NAR Cancer最新文献

A problematic feature of many human cancers is a lack of understanding of mechanisms controlling organ-specific patterns of metastasis, despite recent progress in identifying many mutations and transcriptional programs shown to confer this potential. To address this gap, we developed a methodology that enables different aspects of the metastatic process to be comprehensively characterized at a clonal resolution. Our approach exploits the application of a computational pipeline to analyze and visualize clonal data obtained from transplant experiments in which a cellular DNA barcoding strategy is used to distinguish the separate clonal contributions of two or more competing cell populations. To illustrate the power of this methodology, we demonstrate its ability to discriminate the metastatic behavior in immunodeficient mice of a well-established human metastatic cancer cell line and its co-transplanted LRRC15 knockdown derivative. We also show how the use of machine learning to quantify clone-initiating cell (CIC) numbers and their subsequent metastatic progeny generated in different sites can reveal previously unknown relationships between different cellular genotypes and their initial sites of implantation with their subsequent respective dissemination patterns. These findings underscore the potential of such combined genomic and computational methodologies to identify new clonally-relevant drivers of site-specific patterns of metastasis.

The molecular composition of blood is a signature of human health, reflected in the thousands of blood biomarkers known for human diseases. However, establishing robust disease markers is challenging due to the diversity of individual samples. New sequencing methods have simplified biomarker discovery for circulating DNA and RNA while protein profiling is still laborious and costly. To harness the power of high-throughput sequencing to profile the protein content of a biological sample, we developed a method termed APTASHAPE that uses oligonucleotide aptamers to recognize proteins in complex biofluids. We selected a large pool of 2'Fluoro protected RNA sequences to recognize proteins in human plasma and identified a set of 33 cancer-specific aptamers. Differential enrichment of these aptamers after selection against 1 μl of plasma from individual patients allowed us to differentiate between healthy controls and bladder cancer-diagnosed patients (91% accuracy) and between early non-invasive tumors and late stage tumors (83% accuracy). Affinity purification and mass spectrometry of proteins bound to the predictive aptamers showed the main target proteins to be C4b-binding protein, Complement C3, Fibrinogen, Complement factor H and IgG. The APTASHAPE method thus provides a general, automated and highly sensitive platform for discovering potential new disease biomarkers.

Malignant pleural mesothelioma (MPM) is a rare and incurable cancer, which incidence is increasing in many countries. MPM escapes the classical genetic model of cancer evolution, lacking a distinctive genetic fingerprint. Omics profiling revealed extensive heterogeneity failing to identify major vulnerabilities and restraining development of MPM-oriented therapies. Here, we performed a multilayered analysis based on a functional genome-wide CRISPR/Cas9 screening integrated with patients molecular and clinical data, to identify new non-genetic vulnerabilities of MPM. We identified a core of 18 functionally-related genes as essential for MPM cells. The chromatin reader KAP1 emerged as a dependency of MPM. We showed that KAP1 supports cell growth by orchestrating the expression of a G2/M-specific program, ensuring mitosis correct execution. Targeting KAP1 transcriptional function, by using CDK9 inhibitors resulted in a dramatic loss of MPM cells viability and shutdown of the KAP1-mediated program. Validation analysis on two independent MPM-patients sets, including a consecutive, retrospective cohort of 97 MPM, confirmed KAP1 as new non-genetic dependency of MPM and proved the association of its dependent gene program with reduced patients' survival probability. Overall these data: provided new insights into the biology of MPM delineating KAP1 and its target genes as building blocks of its clinical aggressiveness.

