scholarly journals Increased replication stress and R-loop accumulation in EGFRvIII expressing glioblastoma present new therapeutic opportunities

2021 ◽  
Author(s):  
Nina Struve ◽  
Konstantin Hoffer ◽  
Anna-Sophie Weik ◽  
Britta Riepen ◽  
Leonie Krug ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Growth Factor Receptor ◽  
Replication Stress ◽  
Loop Formation ◽  
Array Analysis ◽  
Stress Markers ◽  
Immunohistochemistry Staining ◽  
Increased Sensitivity ◽  
Epidermal Growth ◽  
Human Gbm

Abstract Background The oncogene epidermal growth factor receptor variant III (EGFRvIII) is expressed in approximately one third of all glioblastoma (GBM). So far it is not clear if EGFRvIII expression induces replication stress in GBM cells, which might serve as a therapeutical target. Methods Isogenetic EGFRvIII- and EGFRvIII+ cell lines with endogenous EGFRvIII expression were used. Markers of oncogenic and replication stress such as γH2AX, RPA, 53BP1, ATR and Chk1 were analysed using western blot, immunofluorescence and flow cytometry. The DNA fiber assay was performed to analyse replication, transcription was measured by incorporation of EU and genomic instability was investigated by micronuclei and CGH-Array analysis. Immunohistochemistry staining was used to detect replication stress markers and R-loops in human GBM samples. Results EGFRvIII+ cells exhibit an activated replication stress response, increased spontaneous DNA damage, elevated levels of single stranded DNA and reduced DNA replication velocity, which are all indicative characteristics for replication stress. Furthermore, we show here that EGFRvIII expression is linked to increased genomic instability. EGFRvIII expressing cells display elevated RNA synthesis and R-loop formation, which could also be confirmed in EGFRvIII positive GBM patient samples. Targeting replication stress by irinotecan resulted in increased sensitivity of EGFRvIII+ cells. Conclusion This study demonstrates that EGFRvIII expression is associated with increased replication stress, R-loop accumulation and genomic instability. This might contribute to intratumoral heterogeneity but may also be exploited for individualized therapy approaches.

scholarly journals Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

Oncogenesis ◽  
2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Yannick P. Kok ◽  
Sergi Guerrero Llobet ◽  
Pepijn M. Schoonen ◽  
Marieke Everts ◽  
Arkajyoti Bhattacharya ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Checkpoint Inhibitors ◽  
Replication Stress ◽  
Underlying Mechanism ◽  
Dna Lesions ◽  
Mitotic Catastrophe ◽  
Replication Checkpoint ◽  
Cyclin E1 ◽  
Checkpoint Kinases ◽  
Increased Sensitivity

Abstract Oncogene-induced replication stress, for instance as a result of Cyclin E1 overexpression, causes genomic instability and has been linked to tumorigenesis. To survive high levels of replication stress, tumors depend on pathways to deal with these DNA lesions, which represent a therapeutically actionable vulnerability. We aimed to uncover the consequences of Cyclin E1 or Cdc25A overexpression on replication kinetics, mitotic progression, and the sensitivity to inhibitors of the WEE1 and ATR replication checkpoint kinases. We modeled oncogene-induced replication stress using inducible expression of Cyclin E1 or Cdc25A in non-transformed RPE-1 cells, either in a TP53 wild-type or TP53-mutant background. DNA fiber analysis showed Cyclin E1 or Cdc25A overexpression to slow replication speed. The resulting replication-derived DNA lesions were transmitted into mitosis causing chromosome segregation defects. Single cell sequencing revealed that replication stress and mitotic defects upon Cyclin E1 or Cdc25A overexpression resulted in genomic instability. ATR or WEE1 inhibition exacerbated the mitotic aberrancies induced by Cyclin E1 or Cdc25A overexpression, and caused cytotoxicity. Both these phenotypes were exacerbated upon p53 inactivation. Conversely, downregulation of Cyclin E1 rescued both replication kinetics, as well as sensitivity to ATR and WEE1 inhibitors. Taken together, Cyclin E1 or Cdc25A-induced replication stress leads to mitotic segregation defects and genomic instability. These mitotic defects are exacerbated by inhibition of ATR or WEE1 and therefore point to mitotic catastrophe as an underlying mechanism. Importantly, our data suggest that Cyclin E1 overexpression can be used to select patients for treatment with replication checkpoint inhibitors.

scholarly journals c-Src Associates with ErbB2 through an Interaction between Catalytic Domains and Confers Enhanced Transforming Potential

