Junctional Adhesion molecule-A Expression Research Articles

Junctional adhesion molecule-A deficient mice are protected from severe experimental autoimmune encephalomyelitis.

In multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), early pathological features include immune cell infiltration into the central nervous system (CNS) and blood-brain barrier (BBB) disruption. We investigated the role of junctional adhesion molecule-A (JAM-A), a tight junction protein, in active EAE (aEAE) pathogenesis. Our study confirms JAM-A expression at the blood-brain barrier and its luminal redistribution during aEAE. JAM-A deficient (JAM-A-/-) C57BL/6J mice exhibited milder aEAE, unrelated to myelin oligodendrocyte glycoprotein-specific CD4+ T-cell priming. While JAM-A absence influenced macrophage behavior on primary mouse brain microvascular endothelial cells (pMBMECs) under flow in vitro, it did not impact T-cell extravasation across primary mouse brain microvascular endothelial cells. At aEAE onset, we observed reduced lymphocyte and CCR2+ macrophage infiltration into the spinal cord of JAM-A-/- mice compared to control littermates. This correlated with increased CD3+ T-cell accumulation in spinal cord perivascular spaces and brain leptomeninges, suggesting JAM-A absence leads to T-cell trapping in central nervous system border compartments. In summary, JAM-A plays a role in immune cell infiltration and clinical disease progression in aEAE.

Expression of Concern: Expression of junctional adhesion molecule-A prevents spontaneous and random motility.

Expression of Concern: Expression of junctional adhesion molecule-A prevents spontaneous and random motility.

Junctional adhesion molecule-A (JAM-A) in gynecological cancers: Current state of knowledge

Junctional adhesion molecule-A (JAM-A), also known as the F11 receptor (F11R), is one of the tight junction components. JAM-A is a transmembrane glycoprotein that regulates many cellular processes, i.e., angiogenesis, leukocyte transendothelial migration, intercellular permeability, epithelial-to-mesenchymal transition, and platelet activation. Of note, it is involved in the pathogenesis of various cancer types, including gynecological cancers. Only a few studies are available about this cancer type. Observed aberrant JAM-A expression in gynecological cancers correlates with poor patient prognosis. To the best of our knowledge, conflicting JAM-A roles in various cancer types suggest that its involvement is complex and tumor-type specific. The underlying molecular mechanisms and pathways responsible for JAM-A functions were not fully elucidated and need to be identified. Finding appropriate novel molecular cancer biomarkers may reduce observed very high mortality rates and could contribute to personalized treatment development. The main aim of the present viewpoint article is to report the current knowledge about JAM-A participation in gynecological malignancies.

Junctional adhesion molecule-A is dispensable for myeloid cell recruitment and diversification in the tumor microenvironment.

Junctional adhesion molecule-A (JAM-A), expressed on the surface of myeloid cells, is required for extravasation at sites of inflammation and may also modulate myeloid cell activation. Infiltration of myeloid cells is a common feature of tumors that drives disease progression, but the function of JAM-A in this phenomenon and its impact on tumor-infiltrating myeloid cells is little understood. Here we show that systemic cancer-associated inflammation in mice enhanced JAM-A expression selectively on circulating monocytes in an IL1β-dependent manner. Using myeloid-specific JAM-A-deficient mice, we found that JAM-A was dispensable for recruitment of monocytes and other myeloid cells to tumors, in contrast to its reported role in inflammation. Single-cell RNA sequencing revealed that loss of JAM-A did not influence the transcriptional reprogramming of myeloid cells in the tumor microenvironment. Overall, our results support the notion that cancer-associated inflammation can modulate the phenotype of circulating immune cells, and we demonstrate that tumors can bypass the requirement of JAM-A for myeloid cell recruitment and reprogramming.

SerpinB3 drives cancer stem cell survival in glioblastoma.

