ABSTRACT
Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.
Subject(s)
Antibodies/therapeutic use , Deep Learning , Diagnosis, Computer-Assisted/methods , Drug Therapy, Computer-Assisted/methods , Neoplasms/pathology , Animals , Humans , MCF-7 Cells , Mice , Mice, Inbred C57BL , Mice, Nude , Mice, SCID , Neoplasm Metastasis , Neoplasms/diagnostic imaging , Neoplasms/drug therapy , Software , Tumor MicroenvironmentABSTRACT
The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfß-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.
Subject(s)
Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Evolution, Molecular , Gene Dosage , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Adaptor Proteins, Signal Transducing/genetics , Alleles , Animals , Carcinogenesis/genetics , Cell Cycle Proteins , Cyclin-Dependent Kinase Inhibitor p16/genetics , Disease Progression , Female , Genes, myc , Genes, p53 , Humans , Male , Mice , Mutation , NF-kappa B p52 Subunit/genetics , Neoplasm Metastasis/genetics , Nuclear Proteins/genetics , Phenotype , Phosphoproteins/genetics , Transcription Factors/genetics , Transcriptome/genetics , Transforming Growth Factor beta1/genetics , YAP-Signaling ProteinsABSTRACT
BACKGROUND: Although the mechanistic target of rapamycin (MTOR) kinase, included in the mTORC1 and mTORC2 signalling hubs, has been demonstrated to be active in a significant fraction of patients with pancreatic ductal adenocarcinoma (PDAC), the value of the kinase as a therapeutic target needs further clarification. METHODS: We used Mtor floxed mice to analyse the function of the kinase in context of the pancreas at the genetic level. Using a dual-recombinase system, which is based on the flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies, we generated a novel cellular model, allowing the genetic analysis of MTOR functions in tumour maintenance. Cross-species validation and pharmacological intervention studies were used to recapitulate genetic data in human models, including primary human 3D PDAC cultures. RESULTS: Genetic deletion of the Mtor gene in the pancreas results in exocrine and endocrine insufficiency. In established murine PDAC cells, MTOR is linked to metabolic pathways and maintains the glucose uptake and growth. Importantly, blocking MTOR genetically as well as pharmacologically results in adaptive rewiring of oncogenic signalling with activation of canonical extracellular signal-regulated kinase and phosphoinositide 3-kinase-AKT pathways. We provide evidence that interfering with such adaptive signalling in murine and human PDAC models is important in a subgroup. CONCLUSIONS: Our data suggest developing dual MTORC1/TORC2 inhibitor-based therapies for subtype-specific intervention.
Subject(s)
Carcinoma, Pancreatic Ductal/metabolism , MAP Kinase Signaling System , Pancreatic Neoplasms/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Animals , Antineoplastic Agents/pharmacology , Benzoxazoles/pharmacology , Bortezomib/pharmacology , Camptothecin/pharmacology , Carcinoma, Pancreatic Ductal/genetics , Cell Line, Tumor , Cell Survival , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 2/antagonists & inhibitors , Mice , Pancreatic Neoplasms/genetics , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitorsABSTRACT
Although BCR-ABL1 tyrosine kinase inhibitors reliably induce disease remission for patients with chronic myeloid leukaemia (CML), unlimited extension of therapy is necessary to prevent relapse from persistent leukaemic cells. Here, we analysed model cell lines and primary CML cells for the expression and functions of the ABC transporter A3 (ABCA3) as well as the embryonic stem cell-associated transcription factor SALL4. ABCA3 protected leukaemic cells from the cytotoxic effects of the tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib. In the surviving cells, exposure to tyrosine kinase inhibitors significantly enhanced ABCA3 expression in vivo and in vitro, and was associated with increased expression of SALL4, which binds the ABCA3 promoter. Inhibition of ABCA3 or SALL4 by genetic silencing or indomethacin, but not interferon gamma, interrupted SALL4-dependent regulation of ABCA3 and restored susceptibility of leukaemic cells to tyrosine kinase inhibition. Tyrosine kinase inhibitor exposure facilitates a protective loop of SALL4 and ABCA3 cooperation in persistent leukaemic cells.
Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Leukemic/drug effects , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Neoplasm Proteins/metabolism , Protein Kinase Inhibitors/pharmacology , Transcription Factors/metabolism , Animals , Benzamides/pharmacology , Dasatinib , Female , HL-60 Cells , Humans , Imatinib Mesylate , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Mice , Piperazines/pharmacology , Pyrimidines/pharmacology , Thiazoles/pharmacologyABSTRACT
S100A8 and S100A9, two heterodimer-forming members of the cytosolic S100 Ca(2+) signaling protein family, are overexpressed in various cancer types, including prostate cancer. They act as proinflammatory danger signals when secreted to the extracellular space and are thought to play an important role during tumorigenesis, affecting inflammatory processes, proliferation, invasion and metastasis of tumor cells. Despite this fact, little is known about tumor environmental factors influencing S100A8/A9 expression. The aim of this study was to test the effect of hypoxia and its master transcriptional regulator hypoxia-inducible factor 1 (HIF-1) on S100A8/A9 expression. Hypoxia treatment resulted in induction of S100A8/A9 protein and mRNA expression in prostate epithelial BPH-1 cells, the latter was also confirmed in the prostate cancer cell lines PC-3 and DU-145. Furthermore, overexpression of HIF-1α caused increase in S100A8/A9 protein and mRNA expression as well as secretion. Functional hypoxia response elements mediating promoter activation on HIF-1α overexpression were identified within the S100A8 and S100A9 promoters using promoter luciferase reporter constructs. Binding of HIF-1α to S100A8 and S100A9 promoters was confirmed by chromatin immunoprecipitation. Immunohistochemical analysis of a prostate cancer tissue array showed clear correlation of S100A8 and S100A9 with HIF-1α expression. Multivariate proportional hazard analysis revealed association of high S100A9 level with time to prostate cancer recurrence. In conclusion, we identified hypoxia and HIF-1 as novel regulators of S100A8/A9 expression in prostate cancer. S100A9 might be useful as prognostic marker for prostate cancer recurrence after radical prostatectomy.
Subject(s)
Calgranulin A/metabolism , Calgranulin B/metabolism , Cell Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Prostatic Neoplasms/metabolism , Base Sequence , Blotting, Western , Calgranulin A/genetics , Calgranulin B/genetics , Cell Line, Tumor , Chromatin Immunoprecipitation , DNA Primers , Humans , Male , Middle Aged , Promoter Regions, Genetic , Prostatectomy , Prostatic Neoplasms/pathology , Prostatic Neoplasms/surgery , Real-Time Polymerase Chain ReactionABSTRACT
KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted and immunotherapies. Among the different PDAC subtypes, basal-like mesenchymal PDAC, which is driven by allelic imbalance, increased gene dosage and subsequent high expression levels of oncogenic KRAS, shows the most aggressive phenotype and strongest therapy resistance. In the present study, we performed a systematic high-throughput combination drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib, which targets KRAS-directed oncogenic signaling in mesenchymal PDAC. This combination treatment induces cell-cycle arrest and cell death, and initiates a context-dependent remodeling of the immunosuppressive cancer cell secretome. Using a combination of single-cell RNA-sequencing, CRISPR screens and immunophenotyping, we show that this combination therapy promotes intratumor infiltration of cytotoxic and effector T cells, which sensitizes mesenchymal PDAC to PD-L1 immune checkpoint inhibition. Overall, our results open new avenues to target this aggressive and therapy-refractory mesenchymal PDAC subtype.
