Targeting ribosome biogenesis reinforces ERK-dependent senescence in pancreatic cancer.

Pancreatic adenocarcinomas (PDAC) often possess mutations in K-Ras that stimulate the ERK pathway. Aberrantly high ERK activation triggers oncogene-induced senescence, which halts tumor progression. Here we report that low-grade pancreatic intraepithelial neoplasia displays very high levels of phospho-ERK consistent with a senescence response. However, advanced lesions that have circumvented the senescence barrier exhibit lower phospho-ERK levels. Restoring ERK hyperactivation in PDAC using activated RAF leads to ERK-dependent growth arrest with senescence biomarkers. ERK-dependent senescence in PDAC was characterized by a nucleolar stress response including a selective depletion of nucleolar phosphoproteins and intranucleolar foci containing RNA polymerase I designated as senescence-associated nucleolar foci (SANF). Accordingly, combining ribosome biogenesis inhibitors with ERK hyperactivation reinforced the senescence response in PDAC cells. Notably, comparable mechanisms were observed upon treatment with the platinum-based chemotherapy regimen FOLFIRINOX, currently a first-line treatment option for PDAC. We thus suggest that drugs targeting ribosome biogenesis can improve the senescence anticancer response in pancreatic cancer.

The clinical landscape of cell-free DNA alterations in 1671 patients with advanced biliary tract cancer.

BACKGROUND: Targeted therapies have transformed clinical management of advanced biliary tract cancer (BTC). Cell-free DNA (cfDNA) analysis is an attractive approach for cancer genomic profiling that overcomes many limitations of traditional tissue-based analysis. We examined cfDNA as a tool to inform clinical management of patients with advanced BTC and generate novel insights into BTC tumor biology. PATIENTS AND METHODS: We analyzed next-generation sequencing data of 2068 cfDNA samples from 1671 patients with advanced BTC generated with Guardant360. We carried out clinical annotation on a multi-institutional subset (n = 225) to assess intra-patient cfDNA-tumor concordance and the association of cfDNA variant allele fraction (VAF) with clinical outcomes. RESULTS: Genetic alterations were detected in cfDNA in 84% of patients, with targetable alterations detected in 44% of patients. Fibroblast growth factor receptor 2 (FGFR2) fusions, isocitrate dehydrogenase 1 (IDH1) mutations, and BRAF V600E were clonal in the majority of cases, affirming these targetable alterations as early driver events in BTC. Concordance between cfDNA and tissue for mutation detection was high for IDH1 mutations (87%) and BRAF V600E (100%), and low for FGFR2 fusions (18%). cfDNA analysis uncovered novel putative mechanisms of resistance to targeted therapies, including mutation of the cysteine residue (FGFR2 C492F) to which covalent FGFR inhibitors bind. High pre-treatment cfDNA VAF was associated with poor prognosis and shorter response to chemotherapy and targeted therapy. Finally, we report the frequency of promising targets in advanced BTC currently under investigation in other advanced solid tumors, including KRAS G12C (1.0%), KRAS G12D (5.1%), PIK3CA mutations (6.8%), and ERBB2 amplifications (4.9%). CONCLUSIONS: These findings from the largest and most comprehensive study to date of cfDNA from patients with advanced BTC highlight the utility of cfDNA analysis in current management of this disease. Characterization of oncogenic drivers and mechanisms of therapeutic resistance in this study will inform drug development efforts to reduce mortality for patients with BTC.

Regulation of autophagy during ECM detachment is linked to a selective inhibition of mTORC1 by PERK.

Adhesion to the extracellular matrix (ECM) is critical for epithelial tissue homeostasis and function. ECM detachment induces metabolic stress and programmed cell death via anoikis. ECM-detached mammary epithelial cells are able to rapidly activate autophagy allowing for survival and an opportunity for re-attachment. However, the mechanisms controlling detachment-induced autophagy remain unclear. Here we uncover that the kinase PERK rapidly promotes autophagy in ECM-detached cells by activating AMP-activated protein kinase (AMPK), resulting in downstream inhibition of mTORC1-p70(S6K) signaling. LKB1 and TSC2, but not TSC1, are required for PERK-mediated inhibition of mammalian target of rapamycinin MCF10A cells and mouse embryo fibroblast cells. Importantly, this pathway shows fast kinetics, is transcription-independent and is exclusively activated during ECM detachment, but not by canonical endoplasmic reticulum stressors. Moreover, enforced PERK or AMPK activation upregulates autophagy and causes luminal filling during acinar morphogenesis by perpetuating a population of surviving autophagic luminal cells that resist anoikis. Hence, we identify a novel pathway in which suspension-activated PERK promotes the activation of LKB1, AMPK and TSC2, leading to the rapid induction of detachment-induced autophagy. We propose that increased autophagy, secondary to persistent PERK and LKB1-AMPK signaling, can robustly protect cells from anoikis and promote luminal filling during early carcinoma progression.

