therapeutic target for HPV + ve HNSCC cancer-like stem cells (CSCs).