Detained introns are a novel, widespread class of post-transcriptionally spliced introns

Deep sequencing of embryonic stem cell RNA revealed many specific internal introns that are significantly more abundant than the other introns within polyadenylated transcripts; we classified these as “detained” introns (DIs). We identified thousands of DIs, many of which are evolutionarily conserved, in human and mouse cell lines as well as the adult mouse liver. DIs can have half-lives of over an hour yet remain in the nucleus and are not subject to nonsense-mediated decay (NMD). Drug inhibition of Clk, a stress-responsive kinase, triggered rapid splicing changes for a specific subset of DIs; half showed increased splicing, and half showed increased intron detention, altering transcript pools of >300 genes. Srsf4, which undergoes a dramatic phosphorylation shift in response to Clk kinase inhibition, regulates the splicing of some DIs, particularly in genes encoding RNA processing and splicing factors. The splicing of some DIs—including those in Mdm4, a negative regulator of p53—was also altered following DNA damage. After 4 h of Clk inhibition, the expression of >400 genes changed significantly, and almost one-third of these are p53 transcriptional targets. These data suggest a widespread mechanism by which the rate of splicing of DIs contributes to the level of gene expression.National Institutes of Health (U.S.) (Grant R01 GM34277-23)American Cancer Society (Novartis Institutes of Biomedical Research Postdoctoral Research Fellowship)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

Inferring genome-scale rearrangement phylogeny and ancestral gene order: a Drosophila case study

A simple, fast, and biologically-inspired computational approach to infer genome-scale rearrangement phylogeny and ancestral gene order has been developed and applied to eight Drosophila genomes, providing insights into evolutionary chromosomal dynamics

Stromal Expression of miR-143/145 Promotes Neoangiogenesis in Lung Cancer Development

The two unrelated miRNAs miR-143 and miR-145, coexpressed from the miR-143/145 cluster, have been proposed to act as tumor suppressors in human cancer, and therapeutic benefits of delivering miR-143 and miR-145 to tumors have been reported. In contrast, we found that tumor-specific deletion of miR-143/145 in an autochthonous mouse model of lung adenocarcinoma did not affect tumor development. This was consistent with the lack of endogenous miR-143/145 expression in normal and transformed lung epithelium. Surprisingly, miR-143/145 in the tumor microenvironment dramatically promoted tumor growth by stimulating the proliferation of endothelial cells. Loss of miR-143/145 in vivo led to derepression of the miR-145 target CAMK1D, an inhibitory kinase, which when overexpressed prevents mitotic entry of endothelial cells. As a consequence, tumors in miR-143/145-deficient animals exhibited diminished neoangiogenesis, increased apoptosis, and their expansion was limited by the tumor’s ability to co-opt the alveolar vasculature. These findings demonstrate that stromal miR-143/145 promotes tumorigenesis and caution against the use of these miRNAs as agents in cancer therapeutics.SIGNIFICANCE: This study shows that miR-143/145 expressed from the tumor microenvironment stimulates neoangiogenesis and supports tumor expansion in the lung, demonstrating a surprising role for the putative tumor suppressor miRNA cluster in promoting tumorigenesis. We propose inhibition of miR-143/145 as a therapeutic avenue to modulate tumor neoangiogenesis.National Institutes of Health (U.S.) (Grant P01-CA42063-26)National Cancer Institute (U.S.) (Grant P30-CA14051

Dicer loss and recovery induce an oncogenic switch driven by transcriptional activation of the oncofetal Imp1–3 family

MicroRNAs(miRNAs) are post-transcriptional regulators of gene expressioncritical for organismal viability. Changes inmiRNAactivity arecommonin cancer, buthowthese changes relate to subsequent alterations in transcription and the process of tumorigenesis is not well understood. Here, we report a deep transcriptional, oncogenic network regulated bymiRNAs. Wepresent analysis of the gene expression and phenotypic changes associated with globalmiRNA restoration in miRNA-deficient fibroblasts. This analysis uncovers a miRNA-repressed network containing oncofetal genesImp1, Imp2, and Imp3(Imp1–3) that is up-regulated primarily transcriptionally > 100-fold uponDicer loss and is resistant to resilencing by complete restoration of miRNA activity. This Dicer-resistant epigenetic switch confers tumorigenicity to these cells. Let-7 targets Imp1–3 are required for this tumorigenicity and feed back to reinforce and sustain expression of the oncogenic network. Together, these Dicer-resistant genes constitute an mRNA expression signature that is present in numerous human cancers and is associated with poor survival.United States. Public Health Service (Grant R01CA133404)National Cancer Institute (U.S.) (Grant P01CA42063)Marie D. and Pierre Casimir-Lamber

