Α5 Integrin Subunit Research Articles

Alternagin-C, an alpha2beta1 integrin ligand, attenuates collagen-based adhesion, stimulating the metastasis suppressor 1 expression in triple-negative breast tumor cells

Triple-negative breast cancer has an aggressive clinical course and its treatment has been challenging due to high metastatic risk. Molecular targets have been sought to provide better strategies for this type of cancer. Integrins are cell adhesion receptors involved in tumor progression and α2β1 integrin, a collagen receptor, has a key role in breast metastasis. Disintegrins, a family of proteins from snake venoms, selectively block the function of integrin receptors. Alternagin-C (ALT-C), a disintegrin-like protein purified from Bothrops alternatus venom, binds to α2β1 integrin, attenuating inflammation and angiogenesis, and decreasing metalloprotease levels in the tumor microenvironment, which suggests anti-metastatic effects. However, its mechanisms of action in metastatic tumor cells have not been fully explored. Here, we investigated ALT-C effects in a triple-negative breast cancer cell line (MDA-MB-231) to elucidate how α2β1 integrin affects cellular adhesion, migration and gene expression related to metastasis. We observed that ALT-C attenuated cell adhesion of MDA-MB-231 cells to collagen I. α2 integrin subunit silencing in MDA-MB-231 cells did not inhibit cell adhesion and migration to collagen I, indicating that other integrins play a crucial role in cell motility for this cell line. ALT-C also stimulated the metastasis suppressor 1 (MTSS1) expression and decreased metalloproteases MMP9 and MMP2. Therefore, we suggest that ALT-C contributes to impair metastasis, preventing extracellular matrix degradation and tumor attachment to collagen I, increasing MTSS1. This study is the first to elucidate the anti-metastatic mechanism involving a disintegrin-like protein from snake venom targeting α2β1 integrin and stimulating a metastasis suppressor.

FoxO3a Inhibits Tamoxifen-Resistant Breast Cancer Progression by Inducing Integrin α5 Expression.

Simple SummaryTamoxifen is a mainstay of adjuvant treatment for estrogen receptor α-positive (ERα+) breast tumors, which account for over 70% of all the diagnosed breast malignancies. Unfortunately, ~30% of patients initially responding to the therapy will eventually relapse with an endocrine-resistant disease. Here, we show that the transcription factor FoxO3a can overcome tamoxifen resistance by inhibiting cell motility and invasiveness, the main features of tumor progression. The underlying mechanism could be ascribed to FoxO3a-dependent transcriptional up-regulation of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion. Indeed, FoxO3a and integrin α5 expression is significantly correlated in ERα+ tumors, and FoxO3a protective effects are lost in cells with reduced levels of integrin α5. Therefore, a pharmacological increase in FoxO3a levels could represent an effective approach to be exploited in combination with tamoxifen treatment in order to restore the sensitivity to the therapy in refractory tumors.Resistance to endocrine therapy is still a major clinical challenge in the management of estrogen receptor α-positive (ERα+) breast cancer (BC). Here, the role of the Forkhead box class O (FoxO)3a transcription factor in tumor progression has been evaluated in tamoxifen-resistant BC cells (TamR), expressing lower levels of FoxO3a compared to sensitive ones. FoxO3a re-expression reduces TamR motility (wound-healing and transmigration assays) and invasiveness (matrigel transwell invasion assays) through the mRNA (qRT-PCR) and protein (Western blot) induction of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion and signaling. The induction occurs through FoxO3a binding to a specific Forkhead responsive core sequence located on the integrin α5 promoter (cloning, luciferase, and ChIP assays). Moreover, FoxO3a failed to inhibit migration and invasion in integrin α5 silenced (siRNA) cells, demonstrating integrin α5 involvement in both processes. Finally, using large-scale gene expression data sets, a strong positive correlation between FoxO3a and integrin α5 in ERα+, but not in ER-negative (ERα−), BC patients emerged. Altogether, our data show how the oncosuppressor FoxO3a, by increasing the expression of its novel transcriptional target integrin α5, reverts the phenotype of endocrine-resistant BC toward a lower aggressiveness.

Expression Analysis of α5 Integrin Subunit Reveals Its Upregulation as a Negative Prognostic Biomarker for Glioblastoma.

