VPS9D1-AS1 overexpression amplifies intratumoral TGF-β signaling and promotes tumor cell escape from CD8+ T cell killing in colorectal cancer
- Medical Research Center, Beijing Chao-Yang Hospital, Capital Medical University, China
- Department of General Surgery, Beijing Chao-Yang Hospital, Capital Medical University, China
- Department of Cellular and Molecular Biology, Beijing Chest Hospital, Capital Medical University & Beijing Tuberculosis and Thoracic Tumor Research Institute, China
Figures
Figure 1 with 1 supplement
VPS9D1-AS1 is significantly upregulated in colorectal cancer (CRC) and activates the TGF-β signaling pathway.
(A) RNAscope stained VPS9D1-AS1 in CRC tissues that were enrolled in OUTDO (upper) and BJCYH cohorts (lower). Semiquantitative analyses of the levels of VPS9D1-AS1 in cancer and normal tissues of …
Figure 1—figure supplement 1
Levels of VPS9D1-AS1 were not related to TGF-β signaling in cancer stromal cells.
(A) Representative image of VPS9D1-AS1 expressed in normal colonic epithelial cells. (B) Clinical pathologic analyses demonstrated that VPS9D1-AS1 was correlated with lymph node metastasis and TNM …
Figure 2 with 1 supplement
VPS9D1-AS1 is associated with reduced T lymphocyte infiltration.
(A) Representative pictures of T cell infiltration (CD4, CD8, FOXP3) in colorectal cancer (CRC) tissues for VPS9D1-AS1 quantification. Tumor cells are marked by cytokeratin. (B) Representative …
Figure 2—figure supplement 1
Integrative analysis of the relationship between VPS9D1-AS1, TGF-β signaling, and T-infiltrating lymphocytes (TILs).
(A) The Cancer Genome Atlas analysis confirmed that the highest VPS9D1-AS1 expression was predominantly in consensus molecular subtype 2 (CMS2)-type colorectal cancer patients. (B) CMS2 patients …
Figure 3 with 3 supplements
VPS9D1-AS1 controls TGF-β signaling and drives cell proliferation and metastasis.
(A) The levels of VPS9D1-AS1 were determined by qRT-PCR in 16 cell lines. (colorectal cancer: HCT116, SW620, HT29, RKO, SW480; cervical cancer: Hela; lung cancer: A549, H1299; gastric cancer: BGC823;…
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Figure 3—source data 1
TGF-β, TGFBR1, SMAD4, SMAD1/5/9, SMAD6, SMAD2/3, β-actin, N-cadherin, E-cadherin, vimentin, CK, ERK, and p-ERK western blot for Figure 3C.
- https://cdn.elifesciences.org/articles/79811/elife-79811-fig3-data1-v2.zip
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Figure 3—source data 2
RPS3 RNA-pull-down western blot for Figure 3K.
- https://cdn.elifesciences.org/articles/79811/elife-79811-fig3-data2-v2.zip
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Figure 3—source data 3
SMAD1/5/9, RPS3, TGF-β, and TGFBR1 western blot for Figure 3L.
- https://cdn.elifesciences.org/articles/79811/elife-79811-fig3-data3-v2.zip
Figure 3—figure supplement 1
VPS9D1-AS1 activated TGF-β signaling.
(A) Northern blotting detected the effectiveness of sgRNA targeting VPS9D1-AS1 in SW480 cells. (B) qRT-PCR was used to determine the levels of VPS9D1-AS1 in stable knockout cells (sgControl vs. …
Figure 3—figure supplement 2
VPS9D1-AS1 regulated TGF-β signaling and promoted tumor proliferation and migration.
(A, B) mRNA levels of TGF-β, TGFBR1, SMAD1, ~5, and ~9 after VPS9D1-AS1 knockout or knockdown. (C) Human recombinant (h) TGF-β- and SB431542-treated SW480 and HCT116 cells. (D) hTGF-β and SB431542 …
Figure 3—figure supplement 3
VPS9D1-AS1 functions as the scaffolding lncRNA.
