Polo-like kinase 1 inhibition diminishes acquired resistance to epidermal growth factor receptor inhibition in non-small cell lung cancer with T790M mutations

Abstract

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective against non-small cell lung cancer (NSCLC) with activating EGFR mutations, but resistance is inevitable. Mechanisms of acquired resistance include T790M mutations and epithelial-mesenchymal transition (EMT). One potential strategy for overcoming this resistance is the inhibition of polo-like kinase 1 (PLK1) based on our previous studies showing that mesenchymal NSCLC cell lines are more sensitive to PLK1 inhibition than epithelial cell lines. To determine the extent to which PLK1 inhibition overcomes EGFR TKI resistance we measured the effects of the PLK1 inhibitor volasertib alone and in combination with the EGFR inhibitor erlotinib in vitro and in vivo in EGFR mutant NSCLC cell lines with acquired resistance to erlotinib. Two erlotinib-resistant cell lines that underwent EMT had higher sensitivity to volasertib, which caused G2/M arrest and apoptosis, than their parental cells. In all NSCLC cell lines with T790M mutations, volasertib markedly reduced erlotinib resistance. All erlotinib-resistant NSCLC cell lines with T790M mutations had higher sensitivity to erlotinib plus volasertib than to erlotinib alone, and the combination treatment caused G2/M arrest and apoptosis. Compared with either agent alone, the combination treatment also caused significantly more DNA damage and greater reductions in tumor size. Our results suggest that PLK1 inhibition is clinically effective against NSCLC that becomes resistant to EGFR inhibition through EMT or the acquisition of a T790M mutation. These results uncover new functions of PLK1 inhibition in the treatment of NSCLC with acquired resistance to EGFR TKIs.

Keywords: drug resistance; epidermal growth factor receptor; epithelial–mesenchymal transition; non-small cell lung cancer; polo-like kinase.

Figure 1. Characteristics of parental and ER NSCLC cell lines

Parental and ER NSCLC cell lines were assessed for protein expression with Western blotting and for viability with the CellTiter-Glo assay. E-cadherin, vimentin, and PLK1 protein expression from the Western blot analysis (A) were quantitated using the Image J software program (B).

Figure 2. PLK1 inhibition is sufficient to induce cell cycle arrest and apoptosis in NSCLC cell lines that have undergone EMT

HCC4006-ER2 (A) and HCC827-ER3 (B) cell lines were treated with 60 nM volasertib and/or 2 μM erlotinib or with vehicle controls and the cell cycle was analyzed with a BrdU FITC flow cytometry kit with 7AAD after 48 h. Apoptosis was assessed with an APO-BrdU TUNEL assay. *p < 0.05 compared with vehicle control after 72 hours.

Figure 3. PLK1 inhibition plus EGFR inhibition is synergistic in ER PC9 cell lines harboring T790M EGFR mutations

The viability of ER PC9 cell lines treated with volasertib and/or erlotinib for 72 h was assessed with the CellTiter-Glo assay. The CI of the two drugs was calculated using the Calcusyn software program. CI depicts synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1).

Figure 4. PLK1 inhibition plus EGFR inhibition induces cell cycle arrest and apoptosis in ER PC9 cell lines harboring T790M EGFR mutations

PC9-ER9 and PC9-ER11 cell lines were treated with 60 nM volasertib and/or 2 μM erlotinib or with vehicle controls for 48 h (A) or 72 h (B). (A) The cell cycle was analyzed with a BrdU FITC flow cytometry kit with 7AAD. (B) Apoptosis was analyzed by assessing typical morphological changes in PC9-ER9 cells (upper left); performing a APO-BrdU TUNEL assay (lower panels); and performing Western blotting for cleaved PARP levels (upper right). *p < 0.05 compared with single-agent volasertib or erlotinib.

Figure 5. PLK1 inhibition plus EGFR inhibition induces DNA damage but does not substantially affect canonical EGFR downstream pathways in ER PC9 cell lines harboring T790M EGFR mutations

PC9-ER9 and PC9-ER11 cell lines were treated with 60 nM volasertib and/or 2 μM erlotinib or with vehicle controls for 2 h (A) or 48 h (B–D). (A, C, D) Cells were lysed and subjected to Western blotting with the indicated antibodies. (B) Representative fluorescence microscopy images of a comet assay (left panel); quantification comet tail moment in individual cells (right panel). *p < 0.05.

Figure 6. Inhibition of both PLK1 and EGFR is more effective than inhibition of either target alone in ER NSCLC xenograft models. Mice bearing PC9-ER9 xenograft tumors with volumes of 150 mm³ were treated with volasertib and/or erlotinib or with vehicle controls

(A) Tumor volume was measured twice weekly and significantly decreased after combination treatment. After 3 weeks of treatment, the mice were humanely killed. Resected tumors were subjected to immunohistochemical analysis for Ki67 (C) and caspase 3 (B) protein expression, which was scored and quantitated.