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 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 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 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

HMGA2 and IGF2BP2 expression in Tsc2+/- tumor tissue.

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

Supplementary Figure 9 - PDF file 83K, TGFBeta 1 mRNA expression in MCF7, MCF-HMGA2, and MDA-MB231 human breast cancer cells as detected by qRT-PCR

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 Tables 1 and 2, Figures S1-16. Supplementary Table 1: Gene lists from DAVID analysis of genes downregulated in RKIP derived TAMs with a Benjamini p-value < 0.5 Supplementary Table 2: Gene lists from DAVID analysis of genes upregulated in RKIP+CCRL5 derived (rescue) TAMs with a Benjamini p-value < 0.5 Figure S1: Immunoblot validation of RKIP expression in 1833 (BM1), MDA-MB-436, and 4T1.2 cells Figure S2: A schematic describing the method used for RNAseq analysis to compare metastatic BM1 with non-metastatic BM1+RKIP Figure S3: Tumor growth in orthotopic xenograft BM1 tumors, significance compared using a 2-way ANOVA. Figure S4: GO categories enriched in BM1+RKIP tumors from RNAseq analysis. Figure S5: Quantile-quantile plots for all stromal cell types tested between metastatic BM1 and non-metastatic BM1+RKIP tumors. Figure S6: Relative TAM infiltration in MDA-MD-436 tumors determined by %pos staining cells for F4/80. Figure S7: Flow cytometric analysis to determine the purity and heterogeneity of BM1 isolated TAMs. Figure S8: CCL5 levels measured by ELISA from BM1 cells in culture. Figure S9: CCL5 expression by qRT-PCR of MDA-MD-436 and 4T1.2 overexpressing RKIP. Figure S10?: RKIP and CCL5 immunoblotts from 1833 (BM1) tumors overexpressing the vectors shown. Figure S11: Tumor growth, tumor weight, and correlation between tumor weight and TAM infiltration in BM1 tumors treated with the CCR5 inhibitor Maraviroc. Figure S12: Invasion assay for BM1 cells overexpressing RKIP, CCL5, or both. Figure S13: Images of Raybiotech L308 arrays. Figure S14: Gene expression data of TAM secreted factors in human patient data sets. Figure S15: Correlation between CCL5 and TAM secreted factors in human patient data sets. Figure S16: Heatmap identifying data sets where breast cancer metastasis free survival is significantly stratified by classifier

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 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

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)

Tsc2+/- extra-renal tumorigenesis is HMGA2 dependent (S1); Summary of human AML LOH analysis (S2).

Legend for Supplemental Figures S1-S2.

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

Tuberin is present in all Tsc2+/- mesenchymal tumors.

AbstractTuberous sclerosis (TSC) is a tumor suppressor gene syndrome that is associated with the widespread development of mesenchymal tumor types. Genetically, TSC is said to occur through a classical biallelic inactivation of either TSC genes (TSC1, hamartin or TSC2, tuberin), an event that is implicated in the induction of the mTOR pathway and subsequent tumorigenesis. High Mobility Group A2 (HMGA2), an architectural transcription factor, is known to regulate mesenchymal differentiation and drive mesenchymal tumorigenesis in vivo. Here, we investigated the role of HMGA2 in the pathogenesis of TSC using the TSC2+/− mouse model that similarly mirrors human disease and human tumor samples. We show that HMGA2 expression was detected in 100% of human and mouse TSC tumors and that HMGA2 activation was required for TSC mesenchymal tumorigenesis in genetically engineered mouse models. In contrast to the current dogma, the mTOR pathway was not activated in all TSC2+/− tumors and was elevated in only 50% of human mesenchymal tumors. Moreover, except for a subset of kidney tumors, tuberin was expressed in both human and mouse tumors. Therefore, haploinsufficiency of one TSC tumor suppressor gene was required for tumor initiation, but further tumorigenesis did not require the second hit, as previously postulated. Collectively, these findings demonstrate that tissue-specific genetic mechanisms are employed to promote tumor pathogenesis in TSC and identify a novel, critical pathway for potential therapeutic targeting. Cancer Res; 76(4); 844–54. ©2016 AACR.

Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer. While five-year survival rates have reached 98% in patients treated with anti-ER or anti-HER2 therapies, patients with TNBC have a five-year survival rate of only 24%. Currently, the only form of therapy for these patients is surgery and platinum based chemotherapy. However, patient outcome is generally poor. Additionally, this disease disproportionately affects African-American women and lower income women, with rates seen approximately three times higher in African-American women compared to the rest of the population. An alternative strategy for treating TNBC patients involves targeting the tumor stroma. To better understand interaction between the tumor and stroma necessary for metastasis and invasion, we employed a triple-negative breast cancer (TNBC) model in which the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP) controls primary tumor invasiveness. RKIP expression, which converts invasive tumors to non-invasive tumors, dramatically inhibits macrophage infiltration. The mechanism, which is dependent on Let-7 suppression of HMGA2, involves decreased expression of the chemokine CCL5. Furthermore, overexpression of CCL5 partially rescued the infiltration of macrophages into the tumor and intravasation of tumor cells into the blood stream. The relationships of the genes in the RKIP, HMGA2, CCL5, and macrophage pathways were observed in multiple sets of expression array data from breast cancer patients. Regulation of macrophage infiltration was observed in tumors from an HMGA2 knockout mouse model. These results show that RKIP regulates macrophage recruitment by tumors and demonstrate for the first time that metastasis suppressor genes can regulate the tumor microenvironment. Citation Format: Daniel C. Rabe, Casey A. Frankenberger, Russell Bainer, Devipriya Sankarasharma, Kiran Chada, Thomas Krausz, Yoav Gilad, Marsha Rich Rosner. The role of tumor associated macrophages (TAMs) in triple-negative breast cancer (TNBC) invasion revealed by species-specific RNA sequencing. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B35. doi:10.1158/1538-7445.CHTME14-B35

Abstract Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis. Because they cannot be treated with targeted therapies, and many do not respond to chemotherapy, they represent a clinically underserved group. TNBC is characterized by reduced expression of metastasis suppressors such as Raf kinase inhibitory protein (RKIP), which inhibits tumor invasiveness. Mechanisms by which metastasis suppressors alter tumor cells are well characterized; however, their ability to regulate the tumor microenvironment and the importance of such regulation to metastasis suppression are incompletely understood. Here, we use species-specific RNA sequencing to show that RKIP expression in tumors markedly reduces the number and metastatic potential of infiltrating tumor-associated macrophages (TAM). TAMs isolated from nonmetastatic RKIP+ tumors, relative to metastatic RKIP− tumors, exhibit a reduced ability to drive tumor cell invasion and decreased secretion of prometastatic factors, including PRGN, and shed TNFR2. RKIP regulates TAM recruitment by blocking HMGA2, resulting in reduced expression of numerous macrophage chemotactic factors, including CCL5. CCL5 overexpression in RKIP+ tumors restores recruitment of prometastatic TAMs and intravasation, whereas treatment with the CCL5 receptor antagonist Maraviroc reduces TAM infiltration. These results highlight the importance of RKIP as a regulator of TAM recruitment through chemokines such as CCL5. The clinical significance of these interactions is underscored by our demonstration that a signature comprised of RKIP signaling and prometastatic TAM factors strikingly separates TNBC patients based on survival outcome. Collectively, our findings identify TAMs as a previously unsuspected mechanism by which the metastasis-suppressor RKIP regulates tumor invasiveness, and further suggest that TNBC patients with decreased RKIP activity and increased TAM infiltration may respond to macrophage-based therapeutics. Cancer Res; 75(19); 4063–73. ©2015 AACR.

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 vivomodulation 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 cellswere identifiedat the invasive frontof humanandmouse tumors. Inaddition, 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 TGFb signaling by activating expression of the TGFb type II receptor, which also localized to the invasive front of tumors. Together our results argued thatHMGA2plays a critical role in EMTbyactivating theTGFb signalingpathway, thereby inducing invasion and metastasis of human epithelial cancers. Cancer Res; 73(14); 4289–99. 2013 AACR.