Cancer Res Treat.  2025 Jan;57(1):212-228. 10.4143/crt.2024.408.

The Oncogenic Role of TNFRSF12A in Colorectal Cancer and Pan-Cancer Bioinformatics Analysis

• Wang C
• Zhao Y
• Chen Y
• Shi Y
• Yang Z
• Wu W
• Ma R
• Wang B
• Sun Y
• Yuan P

• KMID: 2564595
• DOI: http://doi.org/10.4143/crt.2024.408

Abstract

Purpose
Cancer has become a significant major public health concern, making the discovery of new cancer markers or therapeutic targets exceptionally important. Elevated expression of tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) expression has been observed in certain types of cancer. This project aims to investigate the function of TNFRSF12A in tumors and the underlying mechanisms.
Materials and Methods
Various websites were utilized for conducting the bioinformatics analysis. Tumor cell lines with stable knockdown or overexpression of TNFRSF12A were established for cell phenotyping experiments and subcutaneous tumorigenesis in BALB/c mice. RNA-seq was employed to investigate the mechanism of TNFRSF12A.
Results
TNFRSF12A was upregulated in the majority of cancers and associated with a poor prognosis. Knockdown TNFRSF12A hindered the colorectal cancer progression, while overexpression facilitated malignancy both in vitro and in vivo. TNFRSF12A overexpression led to increased nuclear factor кB (NF-κB) signaling and significant upregulation of baculoviral IAP repeat containing 3 (BIRC3), a transcription target of the NF-κB member RELA, and it was experimentally confirmed to be a critical downstream factor of TNFRSF12A. Therefore, we speculated the existence of a TNFRSF12A/RELA/BIRC3 regulatory axis in colorectal cancer.
Conclusion
TNFRSF12A is upregulated in various cancer types and associated with a poor prognosis. In colorectal cancer, elevated TNFRSF12A expression promotes tumor growth, potentially through the TNFRSF12A/RELA/BIRC3 regulatory axis.

Keyword

TNFRSF12A; Computational biology; Neoplasms; Colorectal neoplasms; NF-κB

Figure

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  Fig. 1. Pan-cancer bioinformatic analysis revealed pro-cancer effect of tumor necrosis factor receptor superfamily member 12A (TNFRSF12A). (A) TNFRSF12A mRNA expression in indicated tumor or normal tissues analyzed by TIMER2.0 online tool based on TCGA database. (B) TNFRSF12A protein level in indicated normal and tumor tissues according to Human Protein Atlas database. (C) Kaplan-Meier curves showing relevancy between TNFRSF12A mRNA level and overall survival (OS) for patients with indicated cancers obtained from the KMPlotter website. BCLA, bladder urothelial carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, colon adenocarcinoma; HR, hazard ratio; KIRC, kidney renal clear cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; N, normal; PAAD, pancreatic adenocarcinoma; T, tumor; TPM, transcripts per million.

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  Fig. 2. Tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) effects on growth, colony formation, and migration of colorectal cancer cell lines. (A) Quantitative real-time polymerase chain reaction to assess the mRNA levels of TNFRSF12A in the inducible TNFRSF12A knockdown or overexpression colorectal cancer cell lines. The duration of doxycycline (Dox) treatment was 48 hours. (B) Western blot to assess the protein levels of TNFRSF12A in the inducible TNFRSF12A knockdown or overexpression colorectal cancer cell lines. The duration of Dox treatment was 48 hours. (C) Growth curves of colorectal cancer cell lines with TNFRSF12A knockdown or overexpression. (D) Colony formation results of colorectal cancer cell lines with TNFRSF12A knockdown or overexpression. Scale bars=5 mm. (E) Wound healing results of colorectal cancer cell lines with TNFRSF12A knockdown or overexpression. Scale bars=250 μm. The notation “sh” refers to TNFRSF12A shRNA, and “oeT” indicates overexpressed TNFRSF12A. Statistical significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