(A) Synthesized probes used in RNA pulldown (RPD) assays for targeting the sequence of VPS9D1-AS1. (B) Subcellular localizations of VPS9D1-AS1 were analyzed by online tools (http://www.csbio.sjtu.edu…
Figure 4 with 1 supplement
VPS9D1-AS1 regulates interferon signaling.
(A) Heatmap illustrating the results of RNA sequencing of the genes regulated by VPS9D1-AS1. (B) VPS9D1-AS1 regulated the pathways associated with interferon signaling. (C) Differential expression …
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Figure 4—source data 1
STAT1 and pSTAT1 western blot for Figure 4F.
- https://cdn.elifesciences.org/articles/79811/elife-79811-fig4-data1-v2.zip
Figure 4—figure supplement 1
VPS9D1-AS1 plays a role on interferon signaling.
(A) Volcano plot displaying the differential mRNA profiles of HCT116sgControl and HCT116sgVPS cells. (B) Gene ontology (GO) analyses explored the roles of VPS9D1-AS1 in regulating IFNα/β signaling. …
Figure 5 with 1 supplement
VPS9D1-AS1 mediates crosstalk between T cells and cancer cells by regulating IFNAR1.
(A) Flow cytometry (FCM) revealed the decrease in IFNAR1 after VPS9D1-AS1 knockout in HCT116 cells and (B) in HCT116 sgControl and sgVPS cells after CRISPR/Cas9-mediated inhibition of OAS1. …
Figure 5—figure supplement 1
VPS9D1-AS1 mediates the crosstalk between tumors and T cells through TGF-β and IFN signaling.
(A) Western blotting assay to determine the levels of OAS1, TGFBR1, STAT1, TGF-β, and IFNAR1 in HCT116 cells. (B) IFNAR1 levels in CD4+ cells after coculturing with sgControl, sgVPS, CTRL, and VPS …
Figure 6 with 1 supplement
VPS9D1-AS1 overexpression (OE) cells inhibited T cell function in vivo.
(A) VPS9D1-AS1 OE promoted the expression of TGF-β, TGFBR1, SMAD1, and STAT1 in murine cells. (B) MC38 VPS9D1-AS1 OE and control (CTRL) cells were determined proliferation using CCK8 assays. (C) …
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Figure 6—source data 1
TGF-β, SMAD1, STAT1, TGFBR1, and β-actin western blot for Figure 6A.
- https://cdn.elifesciences.org/articles/79811/elife-79811-fig6-data1-v2.zip
Figure 6—figure supplement 1
VPS9D1-AS1 inhibited T-infiltrating lymphocyte in allograft tumors.
(A) Schematic of human VPS9D1-AS1 and murine NR045849 locations on the chromosome. (B) Fold changes in the expression levels of VPS9D1-AS1 and NR045849 in MC38 and CT26W cells. (C) qRT-PCR confirmed …
Figure 7 with 3 supplements
Transgenic mice validated the inhibitory role of VPS9D1-AS1 on CD8+ T cell infiltration and demonstrated the antitumor effects of targeting VPS9D1-AS1 with an antisense oligonucleotide (ASO) drug.
(A) Treatment scheme of the azoxymethane/dextran sulfate sodium salt (AOM/DSS) colorectal cancer (CRC) model. Endpoint at 12 weeks. (B) Representative images of colorectal tumors in mice of the …
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Figure 7—source data 1
TGF-β, TGFBR1, SMAD1, OAS1, IFI27, ERK, E-cadherin, vimentin, and β-actin western blot for Figure 7K.
- https://cdn.elifesciences.org/articles/79811/elife-79811-fig7-data1-v2.zip
Figure 7—figure supplement 1
VPS9D1-AS1 drives azoxymethane/dextran sulfate sodium salt (AOM/DSS)-induced mouse model of colorectal cancer.
(A) Genotyping of VPS Tg mice. Internal control (IntCon). (B) RNA FISH identified VPS9D1-AS1 expression in intestinal epithelium of VPS Tg and wild-type (WT) mice. A probe targeting GAPDH mRNA was …
Figure 7—figure supplement 2
IFNAR1 knockdown (KD) inhibits tumor proliferation in VPS9D1-AS1 overexpression (OE) cells.
