Six1 promotes breast cancer metastasis via upregulation of the lymphangiogenic factor VEGF-C Free

AMC-03

Homeobox genes represent a superfamily of transcription factors that act as “master regulators”during development, controlling many cellular processes including proliferation, differentiation,apoptosis, cell adhesion, invasion, and neovascularization. These cellular processes thatcontrol embryogenesis, can also promote tumorigenesis when dysregulated. The Six1homeoprotein plays important roles in development, in part by promoting proliferation, inhibitingapoptosis, and affecting cell migration and invasion. Six1 is overexpressed in many cancersincluding breast, ovarian and hepatocellular carcinoma, Wilms’ tumor, and alveolarrhabdomyosarcoma, where it affects transformation, proliferation, survival, and metastasis. Thissuggests that misexpression of Six1, after development is complete, may promotetumorigenesis by aberrantly activating transcriptional programs that normally contribute toorganogenesis. Here, we show that Six1 expression in MCF12A human mammary epithelialcells induces increased in vitro invasion, and furthermore, that in these cells expression of Six1is transforming, leading to highly aggressive, locally invasive tumors in vivo. As Six1 isoverexpressed in an overwhelming 90% of breast carcinoma metastatic lesions, and is a criticalmediator of metastasis in rhabdomyosarcoma, we examined the ability of Six1 to causemetastasis directly through the use of a xenograft model in which Six1 is overexpressed inMCF7 human breast cancer cells. In this model, we show that Six1 induces lymphaticmetastases in vivo, with occasional bone metastases also observed. Since, Six1-overexpressing MCF7 tumors spread primarily to the lymph nodes and lymphatic vasculature,we stained these tumors for lymphatic vessels, revealing an increase in both intra- and peritumorallymphatics. Furthermore, we find that Six1 increases expression of thelymphangiogenic factor VEGF-C, both in breast cancer cell lines and mouse embryo fibroblasts,suggesting that Six1 affects lymphangiogenesis in normal development as well as breastcancer. Together, these data suggest that Six1 promotes lymphatic dissemination of tumorcells by inducing tumor lymphangiogenesis via upregulation of VEGF-C. Finally, to assess theclinical relevance of Six1-induced lymphatic metastases, we examined Six1 expression inbreast carcinoma tissue microarrays and found that Six1 overexpression correlates significantlywith lymph node metastasis (p > 0.05), but not with other clinical parameters examined. Thissupports the relevance of our mouse model for understanding the role of Six1 in human breastcancer progression. As a developmental regulator, Six1 may provide an ideal drug target, as itis absent or low in most adult tissues, and would inhibit tumorigenesis on multiple fronts,including proliferation, survival, and metastasis.