Caspase-2-Mediated Cleavage of Mdm2 Creates a p53-Induced Positive Feedback Loop

Caspase-2 is an evolutionarily conserved caspase, yet its biological function and cleavage targets are poorly understood. Caspase-2 is activated by the p53 target gene product PIDD (also known as LRDD) in a complex called the Caspase-2-PIDDosome. We show that PIDD expression promotes growth arrest and chemotherapy resistance by a mechanism that depends on Caspase-2 and wild-type p53. PIDD-induced Caspase-2 directly cleaves the E3 ubiquitin ligase Mdm2 at Asp 367, leading to loss of the C-terminal RING domain responsible for p53 ubiquitination. As a consequence, N-terminally truncated Mdm2 binds p53 and promotes its stability. Upon DNA damage, p53 induction of the Caspase-2-PIDDosome creates a positive feedback loop that inhibits Mdm2 and reinforces p53 stability and activity, contributing to cell survival and drug resistance. These data establish Mdm2 as a cleavage target of Caspase-2 and provide insight into a mechanism of Mdm2 inhibition that impacts p53 dynamics upon genotoxic stress.Virginia and D.K. Ludwig Fund for Cancer Research (Postdoctoral Fellowship)Human Frontier Science Program (Strasbourg, France) (Fellowship)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051

Survival of pancreatic cancer cells lacking KRAS function

Activating mutations in the proto-oncogene KRAS are a hallmark of pancreatic ductal adenocarcinoma (PDAC), an aggressive malignancy with few effective therapeutic options. Despite efforts to develop KRAS-targeted drugs, the absolute dependence of PDAC cells on KRAS remains incompletely understood. Here we model complete KRAS inhibition using CRISPR/Cas-mediated genome editing and demonstrate that KRAS is dispensable in a subset of human and mouse PDAC cells. Remarkably, nearly all KRAS deficient cells exhibit phosphoinositide 3-kinase (PI3K)-dependent mitogen-activated protein kinase (MAPK) signaling and induced sensitivity to PI3K inhibitors. Furthermore, comparison of gene expression profiles of PDAC cells retaining or lacking KRAS reveal a role of KRAS in the suppression of metastasis-related genes. Collectively, these data underscore the potential for PDAC resistance to even the very best KRAS inhibitors and provide insights into mechanisms of response and resistance to KRAS inhibition

Circadian Rhythm Disruption Promotes Lung Tumorigenesis

Circadian rhythms are 24-hr oscillations that control a variety of biological processes in living systems, including two hallmarks of cancer, cell division and metabolism. Circadian rhythm disruption by shift work is associated with greater risk for cancer development and poor prognosis, suggesting a putative tumor-suppressive role for circadian rhythm homeostasis. Using a genetically engineered mouse model of lung adenocarcinoma, we have characterized the effects of circadian rhythm disruption on lung tumorigenesis. We demonstrate that both physiologic perturbation (jet lag) and genetic mutation of the central circadian clock components decreased survival and promoted lung tumor growth and progression. The core circadian genes Per2 and Bmal1 were shown to have cell-autonomous tumor-suppressive roles in transformation and lung tumor progression. Loss of the central clock components led to increased c-Myc expression, enhanced proliferation, and metabolic dysregulation. Our findings demonstrate that both systemic and somatic disruption of circadian rhythms contribute to cancer progression

