Targeting the FOXO1/KLF6 axis regulates EGFR signaling and treatment response
Jaya Sangodkar, … , Analisa DiFeo, Goutham Narla
Jaya Sangodkar, … , Analisa DiFeo, Goutham Narla
Published June 1, 2012
Citation Information: J Clin Invest. 2012;122(7):2637-2651. https://doi.org/10.1172/JCI62058.
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Research Article Oncology

Targeting the FOXO1/KLF6 axis regulates EGFR signaling and treatment response

Abstract

EGFR activation is both a key molecular driver of disease progression and the target of a broad class of molecular agents designed to treat advanced cancer. Nevertheless, resistance develops through several mechanisms, including activation of AKT signaling. Though much is known about the specific molecular lesions conferring resistance to anti-EGFR–based therapies, additional molecular characterization of the downstream mediators of EGFR signaling may lead to the development of new classes of targeted molecular therapies to treat resistant disease. We identified a transcriptional network involving the tumor suppressors Krüppel-like factor 6 (KLF6) and forkhead box O1 (FOXO1) that negatively regulates activated EGFR signaling in both cell culture and in vivo models. Furthermore, the use of the FDA-approved drug trifluoperazine hydrochloride (TFP), which has been shown to inhibit FOXO1 nuclear export, restored sensitivity to AKT-driven erlotinib resistance through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft models of lung adenocarcinoma. Combined, these findings define a novel transcriptional network regulating oncogenic EGFR signaling and identify a class of FDA-approved drugs as capable of restoring chemosensitivity to anti-EGFR–based therapy for the treatment of metastatic lung adenocarcinoma.

Authors

Jaya Sangodkar, Neil S. Dhawan, Heather Melville, Varan J. Singh, Eric Yuan, Huma Rana, Sudeh Izadmehr, Caroline Farrington, Sahar Mazhar, Suzanna Katz, Tara Albano, Pearlann Arnovitz, Rachel Okrent, Michael Ohlmeyer, Matthew Galsky, David Burstein, David Zhang, Katerina Politi, Analisa DiFeo, Goutham Narla

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Figure 6

Targeted reduction of KLF6 in the erlotinib-sensitive HCC827 cell line confers drug resistance in culture and in vivo.

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Targeted reduction of KLF6 in the erlotinib-sensitive HCC827 cell line c...
(A) qRT-PCR for KLF6 mRNA expression in HCC827 cells treated with 50 nM erlotinib normalized to GAPDH over 24 hours. (B) Western blot for KLF6 and cleaved PARP expression in HCC827 cells treated with erlotinib over for 24 hours. (C) Cell cycle analysis using flow cytometry of the sub-G1 fraction after propidium iodide staining. (D) qRT-PCR for KLF6 mRNA expression in the control cell line shLuc-HCC827 and stable knockdown cell line shKLF6-HCC827 after treatment with 50 nM erlotinib. (E) Western blot for expression of KLF6, cleaved PARP, p-AKT, p-ERK. (F) Cell cycle analysis using flow cytometry of the sub-G1 cell cycle fraction after propidium iodide staining. (G) Clonogenic assay of shLuc-HCC827 and shKLF6-HCC827 cells treated with 0 or 50 nM erlotinib for 7 days; quantification of colonies is presented. (H–J) Growth curves of subcutaneous xenograft tumors generated from 1 × 107 shLuc-HCC827 or shKLF6-HCC827 cells injected into the right posterior flank of nude mice following an initial growth period of 14 days. Group tumor volume (n = 4) averaged 150 mm3 prior to treatment. Tumor measurements were made 48 hours after each injection. (H) Fold change of tumor volume over the duration of treatment described above represented as a box-and-whisker plot. (I) Mean tumor volume of shLuc-HCC827 xenograft tumors treated with DMSO (vehicle control) or erlotinib (25 mg/kg). (J) Mean tumor volume of shKLF6-HCC827 xenograft tumors treated with DMSO or erlotinib (25 mg/kg). *P < 0.05, **P < 0.01, ***P < 0.001.