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Med. Sci. Forum, 2021, IECC 2021

The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response

Online | 1–14 February 2021

Volume Editors:
Nicola Curtin, Newcastle University, UK
Helen E. Bryant, University of Sheffield, UK

Number of Papers: 17
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Cover Story (view full-size image): The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response (IECC2021) brought together researchers in basic, translational, and [...] Read more.
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167 KiB  
Abstract
Development of Small Molecule NUDT22 Inhibitors for Uses in Cancer
by Melanie Walter, Evert Homan, Tobias Koolmeister, Ingrid Almlöf, Oliver Mortusewicz, Thomas Helleday and Patrick Herr
Med. Sci. Forum 2021, 3(1), 1; https://doi.org/10.3390/IECC2021-09197 - 29 Jan 2021
Viewed by 1571
Abstract
Here, we present the characterisation of the so-far-unstudied NUDIX hydrolase family member NUDT22. We previously identified the unique hydrolase activity of NUDT22 towards UDP-glucose from a family-wide biochemical substrate screen. UDP-glucose hydrolysis was found to result in the production of uridine monophosphate (UMP) [...] Read more.
Here, we present the characterisation of the so-far-unstudied NUDIX hydrolase family member NUDT22. We previously identified the unique hydrolase activity of NUDT22 towards UDP-glucose from a family-wide biochemical substrate screen. UDP-glucose hydrolysis was found to result in the production of uridine monophosphate (UMP) and glucose 1-phosphate (G-1-P). We furthermore solved the first crystal structure of NUDT22 in complex with its substrate UDP-glucose [1]. Our mechanistic studies revealed increased replication stress in NUDT22-deficient cells that could be rescued by nucleoside supplementation. We therefore propose the discovery of a novel NUDT22-mediated pyrimidine salvage pathway. Increased replication rates resulting in replication stress is a hallmark of cancer cells, and NUDT22 gene expression alterations are present in several cancer tissues, which makes NUDT22 an interesting new target for the development of small molecule inhibitors for uses in cancer. We employed our NUDT22 crystal structure to perform an in silico docking screen on available small molecule libraries to identify starting points for the development of first-in-class NUDT22 inhibitors. Chemically optimised NUDT22 inhibitors are currently being validated in biochemical assays and cellular target engagement assays, and their cellular activity is being assessed in vitro. Full article
170 KiB  
Abstract
BRAT1 Impairs DNA Damage Repair in Glioblastoma Cell Lines
by Johanna Ertl, Ömer Güllülü, Stephanie Hehlgans, Franz Rödel, Donat Kögel and Benedikt Linder
Med. Sci. Forum 2021, 3(1), 3; https://doi.org/10.3390/IECC2021-09190 - 29 Jan 2021
Cited by 1 | Viewed by 1152
Abstract
Glioblastomas (GBMs) are one of the most malignant brain tumors in adults. This is partly due to the potential presence of so-called glioma stem-like cells (GSCs), which are characterized by the expression of stemness markers and a resistance to radio- and chemotherapy. Previous [...] Read more.
Glioblastomas (GBMs) are one of the most malignant brain tumors in adults. This is partly due to the potential presence of so-called glioma stem-like cells (GSCs), which are characterized by the expression of stemness markers and a resistance to radio- and chemotherapy. Previous work from us showed that after combination treatment of GSCs with arsenic trioxide and gossypol, the protein BRCA1-associated ATM-activator 1 (BRAT1) was one of the most downregulated proteins. This protein is largely undescribed, but it has been shown to regulate DNA damage signaling through interaction with ATM, BRCA1, and DNA-PKcs in initial stages of DNA damage response. An unpublished analysis of The Cancer Genome Atlas and The Human Protein Atlas databases showed an increased expression of BRAT1 in GBMs compared to healthy tissues and that an increased expression is negatively correlated with patient survival. Due to these findings, our goal is to analyze the radio-sensitizing effect of BRAT1 on FCS-grown (i.e., differentiated) highly radio-resistant GBM cells and GSCs. Here, using stable knockdowns of BRAT1, we show that it is needed for effective DNA repair after irradiation using a γH2AX-foci assay, whereas it is dispensable for cellular proliferation. A cell death analysis using Annexin V/propidium iodide staining revealed a first hint that BRAT1 downregulation sensitizes GBM cells to irradiation. Moreover, through immunofluorescent staining, we showed that BRAT1 is needed for BRCA1 recruitment to DNA damage sites. Future experiments will aim at systematically analyzing the downstream effects of BRAT1 depletion and to determine further interactors. Thus, we hope to gain a deeper understanding of the mechanism of radio-resistance in GSCs, also in order to individually determine the effectiveness of radiotherapy. Full article
164 KiB  
Abstract
The Extent of Consequential DNA Damage in Human Tumors from TCGA PanCanAtlas
by Aleksey V. Belikov, Alexey D. Vyatkin, Danila V. Otnykov and Sergey V. Leonov
Med. Sci. Forum 2021, 3(1), 4; https://doi.org/10.3390/IECC2021-09202 - 29 Jan 2021
Viewed by 787
Abstract
DNA damage is crucial for the emergence of cancer cells. If the DNA damage response is defective, the DNA damage is converted to fixed mutations. Some of these mutations drive tumorigenesis and are called driver mutations. However, the extent of consequential DNA damage [...] Read more.
