Collaborative study from the Bladder Cancer Advocacy Network for the genomic analysis of metastatic urothelial cancer.

Urothelial Cancer - Genomic Analysis to Improve Patient Outcomes and Research (NCT02643043), UC-GENOME, is a genomic analysis and biospecimen repository study in 218 patients with metastatic urothelial carcinoma. Here we report on the primary outcome of the UC-GENOME-the proportion of subjects who received next generation sequencing (NGS) with treatment options-and present the initial genomic analyses and clinical correlates. 69.3% of subjects had potential treatment options, however only 5.0% received therapy based on NGS. We found an increased frequency of TP53E285K mutations as compared to non-metastatic cohorts and identified features associated with benefit to chemotherapy and immune checkpoint inhibition, including: Ba/Sq and Stroma-rich subtypes, APOBEC mutational signature (SBS13), and inflamed tumor immune phenotype. Finally, we derive a computational model incorporating both genomic and clinical features predictive of immune checkpoint inhibitor response. Future work will utilize the biospecimens alongside these foundational analyses toward a better understanding of urothelial carcinoma biology.

CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment.

We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC.

The ERK mitogen-activated protein kinase signaling network: the final frontier in RAS signal transduction.

The RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade is aberrantly activated in a diverse set of human cancers and the RASopathy group of genetic developmental disorders. This protein kinase cascade is one of the most intensely studied cellular signaling networks and has been frequently targeted by the pharmaceutical industry, with more than 30 inhibitors either approved or under clinical evaluation. The ERK-MAPK cascade was originally depicted as a serial and linear, unidirectional pathway that relays extracellular signals, such as mitogenic stimuli, through the cytoplasm to the nucleus. However, we now appreciate that this three-tiered protein kinase cascade is a central core of a complex network with dynamic signaling inputs and outputs and autoregulatory loops. Despite our considerable advances in understanding the ERK-MAPK network, the ability of cancer cells to adapt to the inhibition of key nodes reveals a level of complexity that remains to be fully understood. In this review, we summarize important developments in our understanding of the ERK-MAPK network and identify unresolved issues for ongoing and future study.

Comprehensive transcriptomic profiling reveals SOX7 as an early regulator of angiogenesis in hypoxic human endothelial cells.

Endothelial cells (ECs) lining the vasculature of vertebrates respond to low oxygen (hypoxia) by maintaining vascular homeostasis and initiating adaptive growth of new vasculature through angiogenesis. Previous studies have uncovered the molecular underpinnings of the hypoxic response in ECs; however, there is a need for comprehensive temporal analysis of the transcriptome during hypoxia. Here, we sought to investigate the early transcriptional programs of hypoxic ECs by using RNA-Seq of primary cultured human umbilical vein ECs exposed to progressively increasing severity and duration of hypoxia. We observed that hypoxia modulates the expression levels of approximately one-third of the EC transcriptome. Intriguingly, expression of the gene encoding the developmental transcription factor SOX7 (SRY-box transcription factor 7) rapidly and transiently increased during hypoxia. Transcriptomic and functional analyses of ECs following SOX7 depletion established its critical role in regulating hypoxia-induced angiogenesis. We also observed that depletion of the hypoxia-inducible factor (HIF) genes, HIF1A (encoding HIF-1α) and endothelial PAS domain protein 1 (EPAS1 encoding HIF-2α), inhibited both distinct and overlapping transcriptional programs. Our results indicated a role for HIF-1α in down-regulating mitochondrial metabolism while concomitantly up-regulating glycolytic genes, whereas HIF-2α primarily up-regulated the angiogenesis transcriptional program. These results identify the concentration and time dependence of the endothelial transcriptomic response to hypoxia and an early key role for SOX7 in mediating angiogenesis.

Embryo polarity in moth flies and mosquitoes relies on distinct old genes with localized transcript isoforms.

