Junctional Adhesion molecule-A Overexpression Research Articles

MiR-543 promoted the cell proliferation and invasion of nasopharyngeal carcinoma by targeting the JAM-A.

MicroRNAs (miRNAs) are defined as small, non-coding RNAs that act as post-transcriptional regulators of gene expression. Dysfunction of miRNAs was involved in the initiation and progression of nasopharyngeal carcinoma (NPC). Here, we found that miR-543 was markedly overexpressed in NPC tissues and cell lines. Overexpression of miR-543 promoted the proliferation, cell cycle progression and invasion of NPC cells. Down-regulation of miR-543 inhibited the proliferation and induced apoptosis of NPC cells. Bioinformatics analysis suggested the junctional adhesion molecule A (JAM-A) as a potential target of miR-543. Furthermore, molecular study showed that the miR-543 bound the 3'-untranslated region (UTR) of JAM-A and decreased the expression of JAM-A in NPC cells. The expression of JAM-A in NPC tissues was decreased and negatively correlated with that of miR-543. Overexpression of JAM-A attenuated miR-543-induced proliferation of NPC cells. Collectively, these evidence indicated the important roles of miR-543/JAM-A signaling in the progression of NPC, highlighting the potential of miR-543 as a target in the treatment of NPC.

Junctional Adhesion Molecule-a Regulates Vascular Endothelial Growth Factor-a-Dependent Permeability

Introduction: Vascular barrier function is important for maintaining vascular integrity and is regulated by endothelial tight junctions. Breaches in the tight junction result in increased endothelial cell (EC) permeability, which leads to a variety of pathological conditions such as edema, vascular degeneration, diabetic retinopathy, and ischemia reperfusion injury. Junctional adhesion molecule A (JAM-A), is an integral part of the tight junctions of ECs. It has been shown that a lack of Jam-A affects neutrophil extravasation and FGF-2-induced angiogenesis. Role of JAM-A in regulation of vascular permeability is not known.Methods: Jam-A knockout (KO) and age-matched wild-type (WT) control mice of both sexes were used in this study. In vivo vascular permeability was assessed using Miles assay and quantified colorimetrically by extracting Evans blue in formamide at 56oC. The circulating plasma level of vascular endothelial growth factor A (VEGF-A) in WT and Jam-A KO mice was measured by ELISA assay. Surface expression of VEGF receptor 2 (VEGFR2) was analyzed by flow cytometry. Quantitative real- time PCR was used to determine gene transcription. Protein expression was determined by western blotting. VEGF-induced WT and Jam-A KO EC migration was analyzed by both scratch and trans-well assays.Results: Although Jam-A resides at the endothelial tight junctions, genetic ablation of Jam-A does not affect blood vessel structure. However, basal vascular permeability was significantly enhanced (P<0.05) in Jam-A KO mice compared to the WT controls, suggesting that a lack of Jam-A in EC compromises vascular integrity. This was further supported by the fact that there was increased edema in the lungs of the Jam-A KO mice compared to WT controls. Since VEGF is also known as vascular permeability factor, we evaluated the effect of VEGF-A on vascular permeability in Jam-A KO mice. Upon intravenous injection of VEGF-A (100ng/ml), EC permeability was significantly increased in both WT and Jam-A KO mice. In order to determine the cause of this enhanced basal permeability, we evaluated the levels of VEGF-A in WT and Jam-A KO mice. Jam-A deficient mice showed a three-fold (P<0.004) increased level of plasma VEGF-A compared to WT mice. Additionally, VEGF-A protein level was also increased in the ECs from Jam-A KO mice compared to WT controls. Furthermore, mRNA levels of VEGFR2, but not sFlt1, were increased in the Jam-A KO ECs compared to WT ECs. Protein levels as well as surface expression of VEGFR2 were significantly increased (P<0.001) in Jam-A KO ECs compared to WT ECs, suggesting that lack of Jam-A results in increased levels of VEGF-A/VEGFR2. We next evaluated the protein and mRNA levels of both hypoxia-inducible factor-1 α (HIF-1α), a transcription factor known to regulate expression of VEGF-A, and inhibitor of DNA binding 1 (Id1), a transcriptional factor known to regulate expression of VEGFR2. We found that bothHIF-1α and Id1 mRNA as well as protein levels were significantly (P<0.001 and P<0.05 respectively) augmented in Jam-A KO ECs compared to WT ECs. Additionally, overexpression of JAM-A in human umbilical vein endothelial cells (HUVECs) resulted in significantly reduced expression of bothHIF-1α and Id1 proteins. Furthermore, ECs isolated from Jam-A KO mouse showed significantly enhanced (P<0.05) cell migration in response to VEGF-A as compared to WT ECs, suggesting that Jam-A ECs respond favorably to VEGF-A due to increased VEGFR2 on their surface. To confirm that the observed augmented permeability in Jam-A KO mice was primarily due to enhanced VEGFR2 signaling, we inhibited VEGFR2 in mice using DC101 (200μg/kg), a function-blocking antibody. Isotype specific IgG (200μg/kg) was used as control. Inhibition of VEGFR2 completely abrogated basal as well as VEGF-A-induced vascular permeability in both Jam-A KO and WT mice, suggesting that the augmented EC permeability in Jam-A KO mice is not due to structural defect in tight junctions, but due to enhanced VEGF-A/VEGFR2 signaling.Conclusion: Our results suggest that JAM-A positively regulates EC tight junction integrity by suppressing levels of VEGF-A and VEGFR2 through downregulation ofHIF-1α and Id1 transcription factors. DisclosuresNo relevant conflicts of interest to declare.

