No abstract

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...

Tumour metastasis suppressors are inhibitors of metastasis but their mechanisms of action are generally not understood. We previously showed that the suppressor Raf kinase inhibitory protein (RKIP) inhibits breast tumour metastasis in part via let-7. Here, we demonstrate an integrated approach combining statistical analysis of breast tumour gene expression data and experimental validation to extend the signalling pathway for RKIP. We show that RKIP inhibits let-7 targets (HMGA2, BACH1) that in turn upregulate bone metastasis genes (MMP1, OPN, CXCR4). Our results reveal BACH1 as a novel let-7-regulated transcription factor that induces matrix metalloproteinase1 (MMP1) expression and promotes metastasis. An RKIP pathway metastasis signature (designated RPMS) derived from the complete signalling cascade predicts high metastatic risk better than the individual genes. These results highlight a powerful approach for identifying signalling pathways downstream of a key metastasis suppressor and indicate that analysis of genes in the context of their signalling environment is critical for understanding their predictive and therapeutic potential.

BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1a and Hif target genes, including those involved in glycolysis and angiogenesis-two processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunctioncaused by defects in mitophagy-can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

The sources and consequences of nongenetic variability in metastatic progression are largely unknown. To address these questions, we characterized a transcriptional regulatory network for the metastasis suppressor Raf kinase inhibitory protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP and promotes breast cancer metastasis. Here we demonstrate that BACH1 acts in a double-negative (overall positive) feedback loop to inhibit RKIP transcription in breast cancer cells. BACH1 also negatively regulates its own transcription. Analysis of the BACH1 network reveals the existence of an inverse relationship between BACH1 and RKIP involving both monostable and bistable transitions that can potentially give rise to nongenetic variability. Single-cell analysis confirmed monostable and bistablelike behavior. Treatment with histone deacetylase inhibitors or depletion of the polycomb repressor enhancer of zeste homolog 2 altered relative RKIP and BACH1 levels in a manner consistent with a prometastatic state. Together, our results suggest that the mutually repressive relationship between metastatic regulators such as RKIP and BACH1 can play a key role in determining metastatic progression in cancer.ancer progression is an evolutionary process of variant cells competing to expand first locally and then distally within the human body. Ultimately, tumor evolution generates metastatic lesions that account for over 90% of cancer deaths (1). Whereas the requirement of heritably variant cells for tumor progression is undisputed, how they emerge is much less clear. Traditionally, genetic mutations causing oncogene activation or tumor suppressor loss were considered essential (2). However, accumulating evidence for tumor-promoting epigenetic, microenvironmental, and stochastic forms of heritable variation is challenging the traditional view (3, 4). Genetically identical tumor cells show highly variable responses to apoptosis-inducing ligands (5) and chemotherapeutic drugs (6, 7) attributable to cell-cell differences in gene expression and pathway activity. Tumor cells diversify in vitro (8) and in vivo (9) showing different levels of stem cell marker expression. However, the molecular mechanisms and interactions controlling such nongenetic forms of diversity are not fully known (10, 11).Multiple studies have implicated positive feedback loops in amplifying and maintaining stochastic fluctuations, creating heritable diversity in genetically homogenous cell populations (12, 13). This heritable nongenetic diversity then generates random subpopulations that can survive during various forms of environmental stress (14, 15), enabling subsequent evolutionary adaptation (16). Understanding how feedback loops and other network structures may affect nongenetic heterogeneity and contribute to metastatic cancer progression will be crucial for combating the disease (3, 10). However, the regulation of metastasis-related genes is incompletely understood, and the role of regulatory network-mediated n...