DNA damage is crucial for the emergence of cancer cells. If the DNA damage response is defective, the DNA damage is converted to fixed mutations. Some of these mutations drive tumorigenesis and are called driver mutations. However, the extent of consequential DNA damage per tumor, i.e., the number of various kinds of driver mutations, is not known. We utilized the largest database of human cancer mutations (TCGA PanCanAtlas), multiple popular algorithms for cancer driver prediction, and several custom scripts to estimate the number of various kinds of driver mutations in primary tumors. We found that there were on average 22 driver mutations per patient’s tumor, of which 1.7 were point mutations in oncogenes, 8.5 were amplifications of oncogenes, 1.6 were point mutations in tumor suppressors, 3.9 were deletions of tumor suppressors, 1.5 were driver chromosome losses, 1 was driver chromosome gain, 2 were driver chromosome arm losses, and 1.5 were driver chromosome arm gains. The number of driver mutations per tumor increased with age, from 11.2 for 25 y.o. to 28.6 for >85 y.o. The number of driver mutations per tumor varied strongly between cancer types, from 1.9 in thyroid carcinoma to 49 in lung squamous cell carcinoma. Overall, our results provide valuable insights into the extent of functional DNA damage in tumors. Full article
169 KiB  
Abstract
Elucidating the Role of XRN2-Mediated DNA Repair Programs
by Tuyen T. Dang and Julio C. Morales
Med. Sci. Forum 2021, 3(1), 6; https://doi.org/10.3390/IECC2021-09210 - 29 Jan 2021
Viewed by 828
Abstract
Glioblastoma multiforme (GBM) is a highly aggressive brain cancer [...] Full article
203 KiB  
Abstract
The Cytotoxic Effect of 64Cu/NOTA-Terpyridine Platinum Conjugate, as a Novel Chemoradiotherapy Agent
by Meysam Khosravifarsani, Samia Ait-Mohand, Benoit Paquette, Léon Sanche and Brigitte Guérin
Med. Sci. Forum 2021, 3(1), 7; https://doi.org/10.3390/IECC2021-09220 - 31 Jan 2021
Viewed by 783
Abstract
Colorectal cancer is one of the most prevalent cancers worldwide that displays both intrinsic and acquired resistance to platinum-based chemotherapeutic agents (Pt-CAs). To overcome such resistance, new classes of Pt-CAs have been proposed, including terpyridine (TP) compounds that target the G-quadruplex tertiary structure [...] Read more.
Colorectal cancer is one of the most prevalent cancers worldwide that displays both intrinsic and acquired resistance to platinum-based chemotherapeutic agents (Pt-CAs). To overcome such resistance, new classes of Pt-CAs have been proposed, including terpyridine (TP) compounds that target the G-quadruplex tertiary structure of DNA. Additionally, recent studies indicate a maximum chemoradiation benefit when radiation is administered with Pt-CAs at their highest concentrations in cancer cell DNA. Accordingly, we synthesized a novel chemo-radiotheranostic agent by conjugating a TP moiety with 64Cu (64Cu-NOTA-TP). The in vitro cytotoxic effects, cellular uptake, internalization and efflux of 64Cu-NOTA-TP were measured for colorectal cancer (HCT116) and normal fibroblast (GM05757) cells. Radiolabeling NOTA-TP with 64Cu resulted in 17,530-, 40,083- and 66,000-fold enhancements in its cytotoxicity against HCT116 cells (EC50 = 0.017 ± 0.004, 0.012 ± 0.006 and 0.005 ± 0.0002 µM) as compared to coldCu-NOTA-terpyridine (EC50 = 298 ± 2, 481 ± 25 and 330 ± 51 µM) at 24, 48 and 72 h post-administration, respectively. More importantly, the cytotoxicity of the 64Cu-conjugate toward the HCT116 cells was about 3.8-fold higher than that of GM05757 cells at 24 and 72 h. This result was consistent with a 2–3-fold higher internalization of 64Cu-conjugate in HCT116 cells relative to GM05757 cells at similar times. The internalized activity of the 64Cu-conjugate steadily increased from 0.04 ± 0.02% to 18.7 ± 2.8% over a 24 h incubation time. Moreover, the efflux kinetics of the 64Cu-conjugate showed that more than 40% of internalized activity was retained by cancer cells over a 24 h period. In conclusion, this work presents a novel chemo-radiotherapeutic agent with considerable potential for targeted cancer treatment combined with radioisotope imaging. Full article
