Cancer research is not correlated with driver gene mutation burdens.

BACKGROUND: Cancer research is pursued with the goal of positively impacting patients with cancer. Decisions regarding how to allocate research funds reflect a complex balancing of priorities and factors. Even though these are subjective decisions, they should be made with consideration of all available objective facts. An accurate estimate of the affected cancer patient population by mutation is one variable that has only recently become available to inform funding decisions. METHODS: We compared the overall incident burden of mutations within each cancer-associated gene with two measures of cancer research efforts: research grant funding amounts and numbers of academic manuscripts. We ask to what degree the aggregate set of cancer research efforts reflects the relative burdens of the different cancer genetic drivers. We thoroughly investigate the design of our queries to ensure that the presented results are robust and conclusions are well justified. FINDINGS: We find cancer research is generally not correlated with the relative burden of mutation within the different genetic drivers of cancer. CONCLUSIONS: We suggest that cancer research would benefit from incorporating, among other factors, an epidemiologically informed mutation-estimate baseline into a larger framework for funding and research allocation decisions. FUNDING: This work was supported in part by the National Institutes of Health (NIH) P30CA014195 and NIH DP2AT011327.

Dichotomous ovarian cancer-initiating potential of Pax8+ cells revealed by a mouse genetic mosaic model.

Different cellular compartments within a tissue present distinct cancer-initiating capacities. Current approaches to dissect such heterogeneity require cell-type-specific genetic tools based on a well-understood lineage hierarchy, which are lacking for many tissues. Here, we circumvented this hurdle and revealed the dichotomous capacity of fallopian tube Pax8+ cells in initiating ovarian cancer, utilizing a mouse genetic system that stochastically generates rare GFP-labeled mutant cells. Through clonal analysis and spatial profiling, we determined that only clones founded by rare, stem/progenitor-like Pax8+ cells can expand on acquiring oncogenic mutations whereas vast majority of clones stall immediately. Furthermore, expanded mutant clones undergo further attrition: many turn quiescent shortly after the initial expansion, whereas others sustain proliferation and manifest a bias toward Pax8+ fate, underlying early pathogenesis. Our study showcases the power of genetic mosaic system-based clonal analysis for revealing cellular heterogeneity of cancer-initiating capacity in tissues with limited prior knowledge of lineage hierarchy.

Pannexin 1 drives efficient epithelial repair after tissue injury.

Epithelial tissues such as lung and skin are exposed to the environment and therefore particularly vulnerable to damage during injury or infection. Rapid repair is therefore essential to restore function and organ homeostasis. Dysregulated epithelial tissue repair occurs in several human disease states, yet how individual cell types communicate and interact to coordinate tissue regeneration is incompletely understood. Here, we show that pannexin 1 (Panx1), a cell membrane channel activated by caspases in dying cells, drives efficient epithelial regeneration after tissue injury by regulating injury-induced epithelial proliferation. Lung airway epithelial injury promotes the Panx1-dependent release of factors including ATP, from dying epithelial cells, which regulates macrophage phenotype after injury. This process, in turn, induces a reparative response in tissue macrophages that includes the induction of the soluble mitogen amphiregulin, which promotes injury-induced epithelial proliferation. Analysis of regenerating lung epithelium identified Panx1-dependent induction of Nras and Bcas2, both of which positively promoted epithelial proliferation and tissue regeneration in vivo. We also established that this role of Panx1 in boosting epithelial repair after injury is conserved between mouse lung and zebrafish tailfin. These data identify a Panx1-mediated communication circuit between epithelial cells and macrophages as a key step in promoting epithelial regeneration after injury.

Adherence Enables Neisseria gonorrhoeae to Overcome Zinc Limitation Imposed by Nutritional Immunity Proteins.

