FOXK2 promotes ovarian cancer stemness by regulating the unfolded protein response pathway
Yaqi Zhang, … , Mazhar Adli, Daniela Matei
Yaqi Zhang, … , Mazhar Adli, Daniela Matei
Published March 29, 2022
Citation Information: J Clin Invest. 2022;132(10):e151591. https://doi.org/10.1172/JCI151591.
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Research Article Cell biology Oncology

FOXK2 promotes ovarian cancer stemness by regulating the unfolded protein response pathway

Abstract

Understanding the regulatory programs enabling cancer stem cells (CSCs) to self-renew and drive tumorigenicity could identify new treatments. Through comparative chromatin-state and gene expression analyses in ovarian CSCs versus non-CSCs, we identified FOXK2 as a highly expressed stemness-specific transcription factor in ovarian cancer. Its genetic depletion diminished stemness features and reduced tumor initiation capacity. Our mechanistic studies highlight that FOXK2 directly regulated IRE1α (encoded by ERN1) expression, a key sensor for the unfolded protein response (UPR). Chromatin immunoprecipitation and sequencing revealed that FOXK2 bound to an intronic regulatory element of ERN1. Blocking FOXK2 from binding to this enhancer by using a catalytically inactive CRISPR/Cas9 (dCas9) diminished IRE1α transcription. At the molecular level, FOXK2-driven upregulation of IRE1α led to alternative XBP1 splicing and activation of stemness pathways, while genetic or pharmacological blockade of this sensor of the UPR inhibited ovarian CSCs. Collectively, these data establish what we believe is a new function for FOXK2 as a key transcriptional regulator of CSCs and a mediator of the UPR, providing insight into potentially targetable new pathways in CSCs.

Authors

Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Edward J. Tanner, Mazhar Adli, Daniela Matei

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Figure 4

FOXK2 directly regulates IRE1α and activates the unfolded protein response.

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FOXK2 directly regulates IRE1α and activates the unfolded protein respon...
(A) Top 15 canonical pathways identified by Ingenuity Pathway Analysis (IPA) among DEGs determined by RNA-seq in OVCAR5 transduced with shFOXK2-2 (shFOXK2) or control shRNAs (shCtrl). (B) Heatmap shows mRNA expression levels (RNA-seq) of 25 genes involved in the UPR pathway in shCtrl and shFOXK2 OVCAR5 cells. (C and D) Ratios measured by qRT-PCR (n = 3) of the XBP1 mRNA spliced isoform (XBP1s) relative to the unspliced XBP1 (XBP1u) in shCtrl- and shFOXK2-transduced OVCAR5 and OVCAR3 cells (C), and in OVCAR5 cells transduced with dCas9-NT or dCas9-ERN1-1/2 (D). (E and F) RT-PCR products resolved by agarose gel electrophoresis of the XBP1u and the XBP1s in shCtrl- and shFOXK2-transduced OVCAR5 and OVCAR3 cells (E) and in EV (control) and FOXK2-overexpressing (FOXK2-OE) OVCAR5 and OVCAR3 cells (F). (G and H) Western blot of FOXK2, IRE1α, spliced XBP1 (XBP1s), unspliced XBP1 (XBP1u), and GAPDH in shCtrl- and shFOXK2-transduced OC cells (n = 3) (G), and in OVCAR3 and OVCAR5 cells transduced with EV or FOXK2-OE (n = 3) (H). (I) qRT-PCR–measured mRNA levels (n = 3) of XBP1s, HIF1α, VEGFA, and DDIT4 in shCtrl- and shFOXK2-transduced OVCAR5 and OVCAR3 cells. (J) mRNA levels (n = 3) of XBP1, HIF1α, and DDIT4 measured by qRT-PCR in xenografts derived from shCtrl or shFOXK2 OVCAR5 cells. (K) mRNA expression levels (n = 3) of XBP1s, HIF1α, VEGFA, and DDIT4 in OVCAR5 and OVCAR3 transfected with EV or FOXK2-OE. *P < 0.05; **P < 0.01; ****P < 0.0001, by unpaired, 2-tailed Student’s t test.