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Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

Somatic Y chromosome loss in hematopoietic cells is associated with higher mortality in men. However, the status of the Y chromosome in cancer tissue is not fully known due to technical limitations, such as difficulties in labelling and sequencing DNA from the Y chromosome. We have developed a system to quantify Y chromosome gain or loss in patient-derived prostate cancer organoids. Using our system, we observed Y chromosome loss in 4 of the 13 (31%) patient-derived metastatic castration-resistant prostate cancer (mCRPC) organoids; interestingly, loss of Yq (long arm of the Y chromosome) was seen in 38% of patient-derived organoids. Additionally, potential associations were observed between mCRPC and Y chromosome nullisomy. The prevalence of Y chromosome loss was similar in primary and metastatic tissue, suggesting that Y chromosome loss is an early event in prostate cancer evolution and may not a result of drug resistance or organoid derivation. This study reports quantification of Y chromosome loss and gain in primary and metastatic prostate cancer tissue and lays the groundwork for further studies investigating the clinical relevance of Y chromosome loss or gain in mCRPC. [ABSTRACT FROM AUTHOR]

Epigenetic modifications control cancer development and clonal evolution in various cancer types. Here, we show that loss of the male-specific histone demethylase lysine-specific demethylase 5D (KDM5D) encoded on the Y chromosome epigenetically modifies histone methylation marks and alters gene expression, resulting in aggressive prostate cancer. Fluorescent in situ hybridization demonstrated that segmental or total deletion of the Y chromosome in prostate cancer cells is one of the causes of decreased KDM5D mRNA expression. The result of ChIP-sequencing analysis revealed that KDM5D preferably binds to promoter regions with coenrichment of the motifs of crucial transcription factors that regulate the cell cycle. Loss of KDM5D expression with dysregulated H3K4me3 transcriptional marks was associated with acceleration of the cell cycle and mitotic entry, leading to increased DNA-replication stress. Analysis of multiple clinical data sets reproducibly showed that loss of expression of KDM5D confers a poorer prognosis. Notably, we also found stress-induced DNA damage on the serine/threonine protein kinase ATR with loss of KDM5D. In KDM5D-deficient cells, blocking ATR activity with an ATR inhibitor enhanced DNA damage, which led to subsequent apoptosis. These data start to elucidate the biological characteristics resulting from loss of KDM5D and also provide clues for a potential novel therapeutic approach for this subset of aggressive prostate cancer., Introduction Prostate cancer (PC) is the most common cancer among men in the United States and one of the leading causes of cancer death, with approximately 27,000 deaths per year [...]

A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intratumor genetic heterogeneity poses a substantial challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Because of the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh or rapidly frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ show intratumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes., Author(s): Luciano G Martelotto [1]; Timour Baslan [2, 3]; Jude Kendall [2]; Felipe C Geyer [1]; Kathleen A Burke [1]; Lee Spraggon [1]; Salvatore Piscuoglio [1]; Kalyani Chadalavada [4]; Gouri [...]

Glioblastoma (GBM) is among the most aggressive of human cancers (1). A key feature of GBMs is the extensive network of abnormal vasculature characterized by glomeruloid structures and endothelial hyperplasia (2). Yet the mechanisms of angiogenesis and the origin of tumour endothelial cells remain poorly defined (3-5). Here we demonstrate that a subpopulation of endothelial cells within glioblastomas harbour the same somatic mutations identified within tumour cells, such as amplification of EGFR and chromosome 7. We additionally demonstrate that the stem-cell-like [CD133.sup.+] fraction includes a subset of vascular endothelial-cadherin (CD144)-expressing cells that show characteristics of endothelial progenitors capable of maturation into endothelial cells. Extensive in vitro and in vivo lineage analyses, including single cell clonal studies, further show that a subpopulation of the [CD133.sup.+] stem-like cell fraction is multipotent and capable of differentiation along tumour and endothelial lineages, possibly via an intermediate [CD133.sup.+]/[CD144.sup.+] progenitor cell. The findings are supported by genetic studies of specific exons selected from The Cancer Genome Atlas (6), quantitative FISH and comparative genomic hybridization data that demonstrate identical genomic profiles in the [CD133.sup.+] tumour cells, their endothelial progenitor derivatives and mature endothelium. Exposure to the clinical anti-angiogenesis agent bevacizumab (7) or to a γ-secretase inhibitor (8) as well as knockdown shRNA studies demonstrate that blocking VEGF or silencing VEGFR2 inhibits the maturation of tumour endothelial progenitors into endothelium but not the differentiation of [CD133.sup.+] cells into endothelial progenitors, whereas γ-secretase inhibition or NOTCH1 silencing blocks the transition into endothelial progenitors. These data may provide new perspectives on the mechanisms of failure of anti-angiogenesis inhibitors currently in use. The lineage plasticity and capacity to generate tumour vasculature of the putative cancer stem cells within glioblastoma are novel findings that provide new insight into the biology of gliomas and the definition of cancer sternness, as well as the mechanisms of tumour neo-angiogenesis., Blood vessels within GBM express a variety of markers, including CD31 and CD105 (also known as PECAM1 and ENG, respectively); CD105 is a proliferation-associated molecule expressed in angiogenic endothelium (9). [...]