SUMMARYDespite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. Knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a GBM CSC-specific survival mechanism involving a cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of GBM therapies against therapeutically resistant CSCs.

Functional Antagonism of Junctional Adhesion Molecule-A (JAM-A), Overexpressed in Breast Ductal Carcinoma In Situ (DCIS), Reduces HER2-Positive Tumor Progression.

Simple SummarySpecific drug targets for breast ductal carcinoma in situ (DCIS) remain elusive, despite increasing disease prevalence and burden to healthcare services. Estrogen receptor (ER)-negative HER2-positive DCIS, associated with the poorest patient prognosis, is in particular need of novel therapeutic avenues. This report provides the first evidence that a cell surface protein called JAM-A is upregulated on human DCIS patient tissues and can be readily targeted by a novel JAM-A-binding peptide inhibitor in separate in vivo models of DCIS. The anti-tumor efficacy and lack of systemic toxicity of this lead inhibitor, coupled with early indications of potential signaling pathways implicated, support the value of future studies investigating JAM-A as a novel drug target in DCIS patients.Breast ductal carcinoma in situ (DCIS) is clinically challenging, featuring high diagnosis rates and few targeted therapies. Expression/signaling from junctional adhesion molecule-A (JAM-A) has been linked to poor prognosis in invasive breast cancers, but its role in DCIS is unknown. Since progression from DCIS to invasive cancer has been linked with overexpression of the human epidermal growth factor receptor-2 (HER2), and JAM-A regulates HER2 expression, we evaluated JAM-A as a therapeutic target in DCIS. JAM-A expression was immunohistochemically assessed in patient DCIS tissues. A novel JAM-A antagonist (JBS2) was designed and tested alone/in combination with the HER2 kinase inhibitor lapatinib, using SUM-225 cells in vitro and in vivo as validated DCIS models. Murine tumors were proteomically analyzed. JAM-A expression was moderate/high in 96% of DCIS patient tissues, versus 23% of normal adjacent tissues. JBS2 bound to recombinant JAM-A, inhibiting cell viability in SUM-225 cells and a primary DCIS culture in vitro and in a chick embryo xenograft model. JBS2 reduced tumor progression in in vivo models of SUM-225 cells engrafted into mammary fat pads or directly injected into the mammary ducts of NOD-SCID mice. Preliminary proteomic analysis revealed alterations in angiogenic and apoptotic pathways. High JAM-A expression in aggressive DCIS lesions and their sensitivity to treatment by a novel JAM-A antagonist support the viability of testing JAM-A as a novel therapeutic target in DCIS.

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.

Selegiline Protects Against Lipopolysaccharide (LPS)-Induced Impairment of the Blood-Brain Barrier Through Regulating the NF-κB/MLCK/p-MLC Signaling Pathway.

Disruption of the blood-brain barrier (BBB) is an important hallmark of sepsis-associated encephalopathy (SAE). Selegiline, a selective and irreversible inhibitor of monoamine oxidase type B, has been applied for the treatment of nervous disorders. In this study, we aimed to investigate whether selegiline has a protective capacity in the impairment of the BBB in both in vivo and in vitro experiments. In a sepsis mouse model, administration of selegiline ameliorated lipopolysaccharide (LPS)-induced impairment of BBB integrity. Additionally, treatment with selegiline increased the expression of the tight junction protein junctional adhesion molecule A (JAM-A) against LPS. Also, we found that selegiline inhibited the production of the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β. In an in vitro experimental model, bEnd.3 brain endothelial cells were exposed to LPS. Results indicate that stimulation with LPS significantly increased the permeability of bEnd.3 cells and reduced the expression of JAM-A, both of which were rescued by treatment with selegiline. Additionally, selegiline prevented the activation of the NF-κB/MLCK/p-MLC signaling pathway in LPS-challenged bEnd.3 cells. These results indicate that selegiline exerted a protective effect on BBB dysfunction, which might be attributed to the inhibition of the NF-κB/MLCK/p-MLC signaling pathway. These findings provide a basis for further research into the neuroprotective mechanism of selegiline.

