Induction Of Genomic Instability Research Articles

Abstract 415: Keratin-fusion mutation promotes tumor aggressiveness via genomic instability induction

Abstract Keratin intermediate filament is one critical component of cellular architectures, which provides necessary mechanical support to vanquish environmental stresses. Our previous study shows that keratin fusion mutation promotes tumor aggressiveness via enhancing cancer stemness in oral squamous cell carcinoma. It implies proper functions of keratins play a critical role in maintain cell integrity and fate determination. In the present study, keratin fusion mutation functions as a driver to promote genomic instability via centrosome overduplication and trigger the formation of polyaneuploid cancer cell (PACC). Interestingly, genes involved in DNA damage repair were found upregulated in keratin fusion expressing cells, an important feature for mitotic slippage during cancer progression. In addition, PACCs caused by keratin fusion render cancer cells insensitive to cisplatin treatment, which coupled with lower levels of γH2AX induction and higher survival rates. Transcriptome analyses revealed enhanced activity for Regulation_of_Actin_Cytoskeleton in the PACCs, leading to increased actin filament network and higher invasiveness. In summary, keratin fusion mutation promote cancer aggressiveness through genomic instability-mediated PACC formation and cytoskeleton remodeling, which lead to enhanced drug resistance and metastasis. Citation Format: Hsiang-Hao Chuang, Brian Yu-Ting Kuo, Kalpana Sriramadasu, Yi-Che Chen, Jim Jinn-Chyuan Sheu. Keratin-fusion mutation promotes tumor aggressiveness via genomic instability induction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 415.

Balanced spatiotemporal arrangements of histone H3 and H4 posttranslational modifications are necessary for meiotic prophase I chromosome organization.

Dynamic nuclear architecture and chromatin organizations are the key features of the mid-prophase I in mammalian meiosis. The chromatin undergoes major changes, including meiosis-specific spatiotemporal arrangements and remodeling, the establishment of chromatin loop-axis structure, pairing, and crossing over between homologous chromosomes, any deficiencies in these events may induce genome instability, subsequently leading to failure to produce gametes and infertility. Despite the significance of chromatin structure, little is known about the location of chromatin marks and the necessity of their balance during meiosis prophase I. Here, we show a thorough cytological study of the surface-spread meiotic chromosomes of mouse spermatocytes for H3K9,14,18,23,27,36, H4K12,16 acetylation, and H3K4,9,27,36 methylation. Active acetylation and methylation marks on H3 and H4, such as H3K9ac, H3K14ac, H3K18ac, H3K36ac, H3K56ac, H4K12ac, H4K16ac, and H3K36me3 exhibited pan-nuclear localization away from heterochromatin. In comparison, repressive marks like H3K9me3 and H3K27me3 are localized to heterochromatin. Further, taking advantage of the delivery of small-molecule chemical inhibitors methotrexate(heterochromatin enhancer), heterochromatin inhibitor, anacardic acid (histone acetyltransferase inhibitor), trichostatin A (histone deacetylase inhibitor), IOX1 (JmjC demethylases inhibitor), and AZ505 (methyltransferase inhibitor) in seminiferous tubules through the rete testis route, revealed that alteration in histone modifications enhanced the centromere mislocalization, chromosome breakage, altered meiotic recombination and reduced sperm count. Specifically, IOX1 and AZ505 treatment shows severe meiotic phenotypes, including altering chromosome axis length and chromatin loop size via transcriptional regulation of meiosis-specific genes. Our findings highlight the importance of balanced chromatin modifications in meiotic prophase I chromosome organization and instability.

