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Background Currently, there is no in vitro or ex vivo model that can isolate circulating tumor cells (CTCs). Recently, we developed a four-dimensional (4D) lung cancer model that allows for the isolation of CTCs. We postulated that these cells have different properties than parental (2D) cells. Materials and methods We obtained CTCs by growing A549, H1299, 393P, and 344SQ cell lines on the 4D lung model. The CTCs were functionally characterized in vitro and gene expression of the cell adhesion molecules was compared with respective 2D cells. Integrin beta 4 (ITGB4) was further investigated by stably transfecting the A549 and H1299 cells. Results We found that all cell lines produced CTCs, and that CTCs from the 4D model were less adherent to the plastic and have a slower growth rate than respective 2D cells (P < 0.01). Most of the cell adhesion molecules were downregulated (P < 0.05) in CTCs, and ITGB4 was the common molecule, significantly more underexpressed in CTCs from all cell lines than their respective 2D cells. The modulation of ITGB4 led to a differential function of 2D cells. Conclusions CTCs from the 4D model have different transcriptional, translational, and in vitro characteristics than the same cells grown on a petri dish, and these CTCs from the 4D model have the properties of CTCs that are responsible for metastasis. [ABSTRACT FROM AUTHOR]

Summary: Clinical and genomic evidence suggests that the metastatic potential of a primary tumor may be dictated by prometastatic events that have additional oncogenic capability. To test this “deterministic” hypothesis, we adopted a comparative oncogenomics-guided function-based strategy involving: (1) comparison of global transcriptomes of two genetically engineered mouse models with contrasting metastatic potential, (2) genomic and transcriptomic profiles of human melanoma, (3) functional genetic screen for enhancers of cell invasion, and (4) evidence of expression selection in human melanoma tissues. This integrated effort identified six genes that are potently proinvasive and oncogenic. Furthermore, we show that one such gene, ACP5, confers spontaneous metastasis in vivo, engages a key pathway governing metastasis, and is prognostic in human primary melanomas. [Copyright &y& Elsevier]

The complexity of genomic alterations in cancer has made it difficult to identify oncogenic drivers for the development of targeted therapies. The study by Berger et al. in this issue of Cancer Cell demonstrates that high-throughput functional profiling can uncover impactful mutations and oncogenic driver alleles. [ABSTRACT FROM AUTHOR]

Genome-wide copy number analyses of human cancers identified a frequent 5p13 amplification in several solid tumour types, including lung (56%), ovarian (38%), breast (32%), prostate (37%) and melanoma (32%). Here, using integrative analysis of a genomic profile of the region, we identify a Golgi protein, GOLPH3, as a candidate targeted for amplification. Gain- and loss-of-function studies in vitro and in vivo validated GOLPH3 as a potent oncogene. Physically, GOLPH3 localizes to the trans-Golgi network and interacts with components of the retromer complex, which in yeast has been linked to target of rapamycin (TOR) signalling. Mechanistically, GOLPH3 regulates cell size, enhances growth-factor-induced mTOR (also known as FRAP1) signalling in human cancer cells, and alters the response to an mTOR inhibitor in vivo. Thus, genomic and genetic, biological, functional and biochemical data in yeast and humans establishes GOLPH3 as a new oncogene that is commonly targeted for amplification in human cancer, and is capable of modulating the response to rapamycin, a cancer drug in clinical use. [ABSTRACT FROM AUTHOR]

Abstract: Checkpoint Suppressor 1 (CHES1; FOXN3) encodes a member of the forkhead/winged-helix transcription factor family. The human CHES1 cDNA was originally identified by its ability to function as a high-copy suppressor of multiple checkpoint mutants of Saccharomyces cerevisiae. Accumulating expression profile data suggest that CHES1 plays a role in tumorigenicity and responses to cancer treatments, though nothing is known regarding the transcriptional function of CHES1 or other FOXN proteins in human cells. In this report, we find that the carboxyl terminus of CHES1 fused to a heterologous DNA binding domain consistently represses reporter gene transcription in cell lines derived from tumor tissues. Using a cytoplasmic two-hybrid screening approach, we find that this portion of CHES1 interacts with Ski-interacting protein (SKIP; NCoA-62), which is a transcriptional co-regulator known to associate with repressor complexes. We verify this interaction through co-immunoprecipitation experiments performed in mammalian cells. Further analysis of the CHES1/SKIP interaction indicates that CHES1 binds to a region within the final 66 hydrophobic residues of SKIP thus defining a new protein–protein interaction domain of SKIP. These data suggest that CHES1 recruits SKIP to repress genes important for tumorigenesis and the response to cancer treatments. [Copyright &y& Elsevier]

