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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.

and correlates with disease dissemination 4 . More importantly, JAM-A is highly expressed on hematopoietic stem cells with in vivo repopulating activity 6 . However, the role of JAM-A on lymphoma progression needs to be further investigated in DLBCL.
In the present study, we defined JAM-A as a biomarker that bridges lymphoma cell stemness with lymphoma outgrowth in mesoendoderm-derived organs via TGF-β/NODAL signaling. Therefore, therapeutic targeting of JAM-A/NODAL axis by lenalidomide represents a promising strategy to treat DLBCL.

JAM-A was overexpressed and related to disease progression in DLBCL.
We first assessed JAM-A gene and protein expression in tumor samples of 102 de novo DLBCL patients using real-time quantitative RT-PCR and immunohistochemistry. JAM-A was highly expressed in DLBCL, when compared to reactive hyperplasia (N = 20) (gene expression, 3.7 ± 0.5 vs 1.1 ± 0.5, P = 0.0300, protein expression, 1501.0 ± 158.3 vs 640.0 ± 140.4, P = 0.0196, Fig. 1A,B and Supplementary Figure S1). JAM-A gene expression correlated well with JAM-A protein expression (Pearson Correlation Coefficient = 0.6778, P < 0.0001, Fig. 1C).
Of note, when DLBCL patients were classified in terms of involved sites, extranodal involvement group had higher expression of both JAM-A gene and JAM-A protein in tumor samples (P = 0.0051 and P = 0.0342, Fig. 1D). Patients with JAM-A level over and equal to the median value were regarded as high JAM-A expression, whereas those below the median value were included in the low JAM-A expression. Patients with high JAM-A expression tended to have extranodal involvement (P = 0.0421) and multiple extranodal lesions (P = 0.0294), as well as low complete remission rate (P = 0.0126, Table 1). The 2-yr progression-free survival (PFS) of patients in high JAM-A expression group was 53.6%, significantly shorter than those of low JAM-A expression group (73.8%, P = 0.0214, Fig. 1E).

JAM-A indicated lymphoma cell stemness and epithelial-to-mesenchymal transition. To gain
insights into the biological function of JAM-A, we established a JAM-A-transgenic zebrafish model by microinjecting jam-1 (zebrafish JAM-A gene identified in genome data base UCSC) mRNA into zebrafish embryos. Jam-1 overexpression led to a significant elevation of zebrafish hematopoietic stem-cell markers c-myb and runx1 7 , when compared to a wild-type (WT) zebrafish (P = 0.0158 and P = 0.0077, Fig. 2A). We also transfected B-lymphoma cell line DB and SU-DHL-4 with JAM-A vector (JAM-A). Ectopic expression of JAM-A stimulated lymphoma cell colony formation (P = 0.0354 and P = 0.0398, Fig. 2B) and induced higher expression of cancer stem-cell marker CD133 and CD34 than those of control vector (Vector) (P = 0.0003 and P = 0.0061 for CD133, P = 0.0197 and P = 0.0250 for CD34, Fig. 2C). Clinically, DLBCL patients with high JAM-A expression displayed a remarkable increase in tumor CD133 positivity (+++~++++) than those of low JAM-A expression (P = 0.0048, Fig. 2D). Interestingly, high JAM-A group was enriched for a stem cell gene signature, as revealed by RNA-sequencing (P < 0.0001, Fig. 2E).
Moreover, stem cells undergo a process known as epithelial-to-mesenchymal transition (EMT) 8 , defined by downregulated epithelial marker E-Cadherin with upregulated mesenchymal markers Fibronectin and Vimentin 9 . Indeed, EMT was observed in both JAM-A-transfected B-lymphoma cells (P = 0.0025, P = 0.0046 and P = 0.0171, Fig. 3A) and the tumor samples of DLBCL patients with high JAM-A expression (P = 0.0048, P = 0.0126 and P = 0.0101, Fig. 3B). As revealed by cell invasion assay, B-lymphoma cells with JAM-A overexpression achieved a notably higher percentage of cell invasion than those transfected with control vector (P = 0.0201, Fig. 3C). As compared to scramble cells, this invasive ability was inhibited by the molecular silencing of JAM-A using ShRNA (P = 0.0058, Fig. 3D). Thereafter, cancer cells required stemness to potentially reach out distant organs and initiate tumor metastasis 10 .