Abstract A023: High glucose increases DNA damage and elevates the expression of multiple DDR genes

Abstract The DNA Damage Response (DDR) pathways sense DNA damage and coordinate robust DNA repair and bypass mechanisms. A series of repair proteins are recruited depending on the type of breaks and lesions to ensure overall survival. An increase in glucose levels was shown to induce genome instability, yet the links between DDR and glucose are still not well investigated. In this study, we aimed to identify dysregulation in the transcriptome of normal and cancerous breast cell lines upon changing glucose levels. We first performed bioinformatics analysis using a microarray dataset containing the triple-negative breast cancer (TNBC) MDA-MB-231 and the normal human mammary epithelium MCF10A cell lines grown in high glucose (HG) or in the presence of the glycolysis inhibitor 2-deoxyglucose (2DG). Interestingly, multiple DDR genes were significantly upregulated in both cell lines grown in HG. In the wet lab, we remarkably found that HG results in severe DNA damage to TNBC cells as observed using the comet assay. In addition, several DDR genes were confirmed to be upregulated using qPCR analysis in the same cell line. Our results propose a strong need for DDR pathways in the presence of HG to oppose the severe DNA damage induced in cells. Citation Format: Mai A. Rahmoon, Reem A. Elghaish, Aya A. Ibrahim, Zina Alaswad, Mohamed Z. Gad, Sherif F. El-Khamisy, Menattallah Elserafy. High glucose increases DNA damage and elevates the expression of multiple DDR genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A023.

Computational analysis of regulation and expression of plant DNA repair genes under various abiotic stresses reveals a stochastic correlation

Biotic/abiotic stresses are known to induce genome instability in all organisms. Plants being sessile, are more vulnerable to stresses. Thus, they have evolved mechanisms to protect their genome from the unpredictably changing environment. DNA repair genes play a critical role in preserving genome integrity. Plants have about 15 different DNA repair pathways comprising more than 100 genes. To gain a better understanding of how plant DNA repair genes perceive stress and regulate their expression, we performed a computational analysis of the putative binding sites of different abiotic stress-responsive transcription factors (ASRTFs) on their promoters, in the model plant Arabidopsis thaliana. Of the 12 different ASRTFs that we analysed, MYC2 had the highest number of 1876 putative binding sites. Diversity study in 135 DNA repair gene promoters revealed that these had about 6 to 12 types of putative binding sites for the ASRTFs. While RAD51 promoter had the highest number of 207 putative binding sites, RECQ4B had only 11. Comparison of the Arabidopsis eFP browser-based temporal/spatial expression profiles of these genes under nine different abiotic stresses revealed that all of these genes had altered expression under various stresses. Some genes exhibited similar expression profiles. To identify the reasons for their similar expression pattern, we compared the promoter sequences and putative cis-regulatory elements such as binding sites for ASRTFs and methylation-prone nucleotide repeats. Our analysis indicated that the gene expression and gene regulation mediated by ASRTFs, and DNA methylation have a stochastic correlation

NCAPD3 Prompts Diffuse Large B-Cell Lymphoma Progression through Modulating SIRT1 Expression in H3K9 Monomethylation-Dependent Manner

