The online version of this article (doi:10.1186/1476-4598-11-41) contains supplementary material, which is available to authorized users.
Fiona O’Neill, Stephen F Madden, Padraig Doolan and Robert O’Connor contributed equally to this work.
The authors declare that they have no competing interests.
FON and SFM contributed equally to this work. SFM performed all of the bioinformatic/statistical analysis. FON treated the cells with lapatinib extracted the RNA and performed Taqman RT PCR and the proliferation assay. STA participated in the study design, RNA extraction and TaqMan RT PCR and analysis and interpretation of the results. FON, SFM, STA, JC, MC ROC and PD contributed to the result interpretation and manuscript preparation. ROC and PD equally conceived the study, participated in its design, coordination and interpretation of the results and finalized the manuscript. All authors read and approved the final manuscript.
Lapatinib, a tyrosine kinase inhibitor of HER2 and EGFR and is approved, in combination with capecitabine, for the treatment of trastuzumab-refractory metastatic breast cancer. In order to establish a possible gene expression response to lapatinib, a panel of breast cancer cell lines with varying sensitivity to lapatinib were analysed using a combination of microarray and qPCR profiling.
Co-inertia analysis (CIA), a data integration technique, was used to identify transcription factors associated with the lapatinib response on a previously published dataset of 96 microarrays. RNA was extracted from BT474, SKBR3, EFM192A, HCC1954, MDAMB453 and MDAMB231 breast cancer cell lines displaying a range of lapatinib sensitivities and HER2 expression treated with 1 μM of lapatinib for 12 hours and quantified using Taqman RT-PCR. A fold change ≥ ± 2 was considered significant.
A list of 421 differentially-expressed genes and 8 transcription factors (TFs) whose potential regulatory impact was inferred in silico, were identified as associated with lapatinib response. From this group, a panel of 27 genes (including the 8 TFs) were selected for qPCR validation. 5 genes were determined to be significantly differentially expressed following the 12 hr treatment of 1 μM lapatinib across all six cell lines. Furthermore, the expression of 4 of these genes (RB1CC1, FOXO3A, NR3C1 and ERBB3) was directly correlated with the degree of sensitivity of the cell line to lapatinib and their expression was observed to “switch” from up-regulated to down-regulated when the cell lines were arranged in a lapatinib-sensitive to insensitive order. These included the novel lapatinib response-associated genes RB1CC1 and NR3C1. Additionally, Cyclin D1 (CCND1), a common regulator of the other four proteins, was also demonstrated to observe a proportional response to lapatinib exposure.
A panel of 5 genes were determined to be differentially expressed in response to lapatinib at the 12 hour time point examined. The expression of these 5 genes correlated directly with lapatinib sensitivity. We propose that the gene expression profile may represent both an early measure of the likelihood of sensitivity and the level of response to lapatinib and may therefore have application in early response detection.
Additional File 1 : Lapatinib modulated genes responding early or at low dosage. (XLS 84 KB)12943_2012_1015_MOESM1_ESM.xls
Additional File 2 : A ranked list of TFBS associated with a response to lapatinib treatment for each of the 6 comparisons in 0 Table 1. Highlighted in bold are those TFBS consistent across the 6 comparisons. (XLS 35 KB)
Additional File 3 : A full list of the differentially regulated genes and the TFs that are predicted to target them. This file also contains the fold change and the adjusted p-value for each of the six comparisons. (XLS 360 KB)12943_2012_1015_MOESM3_ESM.xls
Additional File 4 : Statistical Overrepresentation of the TFs identified by CIA. (XLS 22 KB)12943_2012_1015_MOESM4_ESM.xls
Additional File 5 : RQ values for all genes tested, including the TFs. (XLS 28 KB)12943_2012_1015_MOESM5_ESM.xls
Additional File 6 : Figure S1. Expression of PIK3C3, ALDH3A2 and CDKN1B across the six cell lines. Figure S2. Basal gene expression (ΔCt) of RB1CC1, FOXO3A, NR3C1, ERBB3 and CCND1 across the six cell lines. (PPT 625 KB)12943_2012_1015_MOESM6_ESM.ppt
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- Gene expression changes as markers of early lapatinib response in a panel of breast cancer cell lines
Stephen F Madden
Sinead T Aherne
- BioMed Central
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