VPS9D1-AS1 overexpression amplifies intratumoral TGF-β signaling and promotes tumor cell escape from CD8+ T cell killing in colorectal cancer
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eLife assessment
This research focuses on the role of a long noncoding RNA VPS9D1-AS1(VPS) in colorectal cancer (CRC) immune evasion and provides evidence on how it is responsible for escape from cytotoxic T cells killing via amplifying intra-tumoral TGF-β signaling. The findings are of considerable translational significance since VPS9D1-AS1 was validated targetable in this work, and it is of broad interest to readers in cancer biology and immunotherapy.
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Abstract
Efficacy of immunotherapy is limited in patients with colorectal cancer (CRC) because high expression of tumor-derived transforming growth factor (TGF)-β pathway molecules and interferon (IFN)-stimulated genes (ISGs) promotes tumor immune evasion. Here, we identified a long noncoding RNA (lncRNA), VPS9D1-AS1, which was located in ribosomes and amplified TGF-β signaling and ISG expression. We show that high expression of VPS9D1-AS1 was negatively associated with T lymphocyte infiltration in two independent cohorts of CRC. VPS9D1-AS1 served as a scaffolding lncRNA by binding with ribosome protein S3 (RPS3) to increase the translation of TGF-β, TGFBR1, and SMAD1/5/9. VPS9D1-AS1 knockout downregulated OAS1, an ISG gene, which further reduced IFNAR1 levels in tumor cells. Conversely, tumor cells overexpressing VPS9D1-AS1 were resistant to CD8 + T cell killing and lowered IFNAR1 expression in CD8 + T cells. In a conditional overexpression mouse model, VPS9D1-AS1 enhanced tumorigenesis and suppressed the infiltration of CD8 + T cells. Treating tumor-bearing mice with antisense oligonucleotide drugs targeting VPS9D1-AS1 significantly suppressed tumor growth. Our findings indicate that the tumor-derived VPS9D1-AS1/TGF-β/ISG signaling cascade promotes tumor growth and enhances immune evasion and may thus serve as a potential therapeutic target for CRC.
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Author Response
Reviewer #1 (Public Review):
In this manuscript, the authors investigated the role of a long noncoding RNA VPS9D1-AS1(VPS) in colorectal cancer (CRC). They found that a high level of VPS was negatively associated with T cell infiltration in CRC patients; in cell line-derived xenograft models or a conditional knock-in mouse model, VPS overexpression enhanced tumor growth and suppressed the infiltration of CD8+ T cells, which was reverted by VPS antisense oligonucleotide (ASO) treatment. They also investigated the molecular mechanisms underlying VPS function and revealed a VPS/TGF-β/ISG signaling cascade in tumor cells and crosstalk between tumors and T cells depending on IFNAR1 level.
The authors had performed extensive analyses on the functions of VPS using patient samples, CRC cell lines, xenograft tumors, and …
Author Response
Reviewer #1 (Public Review):
In this manuscript, the authors investigated the role of a long noncoding RNA VPS9D1-AS1(VPS) in colorectal cancer (CRC). They found that a high level of VPS was negatively associated with T cell infiltration in CRC patients; in cell line-derived xenograft models or a conditional knock-in mouse model, VPS overexpression enhanced tumor growth and suppressed the infiltration of CD8+ T cells, which was reverted by VPS antisense oligonucleotide (ASO) treatment. They also investigated the molecular mechanisms underlying VPS function and revealed a VPS/TGF-β/ISG signaling cascade in tumor cells and crosstalk between tumors and T cells depending on IFNAR1 level.
The authors had performed extensive analyses on the functions of VPS using patient samples, CRC cell lines, xenograft tumors, and drug-induced tumors, and the data were of relatively good quality; they targeted VPS overexpression in cell line-derived xenografts or mouse tumors by ASO treatment as potential therapeutics, although the overexpression level may not be physiologically relevant. The authors also made great efforts to explore the mechanisms in vitro and proposed a very interesting model of ribosomes/VPS/TGF-β/ISG signaling axis in tumor cells and opposing regulation on IFNAR1 in tumor and T cells; however, the mechanistic model was tested in vitro, not in cell line-derived xenografts or mouse tumors used in the study, which undermined the authors' claims.
Thanks for these positive comments from reviewer #1, and we attached great importance to critical comments about in vivo data.
Reviewer #2 (Public Review):
In this paper, Yang et al. seek to show the importance of the lncRNA VPS9D1-AS1 in the biology and pathology of colorectal cancer (CRC). Starting with the analysis of patient data, and proceeding to cellular and animal cancer models.