(A) Western blotting identified the levels of IFNAR1 after transfection of three shRNAs targeting IFNAR1 in HCT116 control (CTRL) and VPS9D1-AS1 OE cells. (B) CCK8 assays determined the cell …
Figure 7—figure supplement 3
Illustration of the mechanism of VPS9D1-AS1 on promoting immune evasion via TGF-β signaling and interferon (IFN)-stimulated genes (OAS1 and IFNAR1).
Author response image 1
Isotype for IFNAR1 in CD4 and CD8 T cells.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | TGF-β (Rabbit polyclonal) | Cell Signaling Technology | Cat# 3711 s | mfIHC(1：800) WB(1:200) IF (1:100) |
| Antibody | TGFBR1 (Rabbit polyclonal) | Abcam | Cat# ab31013 | IF (1:1000) mfIHC(1:2000) WB(1:500) IHC(1:400) |
| Antibody | TGFBR2 (Mouse monoclonal) | Abcam | Cat# ab78419 | WB(1:500) |
| Antibody | SMAD1/5/9 (Rabbit polyclonal) | Abcam | Cat# ab66737 | mfIHC(1:1000) WB(1:400) |
| Antibody | pSMAD1/5/9 (Ser 463/Ser465) (Rabbit polyclonal) | Santa Cruz Biotechnology | Cat# sc-12353 | mfIHC(1：500) |
| Antibody | SMAD4 (Mouse monoclonal) | Cell Signaling Technology | Cat# 46535 | mfIHC(1：1000) |
| Antibody | SMAD2/3 (Rabbit polyclonal) | Santa Cruz Biotechnology | Cat# sc-8332 | mfIHC(1：400) |
| Antibody | pSMAD2/3 (Ser 423/425) (Rabbit polyclonal) | Santa Cruz Biotechnology | Cat# sc-11769 | mfIHC(1：1000) |
| Antibody | SMAD6 (Rabbit polyclonal) | Santa Cruz Biotechnology | Cat# sc-25321 | WB(1:200) |
| Antibody | Cytokeratin (CK) (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-57004 | mfIHC(1：200) WB(1:500) |
| Antibody | STAT1 (Rabbit polyclonal) | Cell Signaling Technology | Cat# 14994 | WB(1:500) |
| Antibody | pSTAT1(Tyr701) (Rabbit polyclonal) | Cell Signaling Technology | Cat# 9167 | WB(1:500) |
| Antibody | ERK (Rabbit polyclonal) | Cell Signaling Technology | Cat# 4695 S | WB(1:200) |
| Antibody | pERK (Rabbit polyclonal) | Cell Signaling Technology | Cat# 4376 S | WB(1:200) |
| Antibody | Vimentin (Rabbit monoclonal) | Abcam | Cat# ab92547 | WB(1:100) |
| Antibody | E-cadherin (Rabbit polyclonal) | Abcam | Cat# ab15148 | WB(1:100) |
| Antibody | N-cadherin (Rabbit polyclonal) | Abcam | Cat# ab12221 | WB(1:100) |
| Antibody | GAPDH (Mouse monoclonal) | Beyotime | Cat# AG019-1 | WB(1:1000) |
| Antibody | β-actin (Mouse monoclonal) | Beyotime | Cat# AF0003 | WB(1:1000) |
| Antibody | CD4 (Rabbit monoclonal) | Abcam | Cat# ab133616 | IHC(1:200) mfIHC(1:1000) |
| Antibody | CD8 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-53212 | IHC(1:500) mfIHC(1:200) |
| Antibody | FOXP3 (Rabbit polyclonal) | Cell Signaling Technology | Cat# 98377 | IHC(1:200) mfIHC(1:800) |
| Antibody | OAS1 (Rabbit polyclonal) | Cell Signaling Technology | Cat# 14498 | IHC(1:100) |
| Antibody | IFI27 (Rabbit polyclonal) | Abclonal | Cat# A14174 | WB(1:100) |
| Antibody | RPS3 (Rabbit monoclonal) | Abcam | Cat# ab128995 | WB(1:200) |
