Inhibiting IRE1α-endonuclease activity decreases tumor burden in a mouse model for hepatocellular carcinoma
- Department of Medical Cell Biology, Uppsala University, Sweden
- Regenerative Medicine & Fibrosis Group, Institute for Liver and Digestive Health, University College London, United Kingdom
- Department of Radiology, Uppsala University Hospital, Sweden
Figures
Figure 1
Inhibiting IRE1α reduces tumor burden in vivo.
(A) Representative images of liver slides stained with hematoxylin and eosin (H and E), Sirius red and αSMA-antibodies. (B) tumor burden of mice with DEN-induced HCC treated with 4μ8C or …
Figure 2 with 2 supplements
Increased expression of ER-stress markers in mice with HCC.
(A) mRNA expression of ER-stress markers Edem1, Ero1b, Grp94, Herp, Atf4, Eif2ak3, Ddit3, and Hspa5 in liver tissue from healthy mice; and tumor tissue and surrounding non-tumoral tissue from mice …
Figure 2—figure supplement 1
Activation of the unfolded protein response is mainly located in the stroma of mice with HCC.
Liver tissue from mice with DEN-induced HCC, stained with αSMA-antibodies and co-stained with antibodies against (A) spliced XBP1, (B) total XBP1, (C) IRE1α (D) phopho-IRE1α, and (E) BIP. Scale bars …
Figure 2—figure supplement 2
Expression of ER-stress markers is localized in close vicinity to αSMA.
Immunofluorescent images from tissue from mice with DEN-induced HCC, stained with αSMA-antibodies and co-stained with antibodies against (A) spliced XBP1, (B) total XBP1, (C) IRE1α, (D) …
Figure 3
Activation of the unfolded protein response pathway is increased in patients with fibrotic HCC.
(A) Heat map showing gene-set enrichment analysis results from samples from fibrous HCC versus non-fibrous HCC. (C) Immunohistochemically stained liver biopsies from HCC-patients obtained from the …
Figure 4 with 1 supplement
Tumor cells secrete factors that induce ER-stress in stellate cells, which contributes to their activation.
(A) mRNA-expression of ER-stress markers ATF6, ATF4, EIF2AK3, GADD34, EDEM1, DDIT3 and HSPA5, in stellate cells (LX2) co-cultured with cancer cells (HepG2 or Huh7) and treated with 4μ8C or control. …
Figure 4—figure supplement 1
Secretion of TGFβ by tumor cells activates stellate cells and induces ER-stress.
(A) Concentration of TGFβ in medium from tumor cells (HepG2 or Huh7) grown in mono-culture or co-cultured with LX2-stellate cells, treated with 4μ8C or control. (B) Concentration of TGFβ in medium …
Figure 5
Inhibiting IRE1α decreases stellate cell activation in human liver 3D scaffolds engrafted with stellate cells and tumor cells.
(A) Representative images of H and E and Sirius red stained slides of decellularized human liver scaffolds engrafted with LX2 stellate cells and HepG2-tumor cells treated with 4μ8C or control. (B) …
Figure 6
Inhibition of IRE1α decreases tumor cell proliferation.
(A) PCNA mRNA-expression of HepG2 or Huh7-cells grown with LX2-cells in transwell inserts and treated with the IRE1α-inhibitor 4μ8C or control. (B) Relative cell number of LX2 and HepG2 or (C) LX2 …
Figure 7
Inhibition of IRE1α decreases cell proliferation and improves liver function in human liver scaffolds engrafted with stellate cells and tumor cells.
(A) PCNA and (B) HNF4A expression of human liver scaffolds engrafted with HepG2-tumor cells and LX2-stellate cells, treated with 4μ8C or control. (C) Representative images of tumor cells (HepG2) and …
Figure 8 with 1 supplement
Inhibition of IRE1α decreases cell migration.
(A) mRNA-expression of pro-metastatic markers MMP9 and (B) MMP1 in HepG2 and Huh7-cells co-cultured with LX2-cells and treated with 4μ8C or control. (C) Scratch wound on HepG2-cells and LX2-cells …
Figure 8—figure supplement 1
Inhibiting IRE1α decreases chemotaxis.
