Lung cancer cells survive epidermal growth factor receptor tyrosine kinase inhibitor exposure through upregulation of cholesterol synthesis.

Shyam Mohapatra,Rajesh Nair,Andrew Hanna,Waise Quarni,Subhra Mohapatra,Mark C. Howell,Roukiah Khalil,Elspeth Foran,Stanley Stevens,Ryan Green,Aleksandr Grinchuk

doi:10.1096/fba.2019-00081

Abstract

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) provide clinical benefits over chemotherapy for lung cancer patients with EGFR activating mutations. Despite initial clinical responses, long‐term efficacy is not possible because of acquired resistance to these therapies. We have developed EGFR TKI drug‐tolerant (DT) human lung cancer cell lines as a model for de novo resistance. Mass spectroscopic analysis revealed that the cytochrome P450 protein, CYP51A1 (Lanosterol 14α‐demethylase), which is directly involved with cholesterol synthesis, was significantly upregulated in the DT cells. Total cellular cholesterol, and more specifically, mitochondrial cholesterol, were found to be upregulated in DT cells. We then used the CYP51A1 inhibitor, ketoconazole, to downregulate cholesterol synthesis. In both parental and DT cells, ketoconazole and EGFR TKIs acted synergistically to induce apoptosis and overcome the development of EGFR tolerance. Lastly, this combination therapy was shown to shrink the growth of tumors in an in vivo mouse model of EGFR TKI resistance. Thus, our study demonstrates for the first time that ketoconazole treatment inhibits upregulation of mitochondrial cholesterol and thereby overcomes EGFR‐TKI resistance in lung cancer cells.

Highlights

About 20% of all non-small cell lung cancer (NSCLC) patients harbor an epidermal growth factor receptor (EGFR) activating mutation.[1]
These results show an increase in total cellular cholesterol levels, as well as enzymes directly involved in cholesterol synthesis in both parental cells treated with EGFR Tyrosine kinase inhibitor (TKI) and EGFR TKI-DT cells
When the cholesterol depleting agent, methyl-β-cyclodextrin (MBCD), was added to the media in addition to the Lipogro, the tolerance to lapatinib was lowered by 2-fold to 18uM (Figure 3H). These results show that increased levels of cholesterol in the cells can lead to increased EGFR TKItolerance, which can be reversed by stopping cholesterol production or depleting cholesterol levels

Summary

Introduction

About 20% of all non-small cell lung cancer (NSCLC) patients harbor an epidermal growth factor receptor (EGFR) activating mutation.[1] EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been shown to provide clinical benefits over chemotherapy for lung cancer patients with EGFR activating mutations.[2] Some first generation-(gefitinib, erlotinib, lapatinib), second generation-(afatinib), and third-generation (osimertinib) EGFR TKIs are clinically approved to treat NSCLC patients.[3] Lapatinib is a special case, as it is qualified as a dual TKI, which interrupts both the HER2 and EGFR pathways, and is commonly used to treat patients with metastatic breast cancer whose tumors overexpress HER2.4 Despite the initial clinical responses to EGFR targeted therapies, acquired drug resistance hampers TKI effectiveness in most patients.[1,3] Target alteration, increased ligand production, increased downstream pathway activation, and alternative pathway activation have all been proposed as mechanisms of resistance to EGFR TKIs.[1,3] Numerous cellular signaling pathways have been implicated in EGFR TKI resistance.[1,5,6,7,8,9,10,11,12,13,14,15,16,17]

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Discussion

Conclusion