scholarly journals Depletion of NK6 Homeobox 3 (NKX6.3) causes gastric carcinogenesis through copy number alterations by inducing impairment of DNA replication and repair regulation

Oncogenesis ◽  
2021 ◽  
Vol 10 (12) ◽  
Author(s):  
Jung Hwan Yoon ◽  
Jung Woo Eun ◽  
Hassan Ashktorab ◽  
Duane T. Smoot ◽  
Jeong kyu Kim ◽  
...  
Keyword(s):  
Dna Replication ◽  
Epithelial Cells ◽  
Genomic Instability ◽  
Copy Number ◽  
Tumor Development ◽  
Genomic Stability ◽  
Gastric Epithelial Cells ◽  
Copy Number Alterations ◽  
Dna Replication And Repair ◽  
Underlying Causes

AbstractGenomic stability maintenance requires correct DNA replication, chromosome segregation, and DNA repair, while defects of these processes result in tumor development or cell death. Although abnormalities in DNA replication and repair regulation are proposed as underlying causes for genomic instability, the detailed mechanism remains unclear. Here, we investigated whether NKX6.3 plays a role in the maintenance of genomic stability in gastric epithelial cells. NKX6.3 functioned as a transcription factor for CDT1 and RPA1, and its depletion increased replication fork rate, and fork asymmetry. Notably, we showed that abnormal DNA replication by the depletion of NKX6.3 caused DNA damage and induced homologous recombination inhibition. Depletion of NKX6.3 also caused copy number alterations of various genes in the vast chromosomal region. Hence, our findings underscore NKX6.3 might be a crucial factor of DNA replication and repair regulation from genomic instability in gastric epithelial cells.

scholarly journals The Human Papillomavirus Type 16 E7 Oncogene Is Required for the Productive Stage of the Viral Life Cycle

2000 ◽  
Vol 74 (14) ◽  
pp. 6622-6631 ◽  
Author(s):  
Elsa R. Flores ◽  
B. Lynn Allen-Hoffmann ◽  
Denis Lee ◽  
Paul F. Lambert
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Dna Replication ◽  
Epithelial Cells ◽  
Dna Synthesis ◽  
Copy Number ◽  
Keratinocyte Differentiation ◽  
Replication Machinery ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16

ABSTRACT The production of the human papillomavirus type 16 (HPV-16) is intimately tied to the differentiation of the host epithelium that it infects. Infection occurs in the basal layer of the epithelium at a site of wounding, where the virus utilizes the host DNA replication machinery to establish itself as a low-copy-number episome. The productive stage of the HPV-16 life cycle occurs in the postmitotic suprabasal layers of the epithelium, where the virus amplifies its DNA to high copy number, synthesizes the capsid proteins (L1 and L2), encapsidates the HPV-16 genome, and releases virion particles as the upper layer of the epithelium is shed. Papillomaviruses are hypothesized to possess a mechanism to overcome the block in DNA synthesis that occurs in the differentiated epithelial cells, and the HPV-16 E7 oncoprotein has been suggested to play a role in this process. To determine whether E7 plays a role in the HPV-16 life cycle, an E7-deficient HPV-16 genome was created by inserting a translational termination linker (TTL) in the E7 gene of the full HPV-16 genome. This DNA was transfected into an immortalized human foreskin keratinocyte cell line shown previously to support the HPV-16 life cycle, and stable cell lines were obtained that harbored the E7-deficient HPV-16 genome episomally, the state of the genome found in normal infections. By culturing these cells under conditions which promote the differentiation of epithelial cells, we found E7 to be necessary for the productive stage of the HPV-16 life cycle. HPV-16 lacking E7 failed to amplify its DNA and expressed reduced amounts of the capsid protein L1, which is required for virus production. E7 appears to create a favorable environment for HPV-16 DNA synthesis by perturbing the keratinocyte differentiation program and inducing the host DNA replication machinery. These data demonstrate that E7 plays an essential role in the papillomavirus life cycle.