Reports that the loss of Sin3/Rpd3 histone deacetylase restores DNA damage response in checkpoint-deficient strains of Saccharomyces cerevisiae. Capability of functional glutathione S-transferase-Ches1 fusion protein to bind in vivo to SIN3; Increase in the resistance to ultraviolet irradiation; Effect of SIN3 on the response to DNA damage.

There are hundreds of genes typically overexpressed in breast cancer cells and it's often assumed that their overexpression contributes to cancer progression. However, the precise proportion of these overexpressed genes contributing to tumorigenicity remains unclear. To address this gap, we undertook a comprehensive screening of a diverse set of seventy-two genes overexpressed in breast cancer. This systematic screening evaluated their potential for inducing malignant transformation and, concurrently, assessed their impact on breast cancer cell proliferation and viability. Select genes including ALDH3B1, CEACAM5, IL8, PYGO2, and WWTR1, exhibited pronounced activity in promoting tumor formation and establishing gene dependencies critical for tumorigenicity. Subsequent investigations revealed that CEACAM5 overexpression triggered the activation of signaling pathways involving β-catenin, Cdk4, and mTOR. Additionally, it conferred a growth advantage independent of exogenous insulin in defined medium and facilitated spheroid expansion by inducing multiple layers of epithelial cells while preserving a hollow lumen. Furthermore, the silencing of CEACAM5 expression synergized with tamoxifen-induced growth inhibition in breast cancer cells. These findings underscore the potential of screening overexpressed genes for both oncogenic drivers and tumor dependencies to expand the repertoire of therapeutic targets for breast cancer treatment. [ABSTRACT FROM AUTHOR]

Identification of cancer driver mutations is critical for advancing cancer research and personalized medicine. Due to inter-tumor genetic heterogeneity, many driver mutations occur at low frequencies, which make it challenging to distinguish them from passenger mutations. Here, we show that a novel Bayesian hierarchical modeling approach, named rDriver can achieve enhanced prediction accuracy by identifying mutations that not only have high functional impact scores but also are associated with systemic variation in gene expression levels. In examining 3,080 tumor samples from 8 cancer types in The Cancer Genome Atlas, rDriver predicted 1,389 driver mutations. Compared with existing tools, rDriver identified more low frequency mutations associated with lineage specific functional properties, timing of occurrence and patient survival. Evaluation of rDriver predictions using engineered cell-line models resulted in a positive predictive value of 0.94 in PIK3CA genes. Our study highlights the importance of integrating multi-omic data in predicting cancer driver mutations and provides a statistically rigorous solution for cancer target discovery and development. [ABSTRACT FROM AUTHOR]

Epithelial tumor cells undergo epithelial-to-mesenchymal transition (EMT) to gain metastatic activity. Competing endogenous RNAs (ceRNAs) have binding sites for a common set of microRNAs (miRs) and regulate each other's expression by sponging miRs. Here, we address whether ceRNAs govern metastasis driven by the EMT-activating transcription factor ZEB1. High miR-181b levels were correlated with an improved prognosis in human lung adenocarcinomas, and metastatic tumor cell lines derived from a murine lung adenocarcinoma model in which metastasis is ZEB1-driven were enriched in miR-181b targets. ZEB1 relieved a strong basal repression of α1 integrin (ITGA1) mRNA, which in turn upregulated adenylyl cyclase 9 mRNA (ADCY9) by sponging miR181b. Ectopic expression of the ITGA1 3'-untranslated region reversed miR-181b-mediated metastasis suppression and increased the levels of adenylyl cyclase 9 protein (AC9), which promoted tumor cell migration and metastasis. In human lung adenocarcinomas, ITGA1 and ADCY9 levels were positively correlated, and an AC9-activated transcriptomic signature had poor-prognostic value. Thus, ZEB1 initiates a miR-181b-regulated ceRNA network to drive metastasis. [ABSTRACT FROM AUTHOR]

