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...

Transfer RNAs (tRNAs) are typically considered housekeeping products with little regulatory function. However, several studies over the past 10 years have linked tRNA misregulation to cancer. We have previously reported that tRNA levels are significantly elevated in breast cancer and multiple myeloma cells. To further investigate the cellular and physiological effects of tRNA overexpression, we overexpressed tRNA i Met in two human breast epithelial cell lines. We then determined tRNA abundance changes and performed phenotypic characterization. Overexpression of tRNA iMet significantly altered the global tRNA expression profile and resulted in increased cell metabolic activity and cell proliferation. Our results extend the relevance of tRNA overexpression in human cells and underscore the complexity of cellular regulation of tRNA expression.

Elucidating targets of physiological tumor metastasis suppressors can highlight key signaling pathways leading to invasion and metastasis. To identify downstream targets of the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP/PEBP1), we utilized an integrated approach based upon statistical analysis of tumor gene expression data combined with experimental validation. Previous studies from our laboratory identified the architectural transcription factor and oncogene, HMGA2, as a target of inhibition by RKIP. Here we identify two signaling pathways that promote HMGA2-driven metastasis. Using both human breast tumor cells and an MMTV-Wnt mouse breast tumor model, we show that RKIP induces and HMGA2 inhibits expression of miR-200b; miR-200b directly inhibits expression of lysyl oxidase (LOX), leading to decreased invasion. RKIP also inhibits syndecan-2 (SDC2), which is aberrantly expressed in breast cancer, via down-regulation of HMGA2; but this mechanism is independent of miR-200. Depletion of SDC2 induces apoptosis and suppresses breast tumor growth and metastasis in mouse xenografts. RKIP, LOX, and SDC2 are coordinately regulated and collectively encompass a prognostic signature for metastasis-free survival in ER-negative breast cancer patients. Taken together, our findings reveal two novel signaling pathways targeted by the metastasis suppressor RKIP that regulate remodeling of the extracellular matrix and tumor survival.

Tumors depend upon angiogenesis for growth and metastasis. It is therefore critical to understand the inhibitory signaling mechanisms in endothelial cells that control angiogenesis. Epac is a cyclic adenosine 5-monophosphate-activated guanine nucleotide exchange factor for Rap1. In this study, we show that activation of Epac or Rap1 leads to potent inhibition of angiogenesis in vivo. Epac/ Rap1 activation down-regulates inhibitor of differentiation 1 (Id1), which negatively

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