Chromosomal instability, including number and structure abnormality, is the critical molecular basis in lymphoid oncogenesis. Condensin, a family of structural maintenance of chromosome complexes, has been shown to regulate chromosome compaction and segregation during mitosis. NCAPD3, a HEAT-repeat subunit of condensin II, plays a dominant role in condensin-mediated chromosome dynamics, whose abnormalities are related to tumorigenesis and developmental disorders but remain unexplored in lymphoma. This study aims to unravel the expression, molecular function and mechanism of NCAPD3 in diffuse large B-cell lymphoma (DLBCL). We first elucidated the expression of NCAPD3 in DLBCL. Lymph node tissues from 70 DLBCL patients and 35 reactive hyperplasia cases were collected with informed consent approved by the Medical Ethical Committee of Shandong Provincial Hospital. Higher NCAPD3 expression was identified in DLBCL tissues and correlated with advanced Ann Arbor stage and higher IPI score. The survival analysis presented patients with higher NCAPD3 suffered shorter survival (P=0.045). Meanwhile, upregulation of NCAPD3 was validated in DLBCL cells compared with normal CD19+ B-cell. We further explored the biological functions of NCAPD3 in DLBCL and constructed NCAPD3-knockdown, knockout and overexpression models. Deficiency of NCAPD3 impeded cell proliferation, induced apoptosis and increased the sensitivity to doxorubicin and ibrutinib. Instead, NCAPD3 overexpression facilitated cell proliferation and therapeutic resistance. In vivo experiments further indicated that xenograft DLBCL mice with deficient NCAPD3 presented delayed tumor growth. These suggested NCAPD3 promoted DLBCL progression. Strikingly, we found knocking down NCAPD3 disturbed the mitosis, triggering the formation of aneuploidy, due to its function in chromatin structure and dynamics. Hence, we further portrayed the transformation of chromosome architecture under different NCAPD3 expression. Chromosome spreads exhibited chromosomes became slim and elongated upon NCAPD3 overexpression, instead, NCAPD3 knockout triggered short and highly-curled transformation with the global upregulation of H3K4me3, an indication of transcriptional activation and chromatin decompaction (Figure 1). This suggested NCAPD3 could modulate transcriptional activity. Therewith, we investigated how NCAPD3 regulated gene transcription. We examined the interacting proteins of NCAPD3 by mass spectrometry and Clusters of Orthologous Groups analysis revealed they were mainly enriched in RNA processing and modification, chromatin dynamics and cell cycle control. Of them, transcription factor, GTF2I, may be responsible for NCAPD3-mediated transcription regulation. Coimmunoprecipitation (co-IP) verified their interaction and chromatin immunoprecipitation (ChIP) presented they were both located on the promoter of target gene, SIRT1. We further examined SIRT1 expression after regulating NCAPD3 expression, showing a significantly positive correlation. This suggested NCAPD3 could support SIRT1 transcription via being anchored by GTF2I on the promoter of SIRT1. We further elucidated the potential mechanism how NCAPD3 regulated SIRT1 expression. Previous study found that NCAPD3 could interact with histone methylation markers in mammalian genome, thus, we explored whether NCAPD3 could modulate the SIRT1 expression via recognizing the histone methylation marker on SIRT1 promoter. Co-IP assay identified the interaction between NCAPD3 and monomethylation of lysine residue K9 of histone H3 (H3K9me1), an active promoter tag, in DLBCL and ChIP assay verified the location of H3k9me1 on SIRT1 promoter. When NCAPD3 downregulated, H3K9me1 on SIRT promoter diminished, ruining the transcription of SIRT. To clarify whether the oncogenic effects of NCAPD3 was mediated by SIRT1, we conducted functional reversion assays, showing regulating SIRT1 partially reversed the effects of NCAPD3. The above suggested NCAPD3 prompted DLBCL progression through modulating SIRT expression in H3k9me1-mediated manner. In conclusion, this study unraveled that dysregulation of NCAPD3 could disturb chromosome compaction and segregation, induce genome instability and alter the transcription of SIRT1 via modulating H3k9 monomethylation, which provided novel insights into NCAPD3-based targeting strategy for DLBCL.

Atypical genotoxicity of carcinogenic nickel(II): Linkage to dNTP biosynthesis, DNA-incorporated rNMPs, and impaired repair of TOP1-DNA crosslinks

Cancer is a genetic disease requiring multiple mutations for its development. However, many carcinogens are DNA-unreactive and nonmutagenic and consequently described as nongenotoxic. One of such carcinogens is nickel, a global environmental pollutant abundantly emitted by burning of coal. We investigated activation of DNA damage responses by Ni and identified this metal as a replication stressor. Genotoxic stress markers indicated the accumulation of ssDNA and stalled replication forks, and Ni-treated cells were dependent on ATR for suppression of DNA damage and long-term survival. Replication stress by Ni resulted from destabilization of RRM1 and RRM2 subunits of ribonucleotide reductase and the resulting deficiency in dNTPs. Ni also increased DNA incorporation of rNMPs (detected by a specific fluorescent assay) and strongly enhanced their genotoxicity as a result of repressed repair of TOP1-DNA protein crosslinks (TOP1-DPC). The DPC-trap assay found severely impaired SUMOylation and K48-polyubiquitination of DNA-crosslinked TOP1 due to downregulation of specific enzymes. Our findings identified Ni as the human carcinogen inducing genome instability via DNA-embedded ribonucleotides and accumulation of TOP1-DPC which are carcinogenic abnormalities with poor detectability by the standard mutagenicity tests. The discovered mechanisms for Ni could also play a role in genotoxicity of other protein-reactive carcinogens.

