Junctional Adhesion molecule-A Protein Research Articles

Zinc alleviates thermal stress-induced damage to the integrity and barrier function of cultured chicken embryonic primary jejunal epithelial cells via the MAPK and PI3K/AKT/mTOR signaling pathways

Zinc (Zn) could alleviate the adverse effect of high temperature (HT) on intestinal integrity and barrier function of broilers, but the underlying mechanisms remain unclear. We aimed to investigate the possible protective mechanisms of Zn on primary cultured broiler jejunal epithelial cells exposed to thermal stress (TS). In Exp.1, jejunal epithelial cells were exposed to 40℃ (normal temperature, NT) and 44℃ (HT) for 1, 2, 4, 6, or 8 h. Cells incubated for 8 h had the lowest transepithelial resistance (TEER) and the highest phenol red permeability under HT. In Exp.2, the cells were preincubated with different Zn sources (Zn sulfate as iZn and Zn proteinate with the moderate chelation strength as oZn) and Zn supplemental levels (50 and 100 µmol/L) under NT for 24 h, and then continuously incubated under HT for another 8 h. TS increased phenol red permeability, lactate dehydrogenase (LDH) activity and p-PKC/PKC level, and decreased TEER, cell proliferation, mRNA levels of claudin-1, occludin, zona occludens-1 (ZO-1), PI3K, AKT and mTOR, protein levels of claudin-1, ZO-1 and junctional adhesion molecule-A (JAM-A), and the levels of p-ERK/ERK, p-PI3K/PI3K and p-AKT/AKT. Under HT, oZn was more effective than iZn in increasing TEER, occludin, ZO-1, PI3K, and AKT mRNA levels, ZO-1 protein level, and p-AKT/AKT level; supplementation with 50 μmol Zn/L was more effective than 100 μmol Zn/L in increasing cell proliferation, JAM-A, PI3K, AKT, and PKC mRNA levels, JAM-A protein level, and the levels of p-ERK/ERK and p-PI3K/PI3K; furthermore, supplementation with 50 μmol Zn/L as oZn had the lowest LDH activity, and the highest ERK, JNK-1, and mTOR mRNA levels. Therefore, supplemental Zn, especially 50 μmol Zn/L as oZn, could alleviate the TS-induced integrity and barrier function damage of broiler jejunal epithelial cells possibly by promoting cell proliferation and tight junction protein expression via the MAPK and PI3K/AKT/mTOR signaling pathways.

Generation and characterization of mAb 61H9 against junctional adhesion molecule-a with potent antitumor activity.

Junctional adhesion molecule-A (JAM-A) is an adhesion molecule that exists on the surface of certain types of cells, including white blood cells, endothelial cells, and dendritic cells. In this study, the cDNA sequences of JAM-A-Fc were chemically synthesized with optimization for mammalian expression. Afterward, we analyzed JAM-A protein expression through transient transfection in HEK293 cell lines. Mice were immunized with JAM-A-Fc protein, and hybridoma was prepared by fusing myeloma cells and mouse spleen cells. Antibodies were purified from the hybridoma supernatant and four monoclonal strains were obtained and numbered 61H9, 70E5, 71A8, and 74H3 via enzyme-linked immunosorbent assay screening. Immunofluorescence staining assay showed 61H9 was the most suitable cell line for mAb production due to its fluorescence signal being the strongest. Flow cytometric analysis proved that 61H9 possessed high affinity. Moreover, antagonism of JAM-A mAb could attenuate the proliferative, migrative, and invasive abilities of ESCC cells and significantly inhibit tumor growth in mice. By examining hematoxylin-eosin staining mice tumor tissues, we found inflammatory cells infiltrated lightly in the anti-JAM-A group. The expression of BCL-2 and IκBα in the anti-JAM-A group were decreased in mice tumor tissues compared to the control group. Ultimately, a method for preparing high-yield JAM-A-Fc protein was created and a high affinity mAb against JAM-A with an antitumor effect was prepared.