| Antibody | IFNAR1 (Rabbit polyclonal) | Abcam | Cat# ab45172 | WB(1:100) |
| Antibody | PDL1 (Rabbit monoclonal) | Abcam | Cat# ab205921 | WB (1:100) |
| Antibody | Ki67 (Rabbit polyclonal) | ZSGB-Bio | Cat# ZM-0166 | IHC (1:1000) |
| Antibody | Human CD3 (OKT3) FG (Mouse monoclonal) | eBioscience | Cat# 16-0037-85 | (1:100) |
| Antibody | Human CD28 FG 16-0289-85 (Mouse monoclonal) | eBioscience | Cat# 16-0289-85 | |
| Antibody | Human FITC CD3 (Mouse monoclonal) | BD Pharmingen | Cat# 555332 | FCM(1:100) |
| Antibody | Human APC CD4 (Mouse monoclonal) | BD Pharmingen | Cat# 555349 | FCM(1:100) |
| Antibody | Human PE CD8 (Mouse monoclonal) | BD Pharmingen | Cat# 555637 | FCM(1:100) |
| Antibody | Ultra-LEAF purified anti-mouse CD3 Antibody (Rabbit monoclonal) | BioLegend | Cat# 100239 | FCM (1:100) |
| Antibody | Ultra-LEAF purified anti-mouse CD28 Antibody (Mouse monoclonal) | BioLegend | Cat# 122021 | (1:100) |
| Antibody | Mouse FITC CD3 (Rabbit monoclonal) | BioLegend | Cat# 100204 | FCM(1:100) |
| Antibody | Mouse PE CD8a (Rabbit monoclonal) | BioLegend | Cat# 100708 | FCM(1:100) |
| Antibody | Mouse APC CD44 (Rabbit monoclonal) | BioLegend | Cat# 103012 | FCM(1:100) |
| Antibody | Mouse BV421 CD62L (Rabbit monoclonal) | BioLegend | Cat# 104435 | FCM(1:100) |
| Antibody | Mouse APC CD279 (PD-1) (Rabbit monoclonal) | BioLegend | Cat# 135209 | FCM(1:100) |
| Antibody | Mouse PE/Cy7 CD107a(LAMP-1) (Rabbit monoclonal) | BioLegend | Cat# 121619 | FCM(1:100) |
| Antibody | Mouse PE/Cy7 CD197 (CCR7) (Rabbit monoclonal) | BioLegend | Cat# 353211 | FCM(1:100) |
| Antibody | InVivoPlus Anti-mouse PD-1 (Mouse monoclonal) | BiXcell | Cat# BP0273 | (1:100) |
| Antibody | InVivoPlus Anti-mouse/human TGF-β (Mouse monoclonal) | BiXcell | Cat# BP0057 | (1:100) |
| Antibody | Human IFN-alpha/beta R1 PE-conjugated antibody (Mouse monoclonal) | R&D | Cat# FAB245P | FCM(1:100) |
| Biological samples (Homo sapiens) | Human colon cancer tissue microarray | OUTDO | HCol-AS-180Su-14 | |
| Biological samples (H. sapiens) | Human rectal cancer tissue microarray | OUTDO | HRec-Ade180Sur-05 | |
| Biological samples (H. sapiens) | Human colorectal cancer tissue cDNA | OUTDO | HcolA30CS01 | |
| Cell lines (H. sapiens) | HCT116 | Dr. L. Yang (Beijing Chaoyang Hospital) | ||
| Cell lines (H. sapiens) | SW480 | Dr. L. Yang (Beijing Chaoyang Hospital) | ||
| Cell lines (H. sapiens) | RKO | Dr. L. Yang (Beijing Chaoyang Hospital) | ||
| Cell lines (H. sapiens) | HEK293T | Dr. L. Yang (Beijing Chaoyang Hospital) | ||
| cell lines (Mus musculus) | MC38 | PUMC | ||
| Cell lines (M. musculus) | CT26W | PUMC | ||
| Commercial assay or kit | QuantiNova SYBR Green PCR kit | Qiagen | Cat# 208054 | |
| Commercial assay or kit | QuantiTect Reverse Transcription kit | Qiagen | Cat# 205314 | |
| Commercial assay or kit | Poerce BCA Protein Assay Kit | Thermo | Cat# RB230514 | |
