Introduction Small Cell Lung Cancer (SCLC) typically responds well to initial chemotherapy with Etoposide and a platinum-containing agent. However survival is poor due to invariable relapse with chemoresistant disease. We used a unique series of SCLC cell lines (GLC-14, 16, 19), derived from a single patient at various time-points during her treatment, to identify genes involved in Etoposide resistance. We then attempted to determine their functional role and validate their importance using patient specimens.
Methods The relationship of the cell lines to each other was confirmed using genomic methods. Genes whose expression pattern could explain the relative response of the cell lines to treatment with Etoposide were identified using cDNA microarray. These candidate Etoposide response genes were cloned from the cell line in which they were expressed at the highest level and transiently over-expressed in the cell line in which they were naturally expressed at the lowest level to determine whether this altered Etoposide resistance. Using immunohistochemistry, expression of the most promising candidates was examined using a tissue microarray (TMA).
Results The two most promising candidate genes were identified as DNA Polymerase β, a DNA repair enzyme and NKX2.2, a neuroendocrine transcription factor. Specific inhibition of DNA Polymerase β with Pamoic Acid reduced the numbers of cells surviving treatment with Etoposide (p=0.029) and increased the amount of DNA damage in the cells (p<0.001). Stable overexpression of NKX2.2 significantly increased cell survival in response to Etoposide in two different SCLC cell lines. In keeping with this, we found that absence of nuclear staining for NKX2.2 in the TMA was an independent predictor of improved outcome in chemotherapy treated SCLC patients (HR 0.52, 95% CI 0.33 to 0.82, p=0.005).
Conclusion Using a biologically plausible model of in vivo acquired resistance to Etopoide we have identified two novel Etoposide resistance factors—DNA Polymerase β and NKX2.2. Our in vitro data, in conjunction with the TMA results, provide justification for further prospective work to confirm the roles of these molecules in chemotherapy resistance in SCLC.
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