2009 ◽  
Vol 29 (21) ◽  
pp. 5858-5871 ◽  
Author(s):  
Richard Marcotte ◽  
Lixin Zhou ◽  
Harold Kim ◽  
Calvin D. Roskelly ◽  
William J. Muller
Keyword(s):  
Kinase Inhibitors ◽  
Catalytic Domain ◽  
Growth Factor Receptor ◽  
Src Tyrosine Kinase ◽  
Increased Sensitivity ◽  
Epidermal Growth ◽  
Egfr Family ◽  
Egfr Mutant

ABSTRACT Previous studies have demonstrated that c-Src tyrosine kinase interacts specifically with ErbB2, but not with other members of the epidermal growth factor receptor (EGFR) family. To identify the site of interaction, we recently used a chimeric EGFR/ErbB2 receptor approach to show that c-Src requires the kinase region of ErbB2 for binding. Here, we demonstrate that retention of a conserved amino acid motif surrounding tyrosine 877 (referred to here as EGFRYHAD) is sufficient to confer binding to c-Src. Surprisingly the association of c-Src was not dependent on its SH2 or SH3 domain or on the phosphorylation or kinase activity of the receptor. We further show that the chimeric EGFRs that contain the Y877 motif are transforming in vitro and in vivo following ligand stimulation. Transformation was also partially dependent on sustained activation of Stat3. Finally, we demonstrate that EGFRs with mutations in the catalytic domain, originally identified in lung cancer and conferring increased sensitivity to gefitinib and erlotinib, two EGFR kinase inhibitors, gained the capacity to bind c-Src. Moreover, transformation by these EGFR mutants was inhibited by Src inhibitors regardless of their sensitivities to gefitinib and erlotinib. These observations have important implications for understanding the molecular basis for resistance to EGFR inhibitors and implicate c-Src as a critical signaling molecule in EGFR mutant-induced transformation.

scholarly journals Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexy Promonet ◽  
Ismaël Padioleau ◽  
Yaqun Liu ◽  
Lionel Sanz ◽  
Anna Biernacka ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Replication Stress ◽  
Dna Supercoiling ◽  
Dependent Manner ◽  
Loop Formation ◽  
Dna Double Strand Breaks ◽  
Topoisomerase 1 ◽  
Stress Markers ◽  
Highly Expressed Genes ◽  
Rna:Dna Hybrids

AbstractR-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.

scholarly journals Ganoderma lucidum Prevents Cisplatin-Induced Nephrotoxicity through Inhibition of Epidermal Growth Factor Receptor Signaling and Autophagy-Mediated Apoptosis

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Yasmen F. Mahran ◽  
Hanan M. Hassan
Keyword(s):  
Ganoderma Lucidum ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Antineoplastic Agent ◽  
Growth Factor Receptor ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Egfr Signaling ◽  
Downstream Signaling ◽  
Stress Markers ◽  
Epidermal Growth

Background. Cisplatin (cis-diaminedichloroplatinum, CDDP) is a broad-spectrum antineoplastic agent. However, CDDP has been blamed for its nephrotoxicity, which is the main dose-limiting adverse effect. Ganoderma lucidum (GL), a medicinal mushroom, has antioxidant and inflammatory activities. Therefore, this study is aimed at finding out the potential nephroprotection of GL against CDDP-induced nephrotoxicity in rats and the possible molecular mechanisms including the EGFR downstream signaling, apoptosis, and autophagy. Methods. Rats were given GL (500 mg/kg) for 10 days and a single injection of CDDP (12 mg/kg, i.p). Results. Nephrotoxicity was evidenced by a significant increase in renal indices and oxidative stress markers. Additionally, CDDP showed a plethora of inflammatory and apoptotic responses as evidenced by a profound increase of HMGB-1, NF-κB, and caspase-3 expressions, whereas administration of GL significantly improved all these indices as well as the histopathological insults. Renal expression of EGFR showed a similar trend after GL administration. Furthermore, activation of autophagy protein, LC3 II, was found to be involved in GL-mediated nephroprotection correlated with the downregulation of apoptotic signaling, caspase-3 and terminal deoxynucleotidyl transferase (TDT) renal expressions. Conclusion. These results suggest that GL might have improved CDDP-induced nephrotoxicity through antioxidant, anti-inflammatory, and autophagy-mediated apoptosis mechanisms and that inhibition of EGFR signaling might be involved in nephroprotection.

Sign in / Sign up

Export Citation Format

Share Document