Junctional adhesion molecule-A deletion increases phagocytosis and improves survival in a murine model of sepsis

Expression of the tight junction–associated protein junctional adhesion molecule-A (JAM-A) is increased in sepsis, although the significance of this is unknown. Here, we show that septic JAM-A –/– mice have increased gut permeability, yet paradoxically have decreased bacteremia and systemic TNF and IL-1β expression. Survival is improved in JAM-A–/– mice. However, intestine-specific JAM-A–/– deletion does not alter mortality, suggesting that the mortality benefit conferred in mice lacking JAM-A is independent of the intestine. Septic JAM-A–/– mice have increased numbers of splenic CD44hiCD4+ T cells, decreased frequency of TNF+CD4+ cells, and elevated frequency of IL-2+CD4+ cells. Septic JAM-A–/– mice have increased numbers of B cells in mesenteric lymph nodes with elevated serum IgA and intraepithelial lymphocyte IgA production. JAM-A–/– × RAG–/– mice have improved survival compared with RAG–/– mice and identical mortality as WT mice. Gut neutrophil infiltration and neutrophil phagocytosis are increased in JAM-A–/– mice, while septic JAM-A–/– mice depleted of neutrophils lose their survival advantage. Therefore, increased bacterial clearance via neutrophils and an altered systemic inflammatory response with increased opsonizing IgA produced through the adaptive immune system results in improved survival in septic JAM-A–/– mice. JAM-A may be a therapeutic target in sepsis via immune mechanisms not related to its role in permeability.

Chronic Ethanol Exposure of Basal Cells causes Barrier Dysfunction of Differentiated Mucocilliary Monolayers

Alcohol use disorder affects over 14 million adults and causes 88,000 deaths each year in the United States. Chronic alcohol use significantly increases people’s risk of developing lung infections and acute respiratory distress syndrome (ARDS). This increased sensitivity to injury is a condition known as alcoholic lung syndrome, and it is caused by dysfunction of the lung immune system and alveolar epithelial barrier. However, little is known about how alcohol impacts epithelial cells in the conducting airway, i.e. the trachea and bronchi, which is the first line of defense against infectious pathogens in the lungs. We hypothesize that in vitro ethanol exposure decreases barrier function of human primary bronchial epithelial cells. To test this, we dosed cells with ethanol at various timepoints: 1) during both the basal‐cell expansion and differentiation stages 2) the first 14 days of differentiation on Transwell permeable supports at air liquid interface (ALI); 3) beginning on day 14 of differentiation and continuing for 10 days. We treated cells with 10mM, 60mM or 100mM ethanol almost daily and measured barrier function by transepithelial electrical resistance (TER). We found that ethanol caused a dose‐dependent decrease in barrier function, but only in the group treated during both the expansion and differentiation stages. This suggests that chronically dosing basal cells with ethanol is required for barrier dysfunction of a fully differentiated cell monolayer. Additionally, we cultured primary airway epithelial cells isolated from healthy and alcoholic patients on Transwell permeable supports and differentiated them in vitro to determine if in vivo exposure caused long‐term changes to tight junction protein levels. It is known that the transmembrane protein junctional adhesion molecule A (JAM‐A) regulates barrier function by controlling the paracellular flow of small molecules as part of the tight junction barrier. Our results show in response to alcohol exposure, cells have lower JAM‐A protein expression compared to healthy cells. Together, these results indicate that chronic alcohol exposure detrimentally alters the conducting airway epithelial cell barrier potentially through the loss of JAM‐A. Future directions include determining if other tight junction proteins are altered by alcohol and whether the alcohol‐induced reduction in JAM‐A is sufficient to cause barrier dysfunction in airway cells.

A Transcriptional Link between HER2, JAM-A and FOXA1 in Breast Cancer.