Abstract 54: Tumor dormancy in breast cancer cells: Identification of junctional adhesion molecule-A as a novel regulator

Abstract In tumor dormancy, a sub-population of tumor cells becomes quiescent but can re-enter the cell cycle upon certain environmental cues. The re-awakening of dormant populations has been associated with tumor cell proliferation and poor patient outcomes. Tumor dormancy can be modelled in vitro by exposing cancer cells on fibronectin-coated plates to bFGF-2, a mammary differentiation factor abundant in the bone marrow stroma, which causes partial re-differentiation, cell spreading and re-expression of integrin-α5β1. We have uncovered a novel potential role for the adhesion protein Junctional Adhesion Molecule-A (JAM-A) in tumor dormancy. JAM-A overexpression has previously been linked to increased risk of metastasis in breast cancer patients, and its expression regulates the angiogenic functions of bFGF-2. Furthermore, since loss of JAM-A downregulates the α5β1 downstream effector FAK, we hypothesized that JAM-A is required for maintenance of tumor dormancy. To investigate this, JAM-A was transiently silenced in MCF-7 breast cancer cells grown on fibronectin-coated plates and exposed to FGF-2. JAM-silenced cells exhibited expressional downregulations in integrin-α5β1 and FAK proteins, and failed to exhibit the cortical actin redistribution and morphological spreading which characterize dormant cells. in parallel, pharmacologically-induced loss of JAM-A (using an anti-tumor antibiotic) prevented tumor dormancy and was cytotoxic to dormant cells. We next investigated a potential relationship between JAM-A expression and pro-inflammatory cytokines linked with the re-awakening of dormant cells in post-menopausal women. Exposure of MCF-7 cells to IL-1β, TNF-α, IL-6 and IL-8 increased the protein expression of matrix metalloproteinase ADAM17, which in turn caused JAM-A cleavage and loss from the cell membrane. Analysis of bone metastasis sections from post-menopausal breast cancer patients revealed significant heterogeneity in JAM-A expression, with some regions strongly positive for JAM-A while other regions were negative. Nonetheless, 20/24 metastatic sections expressed either moderate or high JAM-A levels, suggesting that even if cells transiently lose JAM-A to reawaken from tumor dormancy, they may later acquire high JAM-A expression during disease progression. The fact that JAM-high dormant cells had high Aldefluor activity, a feature of stemness, might also support an important role for JAM-A in tumor evolution or progression. To conclude, our data support a model whereby JAM-A is important for the maintenance of dormancy in breast cancer cells in a simulated bone marrow microenvironment, and represents a novel therapeutic target worthy of investigation in breast cancer. This work was financially supported by Science Foundation Ireland (grant 13/IA/1994 to AMH). Citation Format: Sri HariKrishna Vellanki, Rodrigo G. Cruz, Katherine M. Sheehan, Elaine W. Kay, Ann M. Hopkins. Tumor dormancy in breast cancer cells: Identification of junctional adhesion molecule-A as a novel regulator [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 54.