MicroRNAs in metabolism and metabolic diseases.

Aberrant cholesterol/lipid homeostasis is linked to a number of diseases prevalent in the developed world, including metabolic syndrome, type II diabetes, and cardiovascular disease. We have previously uncovered gene regulatory mechanisms of the sterol regulatory element-binding protein (SREBP) family of transcription factors, which control the expression of genes involved in cholesterol and lipid biosynthesis and uptake. Intriguingly, we recently discovered conserved microRNAs (miR-33a/b) embedded within intronic sequences of the human SREBF genes that act in a concerted manner with their host gene products to regulate cholesterol/lipid homeostasis. Indeed, miR-33a/b control the levels of ATP-binding cassette (ABC) transporter ABCA1, a cholesterol efflux pump critical for high-density lipoprotein (HDL) synthesis and reverse cholesterol transport from peripheral tissues. Importantly, antisense inhibition of miR-33 in mice results in elevated HDL and decreased atherosclerosis. Interestingly, miR-33a/b also act in the fatty acid/lipid homeostasis pathway by controlling the fatty acid ß-oxidation genes carnitine O-octanoyltransferase (CROT), hydroxyacyl-coenzyme A-dehydrogenase (HADHB), and carnitine palmitoyltransferase 1A (CPT1A), as well as the energy sensor AMP-activated protein kinase (AMPKα1), the NAD(+)-dependent sirtuin SIRT6, and the insulin signaling intermediate IRS2, key regulators of glucose and lipid metabolism. These results have revealed a highly integrated microRNA (miRNA)-host gene circuit governing cholesterol/lipid metabolism and energy homeostasis in mammals that may have important therapeutic implications for the treatment of cardiometabolic disorders.

Mst1/2 signalling to Yap: gatekeeper for liver size and tumour development.

The mechanisms controlling mammalian organ size have long been a source of fascination for biologists. These controls are needed to both ensure the integrity of the body plan and to restrict inappropriate proliferation that could lead to cancer. Regulation of liver size is of particular interest inasmuch as this organ maintains the capacity for regeneration throughout life, and is able to regain precisely its original mass after partial surgical resection. Recent studies using genetically engineered mouse strains have shed new light on this problem; the Hippo signalling pathway, first elucidated as a regulator of organ size in Drosophila, has been identified as dominant determinant of liver growth. Defects in this pathway in mouse liver lead to sustained liver overgrowth and the eventual development of both major types of liver cancer, hepatocellular carcinoma and cholangiocarcinoma. In this review, we discuss the role of Hippo signalling in liver biology and the contribution of this pathway to liver cancer in humans.

LKB1; linking cell structure and tumor suppression.

Germ line mutations in the LKB1 tumor suppressor gene are associated with the Peutz-Jeghers polyposis and cancer syndrome. Somatic mutations in Lkb1 are observed in sporadic pulmonary, pancreatic and biliary cancers and melanomas. The LKB1 serine-threonine kinase functionally and biochemically links control of cellular structure and energy utilization through activation of the AMPK family of kinases. Lkb1 regulates cell polarity through downstream kinases including AMPKs, MARKs and BRSKs, and nutrient utilization and cellular metabolism through the AMPK-mTOR pathway. LKB1 has been shown to affect normal chromosomal segregation, TGF-beta signaling in the mesenchyme and WNT and p53 activity. Although each of the LKB1-dependent processes and downstream pathways have been individually delineated through work across a range of experimental systems, how they relate to Lkb1's role as a tumor suppressor remains to be fully explored and elucidated. The recent development of mouse cancer models harboring engineered mutations in Lkb1 have offered insights into how LKB1 may be functioning to restrain tumorigenesis and how its role as a master regulator of polarity and metabolism could contribute to its tumor suppressor function.

Hedgehog inhibition prolongs survival in a genetically engineered mouse model of pancreatic cancer.

