Integrative identification of non-coding regulatory regions driving metastatic prostate cancer.

Large-scale sequencing efforts have been undertaken to understand the mutational landscape of the coding genome. However, the vast majority of variants occur within non-coding genomic regions. We designed an integrative computational and experimental framework to identify recurrently mutated non-coding regulatory regions that drive tumor progression. Applying this framework to sequencing data from a large prostate cancer patient cohort revealed a large set of candidate drivers. We used (1) in silico analyses, (2) massively parallel reporter assays, and (3) in vivo CRISPR interference screens to systematically validate metastatic castration-resistant prostate cancer (mCRPC) drivers. One identified enhancer region, GH22I030351, acts on a bidirectional promoter to simultaneously modulate expression of the U2-associated splicing factor SF3A1 and chromosomal protein CCDC157. SF3A1 and CCDC157 promote tumor growth in vivo. We nominated a number of transcription factors, notably SOX6, to regulate expression of SF3A1 and CCDC157. Our integrative approach enables the systematic detection of non-coding regulatory regions that drive human cancers.

Integrative identification of non-coding regulatory regions driving metastatic prostate cancer.

Large-scale sequencing efforts of thousands of tumor samples have been undertaken to understand the mutational landscape of the coding genome. However, the vast majority of germline and somatic variants occur within non-coding portions of the genome. These genomic regions do not directly encode for specific proteins, but can play key roles in cancer progression, for example by driving aberrant gene expression control. Here, we designed an integrative computational and experimental framework to identify recurrently mutated non-coding regulatory regions that drive tumor progression. Application of this approach to whole-genome sequencing (WGS) data from a large cohort of metastatic castration-resistant prostate cancer (mCRPC) revealed a large set of recurrently mutated regions. We used (i) in silico prioritization of functional non-coding mutations, (ii) massively parallel reporter assays, and (iii) in vivo CRISPR-interference (CRISPRi) screens in xenografted mice to systematically identify and validate driver regulatory regions that drive mCRPC. We discovered that one of these enhancer regions, GH22I030351, acts on a bidirectional promoter to simultaneously modulate expression of U2-associated splicing factor SF3A1 and chromosomal protein CCDC157. We found that both SF3A1 and CCDC157 are promoters of tumor growth in xenograft models of prostate cancer. We nominated a number of transcription factors, including SOX6, to be responsible for higher expression of SF3A1 and CCDC157. Collectively, we have established and confirmed an integrative computational and experimental approach that enables the systematic detection of non-coding regulatory regions that drive the progression of human cancers.

The DNA methylation landscape of advanced prostate cancer.

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.

Genomic Hallmarks and Structural Variation in Metastatic Prostate Cancer.

While mutations affecting protein-coding regions have been examined across many cancers, structural variants at the genome-wide level are still poorly defined. Through integrative deep whole-genome and -transcriptome analysis of 101 castration-resistant prostate cancer metastases (109X tumor/38X normal coverage), we identified structural variants altering critical regulators of tumorigenesis and progression not detectable by exome approaches. Notably, we observed amplification of an intergenic enhancer region 624 kb upstream of the androgen receptor (AR) in 81% of patients, correlating with increased AR expression. Tandem duplication hotspots also occur near MYC, in lncRNAs associated with post-translational MYC regulation. Classes of structural variations were linked to distinct DNA repair deficiencies, suggesting their etiology, including associations of CDK12 mutation with tandem duplications, TP53 inactivation with inverted rearrangements and chromothripsis, and BRCA2 inactivation with deletions. Together, these observations provide a comprehensive view of how structural variations affect critical regulators in metastatic prostate cancer.

Looking "Under the Hood" of Cellular Mechanotransduction with Computational Tools: A Systems Biomechanics Approach across Multiple Scales.

Signal modulation has been developed in living cells throughout evolution to promote utilizing the same machinery for multiple cellular functions. Chemical and mechanical modules of signal transmission and transduction are interconnected and necessary for organ development and growth. However, due to the high complexity of the intercommunication of physical intracellular connections with biochemical pathways, there are many missing details in our overall understanding of mechanotransduction processes, i.e., the process by which mechanical signals are converted to biochemical cascades. Cell-matrix adhesions are mechanically coupled to the nucleus through the cytoskeleton. This modulated and tightly integrated network mediates the transmission of mechanochemical signals from the extracellular matrix to the nucleus. Various experimental and computational techniques have been utilized to understand the basic mechanisms of mechanotransduction, yet many aspects have remained elusive. Recently, in silico experiments have made important contributions to the field of mechanobiology. Herein, computational modeling efforts devoted to understanding integrin-mediated mechanotransduction pathways are reviewed, and an outlook is presented for future directions toward using suitable computational approaches and developing novel techniques for addressing important questions in the field of mechanotransduction.

The interaction of CRM1 and the nuclear pore protein Tpr.

While much has been devoted to the study of transport mechanisms through the nuclear pore complex (NPC), the specifics of interactions and binding between export transport receptors and the NPC periphery have remained elusive. Recent work has demonstrated a binding interaction between the exportin CRM1 and the unstructured carboxylic tail of Tpr, on the nuclear basket. Strong evidence suggests that this interaction is vital to the functions of CRM1. Using molecular dynamics simulations and a newly refined method for determining binding regions, we have identified nine candidate binding sites on CRM1 for C-Tpr. These include two adjacent to RanGTP--from which one is blocked in the absence of RanGTP--and three next to the binding region of the cargo Snurportin. We report two additional interaction sites between C-Tpr and Snurportin, suggesting a possible role for Tpr import into the nucleus. Using bioinformatics tools we have conducted conservation analysis and functional residue prediction investigations to identify which parts of the obtained binding sites are inherently more important and should be highlighted. Also, a novel measure based on the ratio of available solvent accessible surface (RASAS) is proposed for monitoring the ligand/receptor binding process.

Brownian dynamics simulation of nucleocytoplasmic transport: a coarse-grained model for the functional state of the nuclear pore complex.

The nuclear pore complex (NPC) regulates molecular traffic across the nuclear envelope (NE). Selective transport happens on the order of milliseconds and the length scale of tens of nanometers; however, the transport mechanism remains elusive. Central to the transport process is the hydrophobic interactions between karyopherins (kaps) and Phe-Gly (FG) repeat domains. Taking into account the polymeric nature of FG-repeats grafted on the elastic structure of the NPC, and the kap-FG hydrophobic affinity, we have established a coarse-grained model of the NPC structure that mimics nucleocytoplasmic transport. To establish a foundation for future works, the methodology and biophysical rationale behind the model is explained in details. The model predicts that the first-passage time of a 15 nm cargo-complex is about 2.6±0.13 ms with an inverse Gaussian distribution for statistically adequate number of independent Brownian dynamics simulations. Moreover, the cargo-complex is primarily attached to the channel wall where it interacts with the FG-layer as it passes through the central channel. The kap-FG hydrophobic interaction is highly dynamic and fast, which ensures an efficient translocation through the NPC. Further, almost all eight hydrophobic binding spots on kap-ß are occupied simultaneously during transport. Finally, as opposed to intact NPCs, cytoplasmic filaments-deficient NPCs show a high degree of permeability to inert cargos, implying the defining role of cytoplasmic filaments in the selectivity barrier.