Specifically, the authors report higher VPS9D1-AS1 levels in tumor tissues in two independent cohorts of CRC patients. There was a positive association between VPS9D1-AS1 levels and molecules involved in TGFb signaling, yet a negative association between VPS9D1-AS1 levels and levels of tumor-infiltrating CD8+ T cells (and a negative correlation of these levels of tumor-infiltrating CD8+ T cells and protein expression of molecules involved in TGFb signaling). Cell line studies revealed a positive feedback loop between VPS9D1-AS1 and TGFb signaling molecules, with a cell-intrinsic, pro-proliferative, and pro-survival effect of VPS9D1-AS1 on CRC cancer cells. VPS9D1-AS1 also controls the expression of several genes in the IFN pathway, in particular the ISGs IFI27 and OAS1. In addition, IFI27 and OAS1 expression are controlled by TGFb, TGFBR1, and SMAD1, and the promoter of OAS1 is targeted by SMAD4 (but also TGFb), which binds to it. VPS9D1-AS1 expression in tumor cells promotes PD1 expression and negatively affects IFNAR1 on T cells to reduce their effector functions. In vivo, MC38 CRC cells overexpressing VPS9D1-AS1 show increased tumor growth in mice, and animals with transgenic VPS9D1-AS1 expression in the intestine develop larger CRC lesions upon AOM/DSS treatment. Finally, in vivo targeting of VPS9D1-AS1 using anti-sense oligo reduced tumor size. The data indicate a series of intricate molecular and cellular interactions and suggest that VPS9D1-AS1 can help with patient stratification, improving prognostic prediction and allowing for personalized treatment.
Taken together, there is a multitude of datasets and several complementary experiments using patient-derived samples, genetically engineered cell lines, and mouse models. Definitely, the paper includes many avenues of inquiry that cover the broad field of cancer molecular biology, biochemistry, and pathogenesis. However, this broad approach renders the paper difficult to follow at times and also leads to numerous typographical and interpretive (but, largely, not methodological), mistakes. In addition, the quality of some of the figures needs to be improved before they can be properly evaluated.
In methodology, the authors are largely successful, and I would not recommend major changes to the work, other than to recommend a "focusing" of the manuscript objectives, or a paring of the data to better convey the desired story.
The experiments presented herein, particularly those that test the efficacy of the lncRNA as cancer therapeutics are important for the field, and should be of high import to other cancer biologists.
We thank you very much for your constructive comments. We had replied all your concerns
Reviewer #3 (Public Review):
The authors have accomplished large amounts of work to prove the role of VPS9D1-AS1 in promoting immune escape from cytotoxic T cells, and the mechanistic exploration is valid enough to support the conclusions, as well as the translational significance of this target through in vivo experiments. However, the logicality of the diagram requires improvement, and several revisions are warranted.
We thank for reviewer’s positive comments. We revised our manuscript according to your suggestions
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eLife assessment
This research focuses on the role of a long noncoding RNA VPS9D1-AS1(VPS) in colorectal cancer (CRC) immune evasion and provides evidence on how it is responsible for escape from cytotoxic T cells killing via amplifying intra-tumoral TGF-β signaling. The findings are of considerable translational significance since VPS9D1-AS1 was validated targetable in this work, and it is of broad interest to readers in cancer biology and immunotherapy.
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Reviewer #1 (Public Review):
In this manuscript, the authors investigated the role of a long noncoding RNA VPS9D1-AS1(VPS) in colorectal cancer (CRC). They found that a high level of VPS was negatively associated with T cell infiltration in CRC patients; in cell line-derived xenograft models or a conditional knock-in mouse model, VPS overexpression enhanced tumor growth and suppressed the infiltration of CD8+ T cells, which was reverted by VPS antisense oligonucleotide (ASO) treatment. They also investigated the molecular mechanisms underlying VPS function and revealed a VPS/TGF-β/ISG signaling cascade in tumor cells and crosstalk between tumors and T cells depending on IFNAR1 level.
The authors had performed extensive analyses on the functions of VPS using patient samples, CRC cell lines, xenograft tumors, and drug-induced tumors, and …
Reviewer #1 (Public Review):
In this manuscript, the authors investigated the role of a long noncoding RNA VPS9D1-AS1(VPS) in colorectal cancer (CRC). They found that a high level of VPS was negatively associated with T cell infiltration in CRC patients; in cell line-derived xenograft models or a conditional knock-in mouse model, VPS overexpression enhanced tumor growth and suppressed the infiltration of CD8+ T cells, which was reverted by VPS antisense oligonucleotide (ASO) treatment. They also investigated the molecular mechanisms underlying VPS function and revealed a VPS/TGF-β/ISG signaling cascade in tumor cells and crosstalk between tumors and T cells depending on IFNAR1 level.