Integrin α5β1 was suggested to be involved in glioblastoma (GBM) aggressiveness and treatment resistance through preclinical studies and genomic analysis in patients. However, further protein expression data are still required to confirm this hypothesis. In the present study, we investigated by immunofluorescence the expression of integrin α5 and its prognostic impact in a glioblastoma series of patients scheduled to undergo the Stupp protocol as first-line treatment for GBM. The integrin α5 protein expression level was estimated in each tumor by the mean fluorescence intensity (MFI) and allowed us to identify two subpopulations showing either a high or low expression level. The distribution of patients in both subpopulations was not significantly different according to age, gender, recursive partitioning analysis (RPA) prognostic score, molecular markers or surgical and medical treatment. A high integrin α5 protein expression level was associated with a high risk of recurrence (HR = 1.696, 95% CI 1.031–2.792, p = 0.0377) and reduced overall survival (OS), even more significant in patients who completed the Stupp protocol (median OS: 15.6 vs. 22.8 months; HR = 2.324; 95% CI 1.168–4.621, p = 0.0162). In multivariate analysis, a high integrin α5 protein expression level was confirmed as an independent prognostic factor in the subpopulation of patients who completed the temozolomide-based first-line treatment for predicting OS over age, extent of surgery, RPA score and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation (p = 0.029). In summary, for the first time, our study validates that a high integrin α5 protein expression level is associated with poor prognosis in GBM and confirms its potential as a therapeutic target implicated in the Stupp protocol resistance.

In vitro maturation using an agarose matrix with incorporated extracellular matrix proteins improves porcine oocyte developmental competence by enhancing cytoplasmic maturation

Here, we present a novel in vitro maturation (IVM) system comprising an agarose matrix supplemented with extracellular matrix (ECM) proteins for enhanced maturation of immature oocytes within cumulus-oocyte complexes (COCs) derived from porcine medium antral follicles (MAFs). Immunocytochemical analyses of integrin subunit α2 , α5 , α6 , β1 , and β4 expression suggested that integrin α2 β1 , α5 β1 , α6 β1 , and α6 β4 play pivotal roles in IVM of porcine immature oocytes. Combinatorial supplementation of fibronectin interacting with integrin α5 β1 , collagen interacting with integrin α2 β1 , and laminin interacting with integrin α6 β1 and α6 β4 to the agarose matrix had no significant effect on nuclear maturation. However, the number of parthenogenetic embryos that developed into blastocysts increased when oocytes were matured using agarose IVM matrices supplemented with fibronectin, collagen, or laminin. Furthermore, significant increases in cytoplasmic maturation-related parameters (BMP15 level, cumulus cell expansion score, intra-oocyte ATP level, and index of cortical granule distribution) were observed in COCs matured in vitro using ECM protein-incorporated agarose matrices. Our data suggest that mature porcine oocytes with enhanced developmental competence and high-quality cytoplasm can be generated via IVM using agarose matrices supplemented with fibronectin, collagen, or laminin.

Biphasic α2β1 Integrin Expression in Breast Cancer Metastasis to Bone.

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2β1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2β1 in the context of bone metastasis. In this study, we aimed to understand the role of α2β1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2β1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2β1 expression and bone-metastatic potential. Inhibiting α2β1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.

Integrin α2β1 as a negative regulator of the laminin receptors α6β1 and α6β4

Integrin α2β1 is a widely expressed collagen I receptor which also mediates laminin-111 binding in some cell types, but the functional relevance of collagen versus laminin binding for different cell types is poorly understood. Here we use AFM-based singe-cell force spectroscopy (SCFS) to compare α2β1-mediated adhesion strength to collagen and laminin in different cell types. Chinese Hamster Ovary (CHO) cells stably expressing integrin α2β1 (CHO-A2) displayed enhanced adhesion to collagen, but weak adhesion to laminin, consistent with a role of α2β1 as a receptor only for collagen in these cells. Inversely, the α2β1-deficient CHO wildtype cells (CHO-WT) showed weak adhesion to collagen, but strong adhesion to laminin-111, in turn suggesting that integrin α2β1 expression suppresses laminin binding. Analogous results were obtained in a pair of SAOS-2 human osteosarcoma cell lines. Again, wildtype cells (SAOS-WT) adhered strongly to laminin and poorly to collagen, while expression of integrin α2β1 (SAOS-A2) induced strong adhesion to collagen, but reduced adhesion to laminin. Expression of α2β1 also shifted cell spreading preference from laminin to collagen and suppressed laminin-dependent transmigration. In agreement with reduced laminin adhesion, α2β1 expression downregulated transcription and expression of integrin subunits α6 and β4, components of the main laminin-111 binding receptors integrin α6β1 and α6β4 in these cells. Integrin α6 and β4 expression was also reduced when α2 expression was chemically induced using tetradecanoyl-phorbol-acetate (TPA). Our results thus show that integrin α2β1 expression negatively regulates integrin α6β1 and α6β4-mediated adhesion, spreading and invasion on laminin in different cancer cell types. In contrast to SAOS-WT, but similar to SAOS-A2 osteosarcoma cells, primary Human osteoblasts (HOB) cells express α2 but only low levels of β4 integrin, preferentially adhere to and spread on collagen over laminin and show suppressed laminin-dependent transmigration. By enhancing collagen binding directly and suppressing laminin binding indirectly through laminin receptor downregulation, α2β1 expression may thus re-direct migrating cancer cells from laminin-rich to collagenous tissues and partially revert osteosarcoma cells towards an untransformed phenotype.

