Abstract PR04: The Six2 homeoprotein mediates breast cancer metastasis via repressing E-cadherin

Homeobox genes are master regulators of embryogenesis and it is widely known that misexpression of these transcription factors can lead to tumorigenesis/ metastasis. The Six1 homeoprotein has been implicated in tumor onset and metastasis of many different cancers, including breast cancer. However, little is known about whether related homeoproteins in the Six family can affect tumor onset or progression. Recent studies have shown that while Six1 knockdown dramatically decreases metastasis, knockdown cells that are capable of metastasizing have upregulated a related family member, Six2. Interestingly, Six2 regulates stem/ progenitor cell populations and maintains a mesenchymal progenitor phenotype in the developing kidney; however, the role of Six2 in breast cancer progression has not been explored. Here, we demonstrate for the first time that up-regulation of Six2 in breast cancer cells can promote metastasis in a mechanism that differs from its related family member, Six1. Knockdown (KD) of Six2 in 66cl4 mouse mammary carcinoma cells significantly decreases lung metastasis in an immune-competent orthotopic mouse model, as compared to control KD. In contrast to what is seen with Six1 KD in the same orthotopic mouse model, no significant inhibition of primary tumor size was observed with Six2 knockdown, suggesting that unlike Six1, the functions of Six2 may be confined to later stages of tumor progression. Overexpression of Six2 in the weakly metastatic 4TO7 mouse mammary carcinoma cell line led to changes in cell morphology, increased anchorage independent growth and significantly enhanced lung metastasis in an immune competent mouse model. Microarray analysis was performed to determine the mechanism by which Six2 mediates metastasis and the epithelial marker E-Cadherin, whose loss has been implicated in the metastatic potential of many cancers, was dramatically downregulated in the 4TO7-Six2 expressing cells. Importantly, restoration of E-cadherin expression reduced lung metastasis induced by Six2 and increased survival of the animals. We further determine that repression of E-cadherin by Six2 is mediated via multiple mechanisms. SIP1/Zeb2 (zinc finger E box binding homeobox2), an epithelial to mesenchymal transition (EMT) inducer, is increased in cells overexpressing Six2, and Zeb2 KD in these cells reverts E-cadherin expression back to the level as that observed in the control cells and importantly leads to a reduction in metastasis in vivo. In addition, we find that Six2 may also repress E-cadherin though an epigenetic mechanism, since repression of E-cadherin downstream of Six2 can be reversed by inhibiting DNA methylation. Finally, analysis of Six2 expression from public microarray datasets indicates that Six2 is increased in breast cancers compared to normal breast tissue and that high expression of SIX2 correlates with poor prognosis (distant metastasis free survival, overall survival and relapse free survival) in 1881 human breast tumors examined using the GOBO (Gene Expression-Based Outcome for Breast Cancer Online) database. Moreover, a reverse correlation of E-cadherin and Six2 in breast cancer datasets suggest that Six2 promotes loss of E-cadherin in human breast cancer. Together, these data demonstrate that Six2 is a specific regulator of the metastatic process, through regulating E-cadherin expression. This abstract is also presented as Poster B094. Citation Format: Chu-An Wang, Paul Jedlicka, Aik-Choon Tan, Catherine Pham, Vadym Zaberezhnvv, Heide Ford. The Six2 homeoprotein mediates breast cancer metastasis via repressing E-cadherin. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr PR04.