Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest.
Increasing evidence suggests that tumor-initiating cells (TICs), also called cancer stem cells, are partly responsible for resistance to DNA-damaging treatment. Here we addressed if such a phenotype may contribute to radio- and cisplatin resistance in non-small cell lung cancer (NSCLC). We showed that four out of eight NSCLC cell lines (H125, A549, H1299 and H23) possess sphere-forming capacity when cultured in stem cell media and three of these display elevated levels of CD133. Indeed, sphere-forming NSCLC cells, hereafter called TICs, showed a reduced apoptotic response and increased survival after irradiation (IR), as compared with the corresponding bulk cell population. Decreased cytotoxicity and apoptotic signaling manifested by diminished poly (ADP-ribose) polymerase (PARP) cleavage and caspase 3 activity was also evident in TICs after cisplatin treatment. Neither radiation nor cisplatin resistance was due to quiescence as H125 TICs proliferated at a rate comparable to bulk cells. However, TICs displayed less pronounced G2 cell cycle arrest and S/G2-phase block after IR and cisplatin, respectively. Additionally, we confirmed a cisplatin-refractory phenotype of H125 TICs in vivo in a mouse xenograft model. We further examined TICs for altered expression or activation of DNA damage repair proteins as a way to explain their increased radio- and/or chemotherapy resistance. Indeed, we found that TICs exhibited increased basal γH2AX (H2A histone family, member X) expression and diminished DNA damage-induced phosphorylation of DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated (ATM), Krüppel-associated protein 1 (KAP1) and monoubiquitination of Fanconi anemia, complementation group D2 (FANCD2). As a proof of principle, ATM inhibition in bulk cells increased their cisplatin resistance, as demonstrated by reduced PARP cleavage. In conclusion, we show that reduced apoptotic response, altered DNA repair signaling and cell cycle perturbations in NSCLC TICs are possible factors contributing to their therapy resistance, which may be exploited for DNA damage-sensitizing purposes.
Department of Oncology-Pathology, Karolinska Biomics Center, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden.
L
L
Lundholm
P
P
Hååg
D
D
Zong
T
T
Juntti
B
B
Mörk
R
R
Lewensohn
K
K
Viktorsson
D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects / Q000528:radiation effects; D006023:Glycoproteins / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D051379:Mice; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011839:Radiation, Ionizing; D012097:Repressor Proteins / Q000378:metabolism; D014183:Transplantation, Heterologous; D000076183:Tripartite Motif-Containing Protein 28; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D000071916:AC133 Antigen; D015703:Antigens, CD; D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006023:Glycoproteins; D006657:Histones; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28; D011065:Poly(ADP-ribose) Polymerases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D002945:Cisplatin