Epidermal Integrin α3β1 Regulates Tumor-Derived Proteases BMP-1, Matrix Metalloprotease-9, and Matrix Metalloprotease-3

As the major cell surface receptors for the extracellular matrix, integrins regulate adhesion and migration and have been shown to drive tumor growth and progression. Previous studies showed that mice lacking integrin α3β1 in the epidermis fail to form skin tumors during two-step chemical tumorigenesis, indicating a protumorigenic role for α3β1. Furthermore, genetic ablation of α3β1 in established skin tumors caused their rapid regression, indicating an essential role in the maintenance of tumor growth. In this study, analysis of immortalized keratinocyte lines and their conditioned media support a role for α3β1 in regulating the expression of several extracellular proteases of the keratinocyte secretome, namely BMP-1, matrix metalloprotease (MMP)-9, and MMP-3. Moreover, immunofluorescence revealed reduced levels of each protease in α3β1-deficient tumors, and RNA in situ hybridization showed that their expression was correspondingly reduced in α3β1-deficient tumor cells in vivo. Bioinformatic analysis confirmed that the expression of BMP1, MMP9, and MMP3 genes correlate with the expression of ITGA3 (gene encoding the integrin α3 subunit) in human squamous cell carcinoma and that high ITGA3 and MMP3 associate with poor survival outcome in these patients. Overall, our findings identify α3β1 as a regulator of several proteases within the secretome of epidermal tumors and as a potential therapeutic target.

Integrin α3β1 Promotes Invasive and Metastatic Properties of Breast Cancer Cells through Induction of the Brn-2 Transcription Factor.

Simple SummaryMetastatic triple-negative breast cancer (TNBC) is highly lethal with limited therapy options. Integrin α3β1 is a cell surface receptor that interacts with the extracellular matrix and facilitates communication between tumor cells and their microenvironment. α3β1 is implicated in breast cancer progression and metastasis, so understanding mechanisms by which α3β1 promotes invasion and metastasis will facilitate the development of this integrin as a potential therapeutic target. Here we identify a novel role for α3β1 in promoting the expression of the transcription factor Brain-2 (Brn-2) in triple-negative breast cancer cells. We further report that Brn-2 promotes invasion and metastasis and partially restores invasion to cells in which expression of α3β1 has been suppressed. Bioinformatic analysis of patient datasets revealed a positive correlation between the expression of the genes encoding the integrin α3 subunit and Brn-2. In summary, our work identifies α3β1-mediated induction of Brn-2 as a mechanism that regulates invasive and metastatic properties of breast cancer cells.In the current study, we demonstrate that integrin α3β1 promotes invasive and metastatic traits of triple-negative breast cancer (TNBC) cells through induction of the transcription factor, Brain-2 (Brn-2). We show that RNAi-mediated suppression of α3β1 in MDA-MB-231 cells caused reduced expression of Brn-2 mRNA and protein and reduced activity of the BRN2 gene promoter. In addition, RNAi-targeting of Brn-2 in MDA-MB-231 cells decreased invasion in vitro and lung colonization in vivo, and exogenous Brn-2 expression partially restored invasion to cells in which α3β1 was suppressed. α3β1 promoted phosphorylation of Akt in MDA-MB-231 cells, and treatment of these cells with a pharmacological Akt inhibitor (MK-2206) reduced both Brn-2 expression and cell invasion, indicating that α3β1-Akt signaling contributes to Brn-2 induction. Analysis of RNAseq data from patients with invasive breast carcinoma revealed that high BRN2 expression correlates with poor survival. Moreover, high BRN2 expression positively correlates with high ITGA3 expression in basal-like breast cancer, which is consistent with our experimental findings that α3β1 induces Brn-2 in TNBC cells. Together, our study demonstrates a pro-invasive/pro-metastatic role for Brn-2 in breast cancer cells and identifies a role for integrin α3β1 in regulating Brn-2 expression, thereby revealing a novel mechanism of integrin-dependent breast cancer cell invasion.