Neisseria gonorrhoeae (Gc) must overcome the limitation of metals such as zinc to colonize mucosal surfaces in its obligate human host. While the zinc-binding nutritional immunity proteins calprotectin (S100A8/A9) and psoriasin (S100A7) are abundant in human cervicovaginal lavage fluid, Gc possesses TonB-dependent transporters TdfH and TdfJ that bind and extract zinc from the human version of these proteins, respectively. Here we investigated the contribution of zinc acquisition to Gc infection of epithelial cells of the female genital tract. We found that TdfH and TdfJ were dispensable for survival of strain FA1090 Gc that was associated with Ect1 human immortalized epithelial cells, when zinc was limited by calprotectin and psoriasin. In contrast, suspension-grown bacteria declined in viability under the same conditions. Exposure to murine calprotectin, which Gc cannot use as a zinc source, similarly reduced survival of suspension-grown Gc, but not Ect1-associated Gc. We ruled out epithelial cells as a contributor to the enhanced growth of cell-associated Gc under zinc limitation. Instead, we found that attachment to glass was sufficient to enhance bacterial growth when zinc was sequestered. We compared the transcriptional profiles of WT Gc adherent to glass coverslips or in suspension, when zinc was sequestered with murine calprotectin or provided in excess, from which we identified open reading frames that were increased by zinc sequestration in adherent Gc. One of these, ZnuA, was necessary but not sufficient for survival of Gc under zinc-limiting conditions. These results show that adherence protects Gc from zinc-dependent growth restriction by host nutritional immunity proteins.

Cancer gene mutation frequencies for the U.S. population.

Mutations play a fundamental role in the development of cancer, and many create targetable vulnerabilities. There are both public health and basic science benefits from the determination of the proportion of all cancer cases within a population that include a mutant form of a gene. Here, we provide the first such estimates by combining genomic and epidemiological data. We estimate KRAS is mutated in only 11% of all cancers, which is less than PIK3CA (13%) and marginally higher than BRAF (8%). TP53 is the most commonly mutated gene (35%), and KMT2C, KMT2D, and ARID1A are among the ten most commonly mutated driver genes, highlighting the role of epigenetic dysregulation in cancer. Analysis of major cancer subclassifications highlighted varying dependencies upon individual cancer drivers. Overall, we find that cancer genetics is less dominated by high-frequency, high-profile cancer driver genes than studies limited to a subset of cancer types have suggested.

Heme Oxygenase-1 at the Nexus of Endothelial Cell Fate Decision Under Oxidative Stress.

Endothelial cells (ECs) form the inner lining of blood vessels and are central to sensing chemical perturbations that can lead to oxidative stress. The degree of stress is correlated with divergent phenotypes such as quiescence, cell death, or senescence. Each possible cell fate is relevant for a different aspect of endothelial function, and hence, the regulation of cell fate decisions is critically important in maintaining vascular health. This study examined the oxidative stress response (OSR) in human ECs at the boundary of cell survival and death through longitudinal measurements, including cellular, gene expression, and perturbation measurements. 0.5 mM hydrogen peroxide (HP) produced significant oxidative stress, placed the cell at this junction, and provided a model to study the effectors of cell fate. The use of systematic perturbations and high-throughput measurements provide insights into multiple regimes of the stress response. Using a systems approach, we decipher molecular mechanisms across these regimes. Significantly, our study shows that heme oxygenase-1 (HMOX1) acts as a gatekeeper of cell fate decisions. Specifically, HP treatment of HMOX1 knockdown cells reversed the gene expression of about 51% of 2,892 differentially expressed genes when treated with HP alone, affecting a variety of cellular processes, including anti-oxidant response, inflammation, DNA injury and repair, cell cycle and growth, mitochondrial stress, metabolic stress, and autophagy. Further analysis revealed that these switched genes were highly enriched in three spatial locations viz., cell surface, mitochondria, and nucleus. In particular, it revealed the novel roles of HMOX1 on cell surface receptors EGFR and IGFR, mitochondrial ETCs (MTND3, MTATP6), and epigenetic regulation through chromatin modifiers (KDM6A, RBBP5, and PPM1D) and long non-coding RNA (lncRNAs) in orchestrating the cell fate at the boundary of cell survival and death. These novel aspects suggest that HMOX1 can influence transcriptional and epigenetic modulations to orchestrate OSR affecting cell fate decisions.