Distinct genomic instability landscape of lung adenocarcinoma associated with treatment and metastasis.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 9534-9534
Author(s):  
Chuanxin Wu ◽  
Jing Zhang ◽  
Hua Bao ◽  
Ao Wang ◽  
Zhuang Luo ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Genomic Instability ◽  
Copy Number ◽  
Targeted Sequencing ◽  
Early Event ◽  
Copy Number Alterations ◽  
Term Survival ◽  
Tissue Samples ◽  
Primary Tumors ◽  
Increased Risk

9534 Background: Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer (NSCLC). Genomic instability, defined as genome-wide copy number alterations, is a key pathogenic signature which occurs at the early stage of most cancers and is associated with an increased risk of recurrence or death. We examined the pattern of genomic instability in primary and metastatic LUAD. Methods: We performed deep targeted sequencing (425 genes) of 3395 tissue samples and whole exome sequencing (WES) of 60 tissue samples from LUAD patients. Whole-genome doubling (WGD) and arm level aneuploidy were analyzed to uncover correlation with clinical phenotypes and other genomic alterations including driver mutations, tumor mutation burden (TMB), and microsatellite instability (MSI). Results: Overall, targeted sequencing revealed that WGD occurred in 64.33% LUAD samples, which was comparable with WES results. Compared to primary site, metastasis exhibited higher proportion of WGD (1.14 fold). Specifically, liver metastasis has the highest WGD percentage among metastasis sites (~87.5%; 1.40 fold increase compared to primary). Interestingly, patients who received tyrosine kinase inhibitors (TKI) had higher frequency of WGD than patients without TKI treatment. In addition, TMB was higher in WGD+ patients but MSI status was not significantly different between groups. Arm-level aneuploidy was prevalent in this cohort. The most common amplification events were 7p gain (62%), 5p gain (54%), and 8q gain (53%); top deletion events were 19p loss (47%), 15q loss (42%), and 10 q loss (41%). Patients with EGFR or TP53 mutation were more likely to have aneuploidy compared to wildtypes. Subgroup analysis showed distinct patterns of aneuploidy among metastasis sites, suggesting organ-specific alterations. Evolution analysis showed 7p gain was an early event common in primary tumor whereas metastatic tumor had multiple distinct evolutionary trajectories following 7p gain. Several copy number signatures were associated with specific TKI and chemotherapies. For example, TKI-naïve tumors lacked 7p gain but had 19p loss as the most common alteration. Conclusions: The genomic landscape of LUAD was characterized by widespread large-scale copy number alterations including WGD and chromosomal aneuploidy. Metastasis had elevated level of aneuploidy compared to primary tumors which were specific to metastatic site. Copy number signature associated with different treatments may contribute to distinct long-term survival and side effects among patients.

scholarly journals Profiling diverse sequence tandem repeats in colorectal cancer reveals co-occurrence of microsatellite and chromosomal instability involving Chromosome 8

2020 ◽  
Author(s):  
GiWon Shin ◽  
Stephanie U. Greer ◽  
Erik Hopmans ◽  
Susan M. Grimes ◽  
HoJoon Lee ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Genomic Instability ◽  
Copy Number ◽  
Chromosomal Instability ◽  
Tandem Repeats ◽  
Chromosome 8 ◽  
Molecular Testing ◽  
Cancer Genes ◽  
Copy Number Alterations ◽  
High Coverage

ABSTRACTColorectal carcinomas (CRCs) which have lost DNA mismatch repair display hypermutability evident in a molecular phenotype called microsatellite instability (MSI). These mismatch repair deficient tumors are thought to lack widespread genomic instability features, such as copy number changes and rearrangements. To identify MSI for clinical diagnosis, current molecular testing looks for changes in mononucleotide or dinucleotide repeats. However, microsatellites have other types of sequence tandem repeats such as tri- and tetranucleotide motifs. These additional classes of microsatellites are generally not examined for MSI but are known to be unstable in a phenotype known as elevated microsatellite alterations at selected tetranucleotide repeats, or EMAST. We developed a sequencing approach that provides ultra-high coverage (>2500X) of microsatellite targets and cancer genes for profiling genomic instability. We assessed the diverse repeat motifs across 200 microsatellites. Our approach provides highly sensitive detection of MSI with high specificity, evaluates copy number alterations with high accuracy, delineates chromosomal instability (CIN) classification and deconvolutes subclonal architecture. By examining both MSI and CIN, we discovered mutations and copy number alterations that defined mixed genomic instability states of CIN and MSI, which are normally considered exclusive. An increase in copy number of chromosome arm 8q was prevalent among MSI tumors. Moreover, we identified an inter-chromosomal translocation event from a CRC with co-occurrence of MSI. Subclonal analysis demonstrated that mutations which are typically considered to be exclusive in MSI, shows mutual occurrence in MSI tumors with more sensitive characterization. Our approach revealed that MSH3 mutations are a potential source of mixed genomic instability features. Overall, our study demonstrates that some colorectal cancers have features of both microsatellite and chromosomal instability. This result may have implications for immunotherapy treatment.