New perspectives of quercetin and vitamin C effects on fibronectin-binding integrins and chemokine receptors in prostate cancer cell lines.

The aim of this study is to investigate the effect of two abundant dietary supplements, quercetin and vitamin C on some factors involved in metastasis and proliferation of prostate cancer, which are resistant to conventional chemotherapies in late stages. Bone and brain are two common sites of metastases in prostate cancer, nevertheless the factors involved in their metastatic pathways are not well understood. The effect of quercetin (75µM) and vitamin C (100 µM) on CXCR4, CXCR7 chemokine receptors, α4, α5 and β1 integrins, ki-67 proliferation marker and Vascular endothelial growth factor, VEGF was evaluated using Quantitative Reverse Transcription PCR (RT-qPCR). The effect of quercetin and vitamin C alone was different on PC3 and DU145 prostate cancer cell lines, but sequential combination reduced significantly the expression of CXCR and CXCR7 chemokine receptors, α4, α5 and β1 integrin subunits, VEGF and Ki-67 proliferation markers in PC3 and DU145 cell lines. Our results indicated the beneficial effect of quercetin and vitamin C on prostate cancer cells with different metastatic sites and their differential response to the treatment which in turn may lead us to reach suitable therapeutic outcomes to combat cancer (Fig. 3, Ref. 36).

Redistribution of EGF Receptor and α5, β1 Integrins in Response to Infection of Epithelial Cells by Serratia proteamaculans

Eukaryotic cells use surface receptors to interact with the environment. Interacting with the cell receptors, pathogenic and opportunistic bacteria can invade cells that normally do not phagocytize. Bacteria Serratia proteamaculans can invade eukaryotic cells, and this ability correlates with the proteolytic activity of the actin-specific protease protealysin (Tsaplina et al., 2009). However, cellular receptors involved in the mechanism by which these bacteria penetrate the cells are not known. Bacterial outer membrane protein OmpX which is one of the substrates of protealysin is known to interact with EGF receptor and fibronectin. Therefore, in this work, changes in the localization of EGF receptors and fibronectin receptors α5, β1 integrins were monitored in the epithelial M-HeLa and A549 cells infected by bacteria S. proteamaculans. It turned out that in response to the infection α5 and β1 integrin subunits accumulate on the surface of M-HeLa cells, and bacteria attach intensively at the sites of the α5 integrin localization. The α5 integrin was not detected in A549 cells. The β1 integrin, localized evenly along the cell perimeter, accumulated in the cytoplasm in response to contact with bacterial cells, while the EGF receptor formed granules in the cytoplasm in response to invasion. A similar redistribution of EGF receptors occurs in response to EGF binding. These results indicate that invasion of S. proteamaculans into the epithelial cells of different origin occurs as a result of their interaction with different surface receptors.

MiR-27b Suppresses Tongue Squamous Cell Carcinoma Epithelial-Mesenchymal Transition by Targeting ITGA5.