170 KiB  
Abstract
The Pluripotency Transcription Factor Oct4 Contributes to Head and Neck Squamous Cell Carcinoma Radioresistance via Regulation of DNA Repair and the Stem Cell Phenotype
by Jacqueline Nathansen, Vasyl Lukiyanchuk, Linda Hein, Maya-Isabel Stolte, Kerstin Borgmann, Steffen Löck, Ina Kurth, Mechthild Krause, Annett Linge and Anna Dubrovska
Med. Sci. Forum 2021, 3(1), 5; https://doi.org/10.3390/IECC2021-09224 - 31 Jan 2021
Viewed by 1019
Abstract
Despite being the sixth most common cancer type worldwide, head and neck squamous cell carcinoma (HNSCC) exhibits low five-year survival rates for advanced-stage patients. The local control probability after radiotherapy crucially depends on efficient depletion of the pluripotent sub-population of tumor cells. These [...] Read more.
Despite being the sixth most common cancer type worldwide, head and neck squamous cell carcinoma (HNSCC) exhibits low five-year survival rates for advanced-stage patients. The local control probability after radiotherapy crucially depends on efficient depletion of the pluripotent sub-population of tumor cells. These cancer stem cells (CSCs) are characterized by an active DNA repair and, consequently, an enhanced radio(chemo)therapy resistance. This study provides evidence that due to its involvement in the regulation of the DNA damage response and stem cell phenotype, the CSC-related transcription factor Oct4 contributes to HNSCC radioresistance. In a siRNA-mediated Oct4 knockdown model, we observed reduced self-renewal capacity and partial radiosensitization of HNSCC cell lines accompanied by decreased expression of the cell cycle checkpoint kinases Chk-1 and WEE1. Consequently, Oct4 knockdown impaired the G2 checkpoint induction after irradiation, linking Oct4 to the HNSCC DNA damage response. Upon CRISPR/Cas9-mediated knockout of the pluripotency-related isoform Oct4 A, radiosensitization of HNSCC cells could only be achieved in combination treatment with the PARP inhibitor Olaparib. In addition, irradiation-induced up-regulation of DNA repair genes, such as the homologous recombination repair (HRR) gene BRCA1, was abolished in Oct4 A knockout cells, indicating that Oct4 A depletion leads to HRR deficiency in HNSCC cells. Further analysis of the Oct4-correlating gene signature in the HNSCC TCGA patient dataset identified the HRR genes PSMC3IP and RAD54L showing a significant correlation with the overall survival of radiotherapy-treated HNSCC patients. The HNSCC self-renewal capacity and clonogenic cell survival after irradiation was reduced upon siRNA-mediated PSMC3IP and RAD54L knockdown, emphasizing the interplay between DNA repair and the CSC phenotype in HNSCC radioresistance mechanisms. All in all, the involvement of Oct4 in the regulation of DNA repair and cell cycle progression provides new insights into HNSCC radioresistance and opens possibilities for combination therapy with PARP inhibitors. Full article
167 KiB  
Abstract
Does the Modification of Serine 477 of DNA Mismatch Repair Protein MLH1 Play a Role in Cell Proliferation?
by May-Britt Firnau and Angela Brieger
Med. Sci. Forum 2021, 3(1), 8; https://doi.org/10.3390/IECC2021-09228 - 31 Jan 2021
Viewed by 752
Abstract
MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player in the DNA mismatch repair (MMR) system and of great importance to correct incorporation errors that occur during DNA replication. Previously, we identified that posttranslational phosphorylation of MLH1 at amino acid [...] Read more.
MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player in the DNA mismatch repair (MMR) system and of great importance to correct incorporation errors that occur during DNA replication. Previously, we identified that posttranslational phosphorylation of MLH1 at amino acid position serine 477 can switch off MMR activity in vitro. We also found that mutation of serine 477 prevented posttranslational phosphorylation. Since MLH1 is involved in numerous MMR-independent cell processes, including the cell cycle control, we hypothesized that phosphorylation of MLH1 might alter the mediation of cell cycle-associated proteins and thus affect proliferation. To investigate the impact of phosphorylation of MLH1 on proliferation, an MTT assay was used. MutLα-deficient HEK293T cells were transiently cotransfected with pcDNA3.1+/MLH1 and pcDNA3.1+/PMS2 for the expression of the MutLα wildtype. For the expression of the non-phosphorylatable MutLα variant, cells were transiently cotransfected with pcDNA3.1+/MLH1S477A and pcDNA3.1+/PMS2. At 48 h after transfection, cells were treated with calyculin (50 nM), a serine-threonine-phosphatase inhibitor, to enhance the amount of phosphorylated MLH1. In parallel, cells were treated with orthovanadate (50 µM), a competitive inhibitor of protein-phosphotyrosine phosphatases, to exclude inhibitor side effects. DMSO was used as a negative control. After a cultivation period of 15 min to 3 h, cells were incubated with MTT reagent and proliferation was evaluated via an ELISA reader. In summary, significant differences of proliferation could be detected between the differently treated cells. Proliferation of calyculin-treated HEK293T cells overexpressing the non-phosphorylatable MutLα variant, however, was only weakly increased compared to cells overexpressing the MutLα wildtype. Due to the fact that calyculin and orthovanadate are able to influence a multitude of signaling pathways, the role of MLH1 phosphorylation cannot be conclusively answered here. Further experiments are necessary to clarify the function of phosphorylated MLH1 in proliferation. Full article
167 KiB  
Abstract
USP9X Inhibition Enhances Radiosensitisation of Head and Neck Cancer Cells in Response to High-LET Radiation by Destabilising Centrosome Proteins
by Maria Rita Fabbrizi, Catherine M. Nickson, Rachel J. Carter, Jonathan R. Hughes, Andrzej Kacperek, Mark A. Hill and Jason L. Parsons
Med. Sci. Forum 2021, 3(1), 2; https://doi.org/10.3390/IECC2021-09211 - 29 Jan 2021
Viewed by 894
Abstract
Ionising radiation (IR) is widely used in cancer treatment as it induces vast DNA damage, ultimately leading to tumour cell death. The mechanisms involved in X-ray-induced cell death have been deeply studied, while little is known about the impact of IR of higher [...] Read more.
Ionising radiation (IR) is widely used in cancer treatment as it induces vast DNA damage, ultimately leading to tumour cell death. The mechanisms involved in X-ray-induced cell death have been deeply studied, while little is known about the impact of IR of higher linear energy transfer (LET) on cell biology and the critical enzymes and mechanisms that are responsive to this. We recently performed a focused small interfering RNA (siRNA) screen to identify proteins involved in cell survival in response to high-LET α-particles and protons, versus low-LET X-rays and protons. From this screening, we validated that depletion of the ubiquitin-specific protease 9X (USP9X) in HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells using siRNA leads to a significantly decreased survival of cells after exposure to high-LET radiation, whilst no effect was observed after low-LET radiation (protons and X-rays) treatment. We consequently investigated the mechanism through which this occurs and found that USP9X inhibition does not interfere with DNA damage (double-strand breaks and complex DNA damage) repair post-irradiation, nor does it induce apoptosis, autophagy or senescence. Instead, we observed that USP9X depletion destabilises key centrosome proteins (CEP55 and CEP131), causing centrosome amplification and, ultimately, cell death in response to high-LET protons. Full article
177 KiB  
Abstract
Evidence of the Nerve–Cancer Connection in Female Reproductive Cancers
by Sonia M. Rodrigues Oliveira, Hubert Hondermarck and Phillip Jobling
Med. Sci. Forum 2021, 3(1), 9; https://doi.org/10.3390/IECC2021-09199 - 31 Jan 2021
Viewed by 824
Abstract
In recent years, the infiltration of tumors by axons or nerves has been increasingly reported and has been linked to poor prognosis. This includes, among others, publications by Magnon et al. (Science 341, 2013); and our own work (Faulkner et al., FASEB BioAdvances [...] Read more.