JAM-A induced lymphoma cell invasion to mesoendoderm-derived extranodal organs via
TGF-β/NODAL signaling. The EMT is triggered by many extracellular signals in cancer cells and the most potent inducers are members of the TGF-β family. As revealed by PCR gene array and RNA sequencing, TGF-β signaling pathway was activated both in JAM-A-transfected B-lymphoma cells (Fig. 4A) and in DLBCL patients (Fig. 4B). NODAL, an important member of TGF-β family proteins, is involved in embryonic stem cell maintenance and differentiation 11 . Moreover, in zebrafish models, key markers of NODAL signaling, ndr-1 (squint) and ndr-2 (cyclops), were higher in jam-1-overexpressing group than in WT group (P = 0.0201 and P = 0.0029, Fig. 4C). In B-lymphoma cells, transfection of JAM-A increased NODAL expression, while treatment with specific TGF-β/NODAL/Smad inhibitor SB431542 abrogated JAM-A-induced NODAL upregulation and lymphoma cell invasion ( Fig. 4D and Supplementary Figure S3A). Also, molecular silencing of JAM-A by ShRNA counteracted JAM-A-induced NODAL expression ( Fig. 4D and Supplementary Figure S3A). Together, these data confirmed that JAM-A activated TGF-β/NODAL signaling.
Lenalidomide modulated JAM-A/NODAL axis and impeded metastatic lymphoma outgrowth to mesoendoderm-derived organs. Lenalidomide (Fig. 5D), JAM-A-overexpressing cells were responsible for higher frequency of tumor metastasis (Fig. 5E). The metastatic lesions revealed by PET-CT all belonged to endoderm-and mesoderm-derived organs (intestine, n = 2, stomach, n = 1 and liver, n = 1, representative images shown in Fig. 5E). Compared to the untreated mice, tumor size remarkably decreased in lenalidomide-treated high JAM-A expression group and lymphoma invasion was significantly reduced (Fig. 5D,E). In accordance with in vitro data, the expression of JAM-A and NODAL was prohibited in the lenalidomide-treated high JAM-A expression group (P = 0.0013 and P = 0.0186, Fig. 5F).

Discussion
Disease dissemination, presented by multiple extranodal involvement, is an essential adverse prognostic factor of DLBCL patients. Biomarkers related to extranodal involvement are not well determined in lymphoma. Here we showed that JAM-A overexpression is associated with multiple extranodal lesions and poor disease outcome with lower CR rate and shorter PFS. In addition to solid tumors 5 , our results provided a direct link of JAM-A to disease progression in DLBCL.
Recent data indicate that non-stem cancer cells can re-enter the stem state and the metastatic disease in patients is concomitant with the appearance of stemness and EMT in these cells 13 . Overexpressed on hematopoietic stem cells with in vivo repopulating activity 6 and subsequently identified as a cancer stem-cell maintenance NODAL signaling is not only responsible for the specification of endoderm and mesoderm during embryogenesis 15 , but also re-emerges during cancer development [16][17][18] . Mimicking embryogenic development, NODAL stimulates cancer cell growth and promotes metastasis in solid tumors 19 . Overexpression of NODAL in breast cancer induces ectoderm-derived breast tissue to mesoderm-derived lymph nodes 20 . Also, JAM-A is upregulated in mesoendoderm-associated tumors like non-small cell lung cancer and gastric cancer 21,22 . In the present study, JAM-A may facilitate lymphoma progression in DLBCL via TGF-β/NODAL signaling, committing cell invasion towards endoderm-and mesoderm-derived extranodal organs. However, the molecular mechanism of action needs to be further investigated.
Lenalidomide is an oral immunomodulatory drug with direct and indirect antineoplastic activity for aggressive or indolent B-cell lymphoma 23 . With the indirect effect mediating through multiple types of immune cells within the tumor microenvironment, recent studies have identified molecular targets that exert the direct effect of lenalidomide on cancer cells, including adhesion molecules like intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 24,25 . Our data confirmed JAM-A as an important adhesion molecule and also as a potential therapeutic target of lenalidomide.

Conclusions
JAM-A is an unfavorable prognostic biomarker related to stem cell property in DLBCL and contributed to extranodal commitment to mesoendoderm-derived organs through the activation of TGF-β/NODAL signaling. JAM-A indicated the sensitivity of B-lymphoma cells to lenalidomide, and therapeutic targeting of JAM-A/NODAL axis represents a promising clinical strategy to counteract tumor progression in DLBCL.