Myoepithelial carcinoma (MECA) is an aggressive salivary gland cancer with largely unknown genetic features. Here we comprehensively analyze molecular alterations in 40 MECAs using integrated genomic analyses. We identify a low mutational load, and high prevalence (70%) of oncogenic gene fusions. Most fusions involve the PLAG1 oncogene, which is associated with PLAG1 overexpression. We find FGFR1-PLAG1 in seven (18%) cases, and the novel TGFBR3-PLAG1 fusion in six (15%) cases. TGFBR3-PLAG1 promotes a tumorigenic phenotype in vitro, and is absent in 723 other salivary gland tumors. Other novel PLAG1 fusions include ND4-PLAG1; a fusion between mitochondrial and nuclear DNA. We also identify higher number of copy number alterations as a risk factor for recurrence, independent of tumor stage at diagnosis. Our findings indicate that MECA is a fusion-driven disease, nominate TGFBR3-PLAG1 as a hallmark of MECA, and provide a framework for future diagnostic and therapeutic research in this lethal cancer. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is among the most aggressive of human cancers. A key feature of GBMs is the extensive network of abnormal vasculature characterized by glomeruloid structures and endothelial hyperplasia. Yet the mechanisms of angiogenesis and the origin of tumour endothelial cells remain poorly defined. Here we demonstrate that a subpopulation of endothelial cells within glioblastomas harbour the same somatic mutations identified within tumour cells, such as amplification of EGFR and chromosome 7. We additionally demonstrate that the stem-cell-like CD133+ fraction includes a subset of vascular endothelial-cadherin (CD144)-expressing cells that show characteristics of endothelial progenitors capable of maturation into endothelial cells. Extensive in vitro and in vivo lineage analyses, including single cell clonal studies, further show that a subpopulation of the CD133+ stem-like cell fraction is multipotent and capable of differentiation along tumour and endothelial lineages, possibly via an intermediate CD133+/CD144+ progenitor cell. The findings are supported by genetic studies of specific exons selected from The Cancer Genome Atlas, quantitative FISH and comparative genomic hybridization data that demonstrate identical genomic profiles in the CD133+ tumour cells, their endothelial progenitor derivatives and mature endothelium. Exposure to the clinical anti-angiogenesis agent bevacizumab or to a ?-secretase inhibitor as well as knockdown shRNA studies demonstrate that blocking VEGF or silencing VEGFR2 inhibits the maturation of tumour endothelial progenitors into endothelium but not the differentiation of CD133+ cells into endothelial progenitors, whereas ?-secretase inhibition or NOTCH1 silencing blocks the transition into endothelial progenitors. These data may provide new perspectives on the mechanisms of failure of anti-angiogenesis inhibitors currently in use. The lineage plasticity and capacity to generate tumour vasculature of the putative cancer stem cells within glioblastoma are novel findings that provide new insight into the biology of gliomas and the definition of cancer stemness, as well as the mechanisms of tumour neo-angiogenesis. [ABSTRACT FROM AUTHOR]