BackgroundMicroRNA27b-3p (miR-27b) has been reported to be dysregulated in multiple types of human cancer. However, the expression levels, biological roles, and underlying mechanism of miR-27b in tongue squamous cell carcinoma (TSCC) remain to be elucidated.MethodsBioinformatics analyses and quantitative real-time PCR (qRT-PCR) were used to determine miR-27b expression in TSCC tissues and cell lines. The influence of miR-27b overexpression or inhibition on TSCC cell proliferation, migration, and invasion in vitro, and on tumor growth in vivo, was explored via CCK8, colony formation, wound healing, and transwell assays, and in xenograft tumors in nude mice, respectively. Luciferase reporter assays, qRT-PCR, and Western blotting were performed to clarify the potential mechanisms involving miR-27b in TSCC cells.ResultsmiR-27b was significantly downregulated in TSCC tissues and cell lines, and its expression was correlated with cancer status. Overexpression of miR-27b led to diminished proliferation, migration, and invasion, and notably reduced tumor growth in vivo. Bioinformatics analysis followed by luciferase reporter assays demonstrated that miR-27b expression was inversely correlated with that of integrin subunit α5 (ITGA5)and that miR-27b directly bound to the 3ʹ-untranslated region of ITGA5 in TSCC cells. The bioinformatics analysis also indicated that ITGA5 was upregulated in TSCC and that its expression was correlated with epithelial–mesenchymal transition (EMT) and poor prognosis. Moreover, we found that miRNA-27b could reverse ITGA5-induced promotion of TSCC cell proliferation and migration. Finally, we demonstrated that regulation of miR-27b expression in TSCC may result in alterations in the expression of ITGA5 and EMT-related marker genes at the mRNA and protein levels.ConclusionThese results indicate that miR-27b hampers TSCC proliferation and migration via suppressing the EMT process by targeting ITGA5. These findings support consideration of miR-27b/ITGA5 as a valuable marker for the metastatic potential of TSCC, or as a therapeutic target for the treatment of TSCC.

The stiffness-controlled release of interleukin-6 by cardiac fibroblasts is dependent on integrin α2β1.

Cardiac fibroblasts are able to sense the rigidity of their environment. The present study examines whether the stiffness of the substrate in cardiac fibroblast culture can influence the release of interleukin‐6 (IL‐6), interleukin‐11 (IL‐11) and soluble receptor of IL‐6 (sIL‐6R). It also examines the roles of integrin α2β1 activation and intracellular signalling in these processes. Cardiac fibroblasts were cultured on polyacrylamide gels and grafted to collagen, with an elasticity of E = 2.23 ± 0.8 kPa (soft gel) and E = 8.28 ± 1.06 kPa (stiff gel, measured by Atomic Force Microscope). Flow cytometry and ELISA demonstrated that the fibroblasts cultured on the soft gel demonstrated higher expression of the α2 integrin subunit and increased α2β1 integrin count and released higher levels of IL‐6 and sIL‐6R than those on the stiff gel. Substrate elasticity did not modify fibroblast IL‐11 content. The silencing of the α2 integrin subunit decreased the release of IL‐6. Similar effects were induced by TC‐I 15 (an α2β1 integrin inhibitor). The IL‐6 levels in the serum and heart were markedly lower in α2 integrin‐deficient mice B6.Cg‐Itga2tm1.1Tkun/tm1.1Tkun than wild type. Inhibition of Src kinase by AZM 475271 modifies the IL‐6 level. sIL‐6R secretion is not dependent on α2β1 integrin. Conclusion: The elastic properties of the substrate influence the release of IL‐6 by cardiac fibroblasts, and this effect is dependent on α2β1 integrin and kinase Src activation.

Levels of isoforms of fibronectin and α5/CD49e integrin on lymphocytes and in blood plasma in the conditions of chronic diffuse liver diseases

Chronic diffuse liver diseases are characterized by accumulation of complex inflammatory infiltrate in the liver tissues, blood, and lympha, and activation of the immune system. Leukocytes become involved in the area of inflammation after the activation of receptors of blood adhesia, particularly integrins and their ligands. Plasma lymphocytes quickly activate the function of integrins by changing their conformation, leading to high affinity and underlying the formation of strong stable connection between the components of extracellular matrix. A vitally important role in the process of liver fibrogenesis is performed by a pro-fibrogenicic protein fibronectin which induces the expresson of collagen genes and precedes the deposition of other components of matrix. The studies were conducted in the group of patients suffering from chronic diffuse liver diseases of non-viral etiology aged 28–60 years, n = 36 and in the group of 15 practically healthy volunteer donors aged 25 to 52 years without a history of liver diseases using the methods of flow cytofluorometry, immunoenzymatic analysis, and quantitative real-time polymerase chain reaction. The patients of the group with chronic diffuse liver diseases were observed to have statistically significant decrease in the concentration of plasmatic form of fibronectin measuring 27.6% compared with the control group. We determined increase in the concentration of cellular fibronectin in blood plasma of patients with the diseases on average accounting for 63.8% compared with the norm, and the highest increase in this parameter equaling 77.2% was seen in patients suffering from drug-induced hepatitis. Significant increase in the level of exposure of cellular FN on blood lymphocytes was determined in patients with chronic diffuse liver diseases, measuring 231.8%, whereas the level of plasmatic form of fibronectin in these cells was decreased (statistically unreliable). For α5-integrin subunit, we determined a 390.8% increase in the level of its exposure in blood lymphocytes in the surveyed groups compared with the control. Level of blood lymphocytes that express the cellular fibronectin significantly decreased by 140.1%. Statistical characteristics of diagnostic possibility of the parameters of the level of plasmatic and cellular fibronectin in blood, determined over the analysis of ROC-curves, demonstrated excellent informativeness of these tests. Analysis of the possibility of predicting the presence of pathology using the model of logistic regression revealed zero error of prediction and maximum efficiency of the tests: intensity of exposure of α5-integrin receptor on the surface of lymphocytes, intensity of exposure of plasmatic fibronectin on the surface of lymphocytes, intensity of exposure of cellular fibronectin on the surface of lymphocytes, concentration of plasmatic fibronectin in blood, concentration of cellular fibronectin in blood plasma. These parameters may be proposed for further surveys for developing serologic biomarkers based on the parameters for diagnostics of chronic diffuse liver diseases.