BACKGROUND AND AIMS: Pancreatic cancer is among the most dismal of human malignancies. Current therapeutic strategies are virtually ineffective in controlling advanced, metastatic disease. Recent evidence suggests that the Hedgehog signalling pathway is aberrantly reactivated in the majority of pancreatic cancers, and that Hedgehog blockade has the potential to prevent disease progression and metastatic spread. METHODS: Here it is shown that the Hedgehog pathway is activated in the Pdx1-Cre;LsL-Kras(G12D);Ink4a/Arf(lox/lox) transgenic mouse model of pancreatic cancer. The effect of Hedgehog pathway inhibition on survival was determined by continuous application of the small molecule cyclopamine, a smoothened antagonist. Microarray analysis was performed on non-malignant human pancreatic ductal cells overexpressing Gli1 in order to screen for downstream Hedgehog target genes likely to be involved in pancreatic cancer progression. RESULTS: Hedgehog inhibition with cyclopamine significantly prolonged median survival in the transgenic mouse model used here (67 vs 61 days; p = 0.026). In vitro data indicated that Hedgehog activation might at least in part be ascribed to oncogenic Kras signalling. Microarray analysis identified 26 potential Hedgehog target genes that had previously been found to be overexpressed in pancreatic cancer. Five of them, BIRC3, COL11A1, NNMT, PLAU and TGM2, had been described as upregulated in more than one global gene expression analysis before. CONCLUSION: This study provides another line of evidence that Hedgehog signalling is a valid target for the development of novel therapeutics for pancreatic cancer that might be worth evaluating soon in a clinical setting.

K-ras activation generates an inflammatory response in lung tumors.

Activating mutations in K-ras are one of the most common genetic alterations in human lung cancer. To dissect the role of K-ras activation in bronchial epithelial cells during lung tumorigenesis, we created a model of lung adenocarcinoma by generating a conditional mutant mouse with both Clara cell secretory protein (CC10)-Cre recombinase and the Lox-Stop-Lox K-ras(G12D) alleles. The activation of K-ras mutant allele in CC10 positive cells resulted in a progressive phenotype characterized by cellular atypia, adenoma and ultimately adenocarcinoma. Surprisingly, K-ras activation in the bronchiolar epithelium is associated with a robust inflammatory response characterized by an abundant infiltration of alveolar macrophages and neutrophils. These mice displayed early mortality in the setting of this pulmonary inflammatory response with a median survival of 8 weeks. Bronchoalveolar lavage fluid from these mutant mice contained the MIP-2, KC, MCP-1 and LIX chemokines that increased significantly with age. Cell lines derived from these tumors directly produced MIP-2, LIX and KC. This model demonstrates that K-ras activation in the lung induces the elaboration of inflammatory chemokines and provides an excellent means to further study the complex interactions between inflammatory cells, chemokines and tumor progression.

Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis.

The cyclin-dependent kinase inhibitor p16INK4a can induce senescence of human cells, and its loss by deletion, mutation or epigenetic silencing is among the most frequently observed molecular lesions in human cancer. Overlapping reading frames in the INK4A/ARF gene encode p16INK4a and a distinct tumour-suppressor protein, p19ARF (ref. 3). Here we describe the generation and characterization of a p16Ink4a-specific knockout mouse that retains normal p19Arf function. Mice lacking p16Ink4a were born with the expected mendelian distribution and exhibited normal development except for thymic hyperplasia. T cells deficient in p16Ink4a exhibited enhanced mitogenic responsiveness, consistent with the established role of p16Ink4a in constraining cellular proliferation. In contrast to mouse embryo fibroblasts (MEFs) deficient in p19Arf (ref. 4), p16Ink4a-null MEFs possessed normal growth characteristics and remained susceptible to Ras-induced senescence. Compared with wild-type MEFs, p16Ink4a-null MEFs exhibited an increased rate of immortalization, although this rate was less than that observed previously for cells null for Ink4a/Arf, p19Arf or p53 (refs 4, 5). Furthermore, p16Ink4a deficiency was associated with an increased incidence of spontaneous and carcinogen-induced cancers. These data establish that p16Ink4a, along with p19Arf, functions as a tumour suppressor in mice.

The genetics of pancreatic adenocarcinoma: a roadmap for a mouse model.