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  Fig. 3. Tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) promotes colorectal cancer growth in vivo. (A) Quantitative real-time polymerase chain reaction to assess the mRNA levels of TNFRSF12A in the inducible TNFRSF12A knockdown or overexpression CT26 mouse colorectal cancer cells. The duration of Dox treatment was 48 hours. (B) Western blot to assess the protein levels of TNFRSF12A in the inducible TNFRSF12A knockdown or overexpression CT26 mouse colorectal cancer cells. The duration of Dox treatment was 48 hours. (C) Growth curves of CT26 cells with TNFRSF12A knockdown or overexpression. (D) Representative colony formation images of CT26 cells with TNFRSF12A knockdown or overexpression. Scale bars=5 mm. (E) Representative wound healing images of CT26 cells with TNFRSF12A knockdown or overexpression. Scale bars=250 μm. (F) Images of tumors obtained from mice that were subcutaneously transplanted with CT26 cells with either TNFRSF12A knockdown or overexpression (n=5). (G) Growth curves of tumors in mice subcutaneously transplanted with CT26 cells with either TNFRSF12A knockdown or overexpression. (H) The weight of tumors obtained from mice that were subcutaneously transplanted with CT26 cells with either TNFRSF12A knockdown or overexpression. The notation “sh#1” refers to TNFRSF12A shRNA#1, and “oeT” indicates overexpressed TNFRSF12A. Statistical significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

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  Fig. 4. Differential expression genes and enrichment analysis of colorectal cancer cell line with or without tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) overexpression. (A) The heatmap of differential expression genes between TNFRSF12A overexpressed (oeT) DLD-1 cells and the control (oeCtl) DLD-1 cells. (B) The volcano plot of differential expression genes between TNFRSF12A overexpressed (oeT) DLD-1 cells and the control (oeCtl) DLD-1 cells. (C) The results gene ontology (GO) analysis for differential expression genes between TNFRSF12A overexpressed (oeT) DLD-1 cells and the control (oeCtl) DLD-1 cells. BP, biological process; MF, molecular function. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for differential expression genes between TNFRSF12A overexpressed (oeT) DLD-1 cells and the control (oeCtl) DLD-1 cells. (E) The heatmap of the expression of nuclear factor κB (NF-κB)–related genes in TNFRSF12A overexpressed (oeT) DLD-1 cells and the control (oeCtl) DLD-1 cells. (F) The heatmap of the expression of putative NF-κB targets in TNFRSF12A overexpressed (oeT) DLD-1 cells and the control (oeCtl) DLD-1 cells.

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  Fig. 5. Baculoviral IAP repeat containing 3 (BIRC3) is upregulated after tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) overexpression and a potential target of RELA. (A) Quantitative real-time polymerase chain reaction verification of BIRC3 expression in DLD-1 and RKO cells with TNFRSF12A knockdown and overexpression. The doxycycline (Dox) treatment duration was 48 hours. The notation “sh#1” refers to TNFRSF12A shRNA#1, and “oeT” indicates overexpressed TNFRSF12A. Statistical significance levels are denoted as follows: **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Profile of RELA binding peaks on BIRC3 gene in LoVo colorectal cancer cells from ChIP-Atlas database (SRX359916). (C) Potential relationships among TNFRSF12A, BIRC3, and RELA in STRING database.

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  Fig. 6. Tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) overexpression affects colorectal cancer cell growth, colony formation, and migration through TNFRSF12A/RELA/BIRC3 axis. (A) Quantitative real-time polymerase chain reaction verification of the efficiency of transient knockdown BIRC3 in DLD-1 and RKO cells. The doxycycline (Dox) treatment duration was 48 hours. (B) The growth curves of TNFRSF12A overexpression DLD-1 and RKO cells with transient BIRC3 knockdown. (C) Representative colony formation images of TNFRSF12A overexpression DLD-1 and RKO cells with transient BIRC3 knockdown. Scale bars=5 mm. (D) Representative wound healing assay images of TNFRSF12A overexpression DLD-1 and RKO cells with transient BIRC3 knockdown. Scale bars=250 μm. (E) Western blot to detect level of TNFRSF12A and BIRC3, p-RELA and RELA in tumor from TNFRSF12A overexpression group, homograft mouse model mentioned in Fig. 3F. (F) Western blot to detect levels of TNFRSF12A and BIRC3, p-RELA and RELA in DLD-1 and RKO cell lines with or without TNFRSF12A overexpression and RELA inhibitor JSH-23. The treatment duration was 48 hours. The notation “oeT” indicates overexpressed TNFRSF12A. Statistical significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

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  Fig. 7. Scheme of inferred tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) function in colorectal cancer. BIRC3, baculoviral inhibitor of apoptosis protein repeat containing 3.

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