PML-NB-dependent type I interferon memory results in a restricted form of HSV latency.

Herpes simplex virus (HSV) establishes latent infection in long-lived neurons. During initial infection, neurons are exposed to multiple inflammatory cytokines but the effects of immune signaling on the nature of HSV latency are unknown. We show that initial infection of primary murine neurons in the presence of type I interferon (IFN) results in a form of latency that is restricted for reactivation. We also find that the subnuclear condensates, promyelocytic leukemia nuclear bodies (PML-NBs), are absent from primary sympathetic and sensory neurons but form with type I IFN treatment and persist even when IFN signaling resolves. HSV-1 genomes colocalize with PML-NBs throughout a latent infection of neurons only when type I IFN is present during initial infection. Depletion of PML prior to or following infection does not impact the establishment latency; however, it does rescue the ability of HSV to reactivate from IFN-treated neurons. This study demonstrates that viral genomes possess a memory of the IFN response during de novo infection, which results in differential subnuclear positioning and ultimately restricts the ability of genomes to reactivate.

Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage Myf6Cre,Pax3:Foxo1,p53 mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor Tp53 (Myf6Cre,Pax3:Foxo1,p53), exhibit marked heterogeneity in PAX3:FOXO1 (P3F) expression at the single cell level. In mouse RMS cells, P3F expression is directed by the Pax3 promoter and coupled to eYFP YFPlow/P3Flow mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFPlow/P3Flow compared with YFPhigh/P3Fhigh cells. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Heterogeneous expression of PAX3:FOXO1 at the single cell level may provide a critical advantage during tumor progression.

Premature Activation of Immune Transcription Programs in Autoimmune-Predisposed Mouse Embryonic Stem Cells and Blastocysts.

Autoimmune diabetes is a complex multifactorial disease with genetic and environmental factors playing pivotal roles. While many genes associated with the risk of diabetes have been identified to date, the mechanisms by which external triggers contribute to the genetic predisposition remain unclear. Here, we derived embryonic stem (ES) cell lines from diabetes-prone non-obese diabetic (NOD) and healthy C57BL/6 (B6) mice. While overall pluripotency markers were indistinguishable between newly derived NOD and B6 ES cells, we discovered several differentially expressed genes that normally are not expressed in ES cells. Several genes that reside in previously identified insulin-dependent diabetics (Idd) genomic regions were up-regulated in NOD ES cells. Gene set enrichment analysis showed that different groups of genes associated with immune functions are differentially expressed in NOD. Transcriptomic analysis of NOD blastocysts validated several differentially overexpressed Idd genes compared to B6. Genome-wide mapping of active histone modifications using ChIP-Seq supports active expression as the promoters and enhancers of activated genes are also marked by active histone modifications. We have also found that NOD ES cells secrete more inflammatory cytokines. Our data suggest that the known genetic predisposition of NOD to autoimmune diabetes leads to epigenetic instability of several Idd regions.

Prediabetes Induced by a Single Autoimmune B Cell Clone.

While B cells play a significant role in the onset of type-1 diabetes (T1D), little is know about their role in those early stages. Thus, to gain new insights into the role of B cells in T1D, we converted a physiological early pancreas-infiltrating B cell into a novel BCR mouse model using Somatic Cell Nuclear Transfer (SCNT). Strikingly, SCNT-derived B1411 model displayed neither developmental block nor anergy. Instead, B1411 underwent spontaneous germinal center reactions. Without T cell help, B1411-Rag1-/- was capable of forming peri-/intra-pancreatic lymph nodes, and undergoing class-switching. RNA-Seq analysis identified 93 differentially expressed genes in B1411 compared to WT B cells, including Irf7, Usp18, and Mda5 that had been linked to a potential viral etiology of T1D. We also found various members of the oligoadenylate synthase (OAS) family to be enriched in B1411, such as Oas1, which had recently also been linked to T1D. Strikingly, when challenged with glucose B1411-Rag1-/- mice displayed impaired glucose tolerance.

Time varying causal network reconstruction of a mouse cell cycle.

