149P - MicroRNA profiling and CDDP-GPG DNA adduct formation analysis in a panel of cisplatin resistant non-small cell lung cancer cell lines

Date 30 September 2012
Event ESMO Congress 2012
Session Poster presentation II
Topics Basic Science
Lung and other Thoracic Tumours
Presenter Martin Barr
Authors M. Barr1, S.G. Gray2, D. Fennell3, J.J. O'Leary4, D. Richard5, J. Thomale6, A.C. Hoffmann7, K.J. O'Byrne1
  • 1Clinical Medicine, St James's Hospital, D8 - Dublin/IE
  • 2Clinical Medicine, Trinity College Dublin, D8 - Dublin/IE
  • 3Queen's University Belfast, Belfast/UK
  • 4Histopathology, St James's Hospital, D8 - Dublin/IE
  • 5Queensland University Of Technology, Institute of Health and Biomedical Innovation, Brisbane/AU
  • 6Cancer Research, Institute for Cell Biology, University Hospital Essen, Germany, na - Essen/DE
  • 7Dept. Int. Medicine (cancer Research), Molecular oncology Risk-Profile Evaluation (M.O.R.E) University Hospital Essen, Germany, na - Essen/DE

Abstract

Introduction

The outcome of cisplatin therapy in NSCLC has reached a plateau, with the development of resistance being a major obstacle in the use of this drug. Understanding the molecular mechanisms underlying this resistance phenotype may aid in the development of novel agents that enhance the sensitivity of cisplatin. In this study, we examined the microRNA profile and levels of cisplatin-DNA adduct formation in a panel of cisplatin resistant NSCLC cell lines.

Methods

We have previously generated a panel of cisplatin resistant (CisR) cell lines (MOR, H460, A549, SKMES-1, H1299) from original, age-matched parent (PT) cells and extensively characterised these based on a number of functional assays including proliferation (MTT), apoptosis (FACS), cell cycle arrest (PI staining), survival ability (clonogenic assay), DNA copy number gains and losses (CGH arrays) and cancer stem cell marker expression (CD44, CD133, ALDH1, Nanog, Sox-2 and Oct-4) by FACS and Western blot analysis. MicroRNA profiling was carried out using the nCounter miRNA Expression Assay consisting of 749 target miRNA's. Immunofluorescence staining and measurement of specific DNA platination products (CDDP-GpG DNA adducts) was performed at 0, 4, 12 and 24h using immunofluorescence microscopy and quantitative digital image analysis using the ACAS 6.0 CytometryAnalysis System, respectively.

Results

In a panel of five NSCLC cell lines exhibiting a cisplatin resistant phenotype, a 3- and 13-miRNA signature was deduced, based on differential expression of 3 miRNA's (5/5 cell lines) and 13 miRNA's (4/5 cell lines) in the CisR cell lines relative to their parent counterparts. Levels of CDDP-GpG adducts were greatly reduced in resistant cells up to 24h in response to cisplatin compared to that observed in corresponding parent cells.

Conclusion

We have identified a distinct miRNA fingerprint in a clinically relevant, isogenic model of cisplatin resistance that may predict resistance to cisplatin in lung cancer. Furthermore, as cisplatin-DNA adducts correlate with cisplatin resistance in vitro, these may be used as a potential predictive marker in patient response to platinum therapy in NSCLC.

Disclosure

All authors have declared no conflicts of interest.