The authors had performed extensive analyses on the functions of VPS using patient samples, CRC cell lines, xenograft tumors, and drug-induced tumors, and the data were of relatively good quality; they targeted VPS overexpression in cell line-derived xenografts or mouse tumors by ASO treatment as potential therapeutics, although the overexpression level may not be physiologically relevant.
The authors also made great efforts to explore the mechanisms in vitro and proposed a very interesting model of ribosomes/VPS/TGF-β/ISG signaling axis in tumor cells and opposing regulation on IFNAR1 in tumor and T cells; however, the mechanistic model was tested in vitro, not in cell line-derived xenografts or mouse tumors used in the study, which undermined the authors' claims.
-
Reviewer #2 (Public Review):
In this paper, Yang et al. seek to show the importance of the lncRNA VPS9D1-AS1 in the biology and pathology of colorectal cancer (CRC). Starting with the analysis of patient data, and proceeding to cellular and animal cancer models.
Specifically, the authors report higher VPS9D1-AS1 levels in tumor tissues in two independent cohorts of CRC patients. There was a positive association between VPS9D1-AS1 levels and molecules involved in TGFb signaling, yet a negative association between VPS9D1-AS1 levels and levels of tumor-infiltrating CD8+ T cells (and a negative correlation of these levels of tumor-infiltrating CD8+ T cells and protein expression of molecules involved in TGFb signaling). Cell line studies revealed a positive feedback loop between VPS9D1-AS1 and TGFb signaling molecules, with a …
Reviewer #2 (Public Review):
In this paper, Yang et al. seek to show the importance of the lncRNA VPS9D1-AS1 in the biology and pathology of colorectal cancer (CRC). Starting with the analysis of patient data, and proceeding to cellular and animal cancer models.
Specifically, the authors report higher VPS9D1-AS1 levels in tumor tissues in two independent cohorts of CRC patients. There was a positive association between VPS9D1-AS1 levels and molecules involved in TGFb signaling, yet a negative association between VPS9D1-AS1 levels and levels of tumor-infiltrating CD8+ T cells (and a negative correlation of these levels of tumor-infiltrating CD8+ T cells and protein expression of molecules involved in TGFb signaling). Cell line studies revealed a positive feedback loop between VPS9D1-AS1 and TGFb signaling molecules, with a cell-intrinsic, pro-proliferative, and pro-survival effect of VPS9D1-AS1 on CRC cancer cells. VPS9D1-AS1 also controls the expression of several genes in the IFN pathway, in particular the ISGs IFI27 and OAS1. In addition, IFI27 and OAS1 expression are controlled by TGFb, TGFBR1, and SMAD1, and the promoter of OAS1 is targeted by SMAD4 (but also TGFb), which binds to it. VPS9D1-AS1 expression in tumor cells promotes PD1 expression and negatively affects IFNAR1 on T cells to reduce their effector functions. In vivo, MC38 CRC cells overexpressing VPS9D1-AS1 show increased tumor growth in mice, and animals with transgenic VPS9D1-AS1 expression in the intestine develop larger CRC lesions upon AOM/DSS treatment. Finally, in vivo targeting of VPS9D1-AS1 using anti-sense oligo reduced tumor size. The data indicate a series of intricate molecular and cellular interactions and suggest that VPS9D1-AS1 can help with patient stratification, improving prognostic prediction and allowing for personalized treatment.
Taken together, there is a multitude of datasets and several complementary experiments using patient-derived samples, genetically engineered cell lines, and mouse models. Definitely, the paper includes many avenues of inquiry that cover the broad field of cancer molecular biology, biochemistry, and pathogenesis. However, this broad approach renders the paper difficult to follow at times and also leads to numerous typographical and interpretive (but, largely, not methodological), mistakes. In addition, the quality of some of the figures needs to be improved before they can be properly evaluated.In methodology, the authors are largely successful, and I would not recommend major changes to the work, other than to recommend a "focusing" of the manuscript objectives, or a paring of the data to better convey the desired story.
The experiments presented herein, particularly those that test the efficacy of the lncRNA as cancer therapeutics are important for the field, and should be of high import to other cancer biologists.
-
Reviewer #3 (Public Review):
The authors have accomplished large amounts of work to prove the role of VPS9D1-AS1 in promoting immune escape from cytotoxic T cells, and the mechanistic exploration is valid enough to support the conclusions, as well as the translational significance of this target through in vivo experiments. However, the logicality of the diagram requires improvement, and several revisions are warranted.
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