The ten-eleven translocation (TET) family of methylcytosine dioxygenases initiates demethylation of DNA and is associated with tumorigenesis in many cancers; however, the mechanism is mostly unknown. Here we identify upstream activators and downstream effectors of TET1 in breast cancer using human breast cancer cells and a genetically engineered mouse model. We show that depleting the architectural transcription factor high mobility group AThook 2 (HMGA2) induces TET1. TET1 binds and demethylates its own promoter and the promoter of homeobox A (HOXA) genes, enhancing its own expression and stimulating expression of HOXA genes including HOXA7 and HOXA9. Both TET1 and HOXA9 suppress breast tumor growth and metastasis in mouse xenografts. The genes comprising the HMGA2-TET1-HOXA9 pathway are coordinately regulated in breast cancer and together encompass a prognostic signature for patient survival. These results implicate the HMGA2-TET1-HOX signaling pathway in the epigenetic regulation of human breast cancer and highlight the importance of targeting methylation in specific subpopulations as a potential therapeutic strategy. E pigenetic changes play an important role in cancer progression as well as development (1). Recent studies indicate that DNA demethylation can be catalyzed by a class of methylcytosine dioxygenases termed the ten-eleven translocation (TET) family (2-5). TET1 promotes DNA demethylation by catalyzing conversion of 5-methylcytosine (5mC) primarily to 5-hydroxymethylcytosine (5hmC) as well as 5-formylcytosine or 5-carboxylcytosine (3, 5). The modified cytosines are then removed through active or passive mechanisms (2-6). While TET1 is highly expressed in embryonic stem (ES) cells (5, 7-10), loss of TET1 protein and decreased 5hmC levels have been recently shown in solid tumors relative to normal epithelial cells (2,(11)(12)(13)(14). However, the mechanism by which TET1 is suppressed in solid tumors has not been identified. Furthermore, the downstream targets by which TET1 regulates growth and metastasis in cancer are largely unknown.High mobility group AT-hook 2 (HMGA2), a chromatinremodeling factor (15), binds to AT-rich regions in DNA, altering chromatin architecture to either promote or inhibit the action of transcriptional enhancers. HMGA2 is highly expressed in ES cells but is generally low or lacking in normal somatic cells. Interestingly, HMGA2 is highly expressed in most malignant epithelial tumors, including breast (16), pancreas (17), oral squamous cell carcinoma (18), and non-small-cell lung cancer (19). HMGA2 overexpression in transgenic mice causes tumor formation, whereas Hmga2-knockout mice have a pygmy phenotype indicative of a growth defect (20). We have reported that HMGA2 promotes tumor invasion and metastasis in breast cancer in part through regulation of prometastatic genes, including Snail, osteopontin, and CXCR4 (21,22).To systematically identify critical downstream mediators of HMGA2 that regulate invasion and metastasis, we performed gene expression array analysis by knocki...

The non-histone chromatin binding protein HMGA2 is expressed predominantly in the mesenchyme prior to 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 regarding 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. Additionally, in a mouse allograft model, HMGA2 overexpression converted non-metastatic 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 (TGFβRII), 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.

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 is 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 TAMs. TAMs isolated from non-metastatic RKIP+ tumors, relative to metastatic RKIP− tumors, exhibit a reduced ability to drive tumor cell invasion and decreased secretion of pro-metastatic 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 pro-metastatic TAMs and intravasation, while 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 pro-metastatic 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.