(A) Migration plots of LX2-cells co-cultured with HepG2-cells exposed to an FBS-gradient (increasing towards the right) and treated with control or (B) 4μ8C (C) Quantification of total migration and …
Figure 9 with 1 supplement
Silencing IRE1α in LX2-cells mimics 4μ8C.
(A) ERN1-mRNA-expression of LX2-cells transfected with IRE1α-siRNA (si-IRE1α) or mock-transfected (Scr) (B) PCNA-mRNA-expression of HepG2-cells co-cultured with IRE1α-silenced LX2-cells or controls …
Figure 9—figure supplement 1
Proliferation and migration after silencing IRE1α in LX2-cells.
(A) Proliferation of spheroids of HepG2-cells and IRE1α-silenced LX2-cells or controls (B) Images and (C) quantification of αSMA-stained spheroids with HepG2-cells and IRE1α-silenced LX2-cells or …
Figure 10
Inhibiting IRE1α alters generation of ROS.
(A) intracellular ROS-levels in LX2, HepG2, and Huh7 cells treated with 50 μM 4μ8C, 100 μM 4μ8C or controls. (B) intracellular ROS-levels in LX2, HepG2 and Huh7 cells transfected with IRE1α-siRNA …
Author response image 1
Effect of ER-stress on ROS production.
(A) the ER-stress inducer increases ROS levels in HepG2 and Huh7 cells. (B) The ROS-scavenging effect of 4u8C increases over time.
Author response image 2
Effect of silencing IRE1a in different co-culture conditions.
(A) LX2-cells transfected with si-RNA targeting IRE1a or mock-transfected (Scr) in mono-culture or co-culture with tumor cells (HepG2 and Huh7). (B) HepG2 or Huh7-cells transfected with si-RNA …
Author response image 3
Visible bands in lane 2 and 4, which corresponds to LX2-monocultures and LX2+Hep2 co-cultures treated with 4u8C.
Author response image 4
Protein levels of IRE1a in mock transfected and si-IRE1a transfected Huh7 cells.
Tables
Table 1
A proteomics array using the Olink Mouse Exploratory assay – source data Figure 1F.
| CTL | Den | DEN+4 u8c | Statistical significance | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Protein name | Biological process | Mean | St. Dev | Average | St. Dev | Average | St. Dev | DEN vs Ctrl | DEN vs 4 u8C | Ctrl vs 4 u8c |
| Clmp | Not prognostic in HCC | 1.68 | 0.14 | 2.97 | 1.00 | 2.48 | 0.64 | * | ||
| Yes1 | HCC promotor | 7.11 | 0.29 | 7.51 | 0.20 | 7.44 | 0.19 | * | ||
| Foxo1 | Tumor suppressor | 4.15 | 0.06 | 4.12 | 0.73 | 3.87 | 0.49 | |||
| Pla2g4a | HCC promotor | 3.42 | 0.38 | 5.70 | 1.36 | 5.04 | 0.80 | * | * | |
| Prdx5 | HCC promotor | 7.37 | 0.49 | 7.23 | 0.26 | 6.67 | 0.34 | * | ||
| Tgfa | Tumor growth factor | 5.36 | 0.52 | 6.81 | 0.64 | 6.93 | 0.88 | * | * | |
| Epo | Unfavorable prognotic marker | 3.20 | 0.34 | 3.71 | 0.35 | 3.37 | 0.33 | |||
| Axin1 | HCC promotor | 4.24 | 0.38 | 4.80 | 0.37 | 4.39 | 0.35 | |||
| Fst | HCC promotor | 5.87 | 0.31 | 8.04 | 0.73 | 7.50 | 0.71 | * | * | |
