Generation and characterisation of cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature.
BACKGROUND
Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.


METHODS
An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed.


RESULTS
Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.


CONCLUSIONS
Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.
Thoracic Oncology, Institute of Molecular Medicine, Trinity Centre for Health Sciences St. James's Hospital & Trinity College Dublin, Dublin, Ireland. mbarr@stjames.ie
Martin P
MP
Barr
Steven G
SG
Gray
Andreas C
AC
Hoffmann
Ralf A
RA
Hilger
Juergen
J
Thomale
John D
JD
O'Flaherty
Dean A
DA
Fennell
Derek
D
Richard
John J
JJ
O'Leary
Kenneth J
KJ
O'Byrne
D000071916:AC133 Antigen; D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000378:metabolism*; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D018736:DNA Adducts / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D005434:Flow Cytometry; D006023:Glycoproteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D000071317:Nanog Homeobox Protein; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
D000071916:AC133 Antigen; D015703:Antigens, CD; D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D018736:DNA Adducts; D006023:Glycoproteins; D018398:Homeodomain Proteins; D018960:Hyaluronan Receptors; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C000606023:PROM1 protein, human; D010455:Peptides; D055748:SOXB1 Transcription Factors; D051176:beta Catenin; D000444:Aldehyde Dehydrogenase; D019859:Proto-Oncogene Proteins c-met; D002945:Cisplatin