Brain metastasis is a major cause of morbidity and mortality in patients with melanoma. The molecular changes that lead to brain metastasis remain poorly understood. In this study, we developed a model to study human melanoma brain metastasis and found that Stat3 activity was increased in human brain metastatic melanoma cells when compared with that in cutaneous melanoma cells. The expression of activated Stat3 is also increased in human brain metastasis specimens when compared with that in the primary melanoma specimens. Increased Stat3 activation by transfection with a constitutively activated Stat3 enhanced brain metastasis, whereas blockade of Stat3 activation by transfection with a dominant-negative Stat3 suppressed brain metastasis of human melanoma cells in animal models. Furthermore, altered Stat3 activity profoundly affected melanoma angiogenesis in vivo and melanoma cell invasion in vitro and significantly affected the expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-2 (MMP-2) in vivo and in vitro. Finally, Stat3 activity transcriptionally regulated the promoter activity of bFGF in addition to VEGF and MMP-2 in human melanoma cells. These results indicated that Stat3 activation plays an important role in dysregulated expression of bFGF, VEGF, and MMP-2 as well as angiogenesis and invasion of melanoma cells and contributes to brain metastasis of melanoma. Therefore, Stat3 activation might be a new potential target for therapy of human melanoma brain metastases. (Cancer Res 2006; 66(6): 3188-96)

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in malignant glioma. However, the functional importance of this factor in human glioma is not known. In the present study, we found that FoxM1B was the predominant FoxM1 isoform expressed in human glioma but not in normal brain tissue. The level of FoxM1 protein expression in human glioma tissues was directly correlated with the glioma grade. The level of FoxM1 protein expression in human glioblastoma tissues was inversely correlated with patient survival. Enforced FoxM1B expression caused SW1783 and Hs683 glioma cells, which do not form tumor xenografts, to regain tumorigenicity in nude mouse model systems. Moreover, gliomas that arose from FoxM1B-transfected anaplastic astrocytoma SW1783 cells displayed glioblastoma multiforme phenotypes. Inhibition of FoxM1 expression in glioblastoma U-87MG cells suppressed their anchorage-independent growth in vitro and tumorigenicity in vivo. Furthermore, we found that FoxM1 regulates the expression of Skp2 protein, which is known to promote degradation of the cell cycle regulator p27 Kip1 . These results showed that FoxM1 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity. Therefore, FoxM1 might be a new potential target of therapy for human malignant gliomas. (Cancer Res 2006; 66(7): 3593-602)

Polyploidy is much rarer in animals than in plants but it is not known why. The outcome of combining two genomes in vertebrates remains unpredictable, especially because polyploidization seldom shows positive effects and more often results in lethal consequences because viable gametes fail to form during meiosis. Fortunately, the goldfish (maternal) × common carp (paternal) hybrids have reproduced successfully up to generation 22, and this hybrid lineage permits an investigation into the genomics of hybridization and tetraploidization. The first two generations of these hybrids are diploids, and subsequent generations are tetraploids. Liver transcriptomes from four generations and their progenitors reveal chimeric genes (>9%) and mutations of orthologous genes. Characterizations of 18 randomly chosen genes from genomic DNA and cDNA confirm the chimera. Some of the chimeric and differentially expressed genes relate to mutagenesis, repair, and cancer-related pathways in 2nF 1 . Erroneous DNA excision between homologous parental genes may drive the high percentage of chimeric genes, or even more potential mechanisms may result in this phenomenon. Meanwhile, diploid offspring show paternal-biased expression, yet tetraploids show maternal-biased expression. These discoveries reveal that fast and unstable changes are mainly deleterious at the level of transcriptomes although some offspring still survive their genomic abnormalities. In addition, the synthetic effect of genome shock might have resulted in greatly reduced viability of 2nF 2 hybrid offspring. The goldfish × common carp hybrids constitute an ideal system for unveiling the consequences of intergenomic interactions in hybrid vertebrate genomes and their fertility.allopolyploidization | chimeric genes | transcriptomes | sequence validation | vertebrate P olyploidization is much rarer in vertebrates than in plants, and the reasons for this difference remain a mystery (1-3). Traditional explanations include barriers to sex determination, physiological and developmental constraints (especially nuclearcytoplasmic interactions and related factors) (2, 3), and genome shock or dramatic genomic restructuring (2-4). One type of polyploidization, allopolyploidization, involves the genomes of two species. Hybridization, accompanied by polyploidization, triggers vast genetic and genomic imbalances, including abnormal quadrivalent chromosomal groups, dosage imbalances, a high rate of DNA mutations and combinations, and other non-Mendelian phenomena (5-7). The effects of these imbalances are usually deleterious and are rarely advantageous. Imbalances in many plant crops determine the fate of the allopolyploid offspring. Genomic changes immediately follow allopolyploidization. Various and SignificanceWhy is polyploidization rarer in animals than in plants? This question remains unanswered due to the absence of a suitable system in animals for studying instantaneous polyploidization and the crucial changes that immediately follow hybridization. RNA-seq analyses discover exte...