Presenilin-1, mutated in familial Alzheimer’s disease, maintains genome stability via a γ-secretase dependent way

Mutations in Presenilin-1 (PS1) account for over 80 % mutations linked to familial Alzheimer’s disease (AD). However, the mechanisms of action of PS1 mutations in causing familial AD are not fully understood, limiting opportunities to develop targeted disease-modifying therapies for individuals carrying PS1 mutation. To gain more comprehensive insights into the impact of PS1 mutations on genome stability, we knocked down PS1 in SH-SY5Y, HMC3 and A549 cells. This revealed that PS1 knockdown (KD) dramatically induces genome instability (GIN) in all cell types, as indicated by the increased incidence of micronuclei, nucleoplasmic bridges and/or nuclear buds. Although amyloid β (Aβ) was able to induce GIN, PS1-KD was associated with decreased expression of Aβ in SH-SY5Y cells, suggesting Aβ is not the primary cause of GIN in PS1-KD cells. In contrast, inhibiting the PS1 γ-secretase activity by DAPT recapitulated GIN phenotype as seen in PS1-KD cells, indicating that the induction of GIN following PS1 KD can be attributed to the loss of γ-secretase activity. PS1 KD or γ-secretase inhibition markedly sensitizes SH-SY5Y to the genotoxicity of mitomycin C. Interestingly, overexpression of the wildtype PS1 dramatically increased GIN in SH-SY5Y. Collectively, our study demonstrates the potential of PS1 and its γ-secretase activity in maintaining genome stability, highlighting a novel potential link between PS1 loss-of-function or gain-of-function mutations and familial AD through GIN. Several mechanisms by which GIN induced by PS1 dys-expression may contribute to AD are discussed.

Telomerase-independent survival leads to a mosaic of complex subtelomere rearrangements in Chlamydomonas reinhardtii.

Telomeres and subtelomeres, the genomic regions located at chromosome extremities, are essential for genome stability in eukaryotes. In the absence of the canonical maintenance mechanism provided by telomerase, telomere shortening induces genome instability. The landscape of the ensuing genome rearrangements is not accessible by short-read sequencing. Here, we leverage Oxford Nanopore Technologies long-read sequencing to survey the extensive repertoire of genome rearrangements in telomerase mutants of the model green microalga Chlamydomonas reinhardtii In telomerase-mutant strains grown for hundreds of generations, most chromosome extremities were capped by short telomere sequences that were either recruited de novo from other loci or maintained in a telomerase-independent manner. Other extremities did not end with telomeres but only with repeated subtelomeric sequences. The subtelomeric elements, including rDNA, were massively rearranged and involved in breakage-fusion-bridge cycles, translocations, recombinations, and chromosome circularization. These events were established progressively over time and displayed heterogeneity at the subpopulation level. New telomere-capped extremities composed of sequences originating from more internal genomic regions were associated with high DNA methylation, suggesting that de novo heterochromatin formation contributes to the restoration of chromosome end stability in C. reinhardtii The diversity of alternative strategies present in the same organism to maintain chromosome integrity and the variety of rearrangements found in telomerase mutants are remarkable, and illustrate genome plasticity at short timescales.

Nonlethal Furfural Exposure Causes Genomic Alterations and Adaptability Evolution in Saccharomyces cerevisiae.