Abstract A41: Inhibition of aberrant YAP and TAZ activity to prevent metastasis formation and growth

Abstract Metastasis causes most cancer-related deaths, and few effective therapies exist for patients with metastatic disease. The development of such therapies requires the identification of proteins essential for the spread and subsequent growth of metastatic cancer cells. Previous work from us and other groups has clearly demonstrated that aberrant YAP or TAZ activity, negatively regulated by the Hippo pathway, promotes cancer development and metastasis. Moreover, many human cancers have abnormally high YAP/TAZ expression or nuclear localization, and this is associated with poor prognosis. We hypothesize that targeting YAP and TAZ or upstream activators will prevent metastasis formation and growth, and we have developed and optimized in vivo model systems to test this. Using these methods, we show that YAP and TAZ are required for metastasis formation in human melanoma and breast cancer cells. An ongoing study using our in vivo metastatic growth assay and inducible YAP/TAZ RNAi is testing if YAP and TAZ are collectively required for metastatic growth after dissemination, and we hope to include the results of this study on our poster. In addition to directly targeting YAP and TAZ, we are also interested in identifying proteins that promote YAP/TAZ activity in metastatic cells, and this could lead to more therapeutic targets. Our recently published work shows that Src signaling can promote YAP/TAZ activity and enhance tumor growth and metastasis. Since integrins promote Src activity and regulate YAP and TAZ, we want to test if there are specific integrins required for YAP/TAZ activity in metastatic cells. Therefore, we used a YAP/TAZ-TEAD transcriptional activity reporter assay to perform an unbiased RNAi screen to identify integrins that regulate YAP/TAZ activity in metastatic melanoma and breast cancer cells. Knockdown of integrin subunits α2, α3, α6, and αV each reduced YAP/TAZ-TEAD transcriptional activity in metastatic breast cancer cells. These results have been confirmed with qPCR for the YAP/TAZ target genes CTGF and CRY61. Interestingly, a different set of integrin subunits appears to be required for YAP/TAZ-TEAD transcriptional activity in metastatic melanoma cells, suggesting the regulation of YAP and TAZ is not the same in different cancer types. Current work aims to use in vivo multiplex metastasis assays, which our lab has developed, to test if knockdown of each integrin either alone, or in combination, prevents metastasis formation and growth. Concurrently, we are focused on elucidating the mechanism by which these integrins enhance YAP/TAZ activity in metastatic cells. Collectively, these studies show the importance of YAP and TAZ and their upstream regulators in metastasis formation and growth and suggest that targeting YAP/TAZ activity may treat metastatic disease. Citation Format: Janine S.A. Warren, Emily Norton, John M. Lamar. Inhibition of aberrant YAP and TAZ activity to prevent metastasis formation and growth [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A41.

Extrinsic modulation of integrin α6 and progenitor cell behavior in mesenchymal stem cells.