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Our recent study found that activation of signal transducer and activator of transcription 3 (Stat3) is up-regulated in human brain metastatic cells and contributes to brain metastasis of melanoma. However, the molecular mechanisms underlying this increased Stat3 activation and effect on brain metastasis are unknown. In this report, we showed that the expression of Janus-activated kinase 2 (JAK2), a Stat3 activator, was increased, whereas the expression of a negative regulator of Stat3, suppressor of cytokine signaling-1 (SOCS-1), was reduced in the brain metastatic melanoma cell line A375Br, relative to that in the parental A375P cell line. Consistently, SOCS-1 expression was also lower in the human brain metastatic tissues than in the primary melanoma tissues. Mechanistically, increased JAK2 expression in the A375Br cells was due to, at least in part, its decreased degradation, which was directly correlated with low expression of SOCS-1. Moreover, restoration of SOCS-1 expression resulted in the inhibition of Stat3 activation, whereas depletion of SOCS-1 up-regulated Stat3 activation. These clinical, experimental, and mechanistic findings strongly suggest that increased activation of Stat3 in brain metastatic melanoma cells might be due to decreased SOCS-1 expression. Furthermore, restoration of SOCS-1 expression in brain metastatic A375Br cells significantly inhibited brain metastasis in animal models (P < 0.001). Additionally, alterations of SOCS-1 expression profoundly affected the expression of matrix metalloproteinase-2 (MMP-2), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) and the melanoma cell invasion and angiogenesis. Collectively, these data suggest that the loss of SOCS-1 expression is a critical event, leading to elevated Stat3 signaling and overexpression of MMP-2, bFGF, and VEGF, as well as enhanced invasion and angiogenesis of melanoma cells, consequently promoting brain metastasis. [Cancer Res 2008;68(23):9634-42]

A persistent enigma is the rarity of polyploidy in animals, compared to its prevalence in plants. Although animal polyploids are thought to experience deleterious genomic chaos during initial polyploidization and subsequent rediploidization processes, this hypothesis has not been tested. We provide an improved reference-quality de novo genome for allotetraploid goldfish whose origin dates to ~15 million years ago. Comprehensive analyses identify changes in subgenomic evolution from asymmetrical oscillation in goldfish and common carp to diverse stabilization and balanced gene expression during continuous rediploidization. The homoeologs are coexpressed in most pathways, and their expression dominance shifts temporally during embryogenesis. Homoeolog expression correlates negatively with alternation of DNA methylation. The results show that allotetraploid cyprinids have a unique strategy for balancing subgenomic stabilization and diversification. Rediploidization process in these fishes provides intriguing insights into genome evolution and function in allopolyploid vertebrates.

Although complex interactions between hosts and microbial associates are increasingly well documented, we still know little about how and why hosts shape microbial communities in nature. In addition, host genetic effects on microbial communities vary widely depending on the environment, obscuring conclusions about which microbes are impacted and which plant functions are important. We characterized the leaf microbiota of 200 Arabidopsis thaliana genotypes in eight field experiments and detected consistent host effects on specific, broadly distributed microbial species (operational taxonomic unit [OTUs]). Host genetic effects disproportionately influenced central ecological hubs, with heritability of particular OTUs declining with their distance from the nearest hub within the microbial network. These host effects could reflect either OTUs preferentially associating with specific genotypes or differential microbial success within them. Host genetics associated with microbial hubs explained over 10% of the variation in lifetime seed production among host genotypes across sites and years. We successfully cultured one of these microbial hubs and demonstrated its growth-promoting effects on plants in sterile conditions. Finally, genome-wide association mapping identified many putatively causal genes with small effects on the relative abundance of microbial hubs across sites and years, and these genes were enriched for those involved in the synthesis of specialized metabolites, auxins, and the immune system. Using untargeted metabolomics, we corroborate the consistent association between variation in specialized metabolites and microbial hubs across field sites. Together, our results reveal that host genetic variation impacts the microbial communities in consistent ways across environments and that these effects contribute to fitness variation among host genotypes.

Stat3 activation has been implicated as an important driver of brain metastasis in breast cancer, but the critical targets of Stat3 in this process are yet to be fully defined. In this study, we identified the lipid raft organizing protein Caveolin-1 (Cav-1) as a critical genetic target of Stat3 in this process. In human breast cancers, we found that activated Stat3 correlated with attenuation of Cav-1 in brain metastases relative to primary tumors. Cav-1 promoter activity and gene expression was increased by overexpressing an activated form of Stat3, but decreased by attenuation of Stat3 activity or expression. We identified putative Stat3-binding elements in the Cav-1 promoter and demonstrated a direct repression of Cav-1 transcription by Stat3. Reciprocally, we demonstrated that strategies to increase or decrease Cav-1 expression were sufficient to attenuate or promote breast cancer cell invasion. Further, increased expression of Cav-1 phenocopied the effects of Stat3 activation in blocking primary tumor growth and abrogating formation of brain metastases. Collectively, our findings provide clinical and mechanistic evidence that Cav-1 is a critical target for suppression by Stat3 in driving invasion and metastasis of breast cancer cells.