Abstract A2-59: Resistance mechanisms to erlotinib in the non-small cell lung cancer cell line, HCC827 examined by RNA-seq
Authors: Jacobsen Kirstine , Alcaraz Nicolas , Lund Rikke Raaen , Ditzel Henrik Jørn 
Published: November 2015

Abstract
Background: Erlotinib, an EGFR selective reversible inhibitor, has dramatically changed the treatment of non-small cell lung cancer (NSCLC) as approximately 70% of patients show significant tumor regression upon treatment. However, all patients eventually relapse due to development of acquired resistance, which in 43-50% of cases is caused by a secondary mutation (T790M) in EGFR, and in 5-15% of cases is caused by MET amplification. However, a majority of resistance cases are still unexplained. Consequently, our aim was to identify novel resistance mechanisms – and potentially new drug targets - in erlotinib-resistant subclones of the NSCLC cell line HCC827.
Materials and methods: We established 3 erlotinib-resistant subclones (resistant to 10, 20, 30 µM erlotinib, respectively), and prepared cDNA libraries of purified RNA from biological duplicates using TruSeq® Stranded Total RNA Ribo-Zero™ Gold (Illumina) prior to sequencing on an Illumina HiSeq platform (100bp paired end). The resistant subclones were examined both in presence and absence of erlotinib. The data was analyzed by an in-house developed pipeline including quality control by Trim Galore v0.3.3, mapping of reads to HG19 by TopHat2 v.2.0.10 and differential expression analysis by CuffDiff from the Cufflinks package v.2.2.1.
Results: Significant differences in viability between the resistant clones and parental HCC827 were observed when incubated with erlotinib. Importantly, the resistant clones did not acquire the T790M mutation or other EGFR/KRAS mutations, indicating that other mechanisms are responsible for the resistance in these cells.
We identified 303-581 transcripts being &gt;2-fold upregulated (p &lt; 0.001), and 460-679 transcripts being &gt;2-fold downregulated (p &lt; 0.001) in the three resistant clones treated with erlotinib compared to the parental cell line, HCC827. Among others, we found networks such as growth and proliferation, migration and oxidative stress to be upregulated by Ingenuity Pathway Analysis (IPA), whereas adherens junction signaling was downregulated. More specifically, transcripts encoding potential resistance drivers such as AXL kinase and Fibroblast Growth Factor Receptor 1 (FGFR1) were found consistently upregulated together with a number of central metabolic enzymes, which may also be important for resistance. Vimentin and CD44 encoding transcripts involved with epithelial-to-mesenchymal transition were consistently upregulated and CDH1 encoding E-cadherin was downregulated, indicating that EMT may be correlated with resistance to erlotinib. EGFR was either not regulated or downregulated in the three clones. MET was not regulated, whereas p53 expression was consistently downregulated.
Conclusions: We established 3 erlotinib-resistant NSCLC subclones, which did not harbor any of the common resistance mutations, thus allowing for the identification of novel resistance mechanisms in these subclones. Cancer-related networks such as proliferation and migration were upregulated in the resistant clones, supporting the validity of the model. While EGFR was either not regulated or down-regulated, other potential resistance drivers such as AXL kinase and FGFR1, among others, were found consistently upregulated in the three resistant clones, which may indicate a cooperative bypass signaling mechanism to obtain resistance to erlotinib.
Citation Format: Kirstine Jacobsen, Nicolas Alcaraz, Rikke Raaen Lund, Henrik Jørn Ditzel. Resistance mechanisms to erlotinib in the non-small cell lung cancer cell line, HCC827 examined by RNA-seq. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-59.