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 2

FOXK2 regulates tumor initiation and stemness gene expression in OC cells.

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FOXK2 regulates tumor initiation and stemness gene expression in OC cell...
(A) Log-fraction plot of serial dilutions of shCtrl and shFOXK2 OVCAR5 cells (n = 4 mice/group) estimated by ELDA. (B) Xenografts collected from mice in indicated groups (n = 4 mice/group). (C) Growth curves of xenografts from the 5000 cells/mice group in B (n = 4 mice). (D) Percentage of ALDH+ cells determined by flow cytometry in xenografts generated by shCtrl (n = 8) and shFOXK2 (n = 6) cells. (E and F) FOXK2 mRNA levels (n = 3) (E) and representative images of spheroids (original magnification, ×20) (n = 6) (F) of OVCAR5 and OVCAR3 cells transfected with empty vector (EV) or FOXK2 expression vector (FOXK2-OE). (G) Percentages of ALDH+ cells in EV- or FOXK2-OE–transduced OC cells (n = 3). (H) Western blot of protein levels of FOXK2/Foxk2 in OVCAR5 shCtrl and shFOXK2 cells transduced with EV (shCtrl-EV, shFOXK2-EV) or Foxk2 (shCtrl-Foxk2, shFOXK2-Foxk2) (n = 3). (I and J) Percentages of ALDH+ CSCs (n = 3) (I) and cell viability in spheroid cultures (n = 6) formed from OVCAR5 shCtrl and shFOXK2 cells transduced with EV or Foxk2. (K) Log-fraction plot of serial dilutions of shCtrl and shFOXK2 cells transduced with EV or Foxk2 (n = 4 mice/group) generated from ELDA. (L and M) mRNA levels of SOX2, OCT4, NANOG, and ALDH1A1 in shCtrl- or shFOXK2-transduced OVCAR5 cells (n = 3) and cells from HGSOC tumors (n = 3) (L), and in OVCAR5 shCtrl and shFOXK2 cells transduced with EV or Foxk2 (n = 3) (M). (N) Heatmap shows differentially expressed genes (DEGs) measured by RNA-seq in shCtrl versus shFOXK2 cells (269 genes, n = 2). (O) Scatter plot shows overlapping genes among DEGs in CSCs (ALDH+CD133+) vs non-stem cells (ALDH–CD133–) (n = 2) and shCtrl versus shFOXK2 OVCAR5 cells (n = 2). *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001, by unpaired, 2-tailed Student’s t test when comparing 2 groups and 2-way ANOVA with Tukey’s multiple comparisons test when comparing more than 2 groups.