Genomic Stability: A Novel Function of NF-Kb in Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3240-3240
Author(s):  
Sampath Ramachandiran ◽  
Arsene Adone ◽  
Xiangxue Guo ◽  
Albert Liao ◽  
Uston Robert Sunay ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genomic Instability ◽  
Cell Lines ◽  
Nuclear Localization ◽  
Time Course ◽  
Tumor Development ◽  
Genomic Stability ◽  
Centrosome Duplication ◽  
Cyclin G

Abstract Abstract 3240 Poster Board III-177 Lymphomas are heterogeneous diseases comprising multiple clinical and biological subgroups. Several studies have shown that genomic instability and constitutive activation of the NF-kB pathway are key features for lymphoma development (Shen M,Hematologica 2007). However, it remains unclear whether changes in activation of the canonical and non-canonical NF-kB pathways (rel-A/p50 and rel-B/p52, respectively) reflect a response to genomic instability and therefore promotes lymphomagenesis. To answer this question, we first demonstrated in two lymphoma cell lines (Daudi and OCI-Ly3) that DNA damage induced by Doxorubicin (2 mcg/mL) resulted in nuclear localization of rel-A and Rel-B. Then, to determine the role of each NF-kB pathway in DNA repair and centrosome duplication we compared the number of cells positive for phospo-H2aX (pH2aX) and centrosomes numbers (measured by gamma-tubulin) in p105-siRNA (canonical) and P100-siRNA (non-canonical) with luciferase-siRNA (control) expressing cells. Our results showed that the expression of p105 and p100 siRNAs increase the number of pH2aX (+) cells compared to control. Subsequently, a time course measuring pH2aX (+) cells was performed after treating p105, P100 and luciferase siRNA OCI-ly3 expressing cell lines with Doxorubicin (2 mcg/mL). In cells expressing luciferase siRNA, pH2aX (+) cells peak (60%) at 60 minutes (min) and return to normal at 120 min, in p105 siRNA –cells pH2aX peak at 90 min (90% + cells) and then decrease similarly to luciferase siRNA cells. P100 siRNA cells demonstrate a continuous increased in pH2aX (+) cells up to 80%. In addition, p100 siRNA expression was associated with centrosome amplification (>2 centrosomes in 20-30% of the cells vs. < 8% in p105 or Luciferase siRNA expressing cells). Also, the expression of NF-kB siRNAs delayed doxorubicin-induced phosphorylation of p53 (serine 15 – target of ATM) and CHK2. To evaluate whether the genomic instability caused by both NF-kB siRNAs affects tumor development, we performed xenograft experiments. Our results demonstrated that NF-kB siRNAs not only slow down tumor initiation but prevented tumor development (p105 siRNA= 8 days delayed and 22% were tumor free and p100 siRNA= 13.5 days delayed and 55% were tumor free compared to luciferase siRNA). To investigate these findings in primary tissues we measured the number of phospo-H2aX (+) cells and the levels of rel-A and rel-B nuclear localization in 40 primary lymphoma tumor samples. Our results demonstrated that phospo-H2aX levels inversely correlated with rel-B nuclear localization (r=-0.58, p<0.0001). To identify possible explanations for these results, gene expression analysis was performed in cells expressing NF-kB siRNAs. Our results demonstrated that p105 siRNA regulated genes involved in DNA repair (PPP2R5C, ING5, SYF2, SYF2, XRCC6, etc) and p100 siRNA regulated genes involved in both DNA repair and centrosome duplication (GADD45 alpha, cyclin G, REDD1, PCBP4, etc) consistent with our results above. Quantitative PCR for some these genes during a doxorubicin-time course confirmed GADD45 alpha, cyclin G, PCBP4 and SFRS6 to be induced. We explored further the role of GADD45 alpha in lymphomas and found that knock down of this protein increase doxorubicin sensitivity by 50-fold. Overall this study demonstrated that activation of each NF-kB pathway is essential for maintaining genomic stability and therefore promoting tumor resistance to chemotherapy in lymphomas. In addition, we identified that GADD45 alpha is important target of the non-canonical NF-kB pathway for mediating genomic stability. These findings provide the rationale for designing novel agents aiming at targeting key genes involved in genomic stability. Disclosures No relevant conflicts of interest to declare.

The use of whole genome copy number variation (CNV) to measure genomic instability in mCRPC CTCs.