| Chemical compound, drug | Lipofectamine 3000 | Invitrogen | Cat# L3000015 | |
| Recombinant proteins | Human Interleukin-2 (hIL-2) | Cell Signaling Technology | Cat# 8907SC | |
| Commercial assay or kit | PerkinElmer Opal 7-color fIHC Kit | PerkinElmer | Cat# NEL797001KT | |
| Commercial assay or kit | Ficoll reagent | Solarbio | Cat# P8900 | |
| Commercial assay or kit | Tumor Dissociation Kit, mouse | Miltenyi Biotec | Cat# 130-091-376 | |
| Commercial assay or kit | CD8a+ T Cell Isolation Kit, mouse | Miltenyi Biotec | Cat# 130-104-075 | |
| Commercial assay or kit | Anti-Human CD8 Magnetic Particles-DM | BD Pharmingen | Cat# 557766 | |
| Commercial assay or kit | HiScribeTM T7 Quick High Yeild RNA Synthesis Kit | NEB | Cat# E2040S | |
| Commercial assay or kit | Monarch RNA Cleanup Kit | NEB | Cat# T2030S | |
| Commercial assay or kit | IP Lysis Buffer | Thermo Fisher | Cat# 87787 | |
| Commercial assay or kit | Pierce RNA 3’ Desthiobiotinylation kit | Thermo Fisher | Cat# 20163 | |
| Commercial assay or kit | PIERCE MAGNETIC RNA PULL-DOWN KIT | Thermo Fisher | Cat# 20164 | |
| Chemical compound, drug | CX-5461 | MCE | Cat# HY-13323 | |
| Recombinant proteins | Human Interferon-α1 | Cell Signaling Technology | Cat# 8927SC | |
| Commercial assay or kit | DIG Northern Starter | Roche | Cat# 12039672910 | |
| Commercial assay or kit | VPS9D1-AS1 FISH Probes | Biosearch | N/A | |
| Commercial assay or kit | Magna RIP RNA-Binding Protein Immunoprecipitation Kit | Millipore | Cat# 17–700 | |
| Commercial assay or kit | Positively Charged Biodyne Nylon membrane | Invitrogen | Cat# 10100 | |
| Commercial assay or kit | ChIP-IT Express Enzymatic Magnetic Chromatin Immunoprecipitation kit | Active Motif | Cat# 53009 | |
| Commercial assay or kit | Stellaris RNA FISH Wash Buffer A | BIOSEARCH | Cat# SMF-WA1-60 | |
| Commercial assay or kit | Stellaris RNA FISH Wash Buffer B | BIOSEARCH | Cat# SMF-WA1-20 | |
| Commercial assay or kit | Stellaris RNA FISH Hybridization buffers | BIOSEARCH | Cat# SMF-HB1-10 | |
| Commercial assay or kit | Stellaris FISH Probes with Quasar 570 Dye | BIOSEARCH | Cat# SMF-1063–5 | |
| Commercial assay or kit | FITC labeled Goat anti-rabbit secondly antibody | Byotime | Cat# 0652 | |
| Commercial assay or kit | FITC labeled Goat anti-mouse secondly antibody | Byotime | Cat# A0568 | |
| Commercial assay or kit | Antifade mounting medium with DAPI | Solarbio | Cat# S2110 |
Additional files
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Supplementary file 1
Sequences for sgRNA, PCR primer, siRNA and shRNA.
(a) sgRNA sequence. (b) Primers for PCR. (c) siRNA and shRNA sequences. (d) The primer for RNA pull down (RPD) probe synthesized. (e) The Primers for ChIP-PCR.
- https://cdn.elifesciences.org/articles/79811/elife-79811-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/79811/elife-79811-mdarchecklist1-v2.pdf