BACKGROUND: Biochemical networks are often described through static or time-averaged measurements of the component macromolecules. Temporal variation in these components plays an important role in both describing the dynamical nature of the network as well as providing insights into causal mechanisms. Few methods exist, specifically for systems with many variables, for analyzing time series data to identify distinct temporal regimes and the corresponding time-varying causal networks and mechanisms. RESULTS: In this study, we use well-constructed temporal transcriptional measurements in a mammalian cell during a cell cycle, to identify dynamical networks and mechanisms describing the cell cycle. The methods we have used and developed in part deal with Granger causality, Vector Autoregression, Estimation Stability with Cross Validation and a nonparametric change point detection algorithm that enable estimating temporally evolving directed networks that provide a comprehensive picture of the crosstalk among different molecular components. We applied our approach to RNA-seq time-course data spanning nearly two cell cycles from Mouse Embryonic Fibroblast (MEF) primary cells. The change-point detection algorithm is able to extract precise information on the duration and timing of cell cycle phases. Using Least Absolute Shrinkage and Selection Operator (LASSO) and Estimation Stability with Cross Validation (ES-CV), we were able to, without any prior biological knowledge, extract information on the phase-specific causal interaction of cell cycle genes, as well as temporal interdependencies of biological mechanisms through a complete cell cycle. CONCLUSIONS: The temporal dependence of cellular components we provide in our model goes beyond what is known in the literature. Furthermore, our inference of dynamic interplay of multiple intracellular mechanisms and their temporal dependence on one another can be used to predict time-varying cellular responses, and provide insight on the design of precise experiments for modulating the regulation of the cell cycle.

Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer.

Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells.

Targeting CREB Pathway Suppresses Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is the most deadly subtype of lung cancer due to its dismal prognosis. We have developed a lentiviral vector-mediated SCLC mouse model and have explored the role of both the NF-κB and CREB families of transcription factors in this model. Surprisingly, induction of NF-κB activity, which promotes tumor progression in many cancer types including non-small cell lung carcinoma (NSCLC), is dispensable in SCLC. Instead, suppression of NF-κB activity in SCLC tumors moderately accelerated tumor development. Examination of gene expression signatures of both mouse and human SCLC tumors revealed overall low NF-κB but high CREB activity. Blocking CREB activation by a dominant-negative form of PKA (dnPKA) completely abolished the development of SCLC. Similarly, expression of dnPKA or treatment with PKA inhibitor H89 greatly reduced the growth of SCLC tumors in syngeneic transplantation models. Altogether, our results strongly suggest that targeting CREB is a promising therapeutic strategy against SCLC.Implications: Activity of the transcription factor CREB is elevated in SCLC tumors, which helps to maintain its neuroendocrine signature and cell proliferation. Our results highlight the importance of targeting the CREB pathway to develop new therapeutics to combat SCLC. Mol Cancer Res; 16(5); 825-32. ©2018 AACR.

Screening for tumor suppressors: Loss of ephrin receptor A2 cooperates with oncogenic KRas in promoting lung adenocarcinoma.

Lung adenocarcinoma, a major form of non-small cell lung cancer, is the leading cause of cancer deaths. The Cancer Genome Atlas analysis of lung adenocarcinoma has identified a large number of previously unknown copy number alterations and mutations, requiring experimental validation before use in therapeutics. Here, we describe an shRNA-mediated high-throughput approach to test a set of genes for their ability to function as tumor suppressors in the background of mutant KRas and WT Tp53. We identified several candidate genes from tumors originated from lentiviral delivery of shRNAs along with Cre recombinase into lungs of Loxp-stop-Loxp-KRas mice. Ephrin receptorA2 (EphA2) is among the top candidate genes and was reconfirmed by two distinct shRNAs. By generating knockdown, inducible knockdown and knockout cell lines for loss of EphA2, we showed that negating its expression activates a transcriptional program for cell proliferation. Loss of EPHA2 releases feedback inhibition of KRAS, resulting in activation of ERK1/2 MAP kinase signaling, leading to enhanced cell proliferation. Intriguingly, loss of EPHA2 induces activation of GLI1 transcription factor and hedgehog signaling that further contributes to cell proliferation. Small molecules targeting MEK1/2 and Smoothened hamper proliferation in EphA2-deficient cells. Additionally, in EphA2 WT cells, activation of EPHA2 by its ligand, EFNA1, affects KRAS-RAF interaction, leading to inhibition of the RAS-RAF-MEK-ERK pathway and cell proliferation. Together, our studies have identified that (i) EphA2 acts as a KRas cooperative tumor suppressor by in vivo screen and (ii) reactivation of the EphA2 signal may serve as a potential therapeutic for KRas-induced human lung cancers.

