Gene Expression Profiling of Triple Negative Breast Cancer Cells Treated with Afatinib

Purpose: To investigate gene expression profile changes in triple negative breast cancer cells (MDA-MB-231) treated with afatinib.

Methods: Differential expression of 84 genes commonly involved in breast cancer carcinogenesis was examined in MDA-MB-231 cells treated with afatinib (5 µM) and compared to untreated cells. Total RNA was extracted using RNeasy mini kit and subsequently assessed by real-time PCR using the Human Breast Cancer RT2 Profiler PCR Array. Relative gene expression was computed using the ΔΔCt approach and a fold change equal to or greater than 2 was considered significant.

Results: Treatment of MDA-MB-231 cells with afatinib (5 µM) for 24 h resulted in significant differential expression of several genes commonly involved in breast cancer carcinogenesis. Specifically, 33 of the 84 genes examined exhibited greater than two-fold differential expression when MDA-MB-231 cells were exposed to afatinib. Three genes (CTSD, ESR2 and ID1) were upregulated while thirty genes were downregulated in afatinib treated cells compared to control. Core analysis of differentially expressed genes using the Ingenuity Pathway Analysis (IPA) software identified five regulatory networks pertinent to cell cycle, cancer, cellular growth and proliferation. This led to phosphoinositide-3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/ERK being identified as pathways impacted by afatinib.

Conclusions: Our findings elucidate molecular targets with altered expression in MDA-MB-231 cells exposed to afatinib. Based on RT2-PCR array analysis, afatinib increased expression of key tumor suppressor genes and down-regulated expression of pivotal oncogenes. This knowledge could contribute to the design and development of effective afatinib based combination therapies for treating TNBC.