STEM-15. SerpinB3 DRIVES CANCER STEM CELL SURVIVAL IN GLIOBLASTOMA

Abstract Despite therapeutic interventions for glioblastoma (GBM), self-renewing, therapy-resistant populations of cells referred to as cancer stem cells (CSCs) drive recurrence. Previously, we identified the unique expression of junctional adhesion molecule-A (JAM-A) on CSCs and demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. Moreover, we determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the entire signaling network has yet to be fully elucidated. To further delineate this pathway, we immunoprecipitated JAM-A from patient-derived GBM CSCs and performed mass spectrometry to determine JAM-A binding proteins. This led to the identification of the cysteine protease inhibitor SerpinB3 as a putative JAM-A binding partner. Using in vitro CSC functional assays, we show that SerpinB3 is necessary for CSC maintenance and survival. In an in vivo orthotopic xenograft model, knockdown of SerpinB3 extended survival. Mechanistically, knockdown of SerpinB3 led to decreased expression of TGF-β, Myc, WNT, and Notch signaling, known regulators of the CSC state. Additionally, knockdown of SerpinB3 increases susceptibility to radiation therapy. SerpinB3 is essential for buffering cells against cathepsin-mediated cell death, and we found that elevated lysosomal membrane permeability after radiation leads to cathepsin release into the cytoplasm. As a result, SerpinB3 knockdown cells have a diminished capacity to inhibit cathepsin-driven cell death after radiation. The addition of the cathepsin inhibitor E64D partially rescues the SerpinB3 knockdown, however, SerpinB3 mutants that are unable to inhibit cathepsins fail to do the same. Taken together, our findings, identify a novel GBM CSC-specific survival mechanism involving a previously uninvestigated cysteine protease inhibitor, SerpinB3, and provide a potential target to increase the efficacy of standard of care GBM therapies against therapy-resistant CSCs.

OAB-041: Epithelial-mesenchymal-transition regulated by Junctional Adhesion Molecule-A (JAM-A) associates with aggressive extramedullary multiple myeloma disease

<h3>Background</h3> Interactions between multiple myeloma plasma cells (MMPC) and the multiple myeloma (MM) niche control cell proliferation, drug resistance, and disease spreading through various adhesion molecules and chemokine receptors. Among these, junctional adhesion molecule-A (JAM-A) mediates MMPC-endothelial interactions and propagates neovascularization. Here we addressed whether JAM-A signaling mediates MMPC invasive behavior orchestrating intra- and extramedullary MM dissemination. <h3>Methods</h3> We investigated JAM-A expression with flow cytometry and immunohistochemistry in 60 MM patient bone marrow (BM) aspirates and biopsies at different disease stages with and without extramedullary disease (EMD) manifestation. We compared these results with RNA-Seq data from 647 newly diagnosed MM (NDMM) patients collected in the MMRF CoMMpass study. Using bioinformatics, we investigated JAM-A-related pathways with hierarchical cluster analysis. Subsequently, we functionally validated these JAM-A-related pathways regarding epithelial-mesenchymal transition (EMT), invasion, and MM dissemination in vitro. <h3>Results</h3> The median OS differed significantly in subjects with elevated membrane JAM-A expression levels, dividing the patients in the EMD JAM-Ahigh group with a median OS of 84.1 months, whereas median OS in JAM-Alow patients was not reached, irrespective of the EMD status (log-rank=4.19, P=.04). Immunohistochemistry analysis of BM biopsies confirmed these findings. RNA-Seq analysis corroborated the prognostic impact of JAM-A (log-rank=3.8, P=.051). High-risk patients with EMD and JAM-A expression displayed a unique gene-expression signature. Additionally, we constructed a novel expression cluster subgroup model, revealing complex molecular disease patterns and associations between clinical traits and expression markers. Strikingly, MM patients clustered by JAM-A expression levels and EMD manifestation revealed properties of epithelial-mesenchymal-transition and induced focal adhesion pathways. Notably, functional knockdown of JAM-A in MMPC in vitro not only reduced cell viability but also diminished MMPC adhesion molecules and CD138 surface expression. Furthermore, mTOR/PI3K in vitro inhibition with BEZ235 significantly downregulated surface expression in JAM-Ahigh MM cells. Interestingly, when we inhibited mTOR/PI3K in JAM-Alow MM cells, we observed JAM-A induction. Finally, ensuing functional shRNA knockdown of JAM-A halted MM invasion (P<.002), angiogenesis (P<.0001), migration (P<.002), cell survival (P<.001), and expression of cellular-adhesion system molecules such as integrin-beta-1, fibronectin, RAC1 and RHOA (P<.001). Conversely, overexpressing JAM-A in MM.1S cells changed to an EMT-like phenotype compared to empty vector-infected MM.1S cell controls. <h3>Conclusions</h3> Collectively, these data reveal JAM-A-mediated signaling critical for EMT transition and invasive behavior. Based on these findings we propose JAM-A as a promising biomarker and novel theragnostic target in MM patients.

