Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia
Por:
Errazquin, R, Sieiro, E, Moreno, P, Ramirez, MJ, Lorz, C, Peral, J, Ortiz, J, Casado, JA, Roman-Rodriguez, FJ, Hanenberg, H, Rio, P, Surralles, J, Segrelles, C, Garcia-Escudero, R
Publicada:
1 abr 2021
Resumen:
Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.
Filiaciones:
Errazquin, R:
Univ Hosp 12 Octubre, Biomed Res Inst I 12, Madrid 28041, Spain
CIEMAT, Mol Oncol Unit, Madrid 28040, Spain
Sieiro, E:
CIEMAT, Mol Oncol Unit, Madrid 28040, Spain
Moreno, P:
CIEMAT, Mol Oncol Unit, Madrid 28040, Spain
Ramirez, MJ:
Hosp Santa Creu & Sant Pau, Join Res Unit Genom Med UAB St Pau Biomed Res Ins, Barcelona 08041, Spain
Ctr Invest Biomed Enfermedades Raras CIBERER, Madrid 28029, Spain
Lorz, C:
Univ Hosp 12 Octubre, Biomed Res Inst I 12, Madrid 28041, Spain
CIEMAT, Mol Oncol Unit, Madrid 28040, Spain
Ctr Invest Biomed Red Canc CIBERONC, Madrid 28029, Spain
Peral, J:
CIEMAT, Mol Oncol Unit, Madrid 28040, Spain
Ortiz, J:
CIEMAT, Mol Oncol Unit, Madrid 28040, Spain
Casado, JA:
Ctr Invest Biomed Enfermedades Raras CIBERER, Madrid 28029, Spain
CIEMAT, Hematopoiet Innovat Therapies Div, Madrid 28040, Spain
Fdn Jimenez Diaz, Inst Invest Sanitarias, Madrid 28040, Spain
Roman-Rodriguez, FJ:
Ctr Invest Biomed Enfermedades Raras CIBERER, Madrid 28029, Spain
CIEMAT, Hematopoiet Innovat Therapies Div, Madrid 28040, Spain
Fdn Jimenez Diaz, Inst Invest Sanitarias, Madrid 28040, Spain
Hanenberg, H:
Univ Duisburg Essen, Univ Childrens Hosp Essen, D-47057 Essen, Germany
Heinrich Heine Univ, Dept Otorhinolaryngol & Head Neck Surg, D-40225 Dusseldorf, Germany
Rio, P:
Ctr Invest Biomed Enfermedades Raras CIBERER, Madrid 28029, Spain
CIEMAT, Hematopoiet Innovat Therapies Div, Madrid 28040, Spain
Fdn Jimenez Diaz, Inst Invest Sanitarias, Madrid 28040, Spain
Surralles, J:
Hosp Santa Creu & Sant Pau, Join Res Unit Genom Med UAB St Pau Biomed Res Ins, Barcelona 08041, Spain
Ctr Invest Biomed Enfermedades Raras CIBERER, Madrid 28029, Spain
Segrelles, C:
Univ Hosp 12 Octubre, Biomed Res Inst I 12, Madrid 28041, Spain
CIEMAT, Mol Oncol Unit, Madrid 28040, Spain
Ctr Invest Biomed Red Canc CIBERONC, Madrid 28029, Spain
Garcia-Escudero, R:
Univ Hosp 12 Octubre, Biomed Res Inst I 12, Madrid 28041, Spain
CIEMAT, Mol Oncol Unit, Madrid 28040, Spain
Ctr Invest Biomed Red Canc CIBERONC, Madrid 28029, Spain
Green Submitted, Green Published, gold
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