Furfural is a major inhibitor found in lignocellulosic hydrolysate, a promising feedstock for the biofermentation industry. In this study, we aimed to investigate the potential impact of this furan-derived chemical on yeast genome integrity and phenotypic evolution by using genetic screening systems and high-throughput analyses. Our results showed that the rates of aneuploidy, chromosomal rearrangements (including large deletions and duplications), and loss of heterozygosity (LOH) increased by 50-fold, 23-fold, and 4-fold, respectively, when yeast cells were cultured in medium containing a nonlethal dose of furfural (0.6 g/L). We observed significantly different ratios of genetic events between untreated and furfural-exposed cells, indicating that furfural exposure induced a unique pattern of genomic instability. Furfural exposure also increased the proportion of CG-to-TA and CG-to-AT base substitutions among point mutations, which was correlated with DNA oxidative damage. Interestingly, although monosomy of chromosomes often results in the slower growth of yeast under spontaneous conditions, we found that monosomic chromosome IX contributed to the enhanced furfural tolerance. Additionally, terminal LOH events on the right arm of chromosome IV, which led to homozygosity of the SSD1 allele, were associated with furfural resistance. This study sheds light on the mechanisms underlying the influence of furfural on yeast genome integrity and adaptability evolution. IMPORTANCE Industrial microorganisms are often exposed to multiple environmental stressors and inhibitors during their application. This study demonstrates that nonlethal concentrations of furfural in the culture medium can significantly induce genome instability in the yeast Saccharomyces cerevisiae. Notably, furfural-exposed yeast cells displayed frequent chromosome aberrations, indicating the potent teratogenicity of this inhibitor. We identified specific genomic alterations, including monosomic chromosome IX and loss of heterozygosity of the right arm of chromosome IV, that confer furfural tolerance to a diploid S. cerevisiae strain. These findings enhance our understanding of how microorganisms evolve and adapt to stressful environments and offer insights for developing strategies to improve their performance in industrial applications.

6-Regioisomeric 5,8-quinolinediones as potent CDC25 inhibitors against colorectal cancers

Precise and accurate control of cell cycle progression is required to maintain cell identity and proliferation. Failing to keep it will lead to genome instability and tumorigenesis. Cell Division Cycle 25 (CDC25) phosphatases are the key to regulating the activity of the master cell cycle controller, cyclin-dependent kinases (CDKs). Dysregulation of CDC25 has been shown to associate with several human malignancies. Here, we reported a series of derivatives of the CDC25 inhibitor, NSC663284, bearing quinones as core scaffolds and morpholin alkylamino side chains. Among these derivatives, the cytotoxic activity of the 6-isomer of 5,8-quinolinedione derivatives (6b, 16b, 17b, and 18b) displayed higher potency against colorectal cancer (CRC) cells. Compound 6b possessed the most antiproliferative activity, with IC50 values of 0.59 μM (DLD1) and 0.44 μM (HCT116). The treatment of compound 6b resulted in a remarkable effect on cell cycle progression, blocking S-phase progression in DLD1 cells straight away while slowing S-phase progression and accumulated cells in the G2/M phase in HCT116 cells. Furthermore, we showed that compound 6b inhibited CDK1 dephosphorylation and H4K20 methylation in cells. The treatment with compound 6b induced DNA damage and triggered apoptosis. Our study identifies compound 6b as a potent CDC25 inhibitor that induces genome instability and kills cancer cells through an apoptotic pathway, deserving further investigation to fulfill its candidacy as an anti-CRC agent.

A new G-quadruplex-specific photosensitizer inducing genome instability in cancer cells by triggering oxidative DNA damage and impeding replication fork progression.

Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.

The role of transposable elements in aging and cancer.

Transposable elements (TEs) constitute a large portion of the human genome. Various mechanisms at the transcription and post-transcription levels developed to suppress TE activity in healthy conditions. However, a growing body of evidence suggests that TE dysregulation is involved in various human diseases, including age-related diseases and cancer. In this review, we explained how sensing TEs by the immune system could induce innate immune responses, chronic inflammation, and following age-related diseases. We also noted that inflammageing and exogenous carcinogens could trigger the upregulation of TEs in precancerous cells. Increased inflammation could enhance epigenetic plasticity and upregulation of early developmental TEs, which rewires the transcriptional networks and gift the survival advantage to the precancerous cells. In addition, upregulated TEs could induce genome instability, activation of oncogenes, or inhibition of tumor suppressors and consequent cancer initiation and progression. So, we suggest that TEs could be considered therapeutic targets in aging and cancer.