Subject(s)
Adenocarcinoma , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Adenocarcinoma/drug therapy , Carcinoma, Pancreatic Ductal/drug therapy , Humans , Immune Checkpoint Inhibitors , Pancreatic Neoplasms/drug therapy , Tumor MicroenvironmentABSTRACT
In modern oncology, the analysis and evaluation of treatment response are still challenging. Hence, we used a 13C-guided approach to study the impacts of the small molecule dichloroacetate (DCA) upon the metabolic response of pancreatic cancer cells. Two different oncogenic PI3K-driven pancreatic cancer cell lines, 9580 and 10,158, respectively, were treated with 75 mM DCA for 18 h. In the presence of [U-13C6]glucose, the effects of DCA treatment in the core carbon metabolism were analyzed in these cells using gas chromatography-mass spectrometry (GC/MS). 13C-enrichments and isotopologue profiles of key amino acids revealed considerable effects of the DCA treatment upon glucose metabolism. The DCA treatment of the two pancreatic cell lines resulted in a significantly decreased incorporation of [U-13C6]glucose into the amino acids alanine, aspartate, glutamate, glycine, proline and serine in treated, but not in untreated, cancer cells. For both cell lines, the data indicated some activation of pyruvate dehydrogenase with increased carbon flux via the TCA cycle, but also massive inhibition of glycolytic flux and amino acid biosynthesis presumably by inhibition of the PI3K/Akt/mTORC axis. Together, it appears worthwhile to study the early treatment response in DCA-guided or accompanied cancer therapy in more detail, since it could open new avenues for improved diagnosis and therapeutic protocols of cancer.
ABSTRACT
Biliary tract cancer ranks among the most lethal human malignancies, representing an unmet clinical need. Its abysmal prognosis is tied to an increasing incidence and a fundamental lack of mechanistic knowledge regarding the molecular basis of the disease. Here, we show that the Pdx1-positive extrahepatic biliary epithelium is highly susceptible toward transformation by activated PIK3CAH1047R but refractory to oncogenic KrasG12D. Using genome-wide transposon screens and genetic loss-of-function experiments, we discover context-dependent genetic interactions that drive extrahepatic cholangiocarcinoma (ECC) and show that PI3K signaling output strength and repression of the tumor suppressor p27Kip1 are critical context-specific determinants of tumor formation. This contrasts with the pancreas, where oncogenic Kras in concert with p53 loss is a key cancer driver. Notably, inactivation of p27Kip1 permits KrasG12D-driven ECC development. These studies provide a mechanistic link between PI3K signaling, tissue-specific tumor suppressor barriers, and ECC pathogenesis, and present a novel genetic model of autochthonous ECC and genes driving this highly lethal tumor subtype. SIGNIFICANCE: We used the first genetically engineered mouse model for extrahepatic bile duct carcinoma to identify cancer genes by genome-wide transposon-based mutagenesis screening. Thereby, we show that PI3K signaling output strength and p27Kip1 function are critical determinants for context-specific ECC formation. This article is highlighted in the In This Issue feature, p. 2945.
Subject(s)
Bile Duct Neoplasms , Biliary Tract Neoplasms , Animals , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/pathology , Bile Ducts, Intrahepatic/pathology , Biliary Tract Neoplasms/genetics , Genes, Tumor Suppressor , Humans , Mice , Phosphatidylinositol 3-Kinases/geneticsABSTRACT
Molecular profiling of cancer patients and modelling of human cancer in mice revealed cell type and tissue-specific differences in tumor development and evolution. However, the context-dependent determinants of cancer remain poorly understood. A systematic characterization of the biological underpinnings of context-specificity will, therefore, be pivotal to design more effective therapies. In this review article, we focus on recent advances on molecular, cellular and microenvironmental aspects of context-dependency. We highlight new strategies to study this phenomenon in tumorigenesis and tumor evolution. Notably, we elucidate tissue and cell type-specific signaling cues as well as tumor microenvironment niches, using novel next-generation dual and triple recombinase-based mouse models of cancer.