Germline loss of PKM2 promotes metabolic distress and hepatocellular carcinoma

Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of pyruvate kinase (PK), and PKM2 expression is closely linked to embryogenesis, tissue regeneration, and cancer. To interrogate the functional requirement for PKM2 during development and tissue homeostasis, we generated germline PKM2-null mice (Pkm2[superscript −/−]). Unexpectedly, despite being the primary isoform expressed in most wild-type adult tissues, we found that Pkm2[superscript −/−] mice are viable and fertile. Thus, PKM2 is not required for embryonic or postnatal development. Loss of PKM2 leads to compensatory expression of PKM1 in the tissues that normally express PKM2. Strikingly, PKM2 loss leads to spontaneous development of hepatocellular carcinoma (HCC) with high penetrance that is accompanied by progressive changes in systemic metabolism characterized by altered systemic glucose homeostasis, inflammation, and hepatic steatosis. Therefore, in addition to its role in cancer metabolism, PKM2 plays a role in controlling systemic metabolic homeostasis and inflammation, thereby preventing HCC by a non-cell-autonomous mechanism.National Cancer Institute (U.S.) (Cancer Center Support Grant P30CA14051)Howard Hughes Medical InstituteBurroughs Wellcome FundSmith Family FoundationUnited States. Dept. of Health and Human Services (P01CA117969)United States. Dept. of Health and Human Services (R01CA168653)American Society for Engineering Education. National Defense Science and Engineering Graduate FellowshipJane Coffin Childs Memorial Fund for Medical Research (Postdoctoral Fellowship

GKAP Acts as a Genetic Modulator of NMDAR Signaling to Govern Invasive Tumor Growth.

Genetic linkage analysis previously suggested that GKAP, a scaffold protein of the N-methyl-D-aspartate receptor (NMDAR), was a potential modifier of invasion in a mouse model of pancreatic neuroendocrine tumor (PanNET). Here, we establish that GKAP governs invasive growth and treatment response to NMDAR inhibitors of PanNET via its pivotal role in regulating NMDAR pathway activity. Combining genetic knockdown of GKAP and pharmacological inhibition of NMDAR, we implicate as downstream effectors FMRP and HSF1, which along with GKAP demonstrably support invasiveness of PanNET and pancreatic ductal adenocarcinoma cancer cells. Furthermore, we distilled genome-wide expression profiles orchestrated by the NMDAR-GKAP signaling axis, identifying transcriptome signatures in tumors with low/inhibited NMDAR activity that significantly associate with favorable patient prognosis in several cancer types

Global microRNA depletion suppresses tumor angiogenesis

MicroRNAs delicately regulate the balance of angiogenesis. Here we show that depletion of all microRNAs suppresses tumor angiogenesis. We generated microRNA-deficient tumors by knocking out Dicer1. These tumors are highly hypoxic but poorly vascularized, suggestive of deficient angiogenesis signaling. Expression profiling revealed that angiogenesis genes were significantly down-regulated as a result of the microRNA deficiency. Factor inhibiting hypoxia-inducible factor 1 (HIF-1), FIH1, is derepressed under these conditions and suppresses HIF transcription. Knocking out FIH1 using CRISPR/Cas9-mediated genome engineering reversed the phenotypes of microRNA-deficient cells in HIF transcriptional activity, VEGF production, tumor hypoxia, and tumor angiogenesis. Using multiplexed CRISPR/Cas9, we deleted regions in FIH1 3′ untranslated regions (UTRs) that contain microRNA-binding sites, which derepresses FIH1 protein and represses hypoxia response. These data suggest that microRNAs promote tumor responses to hypoxia and angiogenesis by repressing FIH1.Swedish Research CouncilHoward Hughes Medical Institute (International Student Research Fellowship)National Institutes of Health (U.S.) (grant number R01-CA133404)MIT-Harvard Center of Cancer Nanotechnology Excellence (grant no. U54-CA151884)David H. Koch Institute for Integrative Cancer Research at MIT (Marie D. and Pierre Casimir-Lambert Fund)National Cancer Institute (U.S.) (Koch Institute Support (core) Grant P30-CA14051))National Institutes of Health (U.S.) (grant EB016101-01A1)Damon Runyon Cancer Research Foundation (Research Fellow (DRG-2117-12)