Dynamic of centromere associated RNAs and the centromere loading of DNA repair proteins in growing oocytes.

Mammalian centromeres are generally composed of dispersed repeats and the satellites such as α-satellites in human and major/minor satellites in mouse. Transcription of centromeres by RNA polymerase II is evolutionary conserved and critical for kinetochore assembly. In addition, it has been found that the transcribed satellite RNAs can bind DNA repair proteins such as MRE11 and PRKDC, and excessively expressed satellite RNAs could induce genome instability and facilitate tumorigenesis. During the maturation of female oocyte, centromeres are critical for accurate segregation of homologous chromosomes and sister chromatids. However, the dynamics of oocyte centromere transcription and whether it associated with DNA repair proteins are unknown. In this study, we found the transcription of centromeres is active in growing oocytes but it is silenced when oocytes are fully grown. DNA repair proteins like Mlh1, Mre11 and Prkdc are found associated with the minor satellites and this association can be interfered by RNA polymerase II inhibitor α-amanitin. When the growing oocyte is in vitro matured, Mlh1/Mre11/Prkdc foci would release from centromeres to the ooplasm. If the oocytes are treated with Mre11 inhibitor Mirin, the meiosis resumption of growing oocytes with Mre11 foci can be suppressed. These data revealed the dynamic of centromeric transcription in oocytes and its potential association with DNA repair proteins, which provide clues about how oocytes maintain centromere stability and assemble kinetochores.

Inflammatory mediator-induced bystander effects and genomic instability: role of tissue-specificity

Much evidence showed that radiation-induced inflammation can cause cellular damage in direct irradiated and bystander cells, which are in vicinity of irradiated cells underlying radiation-induced bystander effects (BE). As well as radiation-induced genomic instability (GI) is observed within the progeny of irradiated and bystander cells as a delayed damage responses. This study tried to mimic the effects of radiation-induced GI and BE by exploring the inflammatory effect of lipopolysaccharide (LPS) on human primary fibroblast (HF 19) and umbilical vein endothelial (HUVEC) cells. To prove the principles of LPS can cause DNA damage in HF 19; cells were treated with 1 μg/ml LPS, and BE was induced by media transfer. Comet assay was used to estimate DNA damage in the direct treated and bystander cells. The early DNA damage result was significantly observed in the direct treated and bystander HF 19 cells. However, these cells did not show a significant delayed DNA damage within their progeny. Comet data showed that LPS frequently induced only initial DNA damage in HF 19 cells. The experiment was repeated utilising HF 19 and HUVEC cells, in order to investigate whether LPS could be involved in the induction of GI in a tissue-specificity manner. Both HF 19 and HUVEC cells were treated with 1 μg/ml LPS. BE was also induced by media transfer. Chromosomal analysis was used for early and late chromosomal damage estimation. Data showed that LPS treatment could significantly cause early chromosomal aberrations in the direct treated and bystander HF 19 and HUVEC cells. However, delayed chromosomal instability was observed only in the direct treated HUVEC cells. Our finding suggested that LPS frequently induced BE in HF 19 and HUVEC cells, which could be mediated by pro-inflammatory cytokines that stimulated by LPS treatment. Moreover, LPS could be involved in the GI induction in a tissue-specificity manner.Keywords:

High Glucose Increases DNA Damage and Elevates the Expression of Multiple DDR Genes.

The DNA Damage Response (DDR) pathways sense DNA damage and coordinate robust DNA repair and bypass mechanisms. A series of repair proteins are recruited depending on the type of breaks and lesions to ensure overall survival. An increase in glucose levels was shown to induce genome instability, yet the links between DDR and glucose are still not well investigated. In this study, we aimed to identify dysregulation in the transcriptome of normal and cancerous breast cell lines upon changing glucose levels. We first performed bioinformatics analysis using a microarray dataset containing the triple-negative breast cancer (TNBC) MDA-MB-231 and the normal human mammary epithelium MCF10A cell lines grown in high glucose (HG) or in the presence of the glycolysis inhibitor 2-deoxyglucose (2DG). Interestingly, multiple DDR genes were significantly upregulated in both cell lines grown in HG. In the wet lab, we remarkably found that HG results in severe DNA damage to TNBC cells as observed using the comet assay. In addition, several DDR genes were confirmed to be upregulated using qPCR analysis in the same cell line. Our results propose a strong need for DDR pathways in the presence of HG to oppose the severe DNA damage induced in cells.