Mesenchymal stem cells (MSC) are heterogeneous cells of complex nature that show different potentials while different culture conditions can modify their functionalities through interactions with the microenviroment. Here, we found that bone marrow (BM) MSC from different donor sources and passages that expressed higher levels of α6 integrin subunit (ITGA6), showed higher clonogenicity, migration and differentiation potential. ITGA6 showed important roles improving these potentials and regulating proliferation through protein kinase B (AKT) pathway and cell cycle inhibitor proteins p53 and p21. Moreover, ITGA6 downregulation impaired migration. Cell confluence regulated ITGA6, increasing its expression in low density cultures and decreasing in high density cultures. Besides, ITGA6- cells expressed ITGA6 when seeded at low densities. We found higher ITGA6 expression on fibronectin substrates at lower confluency. Fibronectin increased proliferation, clonogenicity, activation of AKT, decreased cell cycle inhibitor proteins and augmented growth factors expression. Spheres-derived MSC showed higher ITGA6 expression and enhanced potentials for migration, clonogenicity and proliferation. In conclusion, though there is an intrinsic regulation of ITGA6 expression, associated to the progenitor potential of BM-MSC, this expression is regulated by culture conditions and is translated in changes in cell behavior and proliferation. This knowledge could be used to enhance the potential of BM-MSC for clinical application.

Integrin-Targeting Dye-Doped PEG-Shell/Silica-Core Nanoparticles Mimicking the Proapoptotic Smac/DIABLO Protein.

Cancer cells demonstrate elevated expression levels of the inhibitor of apoptosis proteins (IAPs), contributing to tumor cell survival, disease progression, chemo-resistance, and poor prognosis. Smac/DIABLO is a mitochondrial protein that promotes apoptosis by neutralizing members of the IAP family. Herein, we describe the preparation and in vitro validation of a synthetic mimic of Smac/DIABLO, based on fluorescent polyethylene glycol (PEG)-coated silica-core nanoparticles (NPs) carrying a Smac/DIABLO-derived pro-apoptotic peptide and a tumor-homing integrin peptide ligand. At low μM concentration, the NPs showed significant toxicity towards A549, U373, and HeLa cancer cells and modest toxicity towards other integrin-expressing cells, correlated with integrin-mediated cell uptake and consequent highly increased levels of apoptotic activity, without perturbing cells not expressing the α5 integrin subunit.

Screening of integrins localized on the surface of human epidermal melanocytes.

In vivo, melanocytes occupy three-dimensional (3D) space. Nevertheless, most experiments involving melanocytes are performed in a two-dimensional microenvironment, resulting in difficulty obtaining accurate results. Therefore, it is necessary to construct an artificial in vivo-like 3D microenvironment. Here, as a step towards engineering a precisely defined acellular 3D microenvironment supporting the maintenance of human epidermal melanocytes (HEMs), we examined the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in HEMs. Real-time PCR and fluorescent immunoassay analyses were used to elucidate the expression of integrin α and β subunit genes at the transcriptional and translational levels, respectively. The functionality of the presumed integrin heterodimers was confirmed using attachment and antibody-inhibition assays. Among the genes encoding 12 integrin subunits (α1, α2, α3, α4, α5, α6, α7, αV, β1, β3, β5, and β8) showing significantly higher transcription levels, proteins translated from the integrin α2, α4, α5, β1, β3, and β5 subunit genes were detected on the surface of HEMs. These HEMs showed significantly increased adhesion to collagen I, fibronectin, laminin, and vitronectin, and functional blockade of the integrin α2 subunits significantly inhibited adhesion to collagen I, fibronectin, and laminin. In addition, there was no significant inhibition of the adhesion to fibronectin or vitronectin in HEMs with functional blockade of the integrin α4, α5, or αV subunits. These results indicate that the active integrin α2β1 heterodimer and the inactive integrin α4, α5, αV, β3, and β5 subunits are all localized on the surface of HEMs.

Evaluation of lumican effects on morphology of invading breast cancer cells, expression of integrins and downstream signaling.