JAM-A overexpression is related to disease progression in diffuse large B-cell lymphoma and downregulated by lenalidomide

Cancer stem cells play an important role on tumor progression. Biomarkers of stem cell property and their relationship to extranodal involvement of malignant lymphocytes are undefined in diffuse large B-cell lymphoma (DLBCL). Here we showed that junctional adhesion molecule-A (JAM-A) was highly expressed in DLBCL patients with multiple extranodal lesions. JAM-A maintained B-lymphoma cell stemness and was associated with cell invasion and epithelial-to-mesenchymal transition both in vitro and in vivo. As mechanism of action, JAM-A overexpression selectively activated transforming growth factor-β (TGF-β)/NODAL signaling, thereby enhanced B-lymphoma cell aggressiveness and induced extranodal involvement to mesoendoderm-derived organs in DLBCL. Lenalidomide downregulated JAM-A and downstream NODAL expression, resulting in inhibition of B-lymphoma cell invasion and epithelial-to-mesenchymal transition. In a murine xenograft model established with subcutaneous injection of JAM-A-overexpressing B-lymphoma cells, lenalidomide retarded tumor growth and prevented cell invasion to mesoendoderm-derived organs, consistent with the downregulation of JAM-A and NODAL expression. Collectively, these findings indicated that JAM-A was related to extranodal involvement in DLBCL through modulating TGF-β/NODAL signaling. Identified as a biomarker of stem cell property, JAM-A indicated the sensitivity of B-lymphoma cells to lenalidomide. Therapeutic targeting of JAM-A/NODAL axis could thus be a promising clinical strategy to impede tumor progression in DLBCL.

Abstract 4921: The adhesion protein JAM-A is important for maintenance of tumor dormancy in breast cancer cells, and JAM-A high-dormant cells have stem cell characteristics

Abstract Breast cancer cells which metastasize to bone marrow can remain dormant and survive multiple rounds of adjuvant chemotherapy. The re-awakening of dormant populations, particularly in estrogen receptor (ER)-positive patients, has been associated with tumor cell proliferation and poor patient outcomes. Tumor dormancy can be modelled in vitro by exposing cancer cells on fibronectin-coated plates to bFGF-2, a mammary differentiation factor abundant in the bone marrow stroma, which causes partial re-differentiation, cell spreading and re-expression of integrin-α5β1. Ligation of integrin-α5β1 by fibronectin and activation of the PI3K pathway both contribute to dormant cell survival. Our study has uncovered a novel potential role for the adhesion protein Junctional Adhesion Molecule-A (JAM-A) in tumor dormancy. JAM-A overexpression has previously been linked to increased risk of metastasis in breast cancer patients, and its expression regulates the angiogenic functions of bFGF-2. Furthermore, since loss of JAM-A causes down-regulation of the α5β1 downstream effector FAK, we hypothesized that JAM-A is required for maintenance of breast cancer cell dormancy. To investigate this, JAM-A was transiently silenced using siRNA in MCF-7 breast cancer cells grown on fibronectin-coated plates and exposed to FGF-2. JAM-silenced cells exhibited downregulations in the protein expression of integrin-α5β1 and FAK in this model, and failed to exhibit the cortical actin redistribution and morphological spreading which characterize dormant cells. We next investigated a potential relationship between JAM-A expression and pro-inflammatory cytokines which have been linked with the re-awakening of dormant cells in post-menopausal women. Exposure of MCF-7 cells to the pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and IL-8 decreased JAM-A expression in in a concentration-dependent fashion, suggesting that the loss of JAM-A could be associated with re-awakening from tumor dormancy. We are currently probing alterations in JAM-A expression and that of integrin-α5β1 in matched human primary breast tumor specimens and bone marrow metastases. Since JAM-high dormant cells express high mRNA levels of the stemness factors (Sox2, Oct3/4, Nanog) and have high Aldefluor activity, our data also suggest that JAM-A may be required for a stem-like phenotype. To conclude, our data support a model whereby JAM-A is important for the maintenance of dormancy in breast cancer cells in a simulated bone marrow microenvironment, and represents a novel therapeutic target worthy of investigation in breast cancer. Citation Format: Sri Harikrishna Vellanki, Rodrigo G. Cruz, Ann M. Hopkins. The adhesion protein JAM-A is important for maintenance of tumor dormancy in breast cancer cells, and JAM-A high-dormant cells have stem cell characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4921. doi:10.1158/1538-7445.AM2017-4921

Overexpression of junctional adhesion molecule-A and EphB2 predicts poor survival in lung adenocarcinoma patients.