The histone methyltransferase KMT2C is among the most frequently mutated epigenetic modifier genes in cancer and plays an essential role in MRE11-dependent DNA replication fork restart. However, the effects of KMT2C deficiency on genomic instability during tumorigenesis are unclear. Analyzing 9,663 tumors from 30 cancer cohorts, we report that KMT2C mutant tumors have a significant excess of APOBEC mutational signatures in several cancer types. We show that KMT2C deficiency promotes APOBEC expression and deaminase activity, and compromises DNA replication speed and delays fork restart, facilitating APOBEC mutagenesis targeting single stranded DNA near stalled forks. APOBEC-mediated mutations primarily accumulate during early replication and tend to cluster along the genome and also in 3D nuclear domains. Excessive APOBEC mutational signatures in KMT2C mutant tumors correlate with elevated genome maintenance defects and signatures of homologous recombination deficiency. We propose that KMT2C deficiency is a likely promoter of APOBEC mutagenesis, which fosters further genomic instability during tumor progression in multiple cancer types.

SET domain-containing protein 4 (SETD4) is a member of a unique class of protein lysine methyltransferases. Here, we introduce the basic features of SETD4 and summarize the key findings from recent studies with emphases on its roles in tissue development and tumorigenesis, and its methylation substrates. SETD4 is expressed in stem/progenitor cells. Ablation of Setd4⁺ cells impedes the repopulation of acinar cells after pancreatic injury. Setd4 deletion in mice promotes the recovery of radiation-induced bone marrow (BM) failure by boosting the function of BM niche, facilitates the generation of endothelial cells and neovascularization of capillary vessels in the heart, enhances the proliferation of BM mesenchymal stem cells and disrupts the TLR4 signaling in BM-derived macrophages. SETD4 expression is also associated with the maintenance of quiescent breast cancer stem cells. While mouse Setd4 knockout delays radiation-induced T-lymphoma formation, elevated SETD4 expression has been observed in some proliferative cancer cells and is associated with a pro-survival potential. Oncogenic fusions of SETD4 have also been identified in cancer, albeit rare. In addition, SETD4 methylates lysine-570 in the C-terminal globular domain of KU70, which enables KU70 translocation to cytoplasm to suppress apoptosis.

DNA polymerase theta (POLQ) is a principal component of the alternative non-homologous end-joining (ANHEJ) DNA repair pathway that ligates DNA double-strand breaks. Utilizing independent models of POLQ insufficiency during telomere-driven crisis, we found that POLQ ^{- /-} cells are resistant to crisis-induced growth deceleration despite sustaining inter-chromosomal telomere fusion frequencies equivalent to wild-type (WT) cells. We recorded longer telomeres in POLQ ^{- / -} than WT cells pre- and post-crisis, notwithstanding elevated total telomere erosion and fusion rates. POLQ ^{- /-} cells emerging from crisis exhibited reduced incidence of clonal gross chromosomal abnormalities in accordance with increased genetic heterogeneity. High-throughput sequencing of telomere fusion amplicons from POLQ-deficient cells revealed significantly raised frequencies of inter-chromosomal fusions with correspondingly depreciated intra-chromosomal recombinations. Long-range interactions culminating in telomere fusions with centromere alpha-satellite repeats, as well as expansions in HSAT2 and HSAT3 satellite and contractions in ribosomal DNA repeats, were detected in POLQ ^{- / -} cells. In conjunction with the expanded telomere lengths of POLQ ^{- /-} cells, these results indicate a hitherto unrealized capacity of POLQ for regulation of repeat arrays within the genome. Our findings uncover novel considerations for the efficacy of POLQ inhibitors in clinical cancer interventions, where potential genome destabilizing consequences could drive clonal evolution and resistant disease.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with low overall survival rates and high molecular heterogeneity; therefore, few targeted therapies are available. The luminal androgen receptor (LAR) is the most consistently identified TNBC subtype, but the clinical utility has yet to be established. Here, we constructed a novel genomic classifier, LAR-Sig, that distinguishes the LAR subtype from other TNBC subtypes and provide evidence that it is a clinically distinct disease. A meta-analysis of seven TNBC datasets (n = 1086 samples) from neoadjuvant clinical trials demonstrated that LAR patients have significantly reduced response (pCR) rates than non-LAR TNBC patients (odds ratio = 2.11, 95% CI: 1.33, 2.89). Moreover, deconvolution of the tumor microenvironment confirmed an enrichment of luminal epithelium corresponding with a decrease in basal and myoepithelium in LAR TNBC tumors. Increased immunosuppression in LAR patients may lead to a decreased presence of cycling T-cells and plasma cells. While, an increased presence of myofibroblast-like cancer-associated cells may impede drug delivery and treatment. In summary, the lower levels of tumor infiltrating lymphocytes (TILs), reduced immune activity in the micro-environment, and lower pCR rates after NAC, suggest that new therapeutic strategies for the LAR TNBC subtype need to be developed.