The small leucine-rich proteoglycan lumican regulates estrogen receptors (ERs)-associated functional properties of breast cancer cells, expression of matrix macromolecules, and epithelial-to-mesenchymal transition. However, it is not known whether the ER-dependent lumican effects on breast cancer cells are related to the expression of integrins and their intracellular signaling pathways. Here, we analyzed the effects of lumican in three breast cancer cell lines: the highly metastatic ERβ-positive MDA-MB-231, cells with the respective ERβ-suppressed (shERβMDA-MB-231), and lowly invasive ERα-positive MCF-7/c breast cancer cells. Scanning electron microscopy, confocal microscopy, real-time PCR, western blot, and cell adhesion assays were performed. Lumican effects on breast cancer cell morphology were also investigated in 3-dimensional collagen cultures. Lumican treatment induced cell-cell contacts and cell grouping and inhibited microvesicles and microvilli formation. The expression of the cell surface adhesion receptor CD44, its isoform and variants, hyaluronan (HA), and HA synthases was also investigated. Lumican inhibited the expression of CD44 and HA synthases, and its effect on cell adhesion revealed a major role of α1, α2, α3, αVβ3, and αVβ5 integrins in MDA-MB-231 cells, but not in MCF-7/c cells. Lumican upregulated the expression of α2 and β1 integrin subunits both in MDA-MB-231 and in shERβMDA-MB-231 as compared to MCF-7/c cells. Downstream signaling pathways for integrins, such as FAK, ERK 1/2 MAPK 42/44, and Akt, were found to be downregulated by lumican. Our data shed light to the molecular mechanisms responsible for the anticancer activity of lumican in invasive breast cancer.

Overexpressed ITGA2 contributes to paclitaxel resistance by ovarian cancer cells through the activation of the AKT/FoxO1 pathway.

Ovarian cancer is one of the most malignant tumors of the female reproductive system, with high invasiveness. The disease is a severe threat to women's health. The ITGA2 gene, which codes for integrin subunit α2, is involved in the proliferation, invasion, and metastasis of cancer cells. Although previous studies have shown that ITGA2 increases in ovarian cancer, the specific molecular mechanism of how ITGA2 promotes ovarian cancer proliferation and metastasis is still unclear. In this study, we confirmed that ITGA2 was elevated in ovarian cancer, which led to poor prognosis and survival. Overexpressed ITGA2 promoted the proliferation of ovarian cancer cells. We also found that ITGA2 regulated the phosphorylation of forkhead box O1 (FoxO1) by mediating AKT phosphorylation, which provided a reasonable explanation for ITGA2’s role in ovarian cancer’s resistance to albumin paclitaxel. In summary, ITGA2 could be used as a new therapeutic target and prognostic indicator in ovarian cancer.

SIRT6对老年人皮肤成纤维细胞迁移能力和增殖能力的影响及机制研究

Objective To assess the effect of silent mating type information regulation 2 homolog 6 (SIRT6) on migratory and proliferative activity of skin fibroblasts in the elderly, and to explore their mechanisms. Methods Circumcised foreskins were obtained from patients of different ages in Department of Urologic Surgery, Second Hospital of Shanxi Medical University, including 8 elderly patients and 8 young patients. Human skin fibroblasts were isolated from the foreskin tissues by using a collagenase digestion method. Western blot analysis was performed to determine the expression of SIRT6 and phosphorylated p65 (p - p65) in human skin fibroblasts in different age groups, scratch assay to evaluate cell migratory activity, and cell counting kit-8 (CCK8) assay to assess cellular proliferative activity. Skin fibroblasts in the elderly group were divided into 2 groups: SIRT6 group transfected with a lentiviral vector overexpressing SIRT6, and control group transfected with an empty lentiviral vector. Then, the cell migratory and proliferative activity as well as p-p65 expression were measured by the above methods, and the mRNA expression of type Ⅰ and Ⅲ collagens, and integrin subunits α3, α5 and β1 was determined by real-time PCR in the SIRT6 group and control group. Statistical analysis was carried out with GraphPad Prism 5 software by using t test for comparison between 2 groups. Results Compared with the young group, the elderly group showed significantly decreased SIRT6 expression in skin fibroblasts (0.434 ± 0.179 vs. 1.000 ± 0.067, t = 3.040, P = 0.012) , migration rate (43.81% ± 18.84% vs. 94.63% ± 12.32%, t = 5.903, P = 0.003) and cellular proliferative activity at 24 and 48 hours (both P < 0.05) , but significantly increased p-p65 expression (1.694 ± 0.148 vs. 1.000 ± 0.093, t = 2.949, P = 0.015) . Compared with the control group, the SIRT6 group showed significantly decreased p-p65 expression (P < 0.05) , but significantly increased migratory and proliferative activity (both P < 0.05) , and elevated mRNA expression of type Ⅲ collagen and integrin subunits α3, α5 and β1 (all P < 0.05) . Conclusion SIRT6 can improve the migratory and proliferative activity of human fibroblasts in the elderly, possibly by inhibiting the nuclear factor-κB pathway. Key words: Fibroblasts; Cell proliferation; Cell movement; NF-kappa B; Aged; Wound healing; SIRT6