Subject(s)
Carcinogenesis/genetics , Clonal Evolution/genetics , Neoplasms/genetics , Animals , Cell Transformation, Neoplastic/genetics , Disease Models, Animal , Humans , Mice , Signal Transduction/genetics , Tumor Microenvironment/geneticsABSTRACT
BACKGROUND: CDK4/6 inhibitors are a promising treatment strategy in tumor therapy but are hampered by resistance mechanisms. This study was performed to reveal predictive markers, mechanisms of resistance and to develop rational combination therapies for a personalized therapy approach in bladder cancer. METHODS: A genome-scale CRISPR-dCas9 activation screen for resistance to the CDK4/6 inhibitor Palbociclib was performed in the bladder cancer derived cell line T24. sgRNA counts were analyzed using next generation sequencing and MAGeCK-VISPR. Significantly enriched sgRNAs were cloned and validated on a molecular and functional level for mediating resistance to Palbociclib treatment. Analysis was done in vitro and in vivo in the chorioallantois membrane model of the chicken embryo. Comparison of screen hits to signaling pathways and clinically relevant molecular alterations was performed using DAVID, Reactome, DGIdb and cBioPortal. RESULTS: In the screen, 1024 sgRNAs encoding for 995 genes were significantly enriched indicative of mediators of resistance. 8 random sgRNAs were validated, revealing partial rescue to Palbociclib treatment. Within this gene panel, members of Receptor-Tyrosine Kinases, PI3K-Akt, Ras/MAPK, JAK/STAT or Wnt signaling pathways were identified. Combination of Palbociclib with inhibitors against these signaling pathways revealed beneficial effects in vitro and in in vivo xenografts. CONCLUSIONS: Identification of potential predictive markers, resistance mechanisms and rational combination therapies could be achieved by applying a CRISPR-dCas9 screening approach in bladder cancer.
Subject(s)
Genomics , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Urinary Bladder Neoplasms/drug therapy , Animals , Apoptosis/drug effects , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Chickens , Chorioallantoic Membrane/drug effects , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/genetics , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Heterografts , Humans , Mice , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathologyABSTRACT
Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research.
Subject(s)
Carcinogenesis/genetics , Pancreas/metabolism , Pancreatic Neoplasms/genetics , Animals , BRCA2 Protein/genetics , CRISPR-Cas Systems , Chromosome Deletion , Electroporation , Genetic Engineering/methods , Genome , High-Throughput Nucleotide Sequencing , Immunohistochemistry , Magnetic Resonance Imaging , Mice , Mutation , Neoplasms, Experimental/genetics , Phylogeny , Polymerase Chain Reaction , Proto-Oncogene Proteins p21(ras)/genetics , Sequence Analysis, DNA , Transfection/methods , Translocation, Genetic/geneticsABSTRACT
Here we describe a conditional piggyBac transposition system in mice and report the discovery of large sets of new cancer genes through a pancreatic insertional mutagenesis screen. We identify Foxp1 as an oncogenic transcription factor that drives pancreatic cancer invasion and spread in a mouse model and correlates with lymph node metastasis in human patients with pancreatic cancer. The propensity of piggyBac for open chromatin also enabled genome-wide screening for cancer-relevant noncoding DNA, which pinpointed a Cdkn2a cis-regulatory region. Histologically, we observed different tumor subentities and discovered associated genetic events, including Fign insertions in hepatoid pancreatic cancer. Our studies demonstrate the power of genetic screening to discover cancer drivers that are difficult to identify by other approaches to cancer genome analysis, such as downstream targets of commonly mutated human cancer genes. These piggyBac resources are universally applicable in any tissue context and provide unique experimental access to the genetic complexity of cancer.
Subject(s)
Cell Transformation, Neoplastic/genetics , DNA Transposable Elements/genetics , Gene Regulatory Networks , Mutagenesis, Insertional , Pancreatic Neoplasms/genetics , Amino Acid Sequence , Animals , Forkhead Transcription Factors/analysis , Forkhead Transcription Factors/antagonists & inhibitors , Forkhead Transcription Factors/genetics , Gene Expression Profiling , Gene Expression Regulation , Gene Knock-In Techniques , Genes, Synthetic , Genes, p16 , Humans , Mice , Mice, Transgenic , Molecular Sequence Data , Moths/genetics , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Pancreatic Neoplasms/chemistry , Pancreatic Neoplasms/pathology , Proton-Translocating ATPases/genetics , RNA, Small Interfering/pharmacology , Repressor Proteins/analysis , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/genetics , Transgenes , Transposases/genetics , Transposases/physiologyABSTRACT
Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP-based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell-autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.