Von Hippel-Landau (VHL) protein is a potent tumor suppressor regulating numerous pathways that drive cancer, but mutations in VHL are restricted to limited subsets of malignancies. Here we identified a novel mechanism for VHL suppression in tumors that do not have inactivating mutations. Using developmental processes to uncover new pathways contributing to tumorigenesis, we found that Daam2 promotes glioma formation. Protein expression screening identified an inverse correlation between Daam2 and VHL expression across a host of cancers, including glioma. These in silico insights guided corroborating functional studies, which revealed that Daam2 promotes tumorigenesis by suppressing VHL expression. Furthermore, biochemical analyses demonstrate that Daam2 associates with VHL and facilitates its ubiquitination and degradation. Together, these studies are the first to define an upstream mechanism regulating VHL suppression in cancer and describe the role of Daam2 in tumorigenesis. [ABSTRACT FROM AUTHOR]

Background Recent molecular advances suggest that Spitz nevi and other spitzoid neoplasms are biologically distinct from melanoma and conventional nevi. The ubiquitin ligase UBE2C and the homeobox transcription factor HOXA1 are candidate oncogenes in melanoma. Methods Using RNA expression analysis and immunohistochemistry, we evaluated these biomarkers in Spitz nevi ( n = 20), melanoma ( n = 20), and by immunohistochemistry in conventional nevi ( n = 20). Results RNA analysis with branched DNA multiplex assay identified upregulation of UBE2C in melanomas vs Spitz nevi ( P = .003), whereas HOXA1 was downregulated in melanoma ( P < .0001). Immunohistochemical analysis confirmed increased nuclear expression of UBE2C in melanoma (mean = 18% of cells; range 3%-44%) when compared with Spitz nevi (mean = 9%; range 2%-28%; P = .001) and conventional nevi (mean = 1.5%; range 0-9%; P < .0001). Strong UBE2C staining was identified in cells undergoing mitosis. UBE2C RNA and protein detection correlated with mitotic rate ( P < .0001). On the other hand, HOXA1 nuclear staining was low in melanoma (mean = 69%; range 5%-100%) when compared with Spitz nevi (mean = 94%; range 66%-100%; P = .0024) and conventional nevi (mean = 94%; range 83%-99%; P = .009). Conclusions UBE2C and HOXA1 RNA and protein are differentially expressed in conventional and Spitz nevi and melanoma. [ABSTRACT FROM AUTHOR]

Oncogenic gene fusions drive many human cancers, but tools to more quickly unravel their functional contributions are needed. Here we describe methodology permitting fusion gene construction for functional evaluation. Using this strategy, we engineered the known fusion oncogenes, BCR-ABL1, EML4-ALK, and ETV6-NTRK3, as well as 20 previously uncharacterized fusion genes identified in The Cancer Genome Atlas datasets. In addition to confirming oncogenic activity of the known fusion oncogenes engineered by our construction strategy, we validated five novel fusion genes involving MET, NTRK2, and BRAF kinases that exhibited potent transforming activity and conferred sensitivity to FDA-approved kinase inhibitors. Our fusion construction strategy also enabled domain-function studies of BRAF fusion genes. Our results confirmed other reports that the transforming activity of BRAF fusions results from truncation-mediated loss of inhibitory domains within the N-terminus of the BRAF protein. BRAF mutations residing within this inhibitory region may provide a means for BRAF activation in cancer, therefore we leveraged the modular design of our fusion gene construction methodology to screen N-terminal domain mutations discovered in tumors that are wild-type at the BRAF mutation hotspot, V600. We identified an oncogenic mutation, F247L, whose expression robustly activated the MAPK pathway and sensitized cells to BRAF and MEK inhibitors. When applied broadly, these tools will facilitate rapid fusion gene construction for subsequent functional characterization and translation into personalized treatment strategies. [ABSTRACT FROM AUTHOR]