Overexpression of the human epidermal growth factor receptor-2 (HER2) is associated with aggressive disease in breast and certain other cancers. At a cellular level, the adhesion protein Junctional Adhesion Molecule-A (JAM-A) has been reported to regulate the expression of HER3 via a transcriptional pathway involving FOXA1. Since FOXA1 is also a suggested transcription factor for HER2, this study set out to determine if JAM-A regulates HER2 expression via a similar mechanism. An integrated tripartite approach was taken, involving cellular expression studies after targeted disruption of individual players in the putative pathway, in silico identification of relevant HER2 promoter regions and, finally, interrogation of cancer patient survival databases to deconstruct functionally important links between HER2, JAM-A and FOXA1 gene expression. The outcome of these investigations revealed a unidirectional pathway in which JAM-A expression transcriptionally regulates that of HER2 by influencing the binding of FOXA1 to a specific site in the HER2 gene promoter. Moreover, a correlation between JAM-A and HER2 gene expression was identified in 75% of a sample of 40 cancer types from The Cancer Genome Atlas, and coincident high mean mRNA expression of JAM-A, HER2 and FOXA1 was associated with poorer survival outcomes in HER2-positive (but not HER2-negative) patients with either breast or gastric tumors. These investigations provide the first evidence of a transcriptional pathway linking JAM-A, HER2 and FOXA1 in cancer settings, and support potential future pharmacological targeting of JAM-A as an upstream regulator of HER2.

Alcohol Decreases the Barrier Function of Airway Epithelial cells through the TGFß1 Pathway

Alcohol use disorder affects over 14 million adults and causes 88,000 deaths each year in the United States. Chronic alcohol use significantly increases people's risk of developing lung infections and acute respiratory distress syndrome (ARDS). This increased sensitivity to injury is a condition known as alcoholic lung syndrome, and it is caused by dysfunction of the lung immune system and alveolar epithelial barrier. However, little is known about how alcohol impacts epithelial cells in the conducting airway, i.e. the trachea and bronchi, which are the first line of defense against infectious pathogens in the lung. We hypothesize that alcohol exposure causes a decrease in barrier function of airway epithelial cells through a TGFß1-dependent mechanism. To test this, we cultured primary airway epithelial cells isolated from rats and humans on Transwell permeable supports and differentiated them in vitro at air liquid interface (ALI). We treated cells in vitro with 60mM or 100mM ethanol changed daily for 2 weeks and measured barrier function by transepithelial electrical resistance (TER). We found that ethanol caused a decrease in barrier function compared to control cells. However, cells treated with ethanol and a TGFß1 inhibitor had barrier function similar to control cells. Additionally, we cultured primary airway epithelial cells isolated from healthy and alcoholic patients on Transwell permeable supports and differentiated them in vitro to determine if in vivo exposure caused long-term changes to tight junction protein levels. It is known that the transmembrane protein junctional adhesion molecule A (JAM-A) regulates barrier function by controlling the paracellular flow of small molecules as part of the tight junction barrier. Our results show in response to alcohol exposure, cells have lower JAM-A protein expression compared to healthy cells. Rat airway cells exposed to ethanol in vitro also show a decrease in JAM-A at both the RNA and protein level. Together, these results indicate that chronic alcohol exposure detrimentally alters the conducting airway epithelial cell barrier potentially through the loss of JAM-A. Future directions include determining if other tight junction proteins are altered by alcohol and whether the alcohol-induced reduction in JAM-A is sufficient to cause barrier dysfunction in airway cells.

JAM‐A signals through the Hippo pathway to regulate intestinal epithelial proliferation

