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Supplementary Figure 10 - PDF file 84K, HMGA2 mRNA expression in MCF7 and MDA-MB231 human breast cancer cells treated with TGFBeta 1 as detected by qRT-PCR

Supplementary Figure 6 - PDF file 132K, (A) HMGA2 was detected at the edge of the metastatic lung tumor (left, arrows) and from the vessel of the liver stroma to the parenchyma in the metastatic liver tumor (right, arrows). Scale bars represent 100 microm. (B) Liver metastases (arrows) were induced in 4TO7 cells stably over-expressing HMGA2 (4TO7-HMGA2)

Supplementary Figure 3 - PDF file 171K, Immunofluorescence analysis of ZEB1 (left), Fibronectin (right) expression in MCF7-Mock and MCF7-HMGA2 cells. Scale bars represent 50 microm. ZEB1 and fibronectin are expressed in the nucleus of MCF7-HMGA2 cells that have undergone the EMT as is consistent with the other EMT markers in Figure 1C

Supplementary Figure 2 - PDF file 77K, Agarose Colony Formation Assay. Anchorage-independent proliferation was performed by plating SW403, HT29, SW620, SW480 and HCT116 (1000 cells per well) in six-well plates, with a bottom layer of 0.6% agar and a top layer of 0.3% agar containing the cells. After 21 days incubation, colonies were counted

Supplementary Figure 7 - PDF file 97K, Schematic of liver metastasis. Metastatic cancer cells (4TO7 cells stably over-expressing HMGA2 and 4T1 cells) are located in Glisson's capsule (GC) and Liver parenchyma (LP)

Supplementary Figure 4 - PDF file 253K, beta-catenin was relocalized to the nucleus at the invasive front of Wnt1-mediated tumor on Hmga2+/+ genotypic backgrounds, but not on the Hmga2-/- genetic background. beta-catenin was detected in the nucleus of the cells at the invasive front consistent with the HMGA2 expressed cells in the Wnt1-mediated tumor on the Hmga2+/+ genetic background (arrows). On the other hand, no beta-catenin relocalization was detected in tumor on the Hmga2-/- genotypic backgrounds. Scale bars represent 100microm

PDF file - 80K, TGFBetaRII was up-regulated in 4T1 cells. TGFBetaRII mRNA expression was highly up-regulated in 4T1 cells compared to 4TO7 parental cells (## = P < 0.0001)

Supplementary Figure 8 - PDF file 82K, There is no statistically significant difference in Snail and Twist mRNA expression in ectopic HMGA2 transfected cells (MCF7-HMGA2 cells)

Supplementary Figure 1 - PDF file 220K, Immunohistochemical analyzes of HMGA2 expression are shown (40x). These are representative sections from staining performed on patient tumor samples described in Table 1. Scale bars represent 250 microm

Supplementary Information - PDF file 81K, Supplementary Figure Legends and Supplementary Materials and Methods

The non-histone chromatin-binding protein HMGA2 is expressed predominantly in the mesenchyme before its differentiation, but it is also expressed in tumors of epithelial origin. Ectopic expression of HMGA2 in epithelial cells induces epithelial–mesenchymal transition (EMT), which has been implicated in the acquisition of metastatic characters in tumor cells. However, little is known about in vivo modulation of HMGA2 and its effector functions in tumor metastasis. Here, we report that HMGA2 loss of function in a mouse model of cancer reduces tumor multiplicity. HMGA2-positive cells were identified at the invasive front of human and mouse tumors. In addition, in a mouse allograft model, HMGA2 overexpression converted nonmetastatic 4TO7 breast cancer cells to metastatic cells that homed specifically to liver. Interestingly, expression of HMGA2 enhanced TGFβ signaling by activating expression of the TGFβ type II receptor, which also localized to the invasive front of tumors. Together our results argued that HMGA2 plays a critical role in EMT by activating the TGFβ signaling pathway, thereby inducing invasion and metastasis of human epithelial cancers. Cancer Res; 73(14); 4289–99. ©2013 AACR.