Abstract 1463: SIX1 induces lymphatic and distant metastasis by upregulating VEGF-C and stimulating lymphangiogenesis in breast cancer
Authors: Wang Chu-An , Jedlicka Paul , Christensen Kimberly L. , Micalizzi Douglas S. , Harrell J. Chuck , Ford Heide L. 
Published: June 2012

Abstract
The homeoprotein Six1 is expressed during embryogenesis and re-expressed in 50% of primary and in 90% of metastatic breast cancer lesions.Using an orthotopic xenograft model of breast cancer, Six1 overexpression results in a 40% increased in lymphatic metastases as compared to controls.Six1 correlates with positive lymph node status in human breast cancer, further suggesting that it influences lymphatic metastasis, perhaps through stimulating lymphangiogenesis.Microarray analysis revealed that the critical lymphangiogenic regulator, VEGF-C, may be a target of Six1.Real-time PCR and VEGF-C promoter-luciferase assays demonstrated that VEGF-C upregulation is mediated transcriptionally by Six1.Chromatin immunoprecipitation analysis demonstrates in vivo binding of Six1 to the VEGF-C promoter.Increased levels of VEGF-C mRNA in response to Six1 overexpression result in increased intracellular and secreted VEGF-C protein.We quantified the number of intra- and peri-tumoral vessels in MCF7-Six1 versus MCF7-Control tumors and the MCF7-Six1 tumors display increased lymphangiogenesis and a 5-fold increase in the frequency of lymphatic invasion compared to the MCF7-Ctrl tumors.To determine whether Six1 depends on VEGF-C to increase lymphangiogenesis and lymphatic metastasis, we knocked down VEGF-C in MCF7-Six1 cells and injected the cells into the mammary gland of nude mice.We observed decreased lymph node metastases in the mice bearing MCF7-Six1/VEGF-C KD tumors compared to the MCF7-Six1 tumors, suggesting Six1-induced lymphatic metastasis is in part through upregulation of VEGF-C.We also used a complementary model, the 66c14 mammary carcinoma cell line which metastasizes primarily through the lymphatic system and expresses high levels of Six1, to study the role of Six1 in lymphatic metastasis.Six1 knockdown (KD) in 66c14 cells resulted in decreased levels of Vegf-c and strikingly, the Six1 KD tumors also contained less lymphatic vessel formation, suggesting a reduction of lymphangiogensis. Finally, Six1 KD dramatically decreased lung metastases in the 66c14 model as seen using bioluminescent imaging. When we rescued Vegf-c expression in 66c14 in the context of Six1 KD, we observed a restoration of lymphatic metastasis, but the rescue failed to restore lung metastasis, suggesting Six1 regulates additional factor/pathways required to accomplish the later stages of metastasis.Regulation of VEGF-C by Six1 is supported by clinical observations that correlation of Six1 and VEGF-C can be extended broadly to breast cancer cell lines (oncomine) and to human breast cancer tissues (tissue array).In conclusion, we demonstrate that Six1 mediates lymphangiogenesis and lymphatic metastasis in multiple mouse models via its ability to regulate VEGF-C, and extend this observation to human tissues where Six1 and VEGF-C overexpression are significantly correlated.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1463. doi:10.1158/1538-7445.AM2011-1463