Phosphoprotein analysis reveals MEK inhibition as a way to target non-small cell lung cancer tumor initiating cells

Abstract

Purpose: We aimed to analyze the activation status of commonly deregulated receptor tyrosine kinases (RTK) in human non-small cell lung cancer (NSCLC) tumor initiating cells (TIC) previously demonstrated to be refractory to ionizing radiation and chemotherapy.

Materials and methods: Phosphorylated RTK and important signaling nodes were assayed using PathScan RTK Signaling Antibody Array Kit in NSCLC TIC and bulk cells 4 h post-irradiation (IR) and validated by Western blot. The effect of mitogen- activated protein kinase kinase (MEK) inhibition combined with IR was analyzed using clonogenic assay.

Results: H125 TIC displayed decreased basal phosphorylation of insulin-like growth factor 1 receptor (IGF-1R) and signal transducer and activator of transcription 1 (STAT1) (Tyr701) as compared to bulk cells. Total IGF-1R levels were significantly lower in NSCLC TIC as compared to bulk cells. A higher degree of extracellular signal-regulated kinase (ERK) phosphorylation was evident in TIC and concordantly MEK inhibition reduced TIC viability. Moreover, MEK inhibition also decreased clonogenicity upon IR suggesting that MEK and downstream signaling impart on TIC radiation response.

Conclusions: We demonstrate reduced basal phosphorylation of several signaling pathways including lower total IGF-1R levels in NSCLC TIC which is of potential concern for RTK inhibitor use. Importantly, MEK inhibition decreased cell viability of NSCLC TIC alone or in combination with IR.

Keywords::

• Tumor initiating cell
• cancer stem cell
• IGF-1R
• ERK
• MEK
• non-small cell lung cancer

Acknowledgements

This research was supported by grants from the Swedish Cancer Society (grant agreement 120761/2012), the Cancer Society in Stockholm (to RL and KV), the Swedish Research Council (to RL, grant agreement 90266701/2009), the Swedish National Board of Health and Welfare, the Stockholm County Council and Karolinska Institutet research funds. The financial support from the Swedish Cancer Society (to LL) as a post-doctoral grant for lung cancer research is greatly appreciated. We are grateful to Mrs Birgitta Mörk for excellent technical assistance.

Declaration of interest:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.