Integration of metabolomics and transcriptomics reveals convergent pathways driving radiation-induced salivary gland dysfunction.

Radiation therapy for head and neck cancer causes damage to the surrounding salivary glands, resulting in salivary gland hypofunction and xerostomia. Current treatments do not provide lasting restoration of salivary gland function following radiation; therefore, a new mechanistic understanding of the radiation-induced damage response is necessary for identifying therapeutic targets. The purpose of the present study was to investigate the metabolic phenotype of radiation-induced damage in parotid salivary glands by integrating transcriptomic and metabolomic data. Integrated data were then analyzed to identify significant gene-metabolite interactions. Mice received a single 5 Gy dose of targeted head and neck radiation. Parotid tissue samples were collected 5 days following treatment for RNA sequencing and metabolomics analysis. Altered metabolites and transcripts significantly converged on a specific region in the metabolic reaction network. Both integrative pathway enrichment using rank-based statistics and network analysis highlighted significantly coordinated changes in glutathione metabolism, energy metabolism (TCA cycle and thermogenesis), peroxisomal lipid metabolism, and bile acid production with radiation. Integrated changes observed in energy metabolism suggest that radiation induces a mitochondrial dysfunction phenotype. These findings validated previous pathways involved in the radiation-damage response, such as altered energy metabolism, and identified robust signatures in salivary glands, such as reduced glutathione metabolism, that may be driving salivary gland dysfunction.

Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis.

Merkel cell carcinoma (MCC) frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT) and an intact Small T antigen (ST). While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC) and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A) subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq) and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells.

Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation.

Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer. The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells. To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST. MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1). Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells. Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells. Both NF-κB and MYC have been shown to regulate MCT1 expression. While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels. Several MCC lines had high levels of MYCL and MYCN but not MYC. Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells. Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis.

Interpreting cancer genomes using systematic host network perturbations by tumour virus proteins.

Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual genomic variations. Genome sequencing efforts have identified numerous germline mutations, and large numbers of somatic genomic alterations, associated with a predisposition to cancer. However, it remains difficult to distinguish background, or 'passenger', cancer mutations from causal, or 'driver', mutations in these data sets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations. Here we test the hypothesis that genomic variations and tumour viruses may cause cancer through related mechanisms, by systematically examining host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways, such as Notch signalling and apoptosis, that go awry in cancer. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on a par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches increase the specificity of cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate the prioritization of cancer-causing driver genes to advance the understanding of the genetic basis of human cancer.

Integration of TP53, DREAM, MMB-FOXM1 and RB-E2F target gene analyses identifies cell cycle gene regulatory networks.

Cell cycle (CC) and TP53 regulatory networks are frequently deregulated in cancer. While numerous genome-wide studies of TP53 and CC-regulated genes have been performed, significant variation between studies has made it difficult to assess regulation of any given gene of interest. To overcome the limitation of individual studies, we developed a meta-analysis approach to identify high confidence target genes that reflect their frequency of identification in independent datasets. Gene regulatory networks were generated by comparing differential expression of TP53 and CC-regulated genes with chromatin immunoprecipitation studies for TP53, RB1, E2F, DREAM, B-MYB, FOXM1 and MuvB. RNA-seq data from p21-null cells revealed that gene downregulation by TP53 generally requires p21 (CDKN1A). Genes downregulated by TP53 were also identified as CC genes bound by the DREAM complex. The transcription factors RB, E2F1 and E2F7 bind to a subset of DREAM target genes that function in G1/S of the CC while B-MYB, FOXM1 and MuvB control G2/M gene expression. Our approach yields high confidence ranked target gene maps for TP53, DREAM, MMB-FOXM1 and RB-E2F and enables prediction and distinction of CC regulation. A web-based atlas at www.targetgenereg.org enables assessing the regulation of any human gene of interest.

Identification of FAM111A as an SV40 host range restriction and adenovirus helper factor.

The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.

The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes.

Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like cyclin B, CDC2 and CDC25C are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in G(0)/G(1). It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and human cyclin B2 promoters in G(0). Association of DREAM and cell cycle-dependent regulation is abrogated when the CHR is mutated. Although E2f4 is part of the complex, a CDE is not essential but can enhance binding of DREAM. We show that the CHR element is not only necessary for repression of gene transcription in G(0)/G(1), but also for activation in S, G(2) and M phases. In proliferating cells, the B-myb-containing MMB complex binds the CHR of both promoters independently of the CDE. Bioinformatic analyses identify many genes which contain conserved CHR elements in promoters binding the DREAM complex. With Ube2c as an example from that screen, we show that inverse CHR sites are functional promoter elements that can bind DREAM and MMB. Our findings indicate that the CHR is central to DREAM/MMB-dependent transcriptional control during the cell cycle.