Specific suppression of long terminal repeat retrotransposon mobilization in plants.

The tissue culture passage necessary for the generation of transgenic plants induces genome instability. This instability predominantly involves the uncontrolled mobilization of LTR retrotransposons (LTR-TEs), which are the most abundant class of mobile genetic elements in plant genomes. Here, we demonstrate that in conditions inductive for high LTR-TE mobilization, like abiotic stress in Arabidopsis (Arabidopsis thaliana) and callus culture in rice (Oryza sativa), application of the reverse transcriptase (RT) inhibitor known as Tenofovir substantially affects LTR-TE RT activity without interfering with plant development. We observed that Tenofovir reduces extrachromosomal DNA accumulation and prevents new genomic integrations of the active LTR-TE ONSEN in heat-stressed Arabidopsis seedlings, and transposons of O. sativa 17 and 19 (Tos17 and Tos19) in rice calli. In addition, Tenofovir allows the recovery of plants free from new LTR-TE insertions. We propose the use of Tenofovir as a tool for studies of LTR-TE transposition and for limiting genetic instabilities of plants derived from tissue culture.

Target Mechanisms of the Cyanotoxin Cylindrospermopsin in Immortalized Human Airway Epithelial Cells.

Cylindrospermopsin (CYN) is a cyanobacterial toxin that occurs in aquatic environments worldwide. It is known for its delayed effects in animals and humans such as inhibition of protein synthesis or genotoxicity. The molecular targets and the cell physiological mechanisms of CYN, however, are not well studied. As inhalation of CYN-containing aerosols has been identified as a relevant route of CYN uptake, we analyzed the effects of CYN on protein expression in cultures of immortalized human bronchial epithelial cells (16HBE14o-) using a proteomic approach. Proteins whose expression levels were affected by CYN belonged to several functional clusters, mainly regulation of protein stability, cellular adhesion and integration in the extracellular matrix, cell proliferation, cell cycle regulation, and completion of cytokinesis. With a few exceptions of upregulated proteins (e.g., ITI inhibitor of serine endopeptidases and mRNA stabilizer PABPC1), CYN mediated the downregulation of many proteins. Among these, centrosomal protein 55 (CEP55) and osteonectin (SPARC) were significantly reduced in their abundance. Results of the detailed semi-quantitative Western blot analyses of SPARC, claudin-6, and CEP55 supported the findings from the proteomic study that epithelial cell adhesion, attenuation of cell proliferation, delayed completion of mitosis, as well as induction of genomic instability are major effects of CYN in eukaryotic cells.

The Drivers, Mechanisms, and Consequences of Genome Instability in HPV-Driven Cancers.

Simple SummaryCells infected with high-risk human papillomaviruses (HPV) can accumulate DNA damage and eventually transform into HPV-driven cancers. Genome instability, or the progressive accumulation of DNA alterations (e.g., mutations), in HPV-infected cells is directly induced by the HPV genes and indirectly promoted by HPV infection through the consequences of chronic infection maintenance, increased cell growth, and accumulation of damaging mutations in genes that themselves affect genome instability. While the HPV genome typically exists as a separate entity within cells, genome instability increases the chances of HPV integrating within the host (human) genome, which is common in HPV-induced cancers. The DNA regions surrounding HPV integrations are unstable and can undergo complex alterations that affect both human and HPV genes. This review discusses HPV-dependent and -independent drivers and mechanisms of genome instability in HPV-driven cancers, both globally and around sites of HPV integration, and describes the changes induced in the tumour genome.Human papillomavirus (HPV) is the causative driver of cervical cancer and a contributing risk factor of head and neck cancer and several anogenital cancers. HPV’s ability to induce genome instability contributes to its oncogenicity. HPV genes can induce genome instability in several ways, including modulating the cell cycle to favour proliferation, interacting with DNA damage repair pathways to bring high-fidelity repair pathways to viral episomes and away from the host genome, inducing DNA-damaging oxidative stress, and altering the length of telomeres. In addition, the presence of a chronic viral infection can lead to immune responses that also cause genome instability of the infected tissue. The HPV genome can become integrated into the host genome during HPV-induced tumorigenesis. Viral integration requires double-stranded breaks on the DNA; therefore, regions around the integration event are prone to structural alterations and themselves are targets of genome instability. In this review, we present the mechanisms by which HPV-dependent and -independent genome instability is initiated and maintained in HPV-driven cancers, both across the genome and at regions of HPV integration.