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.

Human Epidermal Growth Factor Receptor-3 Expression Is Regulated at Transcriptional Level in Breast Cancer Settings by Junctional Adhesion Molecule-A via a Pathway Involving Beta-Catenin and FOXA1

Simple SummarySignaling from the human epidermal growth factor receptor (HER) family of proteins increases in many cancers, including breast. HER2-high breast cancers are successfully treated with anti-HER2 therapies, but these drugs are limited by the fact that patients frequently develop resistance to them. One common mechanism by which resistance develops is when tumors acquire high levels of a family member called HER3. We had previously shown that a protein called JAM-A regulates the level of HER2 in breast cancer cells, and is associated with the development of resistance to HER2-targeted therapies. In this study we show for the first time that JAM-A levels also regulate those of HER3. Using breast cancer cell and tissue models and culminating in patient tissue material, we provide evidence that JAM-A regulates HER3 expression via a pathway involving the transcription factors β-catenin and FOXA1. We suggest that JAM-A merits future investigation as a novel drug target for its potential to reduce HER3 tumorigenic signaling and to offset the development of resistance to HER2-targeted therapies. The success of breast cancer therapies targeting the human epidermal growth factor receptor-2 (HER2) is limited by the development of drug resistance by mechanisms including upregulation of HER3. Having reported that HER2 expression and resistance to HER2-targeted therapies can be regulated by Junctional Adhesion Molecule-A (JAM-A), this study investigated if JAM-A regulates HER3 expression. Expressional alteration of JAM-A in breast cancer cells was used to test expressional effects on HER3 and its effectors, alongside associated functional behaviors, in vitro and semi-in vivo. HER3 transcription factors were identified and tested for regulation by JAM-A. Finally a patient tissue microarray was used to interrogate connections between putative pathway components connecting JAM-A and HER3. This study reveals for the first time that HER3 and its effectors are regulated at gene/protein expression level by JAM-A in breast cancer cell lines; with functional consequences in in vitro and semi-in vivo models. In bioinformatic, cellular and patient tissue models, this was associated with regulation of the HER3 transcription factor FOXA1 by JAM-A via a pathway involving β-catenin. Our data suggest a novel model whereby JAM-A expression regulates β-catenin localization, in turn regulating FOXA1 expression, which could drive HER3 gene transcription. JAM-A merits investigation as a novel target to prevent upregulation of HER3 during the development of resistance to HER2-targeted therapies, or to reduce HER3-dependent tumorigenic signaling.