Junctional adhesion molecules are important components of tight junctions, and Eph/ephrin proteins constitute the largest family of receptor tyrosine kinases. Both junctional adhesion molecules and Eph/ephrin are involved in normal tissue development and cancer progression. However, the expression levels and clinical significances of junctional adhesion molecule-A, a member of junctional adhesion molecules, and EphB2, a member of Eph/ephrin family, in lung adenocarcinoma patients are unclear. Therefore, in this study, we aimed to identify the expression and prognostic values of junctional adhesion molecule-A and EphB2 in lung adenocarcinoma patients' cohort. In our study, 70 (55.6%) showed high expression of junctional adhesion molecule-A protein and 51 (40.5%) showed high expression of EphB2 protein in 126 lung adenocarcinoma tissues. Junctional adhesion molecule-A and EphB2 expressions were both significantly increased in tumor tissues compared with noncancerous lung tissues. Kaplan-Meier analysis and log-rank test indicated that low expression of junctional adhesion molecule-A and EphB2 proteins can predict better survival and low mortality rate of lung adenocarcinomas. In univariate analysis, high expression levels of junctional adhesion molecule-A and EphB2 were both found to be significantly correlated with poor overall survival of lung adenocarcinoma patients (hazard ratio = 1.791, 95% confidence interval = 1.041-3.084, p = 0.035; hazard ratio = 1.762, 95% confidence interval = 1.038-2.992, p = 0.036, respectively). The multivariate Cox proportional hazard model demonstrated that EphB2 expression is an independent prognosis parameter in lung adenocarcinoma patients (hazard ratio = 1.738, 95% confidence interval = 1.023-2.952, p = 0.016). Taken together, high expression of junctional adhesion molecule-A and EphB2 can predict poor overall survival and high mortality rate, and EphB2 is an independent prognostic biomarker in lung adenocarcinoma patients.

Dysregulation of junctional adhesion molecule-A via p63/GATA-3 in head and neck squamous cell carcinoma.

Junctional adhesion molecule-A (JAM-A), which belongs to the IgG superfamily, is a tight junction molecule associated with epithelial and endothelial barrier function. Overexpression of JAM-A is also closely associated with invasion and metastasis of cancers such as breast cancer, lung cancer and pancreatic cancer. However, little is known about the mechanism in overexpression of JAM-A in head and neck squamous cell carcinoma (HNSCC). In the present study, we found high expression of JAM-A at the protein and mRNA levels in HNSCC tissues, including those of the oropharynx, larynx, and hypopharynx, together with high protein expression of β-catenin, p63, ΔNp63 and GATA-3. Furthermore, in ELISA, a significant increase of soluble JAM-A in the sera of HNSCC patients was observed compared to healthy subjects. Knockdown of JAM-A by siRNA inhibited cell proliferation, invasion and migration in the HNSCC cell line Detroit562 in vitro. JAM-A expression in Detroit562 was increased via a distinct signal transduction pathway including NF-κB. Expression of JAM-A, β-catenin, p63 and ΔNp63 in Detroit562 was decreased under hypoxia. Knockdown of p63, ΔNp63 or GATA-3 by siRNAs reduced JAM-A expression in Detroit562. In primary cultured HNSCC cells in which CK7, p63, ΔNp63 and GATA-3 were detected, JAM-A expression was decreased by knockdown of p63 or ΔNp63. These results indicate that JAM-A is a biomarker of malignancy in HNSCC and that plasma soluble JAM-A may contribute to serum-based diagnosis of HNSCC. The mechanism of dysregulation of JAM-A via p63/GATA-3 is important in possible molecular targeted therapy for HNSCC.

Coordination of self-renewal in glioblastoma by integration of adhesion and microRNA signaling.

Cancer stem cells (CSCs) provide an additional layer of complexity for tumor models and targets for therapeutic development. The balance between CSC self-renewal and differentiation is driven by niche components including adhesion, which is a hallmark of stemness. While studies have demonstrated that the reduction of adhesion molecules, such as integrins and junctional adhesion molecule-A (JAM-A), decreases CSC maintenance. The molecular circuitry underlying these interactions has yet to be resolved. MicroRNA screening predicted that microRNA-145 (miR-145) would bind to JAM-A. JAM-A overexpression in CSCs was evaluated both in vitro (proliferation and self-renewal) and in vivo (intracranial tumor initiation). miR-145 introduction into CSCs was similarly assessed in vitro. Additionally, The Cancer Genome Atlas dataset was evaluated for expression levels of miR-145 and overall survival of the different molecular groups. Using patient-derived glioblastoma CSCs, we confirmed that JAM-A is suppressed by miR-145. CSCs expressed low levels of miR-145, and its introduction decreased self-renewal through reductions in AKT signaling and stem cell marker (SOX2, OCT4, and NANOG) expression; JAM-A overexpression rescued these effects. These findings were predictive of patient survival, with a JAM-A/miR-145 signature robustly predicting poor patient prognosis. Our results link CSC-specific niche signaling to a microRNA regulatory network that is altered in glioblastoma and can be targeted to attenuate CSC self-renewal.

Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer.

Advanced cancers display cellular heterogeneity driven by self-renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying and isolating cell populations that possess differences in self-renewal and tumor initiation. To overcome these barriers in triple-negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well-established pluripotency gene NANOG. NANOG-GFP+ cells gave rise to both GFP+ and GFP(-) cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self-renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well-established CSC markers CD24(-) /CD44(+) , CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG-GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule-A (JAM-A). JAM-A was highly expressed in GFP+ cells and patient-derived xenograft ALDH+ CSCs compared with the GFP(-) and ALDH(-) cells, respectively. Depletion of JAM-A compromised self-renewal, whereas JAM-A overexpression induced self-renewal in GFP(-) cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non-CSCs in TNBC that can be used to identify CSC-specific targets for the development of future therapeutic strategies.

Assessing the effect of mutant JAM-A overexpression on downstream signalling in breast cancer cells

Background Breast cancer causes the most number of deaths in females worldwide.[1] It contributed to 14% of cancer deaths in 2008 and is also the most commonly diagnosed cancer in women globally. Overexpression of the tight junction protein junctional adhesion molecule-A (JAM-A) in breast cancer tissue is known to produce less favourable outcomes in breast cancer patients. Overexpressed JAM-A may accomplish this by influencing downstream signalling of key proteins involved in cell proliferation, migration and the reaction of cells to therapeutic agents. This study aims to further investigate the promising role of JAM-A in breast cancer advancement by assessing the consequences of changing the structure of JAM-A on 10 key signalling proteins related to tumour progression.

RNA interference mediated JAM-A gene silencing promotes human epidermal stem cell proliferation.

The objective of the study was to explore the influence of junctional adhesion molecule A (JAM-A) gene decoration on proliferation and differentiation of human epidermal stem cells (hEpSCs). JAM-A gene and JAM-A interference gene lentivirus eukaryotic expression vectors were established. The recombinant lentivirus was introduced into hEpSCs to observe and detect viral transfection by fluorescence microscopy and Western blot, respectively. After confirmation of successful introduction of the target gene, cell growth curves were mapped out by cytometry to detect cell proliferation in different groups. The expression of hEpSCs labeled molecules was detected by immunofluorescence, and cell safety was detected by teratoma test in all groups. (1) Fluorescence microscopy showed that in the JAM-A over-expression (JAM-A(ov) EpSCs) group, the green fluorescence was mainly distributed in the cell membrane; in the JAM-A interference (JAM-A(kd) EpSCs) group and blank vector (GFP EpSCs) group, all cell bodies were luminous. Western blot showed that JAM-A protein was up-regulated in JAM-A(ov) EpSCs and down-regulated in JAM-A(kd) EpSCs. (2) Growth curves showed that hEpSCs entered the quick-growing phase 4 days after inoculation and reached the platform phase at day 7. JAM-A(ov) EpSCs proliferated more slowly than GFP EpSCs, while JAM-A(kd) EpSCs proliferated significantly faster than GFP EpSCs. (3) Immunofluorescence showed that the expression of transient amplification epidermal marker keratin 14, hEpSCs marker keratin I9 and β-integrin was down-regulated in JAM-A(kd) EpSCs group as compared to that in the GFP EpSCs group, and the expression of epidermal terminal differentiation marker K10 was negative in the JAM-A(kd) EpSCs group. There was no significant difference in the expression of specific molecules between JAM-A(ov) EpSCs and hEpSCs. (4) The result of teratoma test was negative in all groups. The proliferative ability of hEpSCs was increased markedly after down-regulation of JAM-A. Cells presented initial differentiation, but retained their stem cell characteristics without evidence of tumorigenesis.

MicroRNA-495 induces breast cancer cell migration by targeting JAM-A.

MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression. The deregulated expression of miRNAs is associated with a variety of diseases, including breast cancer. In the present study, we found that miR-495 was markedly up-regulated in clinical breast cancer samples by quantitative real time-PCR (qRT-PCR). Junctional adhesion molecule A (JAM-A) was predicted to be a potential target of miR-495 by bioinformatics analysis and was subsequently verified by luciferase assay and Western blotting. JAM-A was found to be negatively correlated with the migration of breast cancer cells through loss-of-function and gain-of-function assays, and the inhibition of JAM-A by miR-495 promoted the migration of MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of JAM-A could restore miR-495-induced breast cancer cell migration. Taken together, our findings suggest that miR-495 could facilitate breast cancer progression through the repression of JAM-A, making this miRNA a potential therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0088-2) contains supplementary material, which is available to authorized users.

Mesenchymal Stem Cells With Modification of Junctional Adhesion Molecule A Induce Hair Formation

The junctional adhesion molecule A (JAM-A) has been shown to serve a crucial role in the proliferation, differentiation, and tube-like formation of epithelial cells during angiogenesis. The role of JAM-A in hair follicle (HF) regeneration has not yet been reported. In this study, we used human JAM-A-modified human mesenchymal stem cells (MSCs) to repair HF abnormalities in BALB/c nu/nu mice. The JAM-A gene and JAM-A short hairpin RNA were transfected into cultured human MSCs to generate the JAM-A overexpression MSCs (JAM-A(ov) MSCs) and JAM-A knockdown MSCs (JAM-A(kd) MSCs), respectively. These cells were injected intradermally into the skin of nude mice during the first telogen phase of the HF that occurs 21 days postnatally. We found that JAM-A(ov) MSCs migrated into the HF sheath and remodeled HF structure effectively. The HF abnormalities such as HF curve and HF zigzag were remodeled, and hair formation was improved 7 days following injection in both the JAM-A(ov) MSC and MSC groups, compared with the JAM-A(kd) MSC group or negative control group. Furthermore, the JAM-A(ov) MSC group showed enhanced hair formation in contrast to the MSC group, and the number of curved and zigzagged HFs was reduced by 80% (p < .05). These results indicated that JAM-A(ov) MSCs improved hair formation in nude mice through HF structure remodeling.

Overexpression of JAM-A in Non-Small Cell Lung Cancer Correlates with Tumor Progression

The objective of the current study was to determine the clinical significance of junctional adhesion molecule A (JAM-A) in patients with non-small cell lung cancer (NSCLC) and the biological function of JAM-A in NSCLC cell lines. We showed that JAM-A is predominantly expressed in cell membranes and high expression of JAM-A occurred in 37% of lung tumor specimens compared to corresponding normal tissues. High expression of JAM-A was significantly correlated with TNM stage (P = 0.021), lymph node metastasis (P = 0.007), and decreased overall survival (P = 0.02), In addition, we observed that silencing JAM-A by small interfering RNA inhibited tumor cell proliferation and induced cell cycle arrest at the G1/S boundary. Western blotting analysis revealed that knockdown of JAM-A decreased the protein levels of cyclin D1, CDK4, 6, and P-Rb. Thus, JAM-A plays an important role in NSCLC progression.

Angiogenesis Is Suppressed by Junctional Adhesion Molecule A Through Transcription Factors HIF-1a- and Id1-Dependent Modulation of VEGF/VEGFR2 Expression