| Nadk | Not prognostic in HCC | 10.10 | 0.13 | 10.14 | 0.18 | 10.30 | 0.27 | |||
| Snap29 | Not prognostic in HCC | 7.70 | 0.32 | 7.87 | 0.32 | 7.62 | 0.30 | |||
| S100a4 | HCC promotor | 2.73 | 0.74 | 7.01 | 0.62 | 6.85 | 0.97 | * | * | |
| Kitlg | Metastasis | 2.48 | 0.42 | 3.74 | 0.62 | 3.31 | 0.98 | * | ||
| Gfra1 | HCC promotor | 4.40 | 0.35 | 5.07 | 0.40 | 4.92 | 0.39 | * | ||
| Ppp1r2 | Not prognostic in HCC | 4.37 | 0.16 | 4.86 | 0.46 | 4.47 | 0.43 | |||
| Cyr61 | HCC promotor | 2.40 | 0.53 | 4.14 | 1.64 | 3.13 | 1.22 | * | ||
| Ahr | Not prognostic in HCC | 6.95 | 0.46 | 7.68 | 0.74 | 7.38 | 0.64 | |||
| Ccl2 | HCC promotor | 4.59 | 0.58 | 9.69 | 2.04 | 8.93 | . | * | * | |
| Qdpr | Not prognostic in HCC | 7.71 | 0.11 | 7.72 | 0.14 | 7.54 | 0.15 | |||
| Fas | HCC promotor | 8.66 | 0.18 | 8.83 | 0.18 | 8.70 | 0.18 | |||
| Riox2 | HCC promotor | 7.10 | 0.15 | 7.71 | 0.38 | 7.59 | 0.14 | * | * | |
| Epcam | HCC promotor | 1.56 | 0.33 | 3.16 | 1.14 | 3.27 | 0.89 | * | ||
| Ccl3 | Prognostic marker | 1.49 | 0.39 | 4.42 | 1.86 | 3.73 | 1.07 | * | * | |
| Crim1 | HCC promotor | 2.46 | 0.28 | 3.71 | 1.09 | 3.21 | 0.56 | * | * | |
| Hgf | Tumor growth factor | 6.69 | 0.35 | 7.94 | 1.01 | 7.41 | 0.71 | * | ||
| Sez6l2 | HCC promotor | −0.29 | 0.15 | 0.61 | 0.53 | 0.19 | 0.29 | * | ||
| Il1a | Inflammation and fibrosis | 6.65 | 0.51 | 8.35 | 0.65 | 7.62 | 0.54 | * | * | |
| Ddah1 | HCC promotor | 8.04 | 0.22 | 8.18 | 0.05 | 7.84 | 0.18 | * | ||
| Acvrl1 | Not prognostic in HCC | 2.09 | 0.18 | 3.44 | 1.31 | 2.81 | 0.47 | |||
| Cxcl9 | Inflammation and fibrosis | 3.68 | 0.86 | 7.71 | 1.68 | 6.65 | 1.58 | * | * | |
| Map2k6 | Not prognostic in HCC | 7.75 | 0.15 | 7.98 | 0.41 | 7.88 | 0.28 | |||
| Casp3 | Tumor surrpressor | 9.22 | 0.19 | 9.74 | 0.35 | 9.43 | 0.26 | |||
| Pdgfb | Tumor growth factor | 3.52 | 0.31 | 4.96 | 1.27 | 3.97 | 0.40 | * | ||
| Igsf3 | Unfavorable prognotic marker | 3.12 | 0.28 | 4.19 | 0.82 | 3.64 | 0.72 | |||
| Cxcl1 | HCC promotor | 3.77 | 0.40 | 5.74 | 0.78 | 5.06 | 0.51 | * | * | |
| Pak4 | HCC promotor | 3.47 | 0.42 | 4.39 | 0.68 | 3.93 | 0.54 | |||
| Lpl | Not prognostic in HCC | 1.66 | 0.40 | 2.44 | 0.45 | 2.02 | 0.60 | |||
| Dctn2 | Unfavorable prognotic marker | 5.48 | 1.31 | 5.67 | 0.70 | 4.98 | 0.55 | |||
| Ntf3 | Not prognostic in HCC | 2.16 | 0.27 | 2.80 | 0.71 | 2.27 | 0.40 | |||
| Tnfsf12 | HCC promotor | 5.28 | 0.35 | 6.00 | 0.76 | 5.59 | 0.62 | |||
| Ccl20 | Unfavorable prognotic marker | 5.20 | 0.34 | 5.92 | 0.81 | 5.53 | 0.66 | |||
| Fli1 | HCC promotor | 1.91 | 0.22 | 3.73 | 1.38 | 2.98 | 0.83 | |||
| Tpp1 | Unfavorable prognotic marker | 3.67 | 0.38 | 4.24 | 0.64 | 3.73 | 0.50 | |||
| Parp1 | Unfavorable prognotic marker | 10.30 | 0.72 | 10.93 | 0.49 | 10.51 | 0.62 | |||
Table 2
Genes the contributed to the core-enrichment of the GSEA.