In recent years, the infiltration of tumors by axons or nerves has been increasingly reported and has been linked to poor prognosis. This includes, among others, publications by Magnon et al. (Science 341, 2013); and our own work (Faulkner et al., FASEB BioAdvances 2, 2020). Thus, recent investigations have been defying the old mechanistic, non-participating, view of the role of the nervous system in the tumor microenvironment. The ‘nerve–cancer connection’ now is believed to encompass novel therapeutic targets already reported for breast, prostate and gastric cancers. However, the role of the autonomic nervous system in ovarian cancer development and progression remains unclear. We aimed to characterize this new component in the ovarian tumor microenvironment. We identified the infiltration of peripheral axons in some ovarian tumors. In addition, ovarian tumors expressed neurotrophins, including nerve growth factor (NGF), in particular in the initial onset of the tumor. Our work exposes the need to further comprehend the role of the nervous system in female cancers, namely in the unique microenvironments of ovarian tumors. Full article
168 KiB  
Abstract
Increased Replication Stress Sensitises High Risk Neuroblastoma Cells to ATR and PARP Inhibition
by Harriet Southgate, Lindi Chen, Deborah A. Tweddle and Nicola J. Curtin
Med. Sci. Forum 2021, 3(1), 10; https://doi.org/10.3390/IECC2021-09205 - 29 Jan 2021
Viewed by 813
Abstract
Background: Neuroblastoma (NB) is a rare childhood cancer but accounts for 15% of paediatric cancer deaths. MYCN amplification and/or ATM loss through 11q deletion may cause an increase in replication stress (RS) in a substantial proportion (~80%) of high-risk NB. RS creates a [...] Read more.
Background: Neuroblastoma (NB) is a rare childhood cancer but accounts for 15% of paediatric cancer deaths. MYCN amplification and/or ATM loss through 11q deletion may cause an increase in replication stress (RS) in a substantial proportion (~80%) of high-risk NB. RS creates a dependency on ATR-mediated S and G2 checkpoint control. This study aimed to determine if MYCN amplification or ATM loss identifies cells which are sensitive to ATR inhibition (ATRi). As PARP inhibition causes RS through unrepaired single-strand DNA breaks progressing to replication, we also examined the effect of ATRi on PARP inhibitor (PARPi) cytotoxicity and PARPi-induced RS, cell cycle arrest and homologous recombination repair (HRR) activity. Methods: Cell proliferation in response to 72 h of treatment with the ATR inhibitor, VE-821, was assessed by XTT (Roche) and clonogenic survival assays in a panel of 11 NB cell lines and the effect of VE-821 on growth inhibition caused by the PARPi, Olaparib, in 4 cell lines. CHK1S345 (a marker of ATR activity) and RPA2S8 and H2AXS129 phosphorylation (RS markers) were assessed by Western blotting and immunofluorescent microscopy. HRR was examined by the formation of RAD51 foci by immunofluorescent microscopy. Cell cycle analysis was carried out by flow cytometry. Results: VE-821-induced growth inhibition and cell death was significantly increased in MYCN-amplified cell lines and cell lines with low ATM protein expression (p < 0.05 Mann–Whitney U test). Olaparib (5 µM) treatment increased CHK1S345 and H2AXS129 phosphorylation after 24 h of treatment in all cell lines. ATR inhibition prevented CHK1S345 phosphorylation and reduced Olaparib-induced RAD51 foci formation. VE-821 abrogated Olaparib-induced S and G2 checkpoint arrest. Conclusion: MYCN amplification and low ATM protein expression are determinants of ATRi sensitivity in NB cell lines. ATRi sensitises NB cells to PARPi by abrogating S/G2 checkpoint arrest and impairing HRR. Full article
180 KiB  
Abstract
Differences in Durability of PARP Inhibition by PARP Inhibitors in Ovarian Cancer Cells
by Hannah Louise Smith, Asima Mukhopadhyay, Yvette Drew, Elaine Willmore and Nicola Curtin
Med. Sci. Forum 2021, 3(1), 11; https://doi.org/10.3390/IECC2021-09194 - 29 Jan 2021
Viewed by 1175
Abstract
Background: PARP inhibitors (PARPi) exploit defects in homologous recombination repair (HRR) to selectively kill tumour cells. Continuous PARP inhibition is required for cytotoxicity. PARPis rucaparib, olaparib, and niraparib have been approved for use in ovarian cancer on continuous schedules. Previous studies demonstrate prolonged [...] Read more.