Flexible modeling of regulatory networks improves transcription factor activity estimation.

Transcriptional regulation plays a crucial role in determining cell fate and disease, yet inferring the key regulators from gene expression data remains a significant challenge. Existing methods for estimating transcription factor (TF) activity often rely on static TF-gene interaction databases and cannot adapt to changes in regulatory mechanisms across different cell types and disease conditions. Here, we present a new algorithm - Transcriptional Inference using Gene Expression and Regulatory data (TIGER) - that overcomes these limitations by flexibly modeling activation and inhibition events, up-weighting essential edges, shrinking irrelevant edges towards zero through a sparse Bayesian prior, and simultaneously estimating both TF activity levels and changes in the underlying regulatory network. When applied to yeast and cancer TF knock-out datasets, TIGER outperforms comparable methods in terms of prediction accuracy. Moreover, our application of TIGER to tissue- and cell-type-specific RNA-seq data demonstrates its ability to uncover differences in regulatory mechanisms. Collectively, our findings highlight the utility of modeling context-specific regulation when inferring transcription factor activities.

Viral and host network analysis of the human cytomegalovirus transcriptome in latency.

HCMV genes UL135 and UL138 play opposing roles regulating latency and reactivation in CD34+ human progenitor cells (HPCs). Using the THP-1 cell line model for latency and reactivation, we designed an RNA sequencing study to compare the transcriptional profile of HCMV infection in the presence and absence of these genes. The loss of UL138 results in elevated levels of viral gene expression and increased differentiation of cell populations that support HCMV gene expression and genome synthesis. The loss of UL135 results in diminished viral gene expression during an initial burst that occurs as latency is established and no expression of eleven viral genes from the ULb' region even following stimulation for differentiation and reactivation. Transcriptional network analysis revealed host transcription factors with potential to regulate the ULb' genes in coordination with pUL135. These results reveal roles for UL135 and UL138 in regulation of viral gene expression and potentially hematopoietic differentiation.

Viral perturbations of host networks reflect disease etiology.

Many human diseases, arising from mutations of disease susceptibility genes (genetic diseases), are also associated with viral infections (virally implicated diseases), either in a directly causal manner or by indirect associations. Here we examine whether viral perturbations of host interactome may underlie such virally implicated disease relationships. Using as models two different human viruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), we find that host targets of viral proteins reside in network proximity to products of disease susceptibility genes. Expression changes in virally implicated disease tissues and comorbidity patterns cluster significantly in the network vicinity of viral targets. The topological proximity found between cellular targets of viral proteins and disease genes was exploited to uncover a novel pathway linking HPV to Fanconi anemia.

Metabolomics analysis of pathways underlying radiation-induced salivary gland dysfunction stages.

Salivary gland hypofunction is an adverse side effect associated with radiotherapy for head and neck cancer patients. This study delineated metabolic changes at acute, intermediate, and chronic radiation damage response stages in mouse salivary glands following a single 5 Gy dose. Ultra-high performance liquid chromatography-mass spectrometry was performed on parotid salivary gland tissue collected at 3, 14, and 30 days following radiation (IR). Pathway enrichment analysis, network analysis based on metabolite structural similarity, and network analysis based on metabolite abundance correlations were used to incorporate both metabolite levels and structural annotation. The greatest number of enriched pathways are observed at 3 days and the lowest at 30 days following radiation. Amino acid metabolism pathways, glutathione metabolism, and central carbon metabolism in cancer are enriched at all radiation time points across different analytical methods. This study suggests that glutathione and central carbon metabolism in cancer may be important pathways in the unresolved effect of radiation treatment.

EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons.

Dendritic spines are the postsynaptic compartment of a neuronal synapse and are critical for synaptic connectivity and plasticity. A developmental precursor to dendritic spines, dendritic filopodia (DF), facilitate synapse formation by sampling the environment for suitable axon partners during neurodevelopment and learning. Despite the significance of the actin cytoskeleton in driving these dynamic protrusions, the actin elongation factors involved are not well characterized. We identified the Ena/VASP protein EVL as uniquely required for the morphogenesis and dynamics of DF. Using a combination of genetic and optogenetic manipulations, we demonstrated that EVL promotes protrusive motility through membrane-direct actin polymerization at DF tips. EVL forms a complex at nascent protrusions and DF tips with MIM/MTSS1, an I-BAR protein important for the initiation of DF. We proposed a model in which EVL cooperates with MIM to coalesce and elongate branched actin filaments, establishing the dynamic lamellipodia-like architecture of DF.

Integrative analysis reveals therapeutic potential of pyrvinium pamoate in Merkel cell carcinoma.