We have identified new genomic alterations in the breast cancer cell line HCC1954, using high-throughput transcriptome sequencing. With 120 Mb of cDNA sequences, we were able to identify genomic rearrangement events leading to fusions or truncations of genes including MRE11 and NSD1, genes already implicated in oncogenesis, and 7 rearrangements involving other additional genes. This approach demonstrates that high-throughput transcriptome sequencing is an effective strategy for the characterization of genomic rearrangements in cancers. [ABSTRACT FROM AUTHOR]

Summary The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a "normal" human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor microtubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening. Graphical Abstract Highlights • Glioma stem cells home toward, invade, and proliferate in human cerebral organoids • GLICO (cerebral organoid glioma) tumors phenocopy patient glioblastoma • Tumor microtubes promote the invasion of GLICO tumors into normal host tissue • The GLICO model is a tool to study GBM biology in a human brain environment To address limitations with current preclinical glioblastoma (GBM) models, Linkous et al. establish a "GLICO" (cerebral organoid glioma) model to retro-engineer patient-specific GBMs using patient-derived glioma stem cells and human cerebral organoids. Resulting tumors closely phenocopy patient GBMs and are supported by tumor microtubes that promote invasion into host tissue. [ABSTRACT FROM AUTHOR]

Epigenetic modifications control cancer development and clonal evolution in various cancer types. Here, we show that loss of the male-specific histone demethylase lysine-specific demethylase 5D (KDM5D) encoded on the Y chromosome epigenetically modifies histone methylation marks and alters gene expression, resulting in aggressive prostate cancer. Fluorescent in situ hybridization demonstrated that segmental or total deletion of the Y chromosome in prostate cancer cells is one of the causes of decreased KDM5D mRNA expression. The result of ChIP-sequencing analysis revealed that KDM5D preferably binds to promoter regions with coenrichment of the motifs of crucial transcription factors that regulate the cell cycle. Loss of KDM5D expression with dysregulated H3K4me3 transcriptional marks was associated with acceleration of the cell cycle and mitotic entry, leading to increased DNA-replication stress. Analysis of multiple clinical data sets reproducibly showed that loss of expression of KDM5D confers a poorer prognosis. Notably, we also found stress-induced DNA damage on the serine/threonine protein kinase ATR with loss of KDM5D. In KDM5D-deficient cells, blocking ATR activity with an ATR inhibitor enhanced DNA damage, which led to subsequent apoptosis. These data start to elucidate the biological characteristics resulting from loss of KDM5D and also provide clues for a potential novel therapeutic approach for this subset of aggressive prostate cancer. [ABSTRACT FROM AUTHOR]

Germline mutations in the BRCA genes are associated with a higher risk of carcinogenesis, which is linked to an increased mutation rate and loss of the second unaffected BRCA allele (loss of heterozygosity, LOH). However, the mechanisms triggering mutagenesis are not clearly understood. The BRCA genes contain high numbers of repetitive DNA sequences. We detected replication forks stalling, DNA breaks, and deletions at these sites in haploinsufficient BRCA cells, thus identifying the BRCA genes as fragile sites. Next, we found that stalled forks are repaired by error-prone pathways, such as microhomology-mediated break-induced replication (MMBIR) in haploinsufficient BRCA1 breast epithelial cells. We detected MMBIR mutations in BRCA1 tumor cells and noticed deletions-insertions (>50 bp) at the BRCA1 genes in BRCA1 patients. Altogether, these results suggest that under stress, error-prone repair of stalled forks is upregulated and induces mutations, including complex genomic rearrangements at the BRCA genes (LOH), in haploinsufficient BRCA1 cells. [Display omitted] • Replication forks stall at BRCA1 and 2 genes in haploinsufficient BRCAmut/+ cells • BRCA genes are early-replicating fragile sites that break in BRCAmut/+ cells • Stalled forks are repaired by MMBIR, an error-prone non-HR pathway in BRCA1mut/+ cells • Increase in error-prone repair leaves mutations and explains LOH in BRCA1 patients Deshpande et al. discovered an increase in replication fork stalling and deletions at the BRCA1 and 2 genes in stressed haploinsufficient BRCAmut/+ cells. The stalled forks are repaired increasingly by error-prone DNA repair pathways, such as MMBIR, which leave mutations, thus elevating mutagenesis and LOH in BRCA1 patients. [ABSTRACT FROM AUTHOR]

