Targeting the FOXO1/KLF6 axis regulates EGFR signaling and treatment response
Jaya Sangodkar, … , Analisa DiFeo, Goutham Narla
Jaya Sangodkar, … , Analisa DiFeo, Goutham Narla
Published July 2, 2012; First 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 5

Activated EGFR signaling regulates KLF6 expression via the transcription factor FOXO1.

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Activated EGFR signaling regulates KLF6 expression via the transcription...
qRT-PCR for (A) FOXO1 and (C) KLF6 mRNA expression in A549 cells transiently transfected with pCINEO-FOXO1 construct and analyzed after 48 hours. Data are shown as fold change in mRNA expression compared with control empty vector–transfected cells and normalized to GAPDH. (B) KLF6 promoter activity measured by a dual-reporter assay in the presence of FOXO1 overexpression in A549 cells. Data are shown as fold change compared with empty vector–transfected cells. (D) Western blot for KLF6, FOXO1, and GAPDH protein expression after transfection with pCI-neo-FOXO1 construct and pCI-neo empty control vector in A549 cells. (E) EGFR/FOXO1/KLF6 signaling pathway represented by protein Western blot for p-EGFR, EGFR, p-AKT, AKT, p-FOXO1, FOXO1, KLF6, PARP, and GAPDH in HCC827 cells 24 hours after treatment with erlotinib. (F) Western blot for FOXO1, KLF6, histone H3, and GAPDH in HCC827 cells treated with 50 nM erlotinib for 24 hours and subjected to nuclear/cytoplasmic fractionation. Values represent relative protein expression, normalized to histone H3. (G and H) qRT-PCR for FOXO1 and KLF6 mRNA expression normalized to GAPDH in HCC827 cells after transfection with sequence-specific siRNAs to FOXO1 or small, interfering non-targeting control (siNTC) and subsequent treatment with erlotinib. (I) Western blot for FOXO1, KLF6, and the apoptotic marker caspase-3, normalized to GAPDH in HCC827 cells after transfection with sequence-specific siRNAs to FOXO1 or control siNTC and subsequent treatment with erlotinib. *P < 0.05, **P < 0.01, ***P < 0.001.