2016 ◽  
Vol 34 (2_suppl) ◽  
pp. 307-307
Author(s):  
Yipeng Wang ◽  
Stephanie Greene ◽  
Angel Rodriguez ◽  
Jerry Lee ◽  
Mark Landers ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genomic Instability ◽  
Copy Number ◽  
Parp Inhibitors ◽  
Whole Genome ◽  
Copy Number Alterations ◽  
Genome Copy ◽  
Pten Loss ◽  
Dna Repair Gene ◽  
Genome Copy Number

307 Background: AR targeted therapies in combination with PARP inhibitors have recently shown efficacy in mCRPC patients with specific DNA repair gene mutations in metastatic tissue biopsies. Homologous recombination DNA repair deficiencies (HRD), associated with response to PARP inhibitors, can also be assessed by genomic instability/scarring. Accurate genomic scarring measurement in tumors can be confounded by intra-tumor heterogeneity and/or non-tumor genome contamination. To better identify PARPi sensitivity in mCRPC patients, we developed a genomic instability and scarring assay starting from single CTCs. Methods: VCaP, LnCaP or PC3 cell lines were spiked into healthy donor blood. Individual spiked cells were identified and recovered for genomic analysis using the standard Epic CTC assay. Post recovery, cells were lysed, whole genome amplified, constructed into shotgun libraries and sequenced to ~2M 2x150bp PE reads. Following alignment, whole genome copy number and instability analysis was performed to identify large scale transitions (LST, n of chromosomal breaks between adjacent regions of at least 10 Mb), % of genome altered (percentage of 1Mbp bins with copy number alterations), as well as specific tumor suppressor/oncogene copy number alterations. The association of PTEN loss with increases in genomic instability and scaring was performed. Results: Loss of PTEN function was previously shown to be associated with genomic instability. Our assessment of PTEN deletion was confirmed in PC3, while at least one genomic copy was observed in LnCaP and VCaP. The number of LSTs and % of genome altered was higher in PC3 (n = 19 +/- 3; 9.3% +/- 2.6%) than both VCaP and LnCap (n = 8 +/- 2; 6.1% +/- 0.4%). Conclusions: The association of higher genomic instability in a PTEN null cell line (PC3) vs. those with either heterozygous (LnCaP) or wild type (VCaP) PTEN status, matches published reports associating PTEN loss with increased genomic instability. Although, PC3 only demonstrates mild PARPi sensitivity in vitro, the detection of increased genomic scarring vs. cells with at least 1 functional PTEN allele confirms the assays ability to quantify genome instability at the single cell level from CTCs in a liquid biopsy.

scholarly journals DNA Replication Stress Generates Distinctive Landscapes of DNA Copy Number Alterations and Chromosome Scale Losses

2019 ◽  
Author(s):  
Alice Mazzagatti ◽  
Nadeem Shaikh ◽  
Bjorn Bakker ◽  
Diana Carolina Johanna Spierings ◽  
René Wardenaar ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Cell ◽  
Copy Number ◽  
Chromosomal Instability ◽  
Replication Stress ◽  
Fragile Sites ◽  
Copy Number Alterations ◽  
Dna Copy Number ◽  
Dna Copy Number Alterations ◽  
The Impact

AbstractBackgroundWe previously showed that a major driver of cancer chromosomal instability (CIN) is replication stress, the slowing or stalling of DNA replication. However, the precise drivers of replication stress in cancer and the mechanisms by which these cause CIN and influence tumour evolution remain unclear. Common fragile sites are well-known genomic locations of breakage after aphidicolin-induced replication stress, but their precise causes of fragility are debated, and additional genomic consequences of replication stress are not fully explored.ResultsUsing single cell sequencing we detected DNA copy number alterations (CNAs) caused by one cell cycle under replication stress in diploid non-transformed cells. Aphidicolin-induced replication stress caused multiple types of CNAs associated with different genomic regions and features. Coupling cell type-specific analysis of CNAs to gene expression and single cell replication timing analyses allowed us to pinpoint the causative large genes of the most recurrent chromosome-scale CNAs. In RPE1 cells these were largely confined to three sites on chromosomes 1, 2 and 7 and generated acentric lagging chromatin and micronuclei containing these chromosomes. Different replicative stresses generated distinct profiles of CNAs providing the potential to interpret specific replication stress mechanisms from cancer cells.ConclusionsChromosomal instability driven by replication stress occurs via focal CNAs and chromosome arm-scale changes, with the latter confined to a very small subset of chromosome regions, potentially heavily skewing cancer genome evolution trajectories. Single cell CNA analysis thus reveals new insights into the impact of replication stress on the genome and provides a platform to further dissect molecular mechanisms involved in the replication stress response and to gain insights into how replication stress fuels chromosomal instability in cancer.

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