Pancreatic cancer is among the leading causes of cancer death. Although a genetic profile for pancreatic cancer is emerging, many biological aspects of this disease are poorly understood. Indeed, fundamental questions regarding progenitor cell lineages, host stromal milieu, and the role of specific genetic alterations in tumor progression remain unresolved. A mouse model engineered with signature mutations would provide a powerful ally in the study of pancreatic cancer biology and may guide improved prognostic assessment and treatment for the human disease. In this review, we discuss the molecular basis for normal pancreatic development and the genetics of human pancreatic adenocarcinoma in the hope of charting a course for the development of a faithful mouse model for this lethal cancer.

p19ARF targets certain E2F species for degradation.

p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found to complex with transcription factors E2F1, -2, and -3. Levels of endogenous or ectopically expressed E2F1, -2, and -3, but not E2F6, were reduced after synthesis of p19ARF, through a mechanism involving increased turnover. p19ARF-induced degradation of E2F1 depended on a functional proteasome, and E2F1 was relocalized to nucleoli when coexpressed with p19ARF. Consistent with reduced levels of E2F1 and E2F3, the proliferation of cells defective for p53 function was suppressed by p19ARF, and the effect was partially reversed by ectopic overexpression of E2F1. These results suggest a broader role for p19ARF as a tumor suppressor, in which targeting of certain E2F species may cooperate with stimulation of the p53 pathway to counteract oncogenic growth signals.

Dual inactivation of RB and p53 pathways in RAS-induced melanomas.

The frequent loss of both INK4a and ARF in melanoma raises the question of which INK4a-ARF gene product functions to suppress melanoma genesis in vivo. Moreover, the high incidence of INK4a-ARF inactivation in transformed melanocytes, along with the lack of p53 mutation, implies a cell type-specific role for INK4a-ARF that may not be complemented by other lesions of the RB and p53 pathways. A mouse model of cutaneous melanoma has been generated previously through the combined effects of INK4a(Delta2/3) deficiency (null for INK4a and ARF) and melanocyte-specific expression of activated RAS (tyrosinase-driven H-RAS(V12G), Tyr-RAS). In this study, we made use of this Tyr-RAS allele to determine whether activated RAS can cooperate with p53 loss in melanoma genesis, whether such melanomas are biologically comparable to those arising in INK4a(Delta2/3-/-) mice, and whether tumor-associated mutations emerge in the p16(INK4a)-RB pathway in such melanomas. Here, we report that p53 inactivation can cooperate with activated RAS to promote the development of cutaneous melanomas that are clinically indistinguishable from those arisen on the INK4a(Delta2/3) null background. Genomewide analysis of RAS-induced p53 mutant melanomas by comparative genomic hybridization and candidate gene surveys revealed alterations of key components governing RB-regulated G(1)/S transition, including c-Myc, cyclin D1, cdc25a, and p21(CIP1). Consistent with the profile of c-Myc dysregulation, the reintroduction of p16(INK4a) profoundly reduced the growth of Tyr-RAS INK4a(Delta2/3-/-) tumor cells but had no effect on tumor cells derived from Tyr-RAS p53(-/-) melanomas. Together, these data validate a role for p53 inactivation in melanomagenesis and suggest that both the RB and p53 pathways function to suppress melanocyte transformation in vivo in the mouse.

Essential role for oncogenic Ras in tumour maintenance.

Advanced malignancy in tumours represents the phenotypic endpoint of successive genetic lesions that affect the function and regulation of oncogenes and tumour-suppressor genes. The established tumour is maintained through complex and poorly understood host-tumour interactions that guide processes such as angiogenesis and immune sequestration. The many different genetic alterations that accompany tumour genesis raise questions as to whether experimental cancer-promoting mutations remain relevant during tumour maintenance. Here we show that melanoma genesis and maintenance are strictly dependent upon expression of H-RasV12G in a doxycycline-inducible H-Ras12G mouse melanoma model null for the tumour suppressor INK4a. Withdrawal of doxycycline and H-RasV12G down-regulation resulted in clinical and histological regression of primary and explanted tumours. The initial stages of regression involved marked apoptosis in the tumour cells and host-derived endothelial cells. Although the regulation of vascular endothelial growth factor (VEGF) was found to be Ras-dependent in vitro, the failure of persistent endogenous and enforced VEGF expression to sustain tumour viability indicates that the tumour-maintaining actions of activated Ras extend beyond the regulation of VEGF expression in vivo. Our results provide genetic evidence that H-RasV12G is important in both the genesis and maintenance of solid tumours.

