Downlo ough Wnt1 downstream signaling components have been well studied and activated in human cancer, thways that regulate Wnt1 itself have not been explored in depth. Here, we provide gain-of-function, f function, and molecular evidence supporting functional interactions between metastasis-associated n 1 short-form (MTA1s), metastasis-associated protein 1 (MTA1), and Wnt1 signaling components durmmary gland development and tumorigenesis. Using multiple model systems involving overexpression ckdown of MTA1s or MTA1, we discovered that MTA1s and MTA1 hyperactivate the Wnt1 pathway due reased expression of Wnt1 transcription. MTA1s and MTA1 physically interact with Six3 chromatin, a n product of which is a direct histone deacetylase inhibitor–dependent repressor of Wnt1 transcription. on of the MTA1s and MTA1 allele in murine embryonic fibroblasts resulted in the upregulation of Six3 wnregulation of Wnt signaling. In addition, mammary glands from the MTA1s/MTA1 mice exhibited sed recruitment of Six3 corepressor complex to the Wnt1 promoter and inhibition of Wnt1 pathway in ary glands. These findings identify MTA1s and MTA1 as important upstream modifiers of the Wnt1 mamm transcription, and consequently its functions, by directly inhibiting the transcription of Six3, allowing derepression of Wnt1 transcription. Cancer Res; 70(16); 6649–58. ©2010 AACR.