Background: PARP inhibitors (PARPi) exploit defects in homologous recombination repair (HRR) to selectively kill tumour cells. Continuous PARP inhibition is required for cytotoxicity. PARPis rucaparib, olaparib, and niraparib have been approved for use in ovarian cancer on continuous schedules. Previous studies demonstrate prolonged PARP inhibition by rucaparib [1]. Aim: To determine if persistent PARP inhibition is a class effect. Methods: IGROV-1 (human ovarian cancer) cells were treated with 1 µM of rucaparib, olaparib, niraparib, talazoparib, or pamiparib for 1 h before drug was washed off and replaced with fresh media for 0, 1, 24, 48, or 72 h prior to harvesting. Cellular PARP activity was measured using a GCLP-validated assay [2] in comparison with untreated controls and where 1 µM inhibitor was added to the reaction. Results: rucaparib, olaparib, niraparib, talazoparib, and pamiparib each inhibited PARP activity in permeabilized cells > 99% when 1 µM was present during the reaction. After 2 h in drug-free medium, rucaparib-induced PARP inhibition was maintained at 92.3 ± 4.3%, but was much less with talazoparib (58.6 ± 5.0%), pamiparib (56.0 ± 4.5%) olaparib (48.3 ± 19.8%), and niraparib (37.3 ± 11.6%). PARP inhibition in rucaparib-treated cells was maintained for 72 h in drug-free medium (77.7 ± 12.3%). This sustained PARP inhibition was not observed with the other PARPis. PARP inhibition was only 12.3 ± 5.2% and 12.5 ± 4.9% 72 h after talazoparib and pamiparib, respectively, and undetectable with olaparib and niraparib. Conclusion: Rucaparib is unique in its ability to cause persistent PARP inhibition and it is not a class effect. These data have clinical implications for the different uses of PARPi, as a single agent use to exploit HRR defects vs. chemo and radiosensitization. Full article
194 KiB  
Abstract
Exploring Sensitivity to Replicative Stress in BRCA-Deficient Triple Negative Breast Cancer
by Imene Tabet, Carolina Velázquez, Esin Orhan, Lise Fenou, Laura Boudarel, William Jacot, Claude Sardet and Charles Theillet
Med. Sci. Forum 2021, 3(1), 12; https://doi.org/10.3390/IECC2021-09226 - 31 Jan 2021
Viewed by 929
Abstract
In triple negative breast cancer (TNBC), chemotherapy is the only systemic treatment and sustained remissions are rare. We propose to widen therapeutic options. About 30% of TNBC tumors are BRCA1-deficient, presenting defective DNA repair and increased sensitivity to genotoxic drugs. We hypothesized that [...] Read more.
In triple negative breast cancer (TNBC), chemotherapy is the only systemic treatment and sustained remissions are rare. We propose to widen therapeutic options. About 30% of TNBC tumors are BRCA1-deficient, presenting defective DNA repair and increased sensitivity to genotoxic drugs. We hypothesized that BRCA-deficient TNBC is highly sensitive to replication stress-inducing drugs, thus, opening new therapeutic perspectives. Our preliminary results showed that BRCA1-deficient TNBC cell lines and a CRISPR/Cas9 BRCA1 KO isogenic model display increased sensitivity to gemcitabine. Cell cycle distribution of gemcitabine-treated BRCA1-deficient cells was characterized by an elevated Sub-G1 fraction caused by increased numbers of cells in replication catastrophe. This was illustrated by 80% of BRCA1-deficient cells showing persistent (48–72 h post treatment) gH2AX staining in the absence of RPA32 co-staining, whereas in the isogenic BRCA1 WT model gH2AX and RPA32 positive cell numbers started decreasing at 24 h. Interestingly, we noted that in addition to replication catastrophe, BRCA-deficient cells treated with gemcitabine underwent aberrant mitosis as shown by a clear increase of micro-nuclei. Interestingly, in vivo experiments appear to reproduce in vitro data. Indeed, a BRCA hypermethylated TNBC PDX showed a higher sensitivity to gemcitabine than the BRCA1 WT. In conclusion, our data suggest that BRCA-deficient tumors are more sensitive to the replication poison gemcitabine. Furthermore, this sensitivity seems to be mediated by an accentuated replicative stress response that is not well managed. Upon gemcitabine treatment, the cells undergo important DNA damage that leads to stalled replication forks, and DNA breakage. In the absence of BRCA1, the HR pathway is compromised, which leads to fork collapse and accumulation of single-stranded DNA, therefore exhausting the pool of RPA within the cell and inducing replicative catastrophe. In addition to deficient replication, gemcitabine treated BRCA-deficient, but not BRCA-proficient cells, are subjected to mitotic catastrophe. Full article
183 KiB  
Abstract
Exploiting DNA Repair Defect in Triple Negative Breast Cancer Using CDK Inhibition Strategy
by Carolina Velázquez, Esin Orhan, Imene Tabet, Lise Fenou, Laura Boudarel, William Jacot, Claude Sardet and Charles Theillet
Med. Sci. Forum 2021, 3(1), 13; https://doi.org/10.3390/IECC2021-09212 - 29 Jan 2021
Viewed by 881
Abstract
Triple-negative breast cancer (TNBC), representing 15% of breast carcinomas, is an aggressive breast cancer subtype with a high probability of metastasis and limited treatment options. Noticeably, BRCA-deficiency occurs in 25% of the TNBCs and results in deficient homologous recombination (HR) repair. Interestingly, PARP [...] Read more.