Functional Gene Correction for Cystic Fibrosis in Lung Epithelial Cells Generated from Patient iPSCs.

Lung disease is a major cause of death in the United States, with current therapeutic approaches serving only to manage symptoms. The most common chronic and life-threatening genetic disease of the lung is cystic fibrosis (CF) caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). We have generated induced pluripotent stem cells (iPSCs) from CF patients carrying a homozygous deletion of F508 in the CFTR gene, which results in defective processing of CFTR to the cell membrane. This mutation was precisely corrected using CRISPR to target corrective sequences to the endogenous CFTR genomic locus, in combination with a completely excisable selection system, which significantly improved the efficiency of this correction. The corrected iPSCs were subsequently differentiated to mature airway epithelial cells where recovery of normal CFTR expression and function was demonstrated. This isogenic iPSC-based model system for CF could be adapted for the development of new therapeutic approaches.

Lymphoid regeneration from gene-corrected SCID-X1 subject-derived iPSCs.

X-linked Severe Combined Immunodeficiency (SCID-X1) is a genetic disease that leaves newborns at high risk of serious infection and a predicted life span of less than 1 year in the absence of a matched bone marrow donor. The disease pathogenesis is due to mutations in the gene encoding the Interleukin-2 receptor gamma chain (IL-2Rγ), leading to a lack of functional lymphocytes. With the leukemogenic concerns of viral gene therapy there is a need to explore alternative therapeutic options. We have utilized induced pluripotent stem cell (iPSC) technology and genome editing mediated by TALENs to generate isogenic subject-specific mutant and gene-corrected iPSC lines. While the subject-derived mutant iPSCs have the capacity to generate hematopoietic precursors and myeloid cells, only wild-type and gene-corrected iPSCs can additionally generate mature NK cells and T cell precursors expressing the correctly spliced IL-2Rγ. This study highlights the potential for the development of autologous cell therapy for SCID-X1 subjects.

Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans. Here we show that gliomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Transduction by oncogenic lentiviral vectors of neural stem cells (NSCs), astrocytes, or even mature neurons in the brains of mice can give rise to malignant gliomas. All the tumors, irrespective of the site of lentiviral vector injection (the initiating population), shared common features of high expression of stem or progenitor markers and low expression of differentiation markers. Microarray analysis revealed that tumors of astrocytic and neuronal origin match the mesenchymal GBM subtype. We propose that most differentiated cells in the CNS upon defined genetic alterations undergo dedifferentiation to generate a NSC or progenitor state to initiate and maintain the tumor progression, as well as to give rise to the heterogeneous populations observed in malignant gliomas.

Reduced cell proliferation by IKK2 depletion in a mouse lung-cancer model.

Lung cancer is one of the leading cancer malignancies, with a five-year survival rate of only ~15%. We have developed a lentiviral-vector-mediated mouse model, which enables generation of non-small-cell lung cancer from less than 100 alveolar epithelial cells, and investigated the role of IKK2 and NF-κB in lung-cancer development. IKK2 depletion in tumour cells significantly attenuated tumour proliferation and significantly prolonged mouse survival. We identified Timp1, one of the NF-κB target genes, as a key mediator for tumour growth. Activation of the Erk signalling pathway and cell proliferation requires Timp-1 and its receptor CD63. Knockdown of either Ikbkb or Timp1 by short hairpin RNAs reduced tumour growth in both xenograft and lentiviral models. Our results thus suggest the possible application of IKK2 and Timp-1 inhibitors in treating lung cancer.