SUMMARY The mechanistic underpinnings of metastatic dormancy and reactivation are poorly understood. A gain-of-function cDNA screen reveals that Coco, a secreted antagonist of TGF-β ligands, induces dormant breast cancer cells to undergo reactivation in the lung. Mechanistic studies indicate that Coco exerts this effect by blocking lung-derived BMP ligands. Whereas Coco enhances the manifestation of traits associated with cancer stem cells, BMP signaling suppresses it. Coco induces a discrete gene expression signature, which is strongly associated with metastatic relapse to the lung but not to the bone or brain in patients. Experiments in mouse models suggest that these latter organs contain niches devoid of bioactive BMP. These findings reveal that metastasis-initiating cells need to overcome organ-specific anti-metastatic signals in order to undergo reactivation.

Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel-Lindau ( VHL ) tumor suppressor. Roles for noncoding cis -regulatory aberrations in ccRCC tumorigenesis, however, remain unclear. Analyzing 10 primary tumor/normal pairs and 9 cell lines across 79 chromatin profi les, we observed pervasive enhancer malfunction in ccRCC, with cognate enhancer-target genes associated with tissue-specifi c aspects of malignancy. Superenhancer profi ling identifi ed ZNF395 as a ccRCCspecifi c and VHL-regulated master regulator whose depletion causes near-complete tumor elimination in vitro and in vivo . VHL loss predominantly drives enhancer/superenhancer deregulation more so than promoters, with acquisition of active enhancer marks (H3K27ac, H3K4me1) near ccRCC hallmark genes. Mechanistically, VHL loss stabilizes HIF2α-HIF1β heterodimer binding at enhancers, subsequently recruiting histone acetyltransferase p300 without overtly affecting preexisting promoter-enhancer interactions. Subtype-specifi c driver mutations such as VHL may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression. SIGnIFICAnCE:Comprehensive epigenomic profi ling of ccRCC establishes a compendium of somatically altered cis -regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of VHL , a ccRCC signature event, causes pervasive enhancer malfunction, with binding of enhancer-centric HIF2α and recruitment of histone acetyltransferase p300 at preexisting lineage-specifi c promoter-enhancer complexes. Cancer Discov; 7(11); 1284-305.

Regulatory enhancer elements in solid tumours remain poorly characterized. Here we apply micro-scale chromatin profiling to survey the distal enhancer landscape of primary gastric adenocarcinoma (GC), a leading cause of global cancer mortality. Integrating 110 epigenomic profiles from primary GCs, normal gastric tissues and cell lines, we highlight 36,973 predicted enhancers and 3,759 predicted super-enhancers respectively. Cell-line-defined super-enhancers can be subclassified by their somatic alteration status into somatic gain, loss and unaltered categories, each displaying distinct epigenetic, transcriptional and pathway enrichments. Somatic gain super-enhancers are associated with complex chromatin interaction profiles, expression patterns correlated with patient outcome and dense co-occupancy of the transcription factors CDX2 and HNF4α. Somatic super-enhancers are also enriched in genetic risk SNPs associated with cancer predisposition. Our results reveal a genome-wide reprogramming of the GC enhancer and super-enhancer landscape during tumorigenesis, contributing to dysregulated local and regional cancer gene expression.

We have developed a screening platform for the isolation of genetic entities involved in metastatic reactivation. Retroviral libraries of cDNAs from fully metastatic breast-cancer cells or pooled microRNAs were transduced into breast-cancer cells that become dormant upon infiltrating the lung. Upon inoculation in the tail vein of mice, the cells that had acquired the ability to undergo reactivation generated metastatic lesions. Integrated retroviral vectors were recovered from these lesions, sequenced, and subjected to a second round of validation. By using this strategy, we isolated canonical genes and microRNAs that mediate metastatic reactivation in the lung. To identify genes that oppose reactivation, we screened an expression library encoding shRNAs, and we identified target genes that encode potential enforcers of dormancy. Our screening strategy enables the identification and rapid biological validation of single genetic entities that are necessary to maintain dormancy or to induce reactivation. This technology should facilitate the elucidation of the molecular underpinnings of these processes.forward genetic screens | metastatic reactivation | cDNA library screen | shRNA library screen | microRNA library screen T he majority of cancer-related deaths are caused by metastatic relapse (1). The process through which cancer cells acquire metastatic capacity is complex. Unrestrained proliferation, resistance to proapoptotic insults, and invasion through tissue boundaries are not sufficient for metastasis. To colonize distant organs, cancer cells must also adapt to the local microenvironment of the target organ and finally outgrow (2, 3). Mathematical modeling of clinical data and experiments in mouse models suggest that cancer cells disseminating from prevalent cancers, such as those of the breast and prostate, undergo an extended period of dormancy at premetastatic sites (4). Insights into the mechanisms that enable disseminated cancer cells to survive during dormancy and then outgrow into life-threatening lesions may lead to the identification of novel therapeutic targets for the prevention or treatment of metastatic disease.Advances in genomics and mouse modeling have fostered a renaissance of studies on metastasis (2, 3). Current approaches to the study of metastasis can be divided in two categories. In the first, genetic methods are used to modify the function of a candidate gene in intact mice or in cells that are subsequently transplanted in mice (5, 6). In the second, genomic methods, such as DNA microarray analysis or array comparative genomic hybridization (aCGH), are used to identify a restricted number of candidate genes, which are then tested in appropriate mouse models (7,8). Although these approaches have been extremely successful, they are very laborious and do not necessarily yield biologically potent mediators of metastasis. Functional genetic screens can lead to the rapid identification of strong mediators of a selectable phenotype (9, 10). In agreement with this notion, recent studies have ...

Oral-cavity-only (OCO) identifications of airphase trigeminal stimulus chemicals, i.e., pure chemicals that are often discriminated from their solvents by anosmics and are usually lateralized without sniffing by normosmics, were compared with retronasal identifications made by 20 participants. Participants selected the best possible identification from nine alternatives, but did not respond if they could not provide an identification within the 10-s response interval. It was found that, except for DL-menthol, the frequencies of correct identifications for OCO presentations were significantly different from the frequencies for retronasal presentations. OCO percent correct identifications were: eugenol 7%, heptyl alcohol 5%, nonanal 10%, 1-octanal 18%, and valeric acid 20%, but 58% correct for DL-menthol. Median percent correct OCO identifications were all 0% except 67% for DL-menthol. Modal OCO identification responses were 'no response', except "peppermint" for DL-menthol. In contrast, retronasal overall percent correct identifications, median percent correct identifications, and modal identifications were: eugenol 78%, 100%, "cloves"; heptyl alcohol 49%, 67%, "cleaner"; nonanal 54%, 58%, "citrus"; 1-octanal 71%, 67%, "cleaner"; DL-menthol 80%, 100%, "peppermint"; valeric acid 66%, 67%, "rancid". One implication of the differences between OCO and retronasal responses is that, for many trigeminal stimuli at retronasal-effective concentrations, responses from the oral cavity trigeminal sensory system are not sufficient for identification, suggesting that the oral cavity trigeminal system may be different from and generally provides less differential information than the nasal cavity one. However, because DL-menthol received consistent oral cavity identifications comparable to retronasal identifications, this ten-carbon alcohol may represent a unique class of trigeminal stimuli. Abbreviations ANOVA Analysis of variance dfDegrees of freedom ID Identification OCOOral-cavity-only ODC Odorant delivery container SIR Semi-interquartile range TRPM8 Transient receptor potential melastatine family 8