The intestinal mucosa is lined by a columnar epithelial monolayer that serves as a dynamic interface, regulating barrier function and homeostasis. Perturbation of homeostasis, including changes in intestinal epithelial cell (IEC) proliferation and migration, can give rise to pathological states including ulcerative colitis and cancer. Regulation of proliferation and migration is a complex process coordinated, in part, by signaling events emanating from tight junction proteins at cell-cell contacts. One tight junction protein that has been linked to regulation of cellular proliferation is Junctional Adhesion Molecule A (JAM-A). JAM-A forms homodimers in cis and trans through interactions between its extracellular immunoglobulin-like domains. Loss of JAM-A results in enhanced IEC proliferation that has been linked to loss of homophilic interactions and Akt-dependent, beta-catenin signaling. However, JAM-A contributions to other signaling pathways that regulate cell proliferation are poorly understood. Since the Hippo signaling pathway is a well-known regulator of cellular proliferation, differentiation, and apoptosis, we examined whether loss of JAM-A alters Hippo pathway activity. We assessed activity of the Hippo transcriptional coactivator, Yes-1 Associated Protein (YAP), in JAM-A deficient IEC by gene-reporter activity assay and immunoblotting and observed significant increases, suggesting that JAM-A may interact with Hippo pathway constituents upstream of YAP. Immunolabeling studies with monolayers of IEC revealed association of JAM-A with two Hippo pathway constituents, Large Tumor Suppressor Kinase (LATS1) and Merlin. Co-immunoprecipitation experiments confirmed LATS1 and Merlin association with JAM-A. Moreover, phosphoprotein specific immunoblotting studies on JAM-A deficient IEC revealed decreased Hippo pathway activity characterized by decreased LATS1 activity and subsequent increased YAP activity. Such findings are consistent with JAM-A signaling upstream of Merlin and LATS1 and coordinating downstream Hippo signaling. Since there is evidence suggesting that dimerization of JAM-A is a required signal that suppresses IEC proliferation, we performed experiments using an exogenous overexpression system to study effects of a dimerization deficient JAM-A mutant on Hippo signaling. Phosphoprotein specific immunoblotting and EdU incorporation revealed that overexpression of full-length JAM-A resulted in activation of Hippo signaling, suppressing cell proliferation compared to empty vector control cells. However, overexpression of a dimerization defective JAM-A mutant protein lacking the distal immunoglobulin loop (DL1) was unable to activate Hippo signaling and suppress cell proliferation. Based on these observations, we propose that homodimerization of JAM-A functions as a cell-cell contact sensor, initiating downstream Hippo signaling to suppress IEC proliferation. These findings have important implications for understanding effects of altered proliferation on mucosal wound repair, as well as in morbidity associated with certain cancers which have been reported to express decreased levels of JAM-A protein.

Development of a Novel Weighted Ranking Method for Immunohistochemical Quantification of a Heterogeneously Expressed Protein in Gastro-Esophageal Cancers.

Simple SummaryHigh levels of the protein Junctional Adhesion Molecule-A (JAM-A) have been linked with aggressive disease in patients with several different cancers. However, its distribution is often non-uniform (heterogeneous) across tumors, and can be difficult to quantify. JAM-A has also been linked with high levels of HER2 (an important oncogene) in breast tumors, and the development of resistance to HER2-targeted drugs in those patients. Since gastro-esophageal (GE) cancers are often high in HER2 and have also been approved for HER2-targeted drugs, the aim of this study was to investigate if levels of JAM-A and HER2 are linked in GE cancer. JAM-A was expressed very heterogeneously across miniaturized tissue sections called tissue microarrays (TMAs) of GE cancer patients. In this model, therefore, there was no correlation between JAM-A and HER2 expression. However, when we used larger tissue sections and developed a scoring system to account for heterogeneity, a significant correlation between JAM-A and HER2 levels emerged. This work illustrates the importance of taking intra-tumor heterogeneity into account, particularly in an era when analysis of protein levels by this method is increasingly used to select patients for targeted cancer drugs.High expression of Junctional Adhesion Molecule-A (JAM-A) has been linked with poor prognosis in several cancers, including breast cancers overexpressing the human epidermal growth factor receptor-2 (HER2). Furthermore, JAM-A expression has been linked with regulating that of HER2, and associated with the development of resistance to HER2-targeted therapies in breast cancer patients. The purpose of this study was to establish a potential relationship between JAM-A and HER2 in HER2-overexpressing gastro-esophageal (GE) cancers. Interrogation of gene expression datasets revealed that high JAM-A mRNA expression was associated with poorer survival in HER2-positive gastric cancer patients. However, high intra-tumoral heterogeneity of JAM-A protein expression was noted upon immunohistochemical scoring of a GE cancer tissue microarray (TMA), precluding a simple confirmation of any relationship between JAM-A and HER2 at protein level. However, in a test-set of 25 full-face GE cancer tissue sections, a novel weighted ranking system proved effective in capturing JAM-A intra-tumoral heterogeneity and confirming statistically significant correlations between JAM-A/HER2 expression. Given the growing importance of immunohistochemistry in stratifying cancer patients for the receipt of new targeted therapies, this may sound a cautionary note against over-relying on cancer TMAs in biomarker discovery studies of heterogeneously expressed proteins. It also highlights a timely need to develop validated mechanisms of capturing intra-tumoral heterogeneity to aid in future biomarker/therapeutic target development for the benefit of cancer patients.