| Probe | Description | Rank Gene list | Rank Metric score | Core enrichment | UPR branch |
|---|---|---|---|---|---|
| ASNS | Asparagine synthetase (glutamine-hydrolyzing) [Source:HGNC Symbol;Acc:HGNC:753] | 207 | 0.940 | Yes | Perk |
| PPP2R5B | Protein phosphatase two regulatory subunit B'beta [Source:HGNC Symbol;Acc:HGNC:9310] | 423 | 0.821 | Yes | Ire1a |
| CCL2 | C-C motif chemokine ligand 2 [Source:HGNC Symbol;Acc:HGNC:10618] | 847 | 0.689 | Yes | Ire1a and Perk |
| EXOSC9 | Exosome component 9 [Source:HGNC Symbol;Acc:HGNC:9137] | 1004 | 0.654 | Yes | Ire1a and Perk |
| WIPI1 | WD repeat domain, phosphoinositide interacting 1 [Source:HGNC Symbol;Acc:HGNC:25471] | 1022 | 0.649 | Yes | Ire1a |
| KDELR3 | KDEL endoplasmic reticulum protein retention receptor 3 [Source:HGNC Symbol;Acc:HGNC:6306] | 1106 | 0.635 | Yes | Ire1a |
| SHC1 | SHC adaptor protein 1 [Source:HGNC Symbol;Acc:HGNC:10840] | 2691 | 0.432 | Yes | Ire1a |
| TPP1 | Tripeptidyl peptidase 1 [Source:HGNC Symbol;Acc:HGNC:2073] | 2884 | 0.414 | Yes | Ire1a |
| HDGF | Heparin binding growth factor [Source:HGNC Symbol;Acc:HGNC:4856] | 3235 | 0.386 | Yes | Ire1a |
| TLN1 | Talin 1 [Source:HGNC Symbol;Acc:HGNC:11845] | 3264 | 0.384 | Yes | Ire1a |
| EXTL3 | Exostosin like glycosyltransferase 3 [Source:HGNC Symbol;Acc:HGNC:3518] | 3488 | 0.365 | Yes | Ire1a |
| TSPYL2 | TSPY like 2 [Source:HGNC Symbol;Acc:HGNC:24358] | 3680 | 0.350 | Yes | Ire1a |
| MBTPS1 | Membrane-bound transcription factor peptidase, site 1 [Source:HGNC Symbol;Acc:HGNC:15456] | 3996 | 0.327 | Yes | Atf6 |
| PDIA5 | Protein disulfide isomerase family A member 5 [Source:HGNC Symbol;Acc:HGNC:24811] | 4530 | 0.294 | Yes | Ire1a |
| DCTN1 | Dynactin subunit 1 [Source:HGNC Symbol;Acc:HGNC:2711] | 4638 | 0.287 | Yes | Ire1a |
| DNAJC3 | DnaJ heat-shock protein family (Hsp40) member C3 [Source:HGNC Symbol;Acc:HGNC:9439] | 4761 | 0.281 | Yes | Ire1a |
| SULT1A4 | Sulfotransferase family 1A member 4 [Source:HGNC Symbol;Acc:HGNC:30004] | 4938 | 0.272 | Yes | Ire1a |
| PARN | Poly(A)-specific ribonuclease [Source:HGNC Symbol;Acc:HGNC:8609] | 5037 | 0.266 | Yes | Perk |
| ADD1 | Adducin 1 [Source:HGNC Symbol;Acc:HGNC:243] | 5375 | 0.250 | Yes | Ire1a |
| ERN1 | Endoplasmic reticulum to nucleus signaling 1 [Source:HGNC Symbol;Acc:HGNC:3449] | 5411 | 0.248 | Yes | Ire1a |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Sv129 mice | Taconic | 129S6 | HCC mouse model, Heindryckx et al., 2010; Heindryckx et al., 2012 |
| Cell line (Homo sapiens) | HepG2 | ATCC | HB-8065 | |
| Cell line (Homo sapiens) | Huh7 | Gifted, Karolinska institute | ||