Triple-negative breast cancer (TNBC), representing 15% of breast carcinomas, is an aggressive breast cancer subtype with a high probability of metastasis and limited treatment options. Noticeably, BRCA-deficiency occurs in 25% of the TNBCs and results in deficient homologous recombination (HR) repair. Interestingly, PARP inhibitors (PARPi) have shown synthetic lethality in a BRCA-deficient context; however, their efficacy is frequently hampered by intrinsic or acquired resistance mechanisms involving restoration of the HR. In that regard, the role of some CDKs proven to regulate key HR actors was of interest to us. In this study, we aimed to understand the rewiring pathways determining resistance to PARPi in BRCA-deficient cancers and to assess the role of transcriptional regulating CDKs such as CDK7, CDK9, or CDK12 in the transcriptional regulation of key HR genes. Our ultimate goal was to determine whether and which CDK inhibitors could be effective approaches to repress HR gene expression and induce pharmacological HR-deficiency. As such, these CDK-inhibitors (CDKi) could be molecules of choice allowing sensitization of tumors that would otherwise respond poorly to DNA damaging treatment. With this purpose, we used in vitro and in vivo (PDX) models of TNBC and studied the attenuation of the HR response in tumor cells and PDX models treated with CDK-inhibitors. Our final aim was to determine the most efficient combination of CDKi and PARPi Our HR read outs were RAD51 and BRCA1 foci formation upon PARPi treatment. We also measured the modification of RNA and protein expression levels induced by CDKi treatment on a series of diagnostic HR genes (BRCA2, PALB2, ATR, FANCD2), as a measure of HR repression. We present data comparing the relative efficiency of three 3 CDKi; dinaciclib, NVP-2, and SR-4835, which have different specificities and inhibit different CDKs with variable efficacy. Full article
191 KiB  
Abstract
Changes in Gene Expression of Metalloproteinases-2 and -9 and Their Inhibitors TIMP2 and TIMP3 in Human Glioma Cells Exposed to Low Levels of Fluoride
by Magdalena Nowak, Marta Skórka-Majewicz and Wojciech Żwierełło
Med. Sci. Forum 2021, 3(1), 14; https://doi.org/10.3390/IECC2021-09229 - 31 Jan 2021
Viewed by 837
Abstract
Introduction: Fluorine compounds are common environmental pollutants and may excessively penetrate the human body, especially the brain (fluoride penetrates the blood–brain barrier). Some of the latest studies have shown that fluoride may interfere with some of the metabolic pathways involved in the development [...] Read more.
Introduction: Fluorine compounds are common environmental pollutants and may excessively penetrate the human body, especially the brain (fluoride penetrates the blood–brain barrier). Some of the latest studies have shown that fluoride may interfere with some of the metabolic pathways involved in the development of invasive potential in many types of cancer (e.g., Wnt/catenin or NF-κB). One of the stages of tumor invasion is the degradation of the extracellular matrix by metalloproteinases (MMP-2 and MMP-9), which allows for the migration and metastasis of cancer cells. Taking into account the above facts, we decided to check whether low concentrations of fluoride affect the expression level of genes encoding MMP-2 and MMP-9, as well as their TIMP-2 and TIMP-3 inhibitors, in human glioblastoma cells. Methods: U-87MG human glioblastoma cells were cultured with EMEM medium (10% FBS, 2 mM glutamine, 1% NEAA, 1 mM sodium pyruvate, 100 IU/mL penicillin, 10 μg/mL streptomycin) under optimal conditions (37 °C in an atmosphere of 5% CO2 with 95% humidity). Cells were treated with sodium fluoride (NaF; 1–5 μM) for 24, 48, and 72 h. The analysis of the expression level of the MMP-2, MMP-9, Timp-2, and Timp-3 genes was carried out via RT-PCR. Results: The results indicate that NaF (0.1–5 μM) can disrupt the expression of MMP-2, MMP-9, Timp-2, and Timp-3. In the case of MMP-2, there was an approximately twofold increase in expression at 48 h (5 μM NaF) and about a 2.5-fold increase in expression at 72 h (0.1–5 μM NaF). For MMP-9, an approximately threefold increase in expression was observed at 24 h (0.1 μM NaF) and 48 h (5 μM NaF). Both Timp-2 and Timp-3 showed a significant increase in expression observed at all time points, especially at the highest concentration of NaF (5 μM). Conclusions: The obtained results suggest that even low concentrations of fluorine compounds may have an undesirable influence promoting the invasive potential of human glioblastoma cells. Full article

Proceedings

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695 KiB  
Proceeding Paper
Space and Time in the Universe of the Cell Nucleus after Ionizing Radiation Attacks: A Comparison of Cancer and Non-Cancer Cell Response
by Michael Hausmann, Charlotte Neitzel, Hannes Hahn, Ruth Winter, Iva Falkova, Dieter W. Heermann, Götz Pilarczyk, Georg Hildenbrand, Harry Scherthan and Martin Falk
Med. Sci. Forum 2021, 3(1), 15; https://doi.org/10.3390/IECC2021-09219 - 31 Jan 2021
Cited by 1 | Viewed by 1088
Abstract
DNA double-strand breaks (DSBs) are known to be the most serious lesions in irradiated cells. Several protein pathways exist for repair. The mechanisms by which cells determine a specific pathway for repair remain poorly understood. DSB induction and repair can be spatiotemporally monitored [...] Read more.
