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Conference Paper: Functional characterization of Rad50 germline deleterious variants identified from deep targeted sequencing study of familial esophageal squamous cell carcinoma

TitleFunctional characterization of Rad50 germline deleterious variants identified from deep targeted sequencing study of familial esophageal squamous cell carcinoma
Authors
Issue Date2018
PublisherAmerican Association for Cancer Research. The Journal's web site is located at http://cancerres.aacrjournals.org/
Citation
Proceedings of the 109th American Association for Cancer Research (AACR) Annual Meeting: Driving Innovative Cancer Science to Patient Care, Chicago, Illinois, USA, 14-18 April 2018 In Cancer Research, 2018, v. 78 n. 13, Suppl, p. Abstract 327 How to Cite?
AbstractEsophageal squamous cell carcinoma (ESCC), a histological subtype of esophageal cancer, is a highly prevalent malignancy in Southeast Asia with poor prognosis and high mortality rate. From previous Next-Generation Sequencing (NGS) studies that sampled familial ESCC cases from Henan, a hotspot of ESCC cases in China, rare germline variants of RAD50 homolog, double strand break repair protein (RAD50) were identified and predicted to be deleterious by in silico models. We hypothesized that ESCC patients carrying dominant-negative germline or somatic RAD50 alterations with disrupted MRN (MRE11-RAD50-NBS1) functions may show improved prognosis, as tumor cells are sensitized to genotoxic agents such as cisplatin and ionizing radiation(IR). The current study aims to characterize if these identified germline loss-of-function (LOF) mutants of RAD50 exert dominant-negative impacts on double-strand break (DSB) repair and ATM/ATR signalling. A stop-gain mutation at the zinc-hook domain (RAD50-SG1), and a missense mutation at the C-terminal ATP-binding cassette (RAD50-MS1) were chosen for functional characterization. These mutations were introduced into RAD50 ORF by site-directed mutagenesis. The wildtype (RAD50-WT), RAD50-SG1 and RAD50-MS1 were expressed alongside a vector-alone control (VA) in two cell lines, U2OS and KYSE70(ESCC), using a lentiviral system. DSB repair efficiency was assessed by γH2AX foci recovery. Expression of RAD50-WT reduced the number of γH2AX foci-(+) cells compared to VA at 6 hours post-IR, hinting of an improved DSB repair efficiency while RAD50-MS1 showed no reduction in γH2AX foci-(+) cells. Intriguingly, the number of γH2AX foci-(+) cells was significantly elevated in RAD50-SG1, indicating a reduced DSB repair efficiency in a dominant-negative manner as U2OS expresses wildtype RAD50 endogenously. RAD50-SG1 expressed U2OS was sensitized to Chk-inhibitor AZD7762, when compared to RAD50-WT, RAD50-MS1 and VA control, as measured by MTT assay. Details about the downstream ATM/ATR signalling for reduced phosphorylation of ATM/ATR substrates for impaired checkpoint signalling, data of dominant-negative functional impacts of the RAD50-SG1 mutant regarding cell proliferation in the presence of DSB stimuli (cisplatin treatment/γ irradiation), DSB repair efficiency and sensitivity towards PARP inhibitor will be presented. We expect our study to provide insight into the potential therapeutic impacts of RAD50 LOF mutants with PARP inhibition or Chk inhibition. It should provide a strategy for design of new therapeutic regimens for combined targeted disruption of MRN function with PARPi or Chki, which is expected to benefit ESCC patients, who show poor responses to conventional chemotherapy and radiotheapy.
Persistent Identifierhttp://hdl.handle.net/10722/258141
ISSN
2017 Impact Factor: 9.13
2015 SCImago Journal Rankings: 5.372

 