Unrelated genes establish head-to-tail polarity in embryos of different fly species, raising the question of how they evolve this function. We show that in moth flies (Clogmia, Lutzomyia), a maternal transcript isoform of odd-paired (Zic) is localized in the anterior egg and adopted the role of anterior determinant without essential protein change. Additionally, Clogmia lost maternal germ plasm, which contributes to embryo polarity in fruit flies (Drosophila). In culicine (Culex, Aedes) and anopheline mosquitoes (Anopheles), embryo polarity rests on a previously unnamed zinc finger gene (cucoid), or pangolin (dTcf), respectively. These genes also localize an alternative transcript isoform at the anterior egg pole. Basal-branching crane flies (Nephrotoma) also enrich maternal pangolin transcript at the anterior egg pole, suggesting that pangolin functioned as ancestral axis determinant in flies. In conclusion, flies evolved an unexpected diversity of anterior determinants, and alternative transcript isoforms with distinct expression can adopt fundamentally distinct developmental roles.

Analysis of biological networks in the endothelium with biomimetic microsystem platform.

Here we describe a novel method for studying the protein "interactome" in primary human cells and apply this method to investigate the effect of posttranslational protein modifications (PTMs) on the protein's functions. We created a novel "biomimetic microsystem platform" (Bio-MSP) to isolate the protein complexes in primary cells by covalently attaching purified His-tagged proteins to a solid microscale support. Using this Bio-MSP, we have analyzed the interactomes of unphosphorylated and phosphomimetic end-binding protein-3 (EB3) in endothelial cells. Pathway analysis of these interactomes demonstrated the novel role of EB3 phosphorylation at serine 162 in regulating the protein's function. We showed that phosphorylation "switches" the EB3 biological network to modulate cellular processes such as cell-to-cell adhesion whereas dephosphorylation of this site promotes cell proliferation. This novel technique provides a useful tool to study the role of PTMs or single point mutations in activating distinct signal transduction networks and thereby the biological function of the protein in health and disease.

N-cadherin signaling via Trio assembles adherens junctions to restrict endothelial permeability.

Vascular endothelial (VE)-cadherin forms homotypic adherens junctions (AJs) in the endothelium, whereas N-cadherin forms heterotypic adhesion between endothelial cells and surrounding vascular smooth muscle cells and pericytes. Here we addressed the question whether both cadherin adhesion complexes communicate through intracellular signaling and contribute to the integrity of the endothelial barrier. We demonstrated that deletion of N-cadherin (Cdh2) in either endothelial cells or pericytes increases junctional endothelial permeability in lung and brain secondary to reduced accumulation of VE-cadherin at AJs. N-cadherin functions by increasing the rate of VE-cadherin recruitment to AJs and induces the assembly of VE-cadherin junctions. We identified the dual Rac1/RhoA Rho guanine nucleotide exchange factor (GEF) Trio as a critical component of the N-cadherin adhesion complex, which activates both Rac1 and RhoA signaling pathways at AJs. Trio GEF1-mediated Rac1 activation induces the recruitment of VE-cadherin to AJs, whereas Trio GEF2-mediated RhoA activation increases intracellular tension and reinforces Rac1 activation to promote assembly of VE-cadherin junctions and thereby establish the characteristic restrictive endothelial barrier.

A computational approach to identify cellular heterogeneity and tissue-specific gene regulatory networks.

BACKGROUND: The heterogeneity of cells across tissue types represents a major challenge for studying biological mechanisms as well as for therapeutic targeting of distinct tissues. Computational prediction of tissue-specific gene regulatory networks may provide important insights into the mechanisms underlying the cellular heterogeneity of cells in distinct organs and tissues. RESULTS: Using three pathway analysis techniques, gene set enrichment analysis (GSEA), parametric analysis of gene set enrichment (PGSEA), alongside our novel model (HeteroPath), which assesses heterogeneously upregulated and downregulated genes within the context of pathways, we generated distinct tissue-specific gene regulatory networks. We analyzed gene expression data derived from freshly isolated heart, brain, and lung endothelial cells and populations of neurons in the hippocampus, cingulate cortex, and amygdala. In both datasets, we found that HeteroPath segregated the distinct cellular populations by identifying regulatory pathways that were not identified by GSEA or PGSEA. Using simulated datasets, HeteroPath demonstrated robustness that was comparable to what was seen using existing gene set enrichment methods. Furthermore, we generated tissue-specific gene regulatory networks involved in vascular heterogeneity and neuronal heterogeneity by performing motif enrichment of the heterogeneous genes identified by HeteroPath and linking the enriched motifs to regulatory transcription factors in the ENCODE database. CONCLUSIONS: HeteroPath assesses contextual bidirectional gene expression within pathways and thus allows for transcriptomic assessment of cellular heterogeneity. Unraveling tissue-specific heterogeneity of gene expression can lead to a better understanding of the molecular underpinnings of tissue-specific phenotypes.

Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells.

Human endothelial cells (ECs) are widely used to study mechanisms of angiogenesis, inflammation, and endothelial permeability. Targeted gene disruption induced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) nuclease gene editing is potentially an important tool for definitively establishing the functional roles of individual genes in ECs. We showed that co-delivery of adenovirus encoding EGFP-tagged Cas9 and lentivirus encoding a single guide RNA (sgRNA) in primary human lung microvascular ECs (HLMVECs) disrupted the expression of the Tie2 gene and protein. Tie2 disruption increased basal endothelial permeability and prevented permeability recovery following injury induced by the inflammatory stimulus thrombin. Thus, gene deletion via viral co-delivery of CRISPR-Cas9 in primary human ECs provides a novel platform to investigate signaling mechanisms of normal and perturbed EC function without the need for clonal expansion.

Molecular mechanisms underlying the exceptional adaptations of batoid fins.

Extreme novelties in the shape and size of paired fins are exemplified by extinct and extant cartilaginous and bony fishes. Pectoral fins of skates and rays, such as the little skate (Batoid, Leucoraja erinacea), show a strikingly unique morphology where the pectoral fin extends anteriorly to ultimately fuse with the head. This results in a morphology that essentially surrounds the body and is associated with the evolution of novel swimming mechanisms in the group. In an approach that extends from RNA sequencing to in situ hybridization to functional assays, we show that anterior and posterior portions of the pectoral fin have different genetic underpinnings: canonical genes of appendage development control posterior fin development via an apical ectodermal ridge (AER), whereas an alternative Homeobox (Hox)-Fibroblast growth factor (Fgf)-Wingless type MMTV integration site family (Wnt) genetic module in the anterior region creates an AER-like structure that drives anterior fin expansion. Finally, we show that GLI family zinc finger 3 (Gli3), which is an anterior repressor of tetrapod digits, is expressed in the posterior half of the pectoral fin of skate, shark, and zebrafish but in the anterior side of the pelvic fin. Taken together, these data point to both highly derived and deeply ancestral patterns of gene expression in skate pectoral fins, shedding light on the molecular mechanisms behind the evolution of novel fin morphologies.

Embryo development. A cysteine-clamp gene drives embryo polarity in the midge Chironomus.

In the fruit fly Drosophila, head formation is driven by a single gene, bicoid, which generates head-to-tail polarity of the main embryonic axis. Bicoid deficiency results in embryos with tail-to-tail polarity and no head. However, most insects lack bicoid, and the molecular mechanism for establishing head-to-tail polarity is poorly understood. We have identified a gene that establishes head-to-tail polarity of the mosquito-like midge, Chironomus riparius. This gene, named panish, encodes a cysteine-clamp DNA binding domain and operates through a different mechanism than bicoid. This finding, combined with the observation that the phylogenetic distributions of panish and bicoid are limited to specific families of flies, reveals frequent evolutionary changes of body axis determinants and a remarkable opportunity to study gene regulatory network evolution.

Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis.

Inherited mutations in the folliculin (FLCN) gene cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterized. We identified plakophilin-4 (p0071) as a potential novel folliculin interacting protein by yeast two-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to the regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially co-localized at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin-deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin-deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalization of E-cadherin in mouse inner medullary collecting duct-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and the regulation of RhoA signalling.

Genome-wide matching of genes to cellular roles using guilt-by-association models derived from single sample analysis.

