ps100102  (OriGene)

Journal: Cancer Discovery

Article Title: Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

doi: 10.1158/2159-8290.cd-21-0680

Figure Lengend Snippet: Figure 1. TDO2 as a synthetic essential gene for the mutant APC gene in colorectal cancer (CRC). A, Venn diagram analysis using three different data sets identified TDO2 as a top potential SE gene. B, TDO2 mRNA expression is significantly correlated with the expression of WNT pathway signature genes in TCGA colorectal cancer (COAD + READ, provisional) patients (n = 433). ****, P < 0.0001. C, Representative images of IHC staining for TDO2 in serial sectioned human colorectal cancer tumors with negative (n = 34) and positive nuclear β-catenin (n = 47). Scale bars, × 10 (200 μm) and × 40 (50 μm). D, Colorectal cancer tumors with nuclear β-catenin showed higher TDO2 expression (TDO2 staining score 0–3). Pearson correlation coefficient = 42.342; ****, P < 0.0001. Chi-squared test. E, IHC analysis of colorectal cancer tumors from iAP and iKAP mice showed increased nuclear β-catenin, Ki-67, and TDO2 compared with normal colon tissue. Dotted lines separate normal and tumor regions; arrows indicate Ki-67/β-catenin–posi- tive tumor regions. Scale bar, 100 μm for iAP and 500 μm for iKAP. F, Immunoblotting for TDO2 in organoids isolated from C57BL/6J ileum and ApcMin/+ mice. SI, small intestine. G, Immunoblotting for TDO2 in colonoids isolated from C57BL/6J mice. APC-KO colonoids were APC-deleted by CRISPR/Cas9. LI, large intestine.

Article Snippet: The mutated TDO2 ORF gene insert was subcloned into PS100102 (pLenti-C-mGFP-P2A-BSD Tagged Cloning Vector; Origene; cat. #PS10094).

Techniques: Mutagenesis, Expressing, Immunohistochemistry, Staining, Western Blot, Isolation, CRISPR

Journal: Cancer Discovery

Article Title: Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

doi: 10.1158/2159-8290.cd-21-0680

Figure Lengend Snippet: Figure 2. TCF4/TCF7L2 mediates the upregulation of TDO2 in APC-mutated colorectal cancer (CRC) cells. A, Immunoblots for TDO2 and β-catenin in colorectal cancer cell lines RKO (human) and MC38 (mouse) with their isogenic APC-KO counterparts. At least three independent experiments were performed. B, RT-qPCR showed APC-deleted RKO and MC38 cell lines exhibit increased Tdo2 mRNA expression. At least three independent experiments were performed. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. FC, fold change. C, DNA sequence binding motif for the transcription factor TCF4/TCF7L2. Pro- moter regions of human and mouse TDO2 genes harbor TCF4 binding motifs near the transcription start site. The motif sequence is conserved in human and mouse genes. D, ChIP-seq in APC-WT and APC-KO MC38 cells showed binding peaks for TCF4 on the promoters of the TDO2 gene. E, ChIP-PCR using the TCF4 antibody showed enriched binding to the promoter regions of the TDO2 gene in DLD-1 cells. GAPDH as a negative control; MYC and AXIN2 as positive controls. F, Luciferase activity of the human TDO2 (hTDO2) promoter in HEK 293T cells with a constitutively active form of β-catenin (Δ90) when cotransfected with dominant-negative (DN) TCF4. ***, P < 0.001. Two independent experiments were performed. G, Luciferase activity of the hTDO2 pro- moter in HEK 293T cells with a constitutively active form of β-catenin (Δ90) and dominant-negative TCF4. **, P < 0.01. H, Luciferase activity of the TCF4 binding motif–mutated hTDO2 promoter in HEK 293T cells with a constitutively active form of β-catenin (Δ90) and dominant-negative TCF4. n.s., P > 0.05. I, Immunoblots for TDO2 and TCF4 in APC-KO MC38 cell lysates after transfecting with siControl (siCon) or three different siTCF4s.