Merkel Cell Carcinoma (MCC) is a highly aggressive neuroendocrine cutaneous malignancy arising from either ultraviolet-induced mutagenesis or Merkel cell polyomavirus (MCPyV) integration. It is the only known neuroendocrine tumor (NET) with a virus etiology. Despite extensive research, our understanding of the molecular mechanisms driving the transition from normal cells to MCC remains limited. To address this knowledge gap, we assessed the impact of inducible MCPyV T antigens into normal human fibroblasts by performing RNA sequencing. Our findings suggested that the WNT signaling pathway plays a critical role in the development of MCC. To test this model, we bioinformatically evaluated various perturbagens for their ability to reverse the MCC gene expression signature and identified pyrvinium pamoate, an FDA-approved anthelminthic drug known for its anti-tumor potential in multiple cancers. Leveraging transcriptomic, network, and molecular analyses, we found that pyrvinium effectively targets multiple MCC vulnerabilities. Specifically, pyrvinium not only reverses the neuroendocrine features of MCC by modulating canonical and non-canonical WNT signaling pathways but also inhibits cancer cell growth by activating the p53-mediated apoptosis pathway, disrupting mitochondrial function, and inducing endoplasmic reticulum (ER) stress. Pyrvinium also effectively inhibits tumor growth in an MCC mouse xenograft model. These findings offer new avenues for the development of therapeutic strategies for neuroendocrine cancer and highlight the utility of pyrvinium as a potential treatment for MCC.

The Network Zoo: a multilingual package for the inference and analysis of gene regulatory networks.

Inference and analysis of gene regulatory networks (GRNs) require software that integrates multi-omic data from various sources. The Network Zoo (netZoo; netzoo.github.io) is a collection of open-source methods to infer GRNs, conduct differential network analyses, estimate community structure, and explore the transitions between biological states. The netZoo builds on our ongoing development of network methods, harmonizing the implementations in various computing languages and between methods to allow better integration of these tools into analytical pipelines. We demonstrate the utility using multi-omic data from the Cancer Cell Line Encyclopedia. We will continue to expand the netZoo to incorporate additional methods.

Canonical NF-kappaB activation is essential for Epstein-Barr virus latent membrane protein 1 TES2/CTAR2 gene regulation.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) transforms rodent fibroblasts and is expressed in most EBV-associated malignancies. LMP1 (transformation effector site 2 [TES2]/C-terminal activation region 2 [CTAR2]) activates NF-κB, p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and interferon regulatory factor 7 (IRF7) pathways. We have investigated LMP1 TES2 genome-wide RNA effects at 4 time points after LMP1 TES2 expression in HEK-293 cells. By using a false discovery rate (FDR) of <0.001 after correction for multiple hypotheses, LMP1 TES2 caused >2-fold changes in 1,916 mRNAs; 1,479 RNAs were upregulated and 437 were downregulated. In contrast to tumor necrosis factor alpha (TNF-α) stimulation, which transiently upregulates many target genes, LMP1 TES2 maintained most RNA effects through the time course, despite robust and sustained induction of negative feedback regulators, such as IκBα and A20. LMP1 TES2-regulated RNAs encode many NF-κB signaling proteins and secondary interacting proteins. Consequently, many LMP1 TES2-regulated RNAs encode proteins that form an extensive interactome. Gene set enrichment analyses found LMP1 TES2-upregulated genes to be significantly enriched for pathways in cancer, B- and T-cell receptor signaling, and Toll-like receptor signaling. Surprisingly, LMP1 TES2 and IκBα superrepressor coexpression decreased LMP1 TES2 RNA effects to only 5 RNAs, with FDRs of <0.001-fold and >2-fold changes. Thus, canonical NF-κB activation is critical for almost all LMP1 TES2 RNA effects in HEK-293 cells and a more significant therapeutic target than previously appreciated.

Randomized Crossover Trial Evaluating Detoxification of Tobacco Carcinogens by Broccoli Seed and Sprout Extract in Current Smokers.

Consumption of cruciferous vegetables, rich in the isothiocyanate glucoraphanin, is associated with reduced risk of tobacco-related cancers. Sulforaphane, released by hydrolysis of glucoraphanin, potently induces cytoprotective phase II enzymes. Sulforaphane decreased the incidence of oral cancer in the 4NQO carcinogenesis model. In residents of Qidong, China, broccoli seed and sprout extracts (BSSE) increased detoxification of air pollutants benzene and acrolein, also found in tobacco smoke. This randomized, crossover trial evaluated detoxification of tobacco carcinogens by the BSSE Avmacol® in otherwise healthy smokers. Participants were treated for 2 weeks with both low and higher-dose BSSE (148 µmol vs. 296 µmol of glucoraphanin daily), separated by a 2-week washout, with randomization to low-high vs. high-low sequence. The primary endpoint was detoxification of benzene, measured by urinary excretion of its mercapturic acid, SPMA. Secondary endpoints included bioavailability, detoxification of acrolein and crotonaldehyde, modulation by GST genotype, and toxicity. Forty-nine participants enrolled, including 26 (53%) females with median use of 20 cigarettes/day. Low and higher-dose BSSE showed a mean bioavailability of 11% and 10%, respectively. Higher-dose BSSE significantly upregulated urinary excretion of the mercapturic acids of benzene (p = 0.04), acrolein (p < 0.01), and crotonaldehyde (p = 0.02), independent of GST genotype. Retention and compliance were high resulting in early study completion. In conclusion, BSSE significantly upregulated detoxification of the tobacco carcinogens benzene, acrolein, and crotonaldehyde in current tobacco smokers.

GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors.

The microenvironment of solid tumors is dynamic and frequently contains pockets of low oxygen levels (hypoxia) surrounded by oxygenated tissue. Indeed, a compromised vasculature is a hallmark of the tumor microenvironment, creating both spatial gradients and temporal variability in oxygen availability. Notably, hypoxia associates with increased metastasis and poor survival in patients. Therefore, to aid therapeutic decisions and better understand hypoxia's role in cancer progression, it is critical to identify endogenous biomarkers of hypoxia to spatially phenotype oncogenic lesions in human tissue, whether precancerous, benign, or malignant. Here, we characterize the glucose transporter GLUT3/SLC2A3 as a biomarker of hypoxic prostate epithelial cells and prostate tumors. Transcriptomic analyses of non-tumorigenic, immortalized prostate epithelial cells revealed a highly significant increase in GLUT3 expression under hypoxia. Additionally, GLUT3 protein increased 2.4-fold in cultured hypoxic prostate cell lines and was upregulated within hypoxic regions of xenograft tumors, including two patient-derived xenografts (PDX). Finally, GLUT3 out-performs other established hypoxia markers; GLUT3 staining in PDX specimens detects 2.6-8.3 times more tumor area compared to a mixture of GLUT1 and CA9 antibodies. Therefore, given the heterogeneous nature of tumors, we propose adding GLUT3 to immunostaining panels when trying to detect hypoxic regions in prostate samples.

Molecular drivers of tumor progression in microsatellite stable APC mutation-negative colorectal cancers.

The tumor suppressor gene adenomatous polyposis coli (APC) is the initiating mutation in approximately 80% of all colorectal cancers (CRC), underscoring the importance of aberrant regulation of intracellular WNT signaling in CRC development. Recent studies have found that early-onset CRC exhibits an increased proportion of tumors lacking an APC mutation. We set out to identify mechanisms underlying APC mutation-negative (APCmut-) CRCs. We analyzed data from The Cancer Genome Atlas to compare clinical phenotypes, somatic mutations, copy number variations, gene fusions, RNA expression, and DNA methylation profiles between APCmut- and APC mutation-positive (APCmut+) microsatellite stable CRCs. Transcriptionally, APCmut- CRCs clustered into two approximately equal groups. Cluster One was associated with enhanced mitochondrial activation. Cluster Two was strikingly associated with genetic inactivation or decreased RNA expression of the WNT antagonist RNF43, increased expression of the WNT agonist RSPO3, activating mutation of BRAF, or increased methylation and decreased expression of AXIN2. APCmut- CRCs exhibited evidence of increased immune cell infiltration, with significant correlation between M2 macrophages and RSPO3. APCmut- CRCs comprise two groups of tumors characterized by enhanced mitochondrial activation or increased sensitivity to extracellular WNT, suggesting that they could be respectively susceptible to inhibition of these pathways.

Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning.

While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.

Generating Ensembles of Gene Regulatory Networks to Assess Robustness of Disease Modules.

The use of biological networks such as protein-protein interaction and transcriptional regulatory networks is becoming an integral part of genomics research. However, these networks are not static, and during phenotypic transitions like disease onset, they can acquire new "communities" (or highly interacting groups) of genes that carry out cellular processes. Disease communities can be detected by maximizing a modularity-based score, but since biological systems and network inference algorithms are inherently noisy, it remains a challenge to determine whether these changes represent real cellular responses or whether they appeared by random chance. Here, we introduce Constrained Random Alteration of Network Edges (CRANE), a method for randomizing networks with fixed node strengths. CRANE can be used to generate a null distribution of gene regulatory networks that can in turn be used to rank the most significant changes in candidate disease communities. Compared to other approaches, such as consensus clustering or commonly used generative models, CRANE emulates biologically realistic networks and recovers simulated disease modules with higher accuracy. When applied to breast and ovarian cancer networks, CRANE improves the identification of cancer-relevant GO terms while reducing the signal from non-specific housekeeping processes.