Development of near-infrared imaging agents for detection of junction adhesion molecule-A protein

IntroductionProstate and breast cancer are the most prevalent primary malignant human tumors globally. Prostatectomy and breast conservative surgery remain the most common definitive treatment option for the >500,000 men and women newly diagnosed with localized prostate and breast cancer each year only in the US. Morphological examination is the mainstay of diagnosis but margin under-sampling of the excised cancer tissue may lead to local recurrence. In despite of the progress of non-invasive optical imaging, there is still a clinical need for targeted optical imaging probes that could rapidly and globally visualize cancerous tissues. MethodsElevated expression of junctional adhesion molecule-A (JAM-A) on tumor cells and its multiple pro-tumorigenic activity make the JAM-A a candidate for molecular imaging. Near-infrared imaging probe, which employed anti-JAM-A monoclonal antibody (mAb) phthalocyanine dye IR700 conjugates (JAM-A mAb/IR700), was synthesized and used to identify and visualize heterotopic human prostate and breast tumor mouse xenografts in vivo. ResultsThe intravenously injected JAM-A mAb/IR700 conjugates enabled the non-invasive detection of prostate and breast cancerous tissue by fluorescence imaging. A single dose of JAM-A mAb/IR700 reduced number of mitotic cancer cells in vivo, indicating theranostic ability of this imaging agent. The JAM-A mAb/IR700 conjugates allowed us to image a specific receptor expression in prostate and breast tumors without post-image processing. ConclusionThis agent demonstrates promise as a method to image the extent of prostate and breast cancer in vivo and could assist with real-time visualization of extracapsular extension of cancerous tissue.

LncRNA TP73-AS1 regulates miR-495 expression to promote migration and invasion of nasopharyngeal carcinoma cells through junctional adhesion molecule A.

The main obstacle to the treatment of nasopharyngeal carcinoma (NPC) is metastasis. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are highly involved in the progression of NPC. In this study, we aimed to explore the regulatory role of lncRNA P73 antisense RNA 1 T (TP73-AS1) and miR-495 in migration and invasion of NPC cells. The expression levels of TP73-AS1, miR-495, and junctional adhesion molecule A (JAM-A) in NPC tissue samples and cell lines were examined by quantitative real-time PCR (qRT-PCR) and/or Western blot. NPC cells were transfected with vectors overexpressing TP73-AS1, short hairpin RNA (shRNA) against TP73-AS1, shRNA against JAM-A, miR-495 mimics, miR-495 inhibitor, and their corresponding negative controls as designated. The MTT assay, cell migration assay, and transwell assay were performed to detect cell viability, migration, and invasion, respectively. Dual-luciferase reporter assay was performed to confirm the binding of TP73-AS1 and miR-495, and miR-495 and JAM-A. TP73-AS1 and JAM-A were significantly upregulated while miR-495 was markedly downregulated in NPC tissues and cell lines compared to normal controls. The overexpression of TP73-AS1 promoted migration and invasion of NPC cell line CNE-2. TP73-AS1 targeted miR-495 and negatively regulated its expression. TP73-AS1 upregulated the expression of JAM-A through miR-495. TP73-AS1 mediated migration and invasion of CNE-2 cells via upregulating JAM-A. LncRNA TP73-AS1, miR-495, and JAM-A are involved in migration and invasion of NPC cells. The TP73-AS1/miR-495/JAM-A axis may serve as a therapeutic target for the treatment of NPC.

Aberrant expression of junctional adhesion molecule-A contributes to the malignancy of cervical adenocarcinoma by interaction with poliovirus receptor/CD155.