Purpose: Targeted therapies have improved outcomes for patients with metastatic colorectal cancer, but their impact is limited by rapid emergence of resistance. We hypothesized that an understanding of the underlying genetic mechanisms and intrinsic tumor features that mediate resistance to therapy will guide new therapeutic strategies and ultimately allow the prevention of resistance., Experimental Design: We assembled a series of 52 patients with paired pretreatment and progression samples who received therapy targeting EGFR (n = 17), BRAF V600E (n = 17), KRAS G12C (n = 15), or amplified HER2 (n = 3) to identify molecular and clinical factors associated with time on treatment (TOT)., Results: All patients stopped treatment for progression and TOT did not vary by oncogenic driver (P = 0.5). Baseline disease burden (≥3 vs. <3 sites, P = 0.02), the presence of hepatic metastases (P = 0.02), and gene amplification on baseline tissue (P = 0.03) were each associated with shorter TOT. We found evidence of chromosomal instability (CIN) at progression in patients with baseline MAPK pathway amplifications and those with acquired gene amplifications. At resistance, copy-number changes (P = 0.008) and high number (≥5) of acquired alterations (P = 0.04) were associated with shorter TOT. Patients with hepatic metastases demonstrated both higher number of emergent alterations at resistance and enrichment of mutations involving receptor tyrosine kinases., Conclusions: Our genomic analysis suggests that high baseline CIN or effective induction of enhanced mutagenesis on targeted therapy underlies rapid progression. Longer response appears to result from a progressive acquisition of genomic or chromosomal instability in the underlying cancer or from the chance event of a new resistance alteration., (©2024 The Authors; Published by the American Association for Cancer Research.)

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance., Significance: Clinical resistance to KRASG12C-EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches. This article is highlighted in the In This Issue feature, p. 1., (©2022 The Authors; Published by the American Association for Cancer Research.)

Copy number alterations (CNAs) play an important role in molding the genomes of breast cancers and have been shown to be clinically useful for prognostic and therapeutic purposes. However, our knowledge of intra-tumoral genetic heterogeneity of this important class of somatic alterations is limited. Here, using single-cell sequencing, we comprehensively map out the facets of copy number alteration heterogeneity in a cohort of breast cancer tumors. Ou/var/www/html/elife/12-05-2020/backup/r analyses reveal: genetic heterogeneity of non-tumor cells (i.e. stroma) within the tumor mass; the extent to which copy number heterogeneity impacts breast cancer genomes and the importance of both the genomic location and dosage of sub-clonal events; the pervasive nature of genetic heterogeneity of chromosomal amplifications; and the association of copy number heterogeneity with clinical and biological parameters such as polyploidy and estrogen receptor negative status. Our data highlight the power of single-cell genomics in dissecting, in its many forms, intra-tumoral genetic heterogeneity of CNAs, the magnitude with which CNA heterogeneity affects the genomes of breast cancers, and the potential importance of CNA heterogeneity in phenomena such as therapeutic resistance and disease relapse., Competing Interests: TB, JK, KV, KM, HC, SD, FA, MR, LR, AL, JS, YM, RS, RG, KC, GN, VV, CC, AK, LH, MW, JH No competing interests declared, JW, ND is an employee of Philips Research North America. AG is affiliated with House Gordon Software Company LTD. The author has no other competing interests to declare.