Although exome sequencing data are generated primarily to detect single-nucleotide variants and indels, they can also be used to identify a subset of genomic rearrangements whose breakpoints are located in or near exons. Using >4,600 tumor and normal pairs across 15 cancer types, we identified over 9,000 high confidence somatic rearrangements, including a large number of gene fusions. We find that the 5′ fusion partners of functional fusions are often housekeeping genes, whereas the 3′ fusion partners are enriched in tyrosine kinases. We establish the oncogenic potential of ROR1-DNAJC6 and CEP85L-ROS1 fusions by showing that they can promote cell proliferation in vitro and tumor formation in vivo. Furthermore, we found that ∼4% of the samples have massively rearranged chromosomes, many of which are associated with upregulation of oncogenes such as ERBB2 and TERT . Although the sensitivity of detecting structural alterations from exomes is considerably lower than that from whole genomes, this approach will be fruitful for the multitude of exomes that have been and will be generated, both in cancer and in other diseases. [ABSTRACT FROM AUTHOR]

Large-scale sequencing efforts are uncovering the complexity of cancer genomes, which are composed of causal "driver" mutations that promote tumor progression along with many more pathologically neutral "passenger" events. The majority of mutations, both in known cancer drivers and uncharacterized genes, are generally of low occurrence, highlighting the need to functionally annotate the long tail of infrequent mutations present in heterogeneous cancers. Here we describe a mutation assessment pipeline enabled by high-throughput engineering of molecularly barcoded gene variant expression clones identified by tumor sequencing. We first used this platform to functionally assess tail mutations observed in PIK3CA, which encodes the catalytic subunit alpha of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) frequently mutated in cancer. Orthogonal screening for PIK3CA variant activity using in vitro and in vivo cell growth and transformation assays differentiated driver from passenger mutations, revealing that PIK3CA variant activity correlates imperfectly with its mutation frequency across breast cancer populations. Although PIK3CA mutations with frequencies above 5% were significantly more oncogenic than wild-type in all assays, mutations occurring at 0.07% to 5.0% included those with and without oncogenic activities that ranged from weak to strong in at least one assay. Proteomic profiling coupled with therapeutic sensitivity assays on PIK3CA variant-expressing cell models revealed variantspecific activation of PI3K signaling as well as other pathways that include the MEK1/2 module of mitogen-activated protein kinase pathway. Our data indicate that cancer treatments will need to increasingly consider the functional relevance of specific mutations in driver genes rather than considering all mutations in drivers as equivalent. [ABSTRACT FROM AUTHOR]

Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stroma l stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen crosslinks. Utilizing resected human lung cancertissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde-derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma. [ABSTRACT FROM AUTHOR]

Nuclear genetic disorders causing mitochondrial DNA (mtDNA) depletion are clinically and genetically heterogeneous, and the molecular etiology remains undiagnosed in the majority of cases. Through whole-exome sequencing, we identified recessive nonsense and splicing mutations in FBXL4 segregating in three unrelated consanguineous kindreds in which affected children present with a fatal encephalopathy, lactic acidosis, and severe mtDNA depletion in muscle. We show that FBXL4 is an F-box protein that colocalizes with mitochondria and that loss-of-function and splice mutations in this protein result in a severe respiratory chain deficiency, loss of mitochondrial membrane potential, and a disturbance of the dynamic mitochondrial network and nucleoid distribution in fibroblasts from affected individuals. Expression of the wild-type FBXL4 transcript in cell lines from two subjects fully rescued the levels of mtDNA copy number, leading to a correction of the mitochondrial biochemical deficit. Together our data demonstrate that mutations in FBXL4 are disease causing and establish FBXL4 as a mitochondrial protein with a possible role in maintaining mtDNA integrity and stability. [ABSTRACT FROM AUTHOR]

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGF?/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans. [ABSTRACT FROM AUTHOR]

Summary: When it escapes early detection, malignant melanoma becomes a highly lethal and treatment-refractory cancer. Melastatin is greatly downregulated in metastatic melanomas and is widely believed to function as a melanoma tumor suppressor. Here we report that tumor suppressive activity is not mediated by melastatin but instead by a microRNA (miR-211) hosted within an intron of melastatin. Increasing expression of miR-211 but not melastatin reduced migration and invasion of malignant and highly invasive human melanomas characterized by low levels of melastatin and miR-211. An unbiased network analysis of melanoma-expressed genes filtered for their roles in metastasis identified three central node genes: IGF2R, TGFBR2, and NFAT5. Expression of these genes was reduced by miR-211, and knockdown of each gene phenocopied the effects of increased miR-211 on melanoma invasiveness. These data implicate miR-211 as a suppressor of melanoma invasion whose expression is silenced or selected against via suppression of the entire melastatin locus during human melanoma progression. [ABSTRACT FROM AUTHOR]