| Cell line (Homo sapiens) | LX2 | Sigma-Aldrich | SCC064 | |
| Transfected construct (human) | si-IRE1α | ThermoFisher | s200432 | 0,1–1 µM |
| Transfected construct (human) | Si-Ctrl; Scr | ThermoFisher | 4390843 | 0,1–1 µM |
| Antibody | KI67 (rat monoclonal) | eBioscience | SolA15 | 1:100 |
| Antibody | EPCAM (rabbit polyclonal) | Abcam | ab71916 | 1:100 |
| Antibody | Spliced XBP1 (goat monoclonal) | Abcam | Ab85546 | 1:50 |
| Antibody | Total XBP1 (Rabbit polyclonal) | Abcam | Ab37152 | 5 µg/ml |
| Antibody | IRE1a (rabbit polyclonal) | Abcam | Ab37073 | 1 µg/ml |
| Antibody | p-IRE1 (rabbit polyclonal) | AbNova | PAB12435 | 1:100 |
| Antibody | αSMA (Rabbit Polyclonal) | ThermoFisher | 710487 | 1:200 |
| Antibody | αSMA (Goat monocolonal) | Abcam | Ab21027 | 1–2 µg/ml |
| Antibody | BIP (goat polyclonal) | Abcam | Ab21027 | 1 µg/ml |
| Antibody | Vinculin (Mouse monoclonal) | ThermoFisher | 14-9777-82 | 1–5 µg/ml |
| Peptide, recombinant protein | Pst-I | ThermoFisher | ER0615 | |
| Commercial assay or kit | Pierce BCA-protein assay kit | ThermoFisher | 233225 | |
| Commercial assay or kit | EZNA RNA isolation Kit II | VWR | R6934-02 | |
| Commercial assay or kit | RNeasy Universal Mini Kit | Qiagen | 73404 | |
| Commercial assay or kit | Diva Decloacker solution | Biocare | DV2004 | |
| Commercial assay or kit | DCFDA - Cellular ROS Detection Assay Kit | Abcam | ab113851 | |
| Chemical compound, drug | N-Nitrosodiethylamine, DEN | Sigma-Aldrich | 1002877809 | |
| Chemical compound, drug | 4μ8C | Sigma-Aldrich | SML0949-25MG | Heindryckx et al., 2016 |
| Chemical compound, drug | SB-431541, TGF-ß receptor inhibitor | Tocris | 1614 | 10 μM |
| Chemical compound, drug | Resazurin | Sigma-Aldrich | R7017-1G | 1:80 dilution |
| Commercial assay or kit | Ingenio electroporation solution | Mirus Bio LLC | MIR50114 | Ice-cold |
| Commercial assay or kit | CellTracker Red CMTPX | ThermoFisher | C34552 | 1 μM |
| Commercial assay or kit | CellTracker Green CMFDA | ThermoFisher | C2925 | 1 μM |
| Other | 12-well CorningCostar Transwellplates | Sigma-Aldrich | 3460 | Calitz et al., 2020 |
| Other | Corning CostarUltra-Low attachment 96-well plates | Sigma-Aldrich | CLS3471 | Calitz et al., 2019 |
| Other | CellDirector | GradienTech | 11-001-10 | Fuchs et al., 2020 |
Additional files
-
Supplementary file 1
Table with primer sequences.
- https://cdn.elifesciences.org/articles/55865/elife-55865-supp1-v2.docx
-
Supplementary file 2
Table with antibodies used for staining.
- https://cdn.elifesciences.org/articles/55865/elife-55865-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55865/elife-55865-transrepform-v2.docx