Methods
Patients. One-hundred and two patients diagnosed as DLBCL were included in this study. The histological diagnosis was established according to WHO classification 26 . All the patients were CD20 positive DLBCL with exclusion of mediastinal large B-cell lymphoma. Clinical characteristics of the patients were listed in Table 1. Twenty age-and sex-matched cases with reactive hyperplasia were referred as controls. The study was approved by Shanghai Rui Jin Hospital Review Board and written informed consent were obtained from patients in   RNA sequencing. RNA was extracted using Trizol and RNeasy MinElute Cleanup kit from tissue samples and using PAXgene Blood miRNA kit from blood samples. Globin RNA was removed using Globin-Zero Gold rRNA Removal kit for RNA from blood samples. Following extraction, the RNA quantity was evaluated on Nanodrop and the integrity of total RNA using RNA 6000 Nano kit on Aligent 2100 Bioanalyzer. RNA library was constructed using TruSeq RNA Sample Preparation kit. The poly-A containing mRNA molecules was purified using oligo-dT attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The cDNA fragments went through an end repair process, the addition of a single ' A' base, and ligation of the adapters. The products were purified and enriched with PCR to create the final cDNA library. The clusters of the cDNA library were generated on the flow cell using TruSeq PE Cluster kit and HiSeq PE flow cell. The clusters were finally sequenced on HiSeq. 2000 system using TruSeq SBS kit.
Cloning and plasmid construction in Zebrafish. Adult zebrafish (Danio rerio) were raised following established protocols. Zebrafish jam-1 gene was identified according to homology to human JAM-A. The specific primers were designed based on genomic sequence in the UCSC data base (University of California, Santa Cruz) to amplify part of jam-1 gene. The plasmid was constructed by GENECHEM, Shanghai, China. Animals were used according to the protocols approved by the Shanghai Rui Jin Hospital Animal Care and Use Committee.
Extraction of mRNA and microinjection of Zebrafish embryos. mRNA of jam-1 was extracted and diluted to DEPC water at the concentration of 100 ng/μl. mRNA were microinjected at a volume of 2 nl into one-cell stage embryos using an air pressure injector and glass capillaries. Injection experiments were performed in triplicate.

Whole-mount in Situ
Hybridization in Zebrafish. pCS2+containing part of c-myb, ndr-1 or ndr-2 was used to produce antisense RNA probes using digoxigenin-11-uridine 59-triphosphate. Zebrafish embryos were fixed in 4% paraformaldehyde at the stages indicated and Whole-mount in Situ Hybridization was performed following the manufacturer's protocol.
SCIeNTIFIC REPORTS | 7: 7433 | DOI:10.1038/s41598-017-07964-5 Cell invasion assay. Cell invasion was tested in the Matrigel Invasion Chamber (BD Pharmingen, Franklin Lakes, NJ, USA), composed of the upper and lower compartment separated by the polycarbonate membranes (8 μm pore size). The upper side of the membrane was coated with Matrigel matrix. 6 × 10 4 cells were incubated with RPMI-1640 (FBS-free, 200 μl) for 24 hours and added to the upper compartment, while RPMI-1640 with 10% FBS (500 μl) was added to the lower compartment. After incubation with 5% CO 2 at 37 °C for 24 hours, the non-migratory cells remained on the Matrigel matrix were removed and the membrane was stained by Wright-Giemsa staining. Migratory cells were observed under the microscope at 40× magnifications and counted in different fields of membranes in triplicate.
Murine model. Severe combined immune deficiency mice (5-6-week-old) were obtained from Shanghai Laboratory Animal Center (Shanghai, China) and injected subcutaneously into the right flank with 2 × 10 7 DB cells transfected with JAM-A vector (JAM-A group) or control vector (control group, 10 mice per group). Tumor volumes were calculated as 0.5 × a × b 2 , where 'a' is the length and 'b' is the width. Treatments (10 mice per group) were started after the tumor became about 0.5 × 0.5 cm in surface (day 0). The JAM-A group received distilled water, whereas the JAM-A + lenalidomide group received lenalidomide (25 mg/kg/day) orally for two weeks. Animals were used according to the protocols approved by the Shanghai Rui Jin Hospital Animal Care and Use Committee. Statistical analysis. Differences of JAM-A expression among groups were assessed by the Mann-Whitney U test. In vitro experimental results were expressed as mean ± SEM. of data obtained from three separate experiments and determined using a t-test to compare variance. Chi-square test was used when comparing the constitution ratio between two groups. P < 0.05 was considered statistically significant. Data Availability. All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).