Tight Junction Protein Junctional Adhesion Molecule-A Regulates the Expression of Receptor Tyrosine Kinase EPHA2 In Triple-Negative Breast Cancer Cells

Breast tumors lacking expression of the human epidermal growth factor receptor-2 (HER2), progesterone receptor (PR) and estrogen receptor (ER?) are defined as triple negative breast cancers (TNBC). A lack of targeted therapies has impaired TNBC patient prognosis. It has previously been shown that high expression of Junctional Adhesion Molecule-A (JAM-A) correlates with aggressive breast cancer patient phenotypes, and that JAM-A regulates the expression of HER2 in breast cancer cells. Accordingly, we hypothesized that JAM-A might regulate the expression of other receptor tyrosine kinases. We show for the first time that JAM-A may regulate the expression of the EPHA2 receptor in TNBC cells and propose that this pathway merits deeper investigation for its therapeutic value in TNBC settings.

TGR5-dependent hepatoprotection through the regulation of biliary epithelium barrier function

ObjectiveWe explored the hypothesis that TGR5, the bile acid (BA) G-protein-coupled receptor highly expressed in biliary epithelial cells, protects the liver against BA overload through the regulation of biliary epithelium...

Natural compound Tetrocarcin-A downregulates Junctional Adhesion Molecule-A in conjunction with HER2 and inhibitor of apoptosis proteins and inhibits tumor cell growth

Overexpression of the tight junction protein Junctional Adhesion Molecule-A (JAM-A) has been linked to aggressive disease in breast and other cancers, but JAM-targeting drugs remain elusive. Screening of a natural compound library identified the antibiotic Tetrocarcin-A as a novel downregulator of JAM-A and human epidermal growth factor receptor-2 (HER2) protein expression in breast cancer cells. Lysosomal inhibition partially rescued the downregulation of JAM-A and HER2 caused by Tetrocarcin-A, and attenuated its cytotoxic activity. Tetrocarcin-A treatment or JAM-A silencing reduced AKT and ERK phosphorylation, inhibited c-FOS phosphorylation at Threonine-232 (its transcriptional regulation site), inhibited nuclear localization of c-FOS, and downregulated expression of the inhibitor of apoptosis proteins (IAP). This was accompanied by Tetrocarcin-A-induced caspase-dependent apoptosis. To begin evaluating the potential clinical relevance of our findings, we extended our studies to other models. Encouragingly, Tetrocarcin-A downregulated JAM-A expression and caused cytotoxicity in primary breast cells and lung cancer stem cells, and inhibited the growth of xenografts in a semi-in vivo model involving invasion across the chicken egg chorioallantoic membrane. Taken together, our data suggest that Tetrocarcin-A warrants future evaluation as a novel cancer therapeutic by virtue of its ability to downregulate JAM-A expression, reduce tumorigenic signaling and induce apoptosis.

Abstract 2670: Natural compound tetrocarcin-A downregulates junctional adhesion molecule-A in conjunction with erα, her2 and inhibitor of apoptosis proteins and inhibits tumor cell growth

