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Supplementary Methods and Supplementary Figure 1-20 from SIX1 Promotes Tumor Lymphangiogenesis by Coordinating TGFβ Signals That Increase Expression of VEGF-C

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posted on 2023-03-30, 22:41 authored by Dan Liu, Li Li, Xiao-Xue Zhang, Dong-Yi Wan, Bi-Xin Xi, Zheng Hu, Wen-Cheng Ding, Da Zhu, Xiao-Li Wang, Wei Wang, Zuo-Hua Feng, Hui Wang, Ding Ma, Qing-Lei Gao

Supplementary Methods and Supplementary Figure 1-20: Supplementary Figure 1 shows the growth of the tumors after orthotopic transplantation. Supplementary Figure 2 shows that over-expression of SIX1 promotes lymphangiogenesis and lymph-node metastasis of C33a cells in vivo. Supplementary Figure 3 shows the assay of cytokine and growth factor with antibody-based array. Supplementary Figure 4 shows the expression of the genes coding for the lymphangiogenic factors. Supplementary Figure 5 shows that SIX1 promotes VEGF-C expression in C33a cells. Supplementary Figure 6 shows that SIX1 promotes migration and tube-formation of HLECs. Supplementary Figure 7 shows the knock-down of VEGF-C in cervical cancer cells. Supplementary Figure 8 shows that the promotional effect of SIX1-expressing tumor cells on the migration and tube-formation of HLECs in vitro is mediated by VEGF-C. Supplementary Figure 9 shows that the promotional effect of SIX1-expressing tumor cells on lymphangiogenesis in vivo is mediated by VEGF-C. Supplementary Figure 10 shows that the promotional effect of SIX1-expressing C33a cells on lymphatic-vessel is mediated by VEGF-C. Supplementary Figure 11 shows that the promotional effect of SIX1 on lymph-node metastasis in vivo is mediated by VEGF-C. Supplementary Figure 12 shows the differences in gene expression levels between tumors in vivo and tumor cells cultured in vitro. Supplementary Figure 13 shows the immunohistochemical analysis of TGF-βs in tissue microarray of human CSCC specimens. Supplementary Figure 14 shows that SIX1 enhances the effect of TGF-β1 on VEGF-C production by tumor cells and tube-formation of HLECs in vitro. Supplementary Figure 15 shows that TGF-β contributes to the differences in VEGF-C expression between tumors in vivo and tumor cells cultured in vitro. Supplementary Figure 16 shows that TGF-β signaling is required for SIX1 to promote lymphangiogenesis and lymph-node metastasis in vivo. Supplementary Figure 17 shows that knocking down the expression of SMAD2 or SMAD3 or TβR1 suppresses TGF-β1 induced VEGF-C expression. Supplementary Figure 18 shows that higher expression of SIX1 correlates with higher expression of TβR1 in human CSCC specimens. Supplementary Figure 19 shows the binding of SIX1 to the VEGF-C promoter. Supplementary Figure 20 shows that knocking down the expression of SMAD2 and SMAD3 does not affect the binding of SIX1 to the VEGF-C promoter.

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ARTICLE ABSTRACT

Lymphatic vessels are one of the major routes for the dissemination of cancer cells. Malignant tumors release growth factors such as VEGF-C to induce lymphangiogenesis, thereby promoting lymph node metastasis. Here, we report that sine oculis homeobox homolog 1 (SIX1), expressed in tumor cells, can promote tumor lymphangiogenesis and lymph node metastasis by coordinating with TGFβ to increase the expression of VEGF-C. Lymphangiogenesis and lymph node metastasis in cervical cancer were closely correlated with higher expression of SIX1 in tumor cells. By enhancing VEGF-C expression in tumor cells, SIX1 could augment the promoting effect of tumor cells on the migration and tube formation of lymphatic endothelial cells (LEC) in vitro and lymphangiogenesis in vivo. SIX1 enhanced TGFβ-induced activation of SMAD2/3 and coordinated with the SMAD pathway to modulate VEGF-C expression. Together, SIX1 and TGFβ induced much higher expression of VEGF-C in tumor cells than each of them alone. Despite its effect in promoting VEGF-C expression, TGFβ could inhibit lymphangiogenesis by directly inhibiting tube formation by LECs. However, the increased production of VEGF-C not only directly promoted migration and tube formation of LECs but also thwarted the inhibitory effect of TGFβ on LECs. That is, tumor cells that expressed high levels of SIX1 could promote lymphangiogenesis and counteract the negative effects of TGFβ on lymphangiogenesis by increasing the expression of VEGF-C. These findings provide new insights into tumor lymphangiogenesis and the various roles of TGFβ signaling in tumor regulation. Our results also suggest that SIX1/TGFβ might be a potential therapeutic target for preventing lymph node metastasis of tumor. Cancer Res; 74(19); 5597–607. ©2014 AACR.

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