Beta Human Papillomavirus 8 E6 Induces Micronucleus Formation and Promotes Chromothripsis.

Cutaneous beta genus human papillomaviruses (β-HPVs) are suspected to promote the development of nonmelanoma skin cancer (NMSC) by destabilizing the host genome. Multiple studies have established the genome destabilizing capacities of β-HPV proteins E6 and E7 as a cofactor with UV. However, the E6 protein from β-HPV8 (HPV8 E6) induces tumors in mice without UV exposure. Here, we examined a UV-independent mechanism of HPV8 E6-induced genome destabilization. We showed that HPV8 E6 reduced the abundance of anaphase bridge resolving helicase, Bloom syndrome protein (BLM). The diminished BLM was associated with increased segregation errors and micronuclei. These HPV8 E6-induced micronuclei had disordered micronuclear envelopes but retained replication and transcription competence. HPV8 E6 decreased antiproliferative responses to micronuclei and time-lapse imaging revealed HPV8 E6 promoted cells with micronuclei to complete mitosis. Finally, whole-genome sequencing revealed that HPV8 E6 induced chromothripsis in nine chromosomes. These data provide insight into mechanisms by which HPV8 E6 induces genome instability independent of UV exposure. IMPORTANCE Some beta genus human papillomaviruses (β-HPVs) may promote skin carcinogenesis by inducing mutations in the host genome. Supporting this, the E6 protein from β-HPV8 (8 E6) promotes skin cancer in mice with or without UV exposure. Many mechanisms by which 8 E6 increases mutations caused by UV have been elucidated, but less is known about how 8 E6 induces mutations without UV. We address that knowledge gap by showing that 8 E6 causes mutations stemming from mitotic errors. Specifically, 8 E6 reduces the abundance of BLM, a helicase that resolves and prevents anaphase bridges. This hinders anaphase bridge resolution and increases their frequency. 8 E6 makes the micronuclei that can result from anaphase bridges more common. These micronuclei often have disrupted envelopes yet retain localization of nuclear-trafficked proteins. 8 E6 promotes the growth of cells with micronuclei and causes chromothripsis, a mutagenic process where hundreds to thousands of mutations occur in a chromosome.

Balancing Affinity, Selectivity, and Cytotoxicity of Hydrazone-Based G-Quadruplex Ligands for Activation of Interferon β Genes in Cancer Cells.

G-quadruplex (G4) ligands are investigated to discover new anticancer drugs with increased cell-killing potency. These ligands can induce genome instability and activate innate immune genes at non-cytotoxic doses, opening the discovery of cytostatic immune-stimulating ligands. However, the interplay of G4 affinity/selectivity with cytotoxicity and immune gene activation is not well-understood. We investigated a series of closely related hydrazone derivatives to define the molecular bases of immune-stimulation activity. Although they are closely related to each other, such derivatives differ in G4 affinity, cytotoxicity, genome instability, and immune gene activation. Our findings show that G4 affinity of ligands is a critical feature for immune gene activation, whereas a high cytotoxic potency interferes with it. The balance of G4 stabilization versus cytotoxicity can determine the level of immune gene activation in cancer cells. Thus, we propose a new rationale based on low cell-killing potency and high immune stimulation to discover effective anticancer G4 ligands.