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. 2019 Jun 21;10(7):490.
doi: 10.1038/s41419-019-1729-4.

Expression of UPR effector proteins ATF6 and XBP1 reduce colorectal cancer cell proliferation and stemness by activating PERK signaling

Claudia N Spaan  1 , Wouter L Smit  1 , Jooske F van Lidth de Jeude  1 , Bartolomeus J Meijer  1 , Vanesa Muncan  1 , Gijs R van den Brink  1   2 , Jarom Heijmans  3   4
Affiliations

Expression of UPR effector proteins ATF6 and XBP1 reduce colorectal cancer cell proliferation and stemness by activating PERK signaling

Claudia N Spaan et al. Cell Death Dis. .

Abstract

The unfolded protein response (UPR) acts through its downstream branches, PERK-eIF2α signaling, IRE1α-XBP1 signaling and ATF6 signaling. In the intestine, activation of the UPR through the kinase PERK results in differentiation of intestinal epithelial stem cells and colon cancer stem cells, whereas deletion of XBP1 results in increased stemness and adenomagenesis. How downstream activation of XBP1 and ATF6 influences intestinal stemness and proliferation remains largely unknown. We generated colorectal cancer cells (LS174T) that harbor doxycycline inducible expression of the active forms of either XBP1(s) or ATF61-373. Activation of either XBP1 or ATF6 resulted in reduced cellular proliferation and reduced expression of markers of intestinal epithelial stemness. Moreover, XBP1 and ATF6 activation reduced global protein synthesis and lowered the threshold for UPR activation. XBP1-mediated loss of stemness and proliferation resulted from crossactivation of PERK-eIF2α signaling and could be rescued by constitutive expression of eIF2α phosphatase GADD34. We thus find that enforced activation of XBP1 and ATF6 results in reduction of stemness and proliferation. We expose a novel interaction between XBP1 and PERK-eIF2α signaling.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. LS174T cells expressing XBP1(s) or ATF61–373 upregulate general UPR target genes.
a Quantitative RT-PCR analysis for XBP1(s) and downstream target genes upon induction of XBP1(s) expression. b Protein level of XBP1(s) and GRP78 in LS174T XBP1(s)Tet On cells. Cells were incubated for 0, 1 or 3 h with thapsigargin 200 nM. Note that transgenic XBP1(s) had a slightly lower molecular weight than endogenous XBP1(s). c Immunostaining of XBP1 in LS174T XBP1(s)Tet On cells. d Quantitative RT-PCR analysis for ATF61–373 and downstream target genes upon induction of ATF61–373 expression. e Protein level of GRP78 in LS174T ATF61–373 Tet On cells. Cells were treated with vehicle or doxycycline 1 μg/mL for 18 h. All data are shown as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2. XBP1(s) or ATF61–373 expression results in reduced stemness and cell proliferation in LS174T colorectal cancer cells.
a Quantitative RT-PCR analysis for intestinal stem cell markers LGR5, OLFM4 and ASCL2 or Wnt target genes LGR5 and AXIN2 in LS174T XBP1(s)Tet On cells. b Crystal violet cell viability assay in LS174T XBP1(s)Tet On cells. c FACS-based EdU incorporation assay in LS174T XBP1(s)Tet On cells; the assay was performed after 2 h of EdU incorporation. d Quantitative RT-PCR analysis for intestinal stem cell markers LGR5, OLFM4 and ASCL2 or Wnt target genes LGR5 and AXIN2 in LS174T ATF61–373 Tet On cells. e Crystal violet cell viability assay in LS174T ATF61–373 Tet On cells. f FACS-based EdU incorporation assay in LS174T ATF61–373 Tet On cells; the assay was performed after 2 h of EdU incorporation. All data are shown as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3. XBP1(s) or ATF61–373 expression upregulates PERK-eIF2α and inhibits global translation.
a, c, e LS174T XBP1(s)Tet On cells. b, d, f LS174T ATF61–373 Tet On cells. a, b Protein levels of PERK and phosphorylated EIF2α. Cells were incubated for 0, 1 or 3 h with thapsigargin 200 nM. c, d Global protein translation rate, measured by 35SMethionine incorporation assay. e, f Percentage of apoptotic cells, measured with propidium iodide and Annexin V staining on Flow Cytometry. All data are shown as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4. Inhibition of eIF2α phosphorylation rescues XBP1(s) induced growth arrest.
a Quantitative RT-PCR analysis for hamster GADD34 in cells expressing either a mock or a GADD34 vector. b Protein levels of GRP78 and phosphorylated eIF2α in LS174T XBP1(s)Tet On cells expressing either a mock or a GADD34 vector. c Quantitative RT-PCR analysis for XBP1(s) and downstream target genes upon induction of XBP1(s) expression in the presence of either a mock or a GADD34 vector. d Global protein translation rate, measured by 35SMethionine incorporation assay. e Crystal violet cell viability assay in LS174T XBP1(s)Tet On cells. f FACS-based EdU incorporation assay in LS174T XBP1(s)Tet On cells; assay was performed after 2 h of EdU incorporation. g Quantitative RT-PCR analysis for intestinal stem cell markers LGR5 and OLFM4. h Percentage of apoptotic cells, measured with propidium iodide and Annexin V staining on Flow Cytometry. (i) Quantification of cell cycle analysis performed on Flow Cytometry using propidium iodide. All data are shown as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
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