Article Snippet: The mutated TDO2 ORF gene insert was subcloned into PS100102 (pLenti-C-mGFP-P2A-BSD Tagged Cloning Vector; Origene; cat. #PS10094).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Sequencing, Binding Assay, ChIP-sequencing, Negative Control, Luciferase, Activity Assay, Dominant Negative Mutation

Journal: Cancer Discovery

Article Title: Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

doi: 10.1158/2159-8290.cd-21-0680

Figure Lengend Snippet: Figure 3. TDO2–Kyn–AhR signaling is essential for cell survival in APC-mutated colorectal cancer (CRC) cells. A, Representative images of colony forma- tion assays of APC-WT and APC-KO MC38 cell lines expressing shTDO2. Three independent experiments were performed. B, Quantification of A. n.s., P > 0.05; ***, P < 0.001. FC, fold change; shCont, shControl. C, Immunoblots of cleaved caspase-3 in APC-WT and APC-KO MC38 cell lines with inducible shTDO2 after doxycycline (Dox) treatment. D, Immunoblots for TDO2 and cleaved caspase-3 in ApcMin/+ ishControl and ishTDO2 organoid cell lysates after Dox treatment for 48 hours. Three independent experiments were performed. E, Brightfield images of APC-WT and APC-KO colonoids treated with DMSO or 20 μm TDO2 inhibi- tor (680C91) for 48 hours. Scale bars, × 40 (50 μm). F, Total flux measurement of tumors in Supplementary Fig. S4E. n.s., P > 0.05; *, P < 0.05; **, P < 0.01. G, Survival curves of C57BL/6J mice orthotopically implanted with ishTDO2 APC-WT and APC-KO MC38 cell lines (2 × 105 cells). Dox food was supplied at day 5 after orthotopic injection to induce TDO2 knockdown in vivo. n.s., P > 0.05; **, P < 0.01; ***, P < 0.001. Log-rank (Mantel–Cox) test. H, Survival curves of C57BL/6J mice orthotopically implanted with APC-WT and APC-KO MC38 cell lines (2 × 105 cells). TDO2 inhibitor (TDO2i) treatment (100 mg/kg) was initiated at day 5 after injection twice a day by oral gavage. n.s., P > 0.05; **, P < 0.01; ***, P < 0.001. Log-rank (Mantel–Cox) test. I, Survival curves of iAP mice after tamoxifen induction in the distal colon. Vehicle or TDO2 inhibitor treatment (100 mg/kg) was initiated at day 24 after induction once a day by oral gav- age. Log-rank (Mantel–Cox) test.

Article Snippet: The mutated TDO2 ORF gene insert was subcloned into PS100102 (pLenti-C-mGFP-P2A-BSD Tagged Cloning Vector; Origene; cat. #PS10094).

Techniques: Expressing, Western Blot, Injection, Knockdown, In Vivo

Journal: Cancer Discovery

Article Title: Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

doi: 10.1158/2159-8290.cd-21-0680

Figure Lengend Snippet: Figure 4. TDO2 mediates tumor growth by regulating macrophage infiltration. A, GSEA (Hallmark gene sets) on genes that overlap between RNA-seq data sets of ishTDO2 APC-KO MC38 cell lines (no Dox vs. 48-hour Dox, n = 3) and microarray data sets of allograft tumors established with ishTDO2 APC- KO MC38 cell lines (no Dox vs. Dox treated, n = 3). The blue bars indicate immune response–related pathways. RNA-seq data from APC-KO MC38 ishTDO2 cell lines and tumor microarray data sets from the tumors established by the cell lines in C57BL/6J mice were overlapped and further narrowed down the list using the pathways that are upregulated by APC deletion to identify the pathways regulated by both the WNT pathway and TDO2. B, GSEA correla- tion of TNFA signaling and inflammatory response with alternatively expressed genes in TDO2-depleted APC-KO MC38 cells. Normalized enrichment scores (NES) and nominal P values are shown. C, viSNE analysis of F4/80+ and CD206+ immune cells assessed by CyTOF from colorectal cancer orthotopic ishTDO2 APC-WT and APC-KO MC38 tumors. D, Quantification of macrophages (CD11b+ F4/80+) and M2 macrophages (CD11b+ F4/80+ CD206hi) in CD45+ cells from tumors shown in C. CyTOF data were analyzed by FlowJo. Data, mean ± SD. n.s., P > 0.05; *, P < 0.05. n = 3 per group. E, TDO2 mRNA expression significantly correlates with the expression of total macrophage markers and M2 macrophage markers in TCGA colorectal cancer (COAD + READ, pro- visional) patients (n = 433). ****, P < 0.0001. F, Representative images of IHC staining for CD163 in serial sectioned human colorectal cancer tumors with negative (n = 42) and positive (n = 50) nuclear β-catenin. Scale bars, × 10 (200 μm) and × 20 (100 μm). G, Colorectal cancer tumors with nuclear β-catenin showed higher CD163 expression. Pearson correlation coefficient = 5.074, P = 0.0243. Chi-squared test. *, P < 0.05.

Article Snippet: The mutated TDO2 ORF gene insert was subcloned into PS100102 (pLenti-C-mGFP-P2A-BSD Tagged Cloning Vector; Origene; cat. #PS10094).

Techniques: RNA Sequencing, Microarray, Expressing, Immunohistochemistry