Recent studies have shown that aberrant expression of tight junction proteins (TJP) contributes to malignant potential of various cancers. In the present study, we investigated the expression of junctional adhesion molecule‐A (JAM‐A), one of the transmembrane TJP, in uterine cervical adenocarcinoma and the significance of its expression for malignancy. Immunohistochemistry on human surgical specimens showed that JAM‐A was aberrantly expressed in neoplastic regions including adenocarcinoma in situ (AIS). Knockout of JAM‐A significantly suppressed cell proliferation and colony‐forming and migration abilities. We also showed that an antibody specific to an extracellular region of JAM‐A reduced cell proliferation ability and that loss of JAM‐A increased drug sensitivity of cervical adenocarcinoma cells. Based on a comprehensive proteome analysis, we found that poliovirus receptor (PVR/CD155) was regulated by JAM‐A and formed a physical interaction with JAM‐A. In human surgical specimens, PVR/CD155 expression was significantly correlated with some clinicopathological features and prognosis of cervical adenocarcinoma. Interestingly, most of the PVR/CD155‐positive cases expressed a high level of JAM‐A, and patients with the expression pattern of PVR/CD155 positive/JAM‐A high had significantly shorter periods of relapse‐free survival (P = .00964) and overall survival (P = .0204) than those for the other patients. Our observations suggest that aberrant expression of JAM‐A promotes malignancy of uterine cervical adenocarcinoma by regulation of PVR/CD155, and JAM‐A is therefore a potential therapeutic target for this malignancy.

STEM-07. IDENTIFICATION OF A NOVEL CSC SIGNALING AXIS CONTAINING JAM-A/SERPINB3/TGF-BETA

Abstract Despite therapeutic interventions including surgery, radiation, and chemotherapy, multiple clones of treatment resistant cells repopulate the tumor, resulting in recurrence and a high rate of mortality in glioblastoma. Effective targeting of these cancer stem cells (CSCs) has been limited by our incomplete understanding of the intracellular signaling mechanisms maintaining the CSC state. Our laboratory previously identified junctional adhesion molecule-A (JAM-A) on CSCs and, through functional studies, demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. We determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the intermediate signaling network is yet to be fully elucidated. Therefore, our goal was to delineate the pathway downstream of JAM-A. To achieve this, we immunoprecipitated JAM-A from GBM CSCs and performed mass spectrometry leading to the identification of the serine/cysteine protease inhibitor SerpinB3. While SerpinB3 has a functional role in lung adenocarcinoma, breast, pancreas, as well as in hepatocellular carcinoma, there is limited information in GBM or CSCs, and its relationship to JAM-A is yet to be elucidated. Using in vitro CSC functional assays, we observed that SerpinB3 is necessary for the maintenance of CSCs. Additionally, in an intracranial implantation model of GBM the knockdown of SerpinB3 extended the survival of mice. Knockdown of SerpinB3 in multiple GBM CSCs models also led to decreased expression of TGF-β, a known regulator of the CSC state. We also observed that SerpinB3 stabilizes JAM-A expression and further studies are investigating the mechanism of this stabilization, identifying the specific interaction sites of JAM-A and Serpin B3, and determining how TGF-β alters the association through positive and negative feedback mechanisms. Taken together, these data highlight the role of SerpinB3 in GBM CSCs and provides a novel target to inhibit the JAM-A-mediated CSC maintenance as a next-generation GBM therapy.

MicroRNA-124 inhibits stem-like properties and enhances radiosensitivity in nasopharyngeal carcinoma cells via direct repression of expression of JAMA.

Cancer stem cells (CSCs) are a source of tumour recurrence in patients with nasopharyngeal carcinoma (NPC); however, the function of microRNA‐124 (miR‐124) in NPC CSCs has not been clearly defined. In this study, we investigated the role of miR‐124 in NPC CSCs. qRT‐PCR was performed to measure miR‐124 expression in NPC tissues and cell lines and the effects of miR‐124 on stem‐like properties and radiosensitivity of NPC cells measured. Luciferase reporter assays and rescue experiments were used to investigate the interaction of miR‐124 with the 3′UTR of junctional adhesion molecule A (JAMA). Finally, we examined the effects of miR‐124 in an animal model and clinical samples. Down‐regulation of miR‐124 was detected in cancer tissues and was inversely associated with tumour stage and lymph node metastasis. Overexpression of miR‐124 inhibited stemness properties and enhanced radiosensitivity of NPC cells in vitro and in vivo via targeting JAMA. Up‐regulation of miR‐124 was correlated with superior overall survival of patients with NPC. Our study demonstrates that miR‐124 can inhibit stem‐like properties and enhance radiosensitivity by directly targeting JAMA in NPC. These findings provide novel insights into the molecular mechanisms underlying therapy failure in NPC.

