J Thorac Oncol. 2013 Mar;8(3):259-69. doi: 10.1097/JTO.0b013e318279e942.

Combined treatment with erlotinib and a transforming growth factor-β type I receptor inhibitor effectively suppresses the enhanced motility of erlotinib-resistant non-small-cell lung cancer cells.

Serizawa M1, Takahashi T, Yamamoto N, Koh Y.

1Drug Discovery and Development Division, and †Division of Thoracic Oncology, Shizuoka Cancer Center Hospital, Shizuoka, Japan.

 

Abstract

INTRODUCTION: Despite an initial dramatic response to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, the majority of non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations develop acquired resistance. Therefore, there is an urgent need to elucidate the unknown mechanisms and biological behaviors of EGFR TKI-resistant lung tumors. We investigated the motility of EGFR TKI-resistant cells, as these characteristics are relevant to cancer metastasis.

METHODS: Erlotinib-resistant PC-9ER cells were generated from PC-9 NSCLC cells, which harbor an EGFR-activating mutation, and used in this study. We investigated the involvement of the transforming growth factor beta (TGF-β) pathway in cell motility, and tested the effects of erlotinib and TGF-β type I receptor (RI) inhibition on cell motility.

RESULTS: PC-9ER cells displayed enhanced motility resulting from autocrine activation of the TGF-β pathway. Increased TGF-β2 secretion resulting from TGF-β2 up-regulation at the transcriptional level was suggested to be responsible for the phosphorylation of Smad2 and the subsequently elevated transcriptional regulatory activity in PC-9ER cells. The motility of PC-9ER cells was suppressed by treatment with either the TGF-βRI inhibitor LY364947 or erlotinib, and greater suppression was observed when used in combination. LY364947 or erlotinib exerted no growth-inhibitory effects, suggesting that motility and growth are driven by different signaling pathways in PC-9ER cells.

CONCLUSIONS: Our results imply that blockade of the TGF-β signaling pathway combined with continuous EGFR TKI treatment will be beneficial in preventing metastasis in patients with EGFR TKI-resistant NSCLC without the EGFR T790M resistance mutation.

PMID: 23334091

 

SUPPLEMENTARY

In this study, we demonstrated that erlotinib-resistant PC-9ER cells established from PC-9 non-small cell lung cancer (NSCLC) cells acquired additional phenotypic and biological characteristics when they acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs).

It is widely known that acquired resistance eventually emerges in patients with EGFR-mutant lung tumors and it is not surprising if those tumors acquire additional biological features at the same time, for example, which may lead to more aggressive/malignant phenotype. Our finding shed light on additional biological changes when tumors become resistant to EGFR-TKI and we revealed that PC-9ER cells acquired enhanced cell motility (Figure 1) via constitutive activation of transforming growth factor β (TGF-β)/SMAD family member 2 (Smad2) pathway due to accelerated TGF-β2 secretion (Figure 2). There has not been any report about the correlation between TGF-β2 and tumor progression and/or metastasis in NSCLC and this is the first report on the involvement of TGF-β2 in metastatic process in NSCLC. The motility of PC-9ER cells was suppressed by treatment not only with TGF-β receptor I inhibitor LY364947 but also with erlotinib (Figure 1), suggesting that PC-9ER cells still retain partial sensitivity to erlotinib in terms of cell motility and continuous treatment with erlotinib may be beneficial in preventing metastasis even after the failure of EGFR-TKI monotherapy, especially in the case without the emergence of EGFR T790M resistance mutation. It has been reported that continuous treatment with EGFR-TKIs beyond progressive disease may be beneficial and the use of EGFR-TKIs after disease progression is under clinical evaluation. These clinical findings seem to support the hypothesis that tumors with acquired resistance and/or non-resistant residual tumors still rely on the EGFR signaling, and blockade of EGFR signaling with continuous treatment with EGFR-TKIs may be still clinically beneficial.

Here we report an additional rationale for continuing the use of EGFR-TKIs after disease progression, that resistant tumors may still retain sensitivity to EGFR-TKIs in terms of inhibiting cell motility (Figure 1, 2). These data indicate that combined blockade of EGFR and TGF-β pathways will be beneficial in preventing metastasis in patients with EGFR-TKI-resistant NSCLC though these results need to be further validated using clinical specimens from patients with acquired resistance to EGFR-TKIs.

Yasuhiro Koh-fig1

Figure 1. Effect of combined blockade of EGFR and TGF-β signaling pathways on wound closure.

 

Yasuhiro Koh-fig2

Figure 2. Proposed mechanisms responsible for the enhanced motility and the acquired resistance to erlotinib in PC-9ER cells. (A) The enhanced motility of PC-9ER cells was induced by the activation of TGF-β/Smad pathway due to the increased autocrine secretion of TGF-β2, and CRKL amplification-induced PI3K/AKT activation is suggested to confer resistance to EGFR-TKIs in PC-9ER cells. Enhanced motility of PC-9ER was suppressed by treatment with either erlotinib (B) or LY364947 (C), and more effectively suppressed by erlotinib and LY364947 combined (D).

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