DNA double-strand breaks (DSBs) are known to be the most serious lesions in irradiated cells. Several protein pathways exist for repair. The mechanisms by which cells determine a specific pathway for repair remain poorly understood. DSB induction and repair can be spatiotemporally monitored by ionizing radiation-induced foci (IRIFs) and the formation of repair complexes. IRIF analyses revealed that DSB formation, repair and misrepair are strongly dependent on the radiation characteristics and the microarchitecture of the chromatin environment. However, the IRIF nano-architecture remains unknown, as does its impact on the decision-making process and follow-up protein recruitment. New insights into the relationship between the physical properties of radiation, environmental chromatin architecture, IRIF architecture and DSB repair mechanisms are presented using single-molecule localization microscopy. Ripley distance statistics and persistent homology calculations have shifted our ability to analyze chromatin and IRIF architectures from imaging to topology and structure calculations. We discuss these approaches for cancer treatment-relevant irradiation processes on selected cell systems and consider whether this “structuromics” can enhance our knowledge about the radiation response. Full article
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718 KiB  
Proceeding Paper
The Electrochemical Behavior of Methotrexate upon Binding to the DNA of Different Cell Lines
by Douglas Vieira Thomaz and Pierre Alexandre dos Santos
Med. Sci. Forum 2021, 3(1), 16; https://doi.org/10.3390/IECC2021-09215 - 29 Jan 2021
Cited by 4 | Viewed by 1743
Abstract
Methotrexate (MTX) is a widely used anticancer agent whose DNA binding properties are well known. Despite its consolidated usage in the therapeutics of cancer, the physicochemical features of MTX binding to healthy and neoplastic DNA are still not fully understood. Therefore, this work [...] Read more.
Methotrexate (MTX) is a widely used anticancer agent whose DNA binding properties are well known. Despite its consolidated usage in the therapeutics of cancer, the physicochemical features of MTX binding to healthy and neoplastic DNA are still not fully understood. Therefore, this work showcases the electrochemical study of MTX binding to distinct DNA sequences through voltametric approaches. Full article
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2265 KiB  
Proceeding Paper
Preclinical Investigation of Inhibition of the DNA Damage Response as a Targetted Therapy in Myeloproliferative Neoplasms Shows Synergism of ATR Inhibitors with Standard-of-Care Treatment
by Aleksander Ślusarczyk, Helen E. Bryant, Edwin Chen, Ian Hitchcock, Martin Zeidler, Andrew Chantry and Sally Thomas
Med. Sci. Forum 2021, 3(1), 17; https://doi.org/10.3390/IECC2021-09209 - 29 Jan 2021
Viewed by 1240
Abstract
Myeloproliferative neoplasms (MPNs) are a group of haematological malignancies arising from haematopoietic stem cells (HSCs) with acquired driver mutations in JAK2, MPL and CALR. Current therapies are not selective for the mutant HSC population. Increased replication stress is seen in the [...] Read more.
Myeloproliferative neoplasms (MPNs) are a group of haematological malignancies arising from haematopoietic stem cells (HSCs) with acquired driver mutations in JAK2, MPL and CALR. Current therapies are not selective for the mutant HSC population. Increased replication stress is seen in the presence of mutant JAK2, suggesting DNA damage response inhibitors (DDRi) may differentially affect mutant HSCs over wild-type HSCs to restore normal haematopoiesis. Using JAK2V617F and CALR (del 52) mutant cell lines, we observed that ATR inhibition (ATRi) by AZD6738 and VE-821 significantly reduced viability. The combination of ATRi and a hydroxyurea/a JAK1/2 inhibitor—ruxolitinib—demonstrated high synergism in both apoptosis induction and proliferation arrest. This study provides preliminary evidence that ATRi combined with standard therapies may be exploited in MPNs harbouring JAK2 and CALR mutations. Full article
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