JAM-A Acts via C/EBP-α to Promote Claudin-5 Expression and Enhance Endothelial Barrier Function.

Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive. We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions. Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of β-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A-C/EBP-α-mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer. We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. Graphic Abstract: An graphic abstract is available for this article.

JAM-A functions as a female microglial tumor suppressor in glioblastoma.

Glioblastoma (GBM) is the most aggressive primary brain tumor and has a dismal prognosis. Previously, we identified that junctional adhesion molecule A (JAM-A), a cell adhesion molecule, is highly elevated in human GBM cancer stem cells and predicts poor patient prognosis. While JAM-A is also highly expressed in other cells in the tumor microenvironment, specifically microglia and macrophages, how JAM-A expression in these cells affects tumor growth has yet to be determined. The goal of this study was to understand the role of microenvironmental JAM-A in mediating GBM growth. Male and female wild-type (WT) and JAM-A-deficient mice were transplanted intracranially with the syngeneic glioma cell lines GL261 and SB28 and were assessed for differences in survival and microglial activation in tumors and in vitro. RNA-sequencing was performed to identify differentially regulated genes among all genotypes, and differences were validated in vitro and in vivo. We found that JAM-A-deficient female mice succumbed to GBM more quickly compared with WT females and JAM-A-deficient and male WT mice. Analysis of microglia in the tumors revealed that female JAM-A-deficient microglia were more activated, and RNA-sequencing identified elevated expression of Fizz1 and Ifi202b specifically in JAM-A-deficient female microglia. Our findings suggest that JAM-A functions to suppress pathogenic microglial activation in the female tumor microenvironment, highlighting an emerging role for sex differences in the GBM microenvironment and suggesting that sex differences extend beyond previously reported tumor cell-intrinsic differences.

Halting the vicious cycle within the multiple myeloma ecosystem: blocking JAM-A on bone marrow endothelial cells restores angiogenic homeostasis and suppresses tumor progression.

Interactions of malignant multiple myeloma (MM) plasma cells with the microenvironment control MM plasma-cell growth, survival, drug-resistance and dissemination. As microvascular density increases in the bone marrow in MM, we investigated whether bone marrow MM endothelial cells control disease progression via the junctional adhesion molecule-A (JAM-A). Membrane and cytoplasmic JAM-A levels were upregulated in MM endothelial cells in 111 patients with newly diagnosed MM and in 201 with relapsed/refractory MM compared to the levels in patients with monoclonal gammopathy of undetermined significance and healthy controls. Elevated membrane expression of JAM-A on MM endothelial cells predicted poor clinical outcome. Mechanistically, addition of recombinant JAM-A to MM endothelial cells increased angiogenesis, whereas inhibition of this adhesion molecule impaired angiogenesis and MM growth in two-dimensional and three-dimensional in vitro cell cultures and chorioallantoic membrane assays. To corroborate these findings, we treated MM-bearing mice with a JAM-A-blocking monoclonal antibody and demonstrated impaired MM progression, corresponding to decreased MM-related vascularity. These findings support the concept that JAM-A is an important mediator of MM progression through facilitating MM-associated angiogenesis. Elevated JAM-A expression on bone marrow endothelial cells is an independent prognostic factor for the survival of both patients with newly diagnosed MM and those with relapsed/refractory MM. Blocking JAM-A restricts angiogenesis in vitro, in utero and in vivo and represents a suitable druggable molecule to halt neo-angiogenesis and MM progression.