FOXK2 promotes ovarian cancer stemness by regulating the unfolded protein response pathway
Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Edward J. Tanner, Mazhar Adli, Daniela Matei
Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Edward J. Tanner, Mazhar Adli, Daniela Matei
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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 6

XBP1 splicing is associated with stemness characteristics.

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XBP1 splicing is associated with stemness characteristics. (A) Annexin V...
(A) Annexin V staining and flow cytometry–measured apoptotic cells among sorted ALDH+ and ALDH– OVCAR5 cells treated with DMSO, 2 μg/mL tunicamycin, or 10 μM STF-083010 (n = 3). (B) qRT-PCR determination of the full-length XBP1 transcript (XBP1u) and the spliced isoform (XBP1s) in OVCAR5 and OVCAR3 cells transduced with 2 different shRNAs directed at XBP1 (shXBP1) or with control shRNA (shCtrl). (C) XBP1 mRNA expression levels measured by qRT-PCR (n = 3) in shCtrl and shXBP1 OVCAR5 cells. (D) Percentage of ALDH+ cells (n = 3) in shXBP1- and shCtrl-transduced OVCAR5 cells. (E) Spheroid formation assessed by a cell viability assay in shCtrl- and shXBP1-transduced OVCAR5 cells (n = 5). (F) mRNA expression levels (n = 3) of SOX2, OCT4, NANOG, and ALDH1A1 in shCtrl- and shXBP1-transduced OVCAR5 and OVCAR3 cells measured by qRT-PCR. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001, by unpaired, 2-tailed Student’s t test.