The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-β signaling
Douglas S. Micalizzi, … , William P. Schiemann, Heide L. Ford
Douglas S. Micalizzi, … , William P. Schiemann, Heide L. Ford
Published August 24, 2009
Citation Information: J Clin Invest. 2009;119(9):2678-2690. https://doi.org/10.1172/JCI37815.
View: Text | PDF
Research Article

The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-β signaling

Abstract

Inappropriate activation of developmental pathways is a well-recognized tumor-promoting mechanism. Here we show that overexpression of the homeoprotein Six1, normally a developmentally restricted transcriptional regulator, increases TGF-β signaling in human breast cancer cells and induces an epithelial-mesenchymal transition (EMT) that is in part dependent on its ability to increase TGF-β signaling. TGF-β signaling and EMT have been implicated in metastatic dissemination of carcinoma. Accordingly, we used spontaneous and experimental metastasis mouse models to demonstrate that Six1 overexpression promotes breast cancer metastasis. In addition, we show that, like its induction of EMT, Six1-induced experimental metastasis is dependent on its ability to activate TGF-β signaling. Importantly, in human breast cancers Six1 correlated with nuclear Smad3 and thus increased TGF-β signaling. Further, breast cancer patients whose tumors overexpressed Six1 had a shortened time to relapse and metastasis and an overall decrease in survival. Finally, we show that the effects of Six1 on tumor progression likely extend beyond breast cancer, since its overexpression correlated with adverse outcomes in numerous other cancers including brain, cervical, prostate, colon, kidney, and liver. Our findings indicate that Six1, acting through TGF-β signaling and EMT, is a powerful and global promoter of cancer metastasis.

Authors

Douglas S. Micalizzi, Kimberly L. Christensen, Paul Jedlicka, Ricardo D. Coletta, Anna E. Barón, J. Chuck Harrell, Kathryn B. Horwitz, Dean Billheimer, Karen A. Heichman, Alana L. Welm, William P. Schiemann, Heide L. Ford

×

Figure 3

Six1 induces features of EMT in MCF7 mammary carcinoma cells.

Options: View larger image (or click on image) Download as PowerPoint
Six1 induces features of EMT in MCF7 mammary carcinoma cells. (A) MCF7-S...
(A) MCF7-Six1 cells displayed differential regulation of EMT-associated genes. Gene expression from MCF7-Six1 and MCF7-Ctrl clones was analyzed by microarray analysis performed in duplicate for each clone. A gene list generated by a priori search of EMT-associated genes was filtered for probesets with a “present” call in at least 50% of the microarrays, then clustered using hierarchical clustering. The color scale represents the expression level of a gene above (red), below (green), or at (black) the mean expression level of that gene across all samples. (B) MCF7-Six1 clones displayed increased fibronectin, decreased cytokeratin 18, and no change in total E-cadherin, as determined by Western blot of whole cell lysates. (C) MCF7-Six1 cells showed a shift of E-cadherin and β-catenin from the insoluble (cytoskeleton-associated) fraction to the soluble (cytosolic) fraction. Fractions were analyzed by Western blot using antibodies against E-cadherin, β-catenin, and β-actin. (D and E) Quantification of Western blot of subcellular localization. (F) MCF7-Six1 cells had increased activity of the β-catenin–responsive luciferase reporter TOP-flash. Values were normalized to renilla luciferase activity. (G) Six1 expression decreased cell adhesion to matrix proteins in MCF7 cells. The relative number of adhering cells was quantified by crystal violet staining. For D–G, data are presented as mean ± SD of 3 individual clones. P values represent statistical analysis using a paired t test.