Abstract 618The process of angiogenesis is associated with a number of human pathologies. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor. VEGF expression is upregulated during ischemia and tumor growth. VEGF is also expressed in healthy adult vascular tissue. However, VEGF-dependent angiogenesis is normally suppressed in healthy adults. The mechanism of this suppression is not known. Here we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is an endogenous suppressor of VEGF-induced angiogenesis. Using gene-targeted Jam-A null mice, we found that murine melanoma tumor growth and associated angiogenesis were significantly augmented (P<0.001) in Jam-A null mice compared to wild-type mice. Additionally, Jam-A null mice showed significantly enhanced (P<0.004) vascular permeability as assessed by a Miles assay using Evans blue dye. Furthermore, VEGF-, but not FGF-2-dependent angiogenesis is significantly augmented (P<0.001) in the absence of Jam-A as assessed by Matrigel plug and aortic ring sprouting, two well-accepted in vivo and ex vivo angiogenic assays, respectively. Vascular endothelial cells isolated from Jam-A null mouse aorta showed significantly enhanced (P<0.05) cell migration and tube-like structure formation (P<0.05) in response to VEGF. Additionally, we found the plasma levels of VEGF in Jam-A null mice is significantly (P<0.000001) and age-dependently increased compared to WT mice. Furthermore, both mRNA and protein levels of VEGF and its receptor VEGFR2 were significantly increased (P<0.001) in Jam-A null endothelial cells, suggesting that the VEGF/VEGFR2 signaling axis is enhanced in the absence of Jam-A. To further confirm this finding, we injected anti-VEGFR2 (DC101) into the Jam-A null mice inoculated with murine melanoma (B16F0) cells. Inhibition of VEGF/VEGFR-2 signaling by DC101 significantly reduced (P<0.00003) tumor growth and associated angiogenesis in Jam-A null mice. Additionally, vascular permeability observed in Jam-A null mice was completely abrogated upon DC101 treatment. When tested if the expression of soluble Flt (sFlt), which is known to trap VEGF, is downregulated in the absence of Jam-A, we found no significant difference in sFlt mRNA expression in Jam-A null endothelial cells compared to WT. In order to determine the mechanism of this upregulation of the VEGF signaling axis, we tested the expression of hypoxia inducible factor 1a (HIF1-a) and inhibitor of DNA binding 1 (Id1), two transcription factors known to upregulate VEGF and VEGFR2 gene expression. Interestingly, mRNA and protein levels of both HIF1-a and Id1 were significantly augmented (P<0.02) in endothelial cells lacking Jam-A. Consistent with this finding, the overexpression of JAM-A in HUVECs attenuated the levels of Id1. These results suggest that JAM-A suppresses VEGF/VEGFR2 expression in endothelial cells by attenuating HIF1-a and Id1 expression, thus suppressing adult angiogenesis. During pathological conditions such as ischemia and tumor growth, it is possible that JAM-A levels are downregulated, thus supporting pathological angiogenesis. Disclosures:No relevant conflicts of interest to declare.

Junctional adhesion molecule-A is co-expressed with HER2 in breast tumors and acts as a novel regulator of HER2 protein degradation and signaling

Junctional adhesion molecule-A (JAM-A) is a membranous cell-cell adhesion protein involved in tight-junction formation in epithelial and endothelial cells. Its overexpression in breast tumors has recently been linked with increased risk of metastasis. We sought to identify if JAM-A overexpression was associated with specific subtypes of breast cancer as defined by the expression of human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER) and progesterone receptor. To this end, JAM-A immunohistochemistry was performed in two breast cancer tissue microarrays. In parallel, cross-talk between JAM-A, HER2 and ER was examined in several breast cell lines, using complementary genetic and pharmacological approaches. High JAM-A expression correlated significantly with HER2 protein expression, ER negativity, lower patient age, high-grade breast cancers, and aggressive luminal B, HER2 and basal subtypes of breast cancer. JAM-A and HER2 were co-expressed at high levels in vitro in SKBR3, UACC-812, UACC-893 and MCF7-HER2 cells. Knockdown or functional antagonism of HER2 did not alter JAM-A expression in any cell line tested. Interestingly, however, JAM-A knockdown decreased HER2 and ER-α expression, resulting in reduced levels of phospho-(active) AKT without an effect on the extracellular signal-related kinase phosphorylation. The downstream effects of JAM-A knockdown on HER2 and phospho-AKT were partially reversed upon treatment with the proteasomal inhibitor MG132. We conclude that JAM-A is co-expressed with HER2 and associates with aggressive breast cancer phenotypes. Furthermore, we speculate that JAM-A may regulate HER2 proteasomal degradation and activity, potentially offering a promise as a therapeutic target in HER2-positive breast cancers.

Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase

IntroductionThe adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells.MethodsMCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation.ResultsJAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6 and the Rap1 activator PDZ-GEF2 in MCF7 cells and in primary cultures from breast cancer patients.ConclusionsOur findings provide compelling evidence of a novel role for JAM-A in driving breast cancer cell migration via activation of Rap1 GTPase and β1-integrin. We speculate that JAM-A over-expression in some breast cancer patients may represent a novel therapeutic target to reduce the likelihood of metastasis.

