Tumor-Derived JAGGED-1 Promotes Breast Cancer Lymphovascular Invasion and Lymph Node Metastasis

Metastasis to tumor draining lymph nodes is a defining prognostic marker for breast cancer patients at the time of diagnosis or surgery. Emerging animal models demonstrate that lymph node metastasis can promote breast cancer progression by acting as a vector for secondary metastasis or, perhaps concomitantly, inducing global immune tolerance. Expression of the Notch ligand JAGGED-1 (JAG1) is associated with tumor lymphangiogenesis, lymph node metastasis, and poor patient outcomes, but causality is unclear. Here, we provide the first evidence that tumor derived Jag1 promotes lymphogenous metastasis though the mammary fatpad and the tumor draining lymph node. Moreover, we demonstrate that tumor Jag1 promotes expression of Notch, VEGF, and inflammatory pathway genes that are involved in tumor-lymphatic endothelial cell interactions. We utilized clinical samples in a tumor microarray (TMA) to determine if tumor derived JAG1 is associated with patient lymph node status. Expression of tumor-derived Jag1 was significantly enriched in lymph node samples compared to patient-matched primary tumors. Based on this evidence, we hypothesized that JAG1 promotes lymphogenous metastasis into lymphatic vessels and tumor draining lymph nodes. We addressed this hypothesis using both murine 66cl4 and human MDA231-LN breast cancer cells, the latter of which was treated with JAG1-targeted CRISPR/Cas9 (JAG1KO) to remove JAG1 expression. Using lymphangiography with high molecular weight dextran, we show 66cl4 and MDA231-LN-JAG1KO cell lines with JAG1 overexpression invade the mammary fatpad lymphatic vasculature significantly more than their respective controls. Moreover, JAG1 increases lymph node metastasis to the primary tumor draining lymph node in 66cl4/BALB.c syngeneic models. We conclude that JAG1 promotes early lymphogenous dissemination of breast cancer cells to the lymph node. Patients rarely succumb to complications of the primary tumor, which is often excised at time of diagnoses. In order to recapitulate a clinically meaningful model, we surgically removed 66cl4- and MDA231-LN-JAG1KO derived tumors, with and without JAG1-overexpression, and followed lymph node metastasis after excision. JAG1 increased lymph node metastatic outgrowth in BALB.c syngeneic models, but there were no observable phenotypes in NCG immunosuppressed mouse models. We discuss the differences between the two models and possible explanations that can account for varying phenotypes. We explore two signaling mechanisms JAG1-high tumor cells may use to communicate to lymphatic endothelial cells (LECs). First, tumor cell JAG1 increases tumor Notch, VEGF, and inflammatory signaling pathways. Within these pathways, JAG1 increases expression of cytokines, including WISP1, CXCL1, IGFBP6, CX3CL1, and IL-6, which were identified by both unbiased mRNA sequencing and analysis with a cytokine profiler array. We show that JAG1 overexpressing breast cancer cells produce paracrine factors in conditioned media that activate LEC VEGFR3. Second, we determine gene expression in LECs when exposed to a plate-tethered Jag1 ligand, consisting of the Jag1 ectodomain. This isolates the effects of JAG1 presentation to LECs. Compared to LECs treated with control Fc, LECs interacting with JAG1 have significantly increased expression of inflammatory pathway genes, including those in NF-κB and TLR pathways. Thus, using unbiased profiling, we identified key pathways regulated in LECs directly by JAG1. Since tumor JAG1 increases VEGF signaling pathways and media from JAG1-high breast cancer cells can activate LEC VEGFR3, we hypothesized that VEGFR3-inhibiton could attenuate lymph node metastasis. We administered the small molecule inhibitor MAZ51 to BALB.c mice bearing 66cl4 JAG1-overexpressing and control tumors. Preliminary analysis shows remarkable attenuation of lymph node metastasis in control 66cl4 tumors, but not tumors overexpressing JAG1. These findings indicate that tumor JAG1 overexpression is associated with resistance to VEGFR3 inhibition in vivo. From this body of work, we conclude that JAG1 is a prognostic indicator for patient lymph node metastasis and an active agent in breast cancer metastasis via lymphogenous dissemination. We identified novel JAG1 mediated pathways in which tumor JAG1 can communicate to the lymphatic endothelium and potential mechanisms in which JAG1 high cells can resist VEGFR3-targeting therapy. Given the emerging importance of JAG1-signaling and lymph node metastasis in mediating immune tolerance in breast cancer progression, understanding JAG1-mediated lymph node metastasis will offer much needed insights into understanding and defeating metastasis.