The Wilms' tumor suppressor gene (wt1) product regulates Dax-1 gene expression during gonadal differentiation.

Gonadal differentiation is dependent upon a molecular cascade responsible for ovarian or testicular development from the bipotential gonadal ridge. Genetic analysis has implicated a number of gene products essential for this process, which include Sry, WT1, SF-1, and DAX-1. We have sought to better define the role of WT1 in this process by identifying downstream targets of WT1 during normal gonadal development. We have noticed that in the developing murine gonadal ridge, wt1 expression precedes expression of Dax-1, a nuclear receptor gene. We document here that the spatial distribution profiles of both proteins in the developing gonad overlap. We also demonstrate that WT1 can activate the Dax-1 promoter. Footprinting analysis, transient transfections, promoter mutagenesis, and mobility shift assays suggest that WT1 regulates Dax-1 via GC-rich binding sites found upstream of the Dax-1 TATA box. We show that two WT1-interacting proteins, the product of a Denys-Drash syndrome allele of wt1 and prostate apoptosis response-4 protein, inhibit WT1-mediated transactivation of Dax-1. In addition, we demonstrate that WT1 can activate the endogenous Dax-1 promoter. Our results indicate that the WT1-DAX-1 pathway is an early event in the process of mammalian sex determination.

Overlapping RNA and DNA binding domains of the wt1 tumor suppressor gene product.

The Wilms' tumour suppressor gene (wt1) is mutated in a subset of patients with Wilms' tumour and has a critical role in urogenital development. wt1 encodes a zinc finger transcription factor which regulates expression of several genes involved in cellular proliferation and differentiation. Although a number of studies have characterized the DNA binding properties of the WT1 protein, recent evidence has suggested that WT1 may also have a role in RNA metabolism. We have used an RNA selection method to identify WT1 binding ligands from a random RNA pool. Three groups of RNA ligands specifically recognized by WT1 were identified. Mutational analysis pinpointed ribonucleotide sequences critical for binding. Analysis of truncated WT1 proteins demonstrated that three of four zinc fingers were necessary for RNA-protein interaction. The naturally occurring WT1 isoforms with insertion of lysine, threonine and serine between zinc fingers three and four were unable to bind the selected RNAs. The selected RNA ligands competed with the cognate WT1 DNA binding site for complex formation with WT1. Our findings suggest potential cellular RNA target sequences for WT1 and provide tools for studying the structural and functional properties of this tumour suppressor protein.

Antagonism of WT1 activity by protein self-association.

Germline loss-of-function mutations at the Wilms tumor (WT) suppressor locus WT1 are associated with a predisposition to WTs and mild genital system anomalies. In contrast, germ-line missense mutations within the WT1 gene encoding the DNA-binding domain often yield a more severe phenotype consisting of WT, sexual ambiguity, and renal nephropathy. In this report, we demonstrate that the products of mutant alleles that impair DNA recognition can antagonize WT1-mediated transcriptional repression. We demonstrate that WT1 can self-associate in vitro and in vivo and that the responsible domain maps to the amino-terminal region of the protein. Oligomers of full-length protein form less efficiently or produce less stable complexes than oligomers between truncated polypeptides and full-length protein. Our data suggest a molecular mechanism to explain how WT1 mutations may act in deregulating cellular proliferation and differentiation.

Clonal expansion and attenuated apoptosis in Wilms' tumors are associated with p53 gene mutations.

The p53 gene product is required for activation of an apoptotic pathway triggered by oncogenes and cytotoxic agents. Wilms' tumor, a pediatric renal malignancy, provides a paradigm for evaluating genetic events involved in tumor progression. This malignancy is generally not associated with p53 mutations, and even in advanced disease states is quite responsive to current treatment regimens. The anaplastic histological variant of Wilms' tumor, however, is frequently associated with p53 gene mutations and shows poor prognosis. We analyzed seven Wilms' tumors for which we had paired samples from nonanaplastic and anaplastic regions. p53 mutations were detected in six of these tumors, five of which demonstrated mutations restricted to anaplastic regions. Nonanaplastic cells of the sixth sample were heterozygous for a p53 mutation, whereas the anaplastic area of this tumor showed reduction to homozygosity. These results indicate that progression to anaplasia is associated with clonal expansion of cells which have acquired a p53 mutation. We demonstrated that tumor cells with p53 mutations show attenuated apoptosis, suggesting that such lesions may provide a selective advantage in vivo by decreasing cell death.