DC FieldValueLanguage
dc.contributor.authorLam, SY-
dc.contributor.authorNing, L-
dc.contributor.authorNg, HY-
dc.contributor.authorLung, ML-
dc.contributor.authorKo, JMY-
dc.date.accessioned2018-08-22T01:33:38Z-
dc.date.available2018-08-22T01:33:38Z-
dc.date.issued2018-
dc.identifier.citationProceedings of the 109th American Association for Cancer Research (AACR) Annual Meeting: Driving Innovative Cancer Science to Patient Care, Chicago, Illinois, USA, 14-18 April 2018 In Cancer Research, 2018, v. 78 n. 13, Suppl, p. Abstract 327-
dc.identifier.issn0008-5472-
dc.identifier.urihttp://hdl.handle.net/10722/258141-
dc.description.abstractEsophageal squamous cell carcinoma (ESCC), a histological subtype of esophageal cancer, is a highly prevalent malignancy in Southeast Asia with poor prognosis and high mortality rate. From previous Next-Generation Sequencing (NGS) studies that sampled familial ESCC cases from Henan, a hotspot of ESCC cases in China, rare germline variants of RAD50 homolog, double strand break repair protein (RAD50) were identified and predicted to be deleterious by in silico models. We hypothesized that ESCC patients carrying dominant-negative germline or somatic RAD50 alterations with disrupted MRN (MRE11-RAD50-NBS1) functions may show improved prognosis, as tumor cells are sensitized to genotoxic agents such as cisplatin and ionizing radiation(IR). The current study aims to characterize if these identified germline loss-of-function (LOF) mutants of RAD50 exert dominant-negative impacts on double-strand break (DSB) repair and ATM/ATR signalling. A stop-gain mutation at the zinc-hook domain (RAD50-SG1), and a missense mutation at the C-terminal ATP-binding cassette (RAD50-MS1) were chosen for functional characterization. These mutations were introduced into RAD50 ORF by site-directed mutagenesis. The wildtype (RAD50-WT), RAD50-SG1 and RAD50-MS1 were expressed alongside a vector-alone control (VA) in two cell lines, U2OS and KYSE70(ESCC), using a lentiviral system. DSB repair efficiency was assessed by γH2AX foci recovery. Expression of RAD50-WT reduced the number of γH2AX foci-(+) cells compared to VA at 6 hours post-IR, hinting of an improved DSB repair efficiency while RAD50-MS1 showed no reduction in γH2AX foci-(+) cells. Intriguingly, the number of γH2AX foci-(+) cells was significantly elevated in RAD50-SG1, indicating a reduced DSB repair efficiency in a dominant-negative manner as U2OS expresses wildtype RAD50 endogenously. RAD50-SG1 expressed U2OS was sensitized to Chk-inhibitor AZD7762, when compared to RAD50-WT, RAD50-MS1 and VA control, as measured by MTT assay. Details about the downstream ATM/ATR signalling for reduced phosphorylation of ATM/ATR substrates for impaired checkpoint signalling, data of dominant-negative functional impacts of the RAD50-SG1 mutant regarding cell proliferation in the presence of DSB stimuli (cisplatin treatment/γ irradiation), DSB repair efficiency and sensitivity towards PARP inhibitor will be presented. We expect our study to provide insight into the potential therapeutic impacts of RAD50 LOF mutants with PARP inhibition or Chk inhibition. It should provide a strategy for design of new therapeutic regimens for combined targeted disruption of MRN function with PARPi or Chki, which is expected to benefit ESCC patients, who show poor responses to conventional chemotherapy and radiotheapy.-
dc.languageeng-
dc.publisherAmerican Association for Cancer Research. The Journal's web site is located at http://cancerres.aacrjournals.org/-
dc.relation.ispartofCancer Research-
dc.relation.ispartofAmerican Association for Cancer Research 109th Annual Meeting-
dc.titleFunctional characterization of Rad50 germline deleterious variants identified from deep targeted sequencing study of familial esophageal squamous cell carcinoma-
dc.typeConference_Paper-
dc.identifier.emailNg, HY: hyng0812@hku.hk-
dc.identifier.emailLung, ML: mlilung@hku.hk-
dc.identifier.emailKo, JMY: joko@hku.hk-
dc.identifier.authorityLung, ML=rp00300-
dc.identifier.authorityKo, JMY=rp02011-
dc.identifier.doi10.1158/1538-7445.AM2018-327-
dc.identifier.hkuros286597-
dc.identifier.volume78-
dc.identifier.issue13, Suppl-
dc.identifier.spageAbstract 327-
dc.identifier.epageAbstract 327-
dc.publisher.placeUnited States-

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