Recently, targeted protein degradation systems have been developed using the ubiquitin-proteasome system. Here, we established Programmed cell death-1 (PD-1) knockdown mice as a model system for subjecting endogenous mouse proteins to the small molecule-assisted shutoff (SMASh) degron system. SMASh degron-tagged PD-1-mCherry in Jurkat cells and CD3⁺ splenocytes were degraded by the NS3/4A protease inhibitors, asunaprevir (ASV) or grazoprevir (GRV). Growth of MC-38 colon adenocarcinoma cells injected in Pdcd1-mCherry-SMASh homozygous knock-in (KI) mice was repressed by ASV or GRV. Moreover, growth of MC-38 cells was suppressed in wild-type mice transplanted with KI bone marrow cells after GRV treatment. This is the first study to use a degron tag targeting an endogenous mouse protein in vivo. Our experimental system using the SMASh degron may be employed for treating diseases and characterizing the cellular functions of essential proteins.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous disease with relatively high morbidity and mortality rates. The lack of effective therapies, high recurrence rates and drug resistance driven in part, by tumor heterogeneity, contribute to the poor prognosis for patients diagnosed with this cancer. This problem is further exacerbated by the fact that key regulatory factors contributing to the disease diversity remains largely elusive. Here, we have identified EHF as an important member of the ETS family of transcription factors that is highly expressed in normal oral tissues, but lost during HNSCC progression. Interestingly, HNSCC tumors and cell lines exhibited a dichotomy of high and low EHF expression, and patients whose tumors retained EHF expression showed significantly better prognosis, suggesting a potential tumor suppressive role for EHF. To address this, we have performed gain and loss of function studies and leveraged bulk and single-cell cancer genomic datasets to identify global EHF targets by RNA-sequencing (RNA-seq) and Chromatin Immunoprecipitation and next generation sequencing (ChIP-seq) experiments of HNSCC cell lines. These mechanistic studies have revealed that EHF, acts as a regulator of a broad spectrum of metabolic processes, specifically targeting regulators of redox homeostasis such as NRF2 and SOX2. Our immunostaining results confirm the mutually exclusive expression patterns of EHF and SOX2 in HNSCC tumors and suggest a possible role for these two factors in establishing discrete metabolic states within the tumor microenvironment. Taken together, EHF may serve as a novel prognostic marker for classifying HNSCC patients for actionable and targeted therapeutic intervention.

Sequencing of cell-free DNA (cfDNA) in cancer patients' plasma offers a minimally-invasive solution to detect tumor cell genomic alterations to aid real-time clinical decision-making. The reliability of copy number detection decreases at lower cfDNA tumor fractions, limiting utility at earlier stages of the disease. To test a novel strategy for detection of allelic imbalance, we developed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation. First, we assessed it on plasma samples from 50 advanced prostate cancer patients. We then confirmed improved detection of genomic alterations in samples with <10% tumor fractions when compared against an independent assay. Finally, we applied PCF_SELECT to serial plasma samples intensively collected from three patients previously characterized as harboring alterations involving DNA repair genes and consequently offered PARP inhibition. We identified more extensive pan-genome allelic imbalance than previously recognized in prostate cancer. We confirmed high sensitivity detection of BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. Overall, we present a framework for sensitive detection of allele-specific copy number changes in cfDNA.