JAM‐A expression positively correlates with poor prognosis in breast cancer patients

The cell-cell adhesion protein junctional adhesion molecule-A (JAM-A) influences epithelial cell morphology and migration. As migration is required for tumor cell invasion and metastasis, we sought to elucidate the role of JAM-A in invasive breast cancer. A breast cancer tissue microarray was analyzed for JAM-A protein expression, in parallel with analysis of JAM-A gene expression data from a breast cancer clinical dataset. Our data demonstrate a novel association between JAM-A gene and protein upregulation and poor prognosis in breast cancer. To mechanistically dissect this process, we used lentiviral technology to stably knock down JAM-A gene expression by shRNA in MCF7 breast cancer cells, which express high-endogenous levels of JAM-A. We also antagonized JAM-A function in wild-type MCF7 cells using an inhibitory antibody that blocks JAM-A dimerization. Knockdown or functional antagonism of JAM-A decreased breast cancer cell migration in scratch-wound assays. Reductions in beta1-integrin protein levels were observed after JAM-A-knockdown in MCF7 cells, suggesting a mechanism for reduced motility after loss of JAM-A. Consistent with this hypothesis, tissue microarray analysis of beta1-integrin protein expression in invasive breast cancer tissues revealed a trend toward high beta1-integrin protein levels being indicative of poor prognosis. Twenty-two percent of patients were observed to coexpress high levels of JAM-A and beta1-integrin protein, and MDA-MB-231 breast cells stably overexpressing JAM-A showed an increase in beta1-integrin protein expression. Our results are consistent with a previously unreported role for JAM-A overexpression as a possible mechanism contributing to progression in primary breast cancer; and a potential therapeutic target.

Attenuation of Junctional Adhesion Molecule-A Is a Contributing Factor for Breast Cancer Cell Invasion

The metastatic potential of cancer cells is directly attributed to their ability to invade through the extracellular matrix. The mechanisms regulating this cellular invasiveness are poorly understood. Here, we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is a key negative regulator of cell migration and invasion. JAM-A is robustly expressed in normal human mammary epithelium, and its expression is down-regulated in metastatic breast cancer tumors. In breast cancer cell lines, an inverse relationship between JAM-A expression and the ability of these cells to migrate on a collagen matrix was observed, which correlates with the known ability of these cells to metastasize. The T47D and MCF-7 cells, which migrate least, are found to express high levels of JAM-A, whereas the more migratory MDA-MB-468 cells have lower levels of JAM-A on the cell surface. MDA-MB-231 cells, which are highly migratory, express the least amount of JAM-A. Overexpression of JAM-A in MDA-MB-231 cells inhibited both migration and invasion through collagen gels. Furthermore, knockdown of JAM-A using short interfering RNAs enhanced the invasiveness of MDA-MB-231 cells as well as T47D cells. The ability of JAM-A to attenuate cell invasion correlated with the formation of increased numbers of focal adhesions and the formation of functional tight junctions. These results show for the first time that an immunoglobulin superfamily cell adhesion protein expressed at tight junctions could serve as a key negative regulator of breast cancer cell invasion and possibly metastasis. Furthermore, loss of JAM-A could be used as a biomarker for aggressive breast cancer.

Junctional adhesion molecule‐A regulates cell migration and resistance to shear stress

Junctional adhesion molecule-A (JAM-A) is an adhesive protein expressed in endothelial cells, epithelial cells, platelets, and some leukocytes. JAM-A localizes to the tight junctions between contacting endothelial and epithelial cells, where it contributes to cell-cell adhesion and to the control of paracellular permeability. JAM-A also regulates cell motility, even though the quantitative biophysical features have not been characterized. In this study, we evaluated the role of JAM-A in the regulation of cell motility using JAM-A-expressing and JAM-A-deficient murine endothelial cells. We report that, in the absence of shear stress, JAM-A absence increases cell motility by increasing directional persistence but not cell speed. In addition, in the presence of shear stress, JAM-A absence increases protrusion extension in the direction of flow and increased downstream cellular displacement (while, conversely, decreasing upstream displacement). All these effects of JAM-A absence are mitigated by the microtubule-stabilizing compound taxol. A motility- and microtubule-related function, integrin-mediated adhesiveness, was only slightly reduced in JAM-A-deficient cells compared with JAM-A-expressing cells. However, overexpression of JAM-A in the JAM-A-deficient cells increased integrin adhesiveness to the same levels as those observed in taxol-treated JAM-A-deficient cells. Taken together, these data indicate that JAM-A regulates cell motility by cooperating with microtubule-stabilizing pathways.