BACKGROUND: High-throughput methods that ascribe a cellular or physiological function for each gene product are useful to understand the roles of genes that have not been extensively characterized by molecular or genetic approaches. One method to infer gene function is "guilt-by-association", in which the expression pattern of a poorly characterized gene is shown to co-vary with the expression of better-characterized genes. The function of the poorly characterized gene is inferred from the known function(s) of the well-described genes. For example, genes co-expressed with transcripts that vary during the cell cycle, development, environmental stresses, and with oncogenesis have been implicated in those processes. FINDINGS: While examining the expression characteristics of several poorly characterized genes, we noted that we could associate each of the genes with a cellular phenotype by correlating individual gene expression changes with gene set enrichment scores from individual samples. We evaluated the effectiveness of this approach using a modest sized gene expression data set (expO) and a compendium of gene expression phenotypes (MSigDBv3.0). We found the transcripts that correlated best with enrichment in mitochondrial and lysosomal gene sets were mostly related to those processes (89/100 and 44/50, respectively). The reciprocal evaluation, ranking gene sets according to correlation of enrichment with an individual gene's expression, also reflected known associations for prominent genes in the biomedical literature (16/19). In evaluating the model, we also found that 4% of the genome encodes proteins that are associated with small molecule and small peptide signal transduction gene sets, implicating a large number of genes in both internal and external environmental sensing. CONCLUSIONS: Our results show that this approach is useful to infer functions of disparate sets of genes. This method mirrors the biological experimental approaches used by others to associate individual genes with defined gene expression changes. Moreover, the approach can be used beyond discovering genes related to a cellular process to discover meaningful expression phenotypes from a compendium that are associated with a given gene. The effectiveness, versatility, and breadth of this approach make possible its application in a variety of contexts and with a variety of downstream analyses.

MK-STYX, a catalytically inactive phosphatase regulating mitochondrially dependent apoptosis.

Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism.

Birt-Hogg-Dubé renal tumors are genetically distinct from other renal neoplasias and are associated with up-regulation of mitochondrial gene expression.

BACKGROUND: Germline mutations in the folliculin (FLCN) gene are associated with the development of Birt-Hogg-Dubé syndrome (BHDS), a disease characterized by papular skin lesions, a high occurrence of spontaneous pneumothorax, and the development of renal neoplasias. The majority of renal tumors that arise in BHDS-affected individuals are histologically similar to sporadic chromophobe renal cell carcinoma (RCC) and sporadic renal oncocytoma. However, most sporadic tumors lack FLCN mutations and the extent to which the BHDS-derived renal tumors share genetic defects associated with the sporadic tumors has not been well studied. METHODS: BHDS individuals were identified symptomatically and FLCN mutations were confirmed by DNA sequencing. Comparative gene expression profiling analyses were carried out on renal tumors isolated from individuals afflicted with BHDS and a panel of sporadic renal tumors of different subtypes using discriminate and clustering approaches. qRT-PCR was used to confirm selected results of the gene expression analyses. We further analyzed differentially expressed genes using gene set enrichment analysis and pathway analysis approaches. Pathway analysis results were confirmed by generation of independent pathway signatures and application to additional datasets. RESULTS: Renal tumors isolated from individuals with BHDS showed distinct gene expression and cytogenetic characteristics from sporadic renal oncocytoma and chromophobe RCC. The most prominent molecular feature of BHDS-derived kidney tumors was high expression of mitochondria-and oxidative phosphorylation (OXPHOS)-associated genes. This mitochondria expression phenotype was associated with deregulation of the PGC-1α-TFAM signaling axis. Loss of FLCN expression across various tumor types is also associated with increased nuclear mitochondrial gene expression. CONCLUSIONS: Our results support a genetic distinction between BHDS-associated tumors and other renal neoplasias. In addition, deregulation of the PGC-1α-TFAM signaling axis is most pronounced in renal tumors that harbor FLCN mutations and in tumors from other organs that have relatively low expression of FLCN. These results are consistent with the recently discovered interaction between FLCN and AMPK and support a model in which FLCN is a regulator of mitochondrial function.