Abstract Overexpression of the tight junction protein Junctional Adhesion Molecule-A (JAM-A) has been linked to aggressive disease in breast and several other cancers, but JAM-targeting drugs remain elusive. Screening of a natural compound library identified the cytotoxic antibiotic Tetrocarcin-A as a novel downregulator of JAM-A protein expression in breast cancer cells. Specifically, Tetrocarcin-A-induced cytotoxicity was accompanied by downregulation of JAM-A protein expression in conjunction with that of estrogen receptor alpha (ERα) and human epidermal growth factor receptor 2 (HER2) in MCF7-HER2-overexpressing breast cells. Lysosomal inhibition partially rescued the downregulation of JAM-A, HER2 and ERα caused by Tetrocarcin-A, and attenuated its cytotoxicity. Interestingly, Tetrocarcin-A repressed the transcriptional activity of c-FOS by inhibiting ERK1/2-mediated phosphorylation at Threonine-232, and downregulated protein expression of the inhibitor of apoptosis proteins (IAP). Investigation of cell death pathways revealed that Tetrocarcin-A induced caspase-dependent apoptosis. To begin testing the potential clinical relevance of our findings, we extended our studies into various other models. Encouragingly, Tetrocarcin-A downregulated JAM-A expression and caused cytotoxicity in primary breast cancer and lung cancer stem cells, and inhibited the growth of model human breast tumors in an in vivo model involving invasion across the chicken egg chorioallantoic membrane. Taken together, our data suggest that Tetrocarcin-A merits future evaluation as a novel cancer therapeutic by virtue of its ability to downregulate JAM-A expression, induce apoptosis and reduce tumorigenic signalling. This work was financially supported by Science Foundation Ireland (grant 13/IA/1994 to AMH). Tetrocarcin-A was provided by the NCI/DTP Open Chemical Repository https://dtp.cancer.gov. (NSC 333856) Citation Format: Sri Harikrishna Vellanki, Lance Hudson, Hanne Jahns, Rodrigo G.B. Cruz, Giovanni Sette, Adriana Eramo, Ann M. Hopkins. Natural compound tetrocarcin-A downregulates junctional adhesion molecule-A in conjunction with erα, her2 and inhibitor of apoptosis proteins and inhibits tumor cell growth [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 2670.

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 as a prognostic factor and new therapeutic target in multiple myeloma

Cell adhesion in the multiple myeloma (MM) microenvironment has been recognized as a major mechanism of MM cell survival and the development of drug resistance. Here we addressed the hypothesis that the protein junctional adhesion molecule-A (JAM-A) may represent a novel target and a clinical biomarker in MM. We evaluated JAM-A expression in MM cell lines and in 147 MM patient bone marrow aspirates and biopsies at different disease stages. Elevated JAM-A levels in patient-derived plasma cells were correlated with poor prognosis. Moreover, circulating soluble JAM-A (sJAM-A) levels were significantly increased in MM patients as compared with controls. Notably, in vitro JAM-A inhibition impaired MM migration, colony formation, chemotaxis, proliferation and viability. In vivo treatment with an anti-JAM-A monoclonal antibody (αJAM-A moAb) impaired tumor progression in a murine xenograft MM model. These results demonstrate that therapeutic targeting of JAM-A has the potential to prevent MM progression, and lead us to propose JAM-A as a biomarker in MM, and sJAM-A as a serum-based marker for clinical stratification.

Expression and prognostic value of JAM-A in gliomas

Gliomas are among the most lethal cancers, being highly resistant to both chemo- and radiotherapy. The expression of junctional adhesion molecule-A (JAM-A) was recently identified on the surface of stem cell-like brain tumor-initiating cells and suggested to function as a unique glioblastoma niche adhesion factor influencing the tumorigenic potential of brain tumor-initiating cells. We have recently identified high JAM-A expression to be associated with poor outcome in glioblastomas, and our aim was to further investigate the expression of JAM-A in gliomas focusing especially on the prognostic value in WHO grade II and III gliomas. JAM-A protein expression was evaluated by immunohistochemistry and advanced quantitative image analysis with continuous estimates of staining intensity. The JAM-A antibody stained tumor cell membranes and cytoplasm to various extent in different glioma subtypes, and the intensity was higher in glioblastomas than low-grade gliomas. We could not detect an association with overall survival in patients with grade II and III tumors. Double-immunofluorescence stainings in glioblastomas revealed co-expression of JAM-A with CD133, SOX2, nestin, and GFAP in tumor cells as well as some co-expression with the microglial/macrophage marker IBA-1. In conclusion, JAM-A expression was higher in glioblastomas compared to low-grade gliomas and co-localized with recognized stem cell markers suggesting an association of JAM-A with glioma aggressiveness. No significant association between JAM-A expression and overall survival was found in grade II and III gliomas. Further research is needed to determine the function and clinical impact of JAM-A in gliomas.

