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Purpose: Lymphatic vessels are mainly regarded as passive conduits for the dissemination of cancer cells. In this study, we investigate whether and how the tumor-associated lymphatic vessels may play an active role in tumor metastasis.Experimental Design: In situ laser capture microdissection of lymphatic vessels followed by cDNA microarray analysis was used to determine the expression profiling of lymphatic endothelial cells (LEC). Gene expression levels and activity of signaling pathways were measured by real-time RT-PCR, ELISA, or immunoblotting. Lymphangiogenesis was assessed by IHC. Lymph node metastasis was measured using fluorescence imaging. The effects of SEMA4C on lymphangiogenesis in vitro were evaluated using migration assay and tube-formation assay of LECs.Results: Tumor-associated LECs are molecularly and functionally different from their normal counterparts. In addition to expressing high levels of membrane-bound SEMA4C, tumorassociated LECs also produced soluble SEMA4C (sSEMA4C). Increased serum sSEMA4C was detected in patients with breast cancer and cervical cancer. Patients with metastasis had much higher levels of serum sSEMA4C. sSEMA4C promoted lymphangiogenesis by activating PlexinB2-ERBB2 signaling in LECs, and promoted the proliferation and migration of tumor cells by activating PlexinB2-MET signaling, thus promoting lymphatic metastasis. Although the SEMA4C signaling pathways differ between LECs and tumor cells, RHOA activation was necessary for the effects of SEMA4C in both types of cells.Conclusions: Tumor-associated LECs produce sSEMA4C to promote lymphatic metastasis of tumors. Our results suggest that SEMA4C and RHOA might be potential therapeutic targets, and that higher serum sSEMA4C could be a marker for breast cancer and cervical cancer.

Semaphorins are a large family of evolutionarily conserved morphogenetic molecules that are associated with repelling axonal guidance. Intriguingly, recent researches indicate that semaphorins are involved in cancer progression. Semaphorin 4C (SEMA4C) has long been considered a neuronal migration gene, but we detected that it is also highly expressed in many malignant human cancers. During an investigation of subcutaneous tumor models, we found that SEMA4C expression promoted tumor growth and progression. We discovered that SEMA4C was involved in maintaining tumor cell self-renewal, likely by regulating the p53 pathway. Inhibiting the expression of endogenous SEMA4C in tumor cells impaired growth and induced senescence and cell-cycle arrest in the G 2 -phase. In addition, we found that SEMA4C induced the production of angiogenin and colony-stimulating factor-1 (CSF-1) in tumor cells by activating the NF-kB pathway in a plexinB2-dependent manner. In conclusion, SEMA4C expression in breast cancer cells promotes cancer cell proliferation, macrophage recruitment, and angiogenesis. Thus, inhibition of SEMA4C activity may be a novel therapeutic strategy for human breast cancer.Implications: In breast cancer, therapeutic targeting of the SEMA4C pathway may prevent tumor growth, angiogenesis, metastasis, and progression.