Purpose: Previous sequencing studies revealed that alterations of genes associated with DNA damage response (DDR) are enriched in men with metastatic castration-resistant prostate cancer (mCRPC). BRCA2 , a DDR and cancer susceptibility gene, is frequently deleted (homozygous and heterozygous) in men with aggressive prostate cancer. Here we show that patients with prostate cancer who have lost a copy of BRCA2 frequently lose a copy of tumor suppressor gene RB1 ; importantly, for the first time, we demonstrate that co-loss of both genes in early prostate cancer is sufficient to induce a distinct biology that is likely associated with worse prognosis., Experimental Design: We prospectively investigated underlying molecular mechanisms and genomic consequences of co-loss of BRCA2 and RB1 in prostate cancer. We used CRISPR-Cas9 and RNAi-based methods to eliminate these two genes in prostate cancer cell lines and subjected them to in vitro studies and transcriptomic analyses. We developed a 3-color FISH assay to detect genomic deletions of BRCA2 and RB1 in prostate cancer cells and patient-derived mCRPC organoids., Results: In human prostate cancer cell lines (LNCaP and LAPC4), loss of BRCA2 leads to the castration-resistant phenotype. Co-loss of BRCA2 - RB1 in human prostate cancer cells induces an epithelial-to-mesenchymal transition, which is associated with invasiveness and a more aggressive disease phenotype. Importantly, PARP inhibitors attenuate cell growth in human mCRPC-derived organoids and human CRPC cells harboring single-copy loss of both genes., Conclusions: Our findings suggest that early identification of this aggressive form of prostate cancer offers potential for improved outcomes with early introduction of PARP inhibitor-based therapy. See related commentary by Mandigo and Knudsen, p. 1784 ., (©2019 American Association for Cancer Research.)

Pancreatic cancer expression profiles largely reflect a classical or basal-like phenotype. The extent to which these profiles vary within a patient is unknown. We integrated evolutionary analysis and expression profiling in multiregion-sampled metastatic pancreatic cancers, finding that squamous features are the histologic correlate of an RNA-seq-defined basal-like subtype. In patients with coexisting basal and squamous and classical and glandular morphology, phylogenetic studies revealed that squamous morphology represented a subclonal population in an otherwise classical and glandular tumor. Cancers with squamous features were significantly more likely to have clonal mutations in chromatin modifiers, intercellular heterogeneity for MYC amplification and entosis. These data provide a unifying paradigm for integrating basal-type expression profiles, squamous histology and somatic mutations in chromatin modifier genes in the context of clonal evolution of pancreatic cancer.

Micropapillary urothelial carcinoma (MPUC) is a rare but an aggressive variant of urothelial carcinoma. MPUC has been shown to commonly exhibit ERBB2 amplification and HER2 protein overexpression, but the frequency and distribution of these findings within micropapillary (MP) and not otherwise specified (NOS) components of tumors with mixed histology have not been addressed. Therefore, we evaluated ERBB2 amplification and HER2 expression in 43 MPUC cases by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Of the 35 tumors containing both MP and NOS components, ERBB2 amplification was present in both the MP and NOS components of 12 tumors (34.3%), in only the MP component of 11 tumors (31.4%), and exclusively in the NOS component of 4 tumors (11.4%). HER2 protein overexpression was significantly more commonly present in the MP component compared to the NOS component within the same tumor (68.6% versus 34.3%, P = .012). Overall, there was a moderately positive correlation between HER2 protein expression and ERBB2 amplification in both MP (ρ = 0.59, P < .001) and NOS (ρ = 0.70, P < .001) components. All MP/NOS areas with IHC score 3+ and none of MP/NOS areas with IHC score 0 were associated with ERBB2 amplification. We conclude that ERBB2 amplification and HER2 overexpression are preferentially but not exclusively identified in the MP component compared to the NOS component within the same tumor. Our findings identify the presence of intratumoral heterogeneity of ERBB2 amplification and HER2 expression in MPUC and provide grounds for further investigation into the mechanisms underlying the development of MPUC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The principles that govern the evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of an amplification in the BRAF oncogene (BRAF amp ) in patient-derived tumor xenografts (PDXs) that were treated with a direct inhibitor of the kinase ERK, either alone or in combination with other ERK signaling inhibitors. Single-cell sequencing and multiplex fluorescence in situ hybridization analyses mapped the emergence of extra-chromosomal amplification in parallel evolutionary trajectories that arose in the same tumor shortly after treatment. The evolutionary selection of BRAF amp was determined by the fitness threshold, the barrier that subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for inhibitors of ERK signaling, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number. Concurrent targeting of the RAF, MEK and ERK kinases, however, imposed a sufficiently high fitness threshold to prevent the propagation of subclones with high-level BRAF amp . When administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11 PDXs of lung cancer or melanoma without apparent toxicity in mice. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and, thus, merit testing in patients.