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 5

IRE1α/XBP1s promotes stemness features.

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IRE1α/XBP1s promotes stemness features. (A) Heatmap shows levels of DEGs...
(A) Heatmap shows levels of DEGs (from RNA-seq) among those listed in the “hallmark of unfolded protein response” GSEA gene set in ovarian CSCs (ALDH+CD133+) versus non-CSCs (ALDH–CD133–) sorted by FACS from OVCAR5 cells (n = 2). (B) RT-PCR products resolved by agarose gel electrophoresis of the full-length XBP1 transcript (XBP1u) and the spliced isoform (XBP1s) in ALDH+ and ALDH– cells sorted by FACS from OVCAR5 and COV362 cells. (C) Representative pictures of spheroids formed from OVCAR5 and OVCAR3 cells treated with the IRE1α inhibitor STF-083010 (STF) or DMSO (original magnification, ×20) (n = 5). (D) Effects of IRE1α inhibition on spheroid formation assessed by measuring cell viability in OVCAR5 and OVCAR3 cells (n = 5 cultures) and in cells isolated from 3 HGSOC tumors (n = 5 per dose). (E) Percentage of ALDH+ cells measured by flow cytometry (n = 3) in OVCAR5 and OVCAR3 cells treated with STF-083010 or vehicle (DMSO). (F) mRNA expression levels of SOX2, OCT4, NANOG, and ALDH1A1 measured by qRT-PCR in OVCAR5 cells treated with STF-083010 or DMSO (n = 3). (G) qRT-PCR–measured mRNA expression levels (n = 3) of XBP1s, HIF1α, VEGFA, and DDIT4 in OVCAR5 cells treated with STF-083010 or DMSO. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001, by unpaired, 2-tailed Student’s t test.