A cardinal feature of malignant melanoma is its metastatic propensity. An incomplete view of the genetic events driving metastatic progression has been a major barrier to rational development of effective therapeutics and prognostic diagnostics for melanoma patients. In this study, we conducted global genomic characterization of primary and metastatic melanomas to examine the genomic landscape associated with metastatic progression. In addition to uncovering three genomic subclasses of metastastic melanomas, we delineated 39 focal and recurrent regions of amplification and deletions, many of which encompassed resident genes that have not been implicated in cancer or metastasis. To identify progressionassociated metastasis gene candidates, we applied a statistical approach, Integrative Genome Comparison (IGC), to define 32 genomic regions of interest that were significantly altered in metastatic relative to primary melanomas, encompassing 30 resident genes with statistically significant expression deregulation. Functional assays on a subset of these candidates, including MET, ASPM, AKAP9, IMP3, PRKCA, RPA3, and SCAP2, validated their pro-invasion activities in human melanoma cells. Validity of the IGC approach was further reinforced by tissue microarray analysis of Survivin showing significant increased protein expression in thick versus thin primary cutaneous melanomas, and a progression correlation with lymph node metastases. Together, these functional validation results and correlative analysis of human tissues support the thesis that integrated genomic and pathological analyses of staged melanomas provide a productive entry point for discovery of melanoma metastases genes. [ABSTRACT FROM AUTHOR]

The Tuberous Sclerosis Complex component, TSC1, functions as a tumor suppressor via its regulation of diverse cellular processes. particularly cell growth. TSC1 exists in a complex with TSC2 and functions primarily as a key negative regulator of mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis, although the TSC1/TSC2 complex also shows mTORC1- independent outputs to other pathways. Here, we explored the role of TSC1 in various aspects of stem cell biology and dissected the extent to which TSC1 functions are executed via mTORC1- dependent versus mTORC1-independent pathways. Using hema- topoietic stem cells (HSCs) as a model system, we demonstrate that somatic deletion of TSC1 produces striking stem cell and derivative effector cell phenotypes characterized by increased HSC cell cy- cling, mobilization,-marked progressive depletion, defective long- term repopulating potential, and hematopoietic lineage develop- mental aberrations. On the mechanistic level, we further establish that TSC1 regulation of HSC quiescence and long-term repopulat- ing potential and hematopoietic lineage development is mediated through mTORC1 signaling. In contrast, TSC1 regulation of HSC mobilization is effected in an mTORC1-independent manner, and gene profiling and functional analyses reveals the actin-bundling protein FSCN1 as a key TSC1/TSC2 target in the regulation of HSC mobilization. Thus, TSC1 is a critical regulator of HSC self-renewal, mobilization, and multilineage development and executes these actions via both mTORC1 -dependent and -independent pathways. [ABSTRACT FROM AUTHOR]

A correction to the article "The epithelial-to-mesenchymal transition activator ZEB1 initiates a prometastatic competing endogenous RNA network," by Xiaochao Tan and colleagues published in a 2018 issue of the journal is presented.

Summary Adenosine (A) to inosine (I) RNA editing introduces many nucleotide changes in cancer transcriptomes. However, due to the complexity of post-transcriptional regulation, the contribution of RNA editing to proteomic diversity in human cancers remains unclear. Here, we performed an integrated analysis of TCGA genomic data and CPTAC proteomic data. Despite limited site diversity, we demonstrate that A-to-I RNA editing contributes to proteomic diversity in breast cancer through changes in amino acid sequences. We validate the presence of editing events at both RNA and protein levels. The edited COPA protein increases proliferation, migration, and invasion of cancer cells in vitro . Our study suggests an important contribution of A-to-I RNA editing to protein diversity in cancer and highlights its translational potential. [ABSTRACT FROM AUTHOR]