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

JAM-A as a Prognostic Factor and New Therapeutic Target in Multiple Myeloma

Cell adhesion in the multiple myeloma (MM) microenvironment is a mechanism by which MM plasma cells escape the effects of therapy and survive. To improve clinical strategies and overcome drug resistance, approaches directed to both MMPCs and bone marrow microenvironment are under investigation. Here, we examined the cell membrane protein Junctional adhesion molecule-A (JAM-A) as a clinical biomarker and novel therapeutic target for MM.We evaluated JAM-A expression by real time PCR (RT-PCR), flow cytometry and immunofluorescence microscopy in 132 MM patients at different stages and various MM cell lines. Next, we measured the concentrations of soluble JAM-A from MM and healthy subjects sera by enzyme linked immune assay (ELISA). We investigated JAM-A functionally in vitro and in vivo by transient gene silencing (siRNA) and with blocking antibodies.Patient-derived plasma cells (MMPCs) expressed increased JAM-A expression levels when compared to control PC from healthy individuals. Elevated JAM-A expression correlated with poor prognosis (Figure 1A,B). Furthermore, soluble JAM-A was significantly increased in MM patient sera when compared to healthy subjects. Additionally, MM cell lines showed high expression of both membrane and cytoplasmic JAM-A. Consequently, inhibition of JAM-A using specific siRNA treatment resulted in diminished tumorigenic potential, including decreased colony formation, chemotaxis and migration. Importantly, treatment of luciferase+RPMI-8226 MM bearing NSG with a JAM-A blocking monoclonal antibody reduced significantly MM progression and dissemination in vivo when compared to MM bearing mice that received an non-specific isotype control antibody (Figure 1C).Conclusively, our data suggest that JAM-A can serve as a biomarker of malignancy in MM patients. Soluble plasma JAM-A could contribute to serum-based clinical stratification. Furthermore, therapeutic targeting of JAM-A appears attractive for clinical translation. [Display omitted] DisclosuresEinsele:Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria.

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.

Abstract 3328: Novel regulation of HER2 degradation and breast cancer cell proliferation by junctional adhesion molecule-a (JAM-A)

Abstract Background: We have previously shown that the adhesion protein Junctional Adhesion Molecule-A (JAM-A) is over-expressed in a population of aggressive and metastatic breast tumours, and that it regulates the migration of breast cells. JAM-dependent regulation of migration could contribute to the likelihood of metastatic disease, since migration is an early event in metastasis. Our objective was to further interrogate the links between JAM-A signalling and markers of disease progression in breast cancer. Approach and Results: Immunohistochemical analysis of JAM-A in two breast cancer tissue microarrays (TMA) revealed that high JAM-A expression correlated significantly with HER2 protein expression, ERnegativity, high grade breast cancers and aggressive luminal B, HER2 and basal subtypes of breast cancer. Using LCC1 and MCF7-HER2 cells which co-express high levels of JAM-A and HER2, we silenced the gene expression of each one in order to examine potential consequences for protein expression of the other. Knockdown of HER2 had no effect on JAM-A protein levels, but knockdown of JAM-A significantly reduced the protein expression of HER2 and its downstream pro-survival effector pAkt. Accordingly, JAM-A knockdown increased apoptosis via a pathway involving Akt activation, as evidenced by apoptosis normalisation upon pharmacological inhibition of Akt. The expressional regulation of HER2 and pAkt by JAM-A was sensitive to inhibition by the proteasomal inhibitor MG132, suggesting that JAM-A regulates the proteasomal degradation of HER2. Finally, over-expression of wild-type JAM-A was sufficient to significantly enhance proliferative and migratory behaviour in breast cell lines. Conclusions: Our results are consistent with a model whereby JAM-A expression inhibits the proteasomal degradation of HER2, facilitating a pro-survival phenotype through increased Akt activity. Therefore JAM-A antagonists may have future therapeutic value in the treatment of breast tumours overexpressing HER2. Citation Format: Kieran Brennan, Elaine A. McSherry, Lance Hudson, Elaine W. Kay, Leonie S. Young, Arnold DK Hill, Ann M. Hopkins. Novel regulation of HER2 degradation and breast cancer cell proliferation by junctional adhesion molecule-a (JAM-A). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3328. doi:10.1158/1538-7445.AM2014-3328