The Oncogenic Role of TNFRSF12A in Colorectal Cancer and Pan-Cancer Bioinformatics Analysis
Article information
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.
Introduction
Cancer has become a major public health issue, with the National Cancer Center of China (NCC) reporting approximately 4,824,700 new cancer cases and 2,574,200 new cancer-related deaths in China in 2022 [1]. Moreover, the International Agency for Research on Cancer (IARC) estimates a global cancer burden of 28.4 million cases by 2040 [2]. In China, colorectal cancer ranks second in new cancer cases and fourth in cancer-related deaths in 2022 [1].
Even though there are established and effective treatment for various types of cancer, patients still face issues related to delayed detection, treatment tolerance, and the recurrence and metastasis of tumors [2]. Thus, the search of new therapeutic targets remains a long-term endeavor. Many public databases, including the Cancer Genome Atlas (TCGA), the Gene Expression Database (GEO) [3,4], and the Human Protein Atlas (HPA) [5], along with their derived secondary analysis websites, have significantly enhanced global cancer research effort.
Tumor necrosis factor receptor superfamily member 12A (TNFRSF12A), also known as Fn14, belongs to the tumor necrosis factor (TNF) receptor superfamily. The intracellular segment of TNFRSF12A contains a TRAF-binding motif. When its extracellular cysteine-rich domain binds to the trimeric form of a unique ligand TNFRSF12, also known as TWEAK [6], TNFRSF12A becomes trimerized and activated. This activation sets off downstream signaling via TRAF. It is also suggested that when TNFRSF12A reaches a specific threshold, it can spontaneously trimerize, leading to signaling independent of its ligand [7]. The downstream effects of TNFRSF12A activation include the activation of NF-κB signaling pathway, as well as pathways such as mitogen-activated protein kinase/ERK/JNK and phosphoinositide 3-kinase/AKT [8].
TNFRSF12A is widely present in various normal tissues at low expression levels. However, under abnormal conditions such as tissue damage, its expression can increase, but excessive TNFRSF12A levels can be detrimental [9]. Activation of TNFRSF12A has been shown to promote cell proliferation and tumor formation in cutaneous squamous cell carcinoma in vivo [10]. Upregulation of TNFRSF12A has also been linked to promoting bone metastasis in prostate cancer [11] and non–small cell lung cancer (NSCLC) [12]. Additionally, TNFRSF12A has been implicated in the tumor microenvironment, playing a role in inducing cytokine secretion by cholangiocarcinoma cells, promoting pro-inflammatory macrophage polarization, and driving the proliferation of cancer-associated fibroblast and collagen deposition [13]. On the other hand, knockdown of TNFRSF12A has been found to inhibit the proliferation of hepatocellular carcinoma cells [14] and the metastasis of prostate cancer [11] and NSCLC [12]. Therefore, TNFRSF12A may be a potential therapeutic target in cancer, and further exploration of its regulatory mechanisms may offer fresh insights into the development of novel cancer treatment strategies.
NF-κB transcription factor family consists of five members: NFKB1 (p105, p50 precursor), NFKB2 (p100, p52 precursor), RELA (RelA, also known as p65), RELB (RelB), and REL (c-Rel). These members form homologous or heterodimer complexes that translocate from cytoplasm into the nuclear to regulate transcription upon activation. In the canonic NF-κB pathway, the NF-κB complex is composed of p50 and p65 [15], whereas in the non-canonic pathway, the NF-κB complex is made up of p52 and RelB. The NF-κB signaling pathway plays an important role in tumorigenesis and cancer progression [16].
Baculoviral inhibitor of apoptosis protein (IAP) repeat containing 3 (BIRC3), a member of the IAP family, is associated with various tumors and frequently linked to tumor drug resistance [17]. In addition, in glioma [18], long non-coding RNA SNHG1 has been observed to promote tumorigenesis by increasing BIRC3 expression. Similarly, in liver cancer [19], BIRC3 has been found to promotes the proliferation and metastasis of tumor cells both in vivo and in vitro.
In this study, we found that TNFRSF12A was upregulated in cancer and associated with a worse prognosis. In vitro and in vivo experiments demonstrated that knockdown of TNFRSF12A inhibited the growth, colony formation, and migration of colorectal cancer cells, while overexpression of TNFRSF12A promoted these activities. Further investigation into the mechanism of TNFRSF12A, we observed that its overexpression led to an increase in NF-κB signaling and transcriptional dysregulation. We also identified BIRC3 as a potential target of the NF-κB component RELA. The enhanced oncogenic effects induced by TNFRSF12A could be reduced by suppressing BIRC3, indicating the existence of a TNFRSF12A/RELA/BIRC3 regulatory axis in colorectal cancer. Disrupting this regulatory axis could potentially impede the progression of colorectal cancer.
Materials and Methods
1. Bioinformatic data sources
Except for the HPA database, which provides data in the form of pictures, other bioinformatics analyses rely directly or indirectly on data from the TCGA database and the GEO database. The relevant TCGA cancer abbreviations are described in S1 Table. All information is publicly available for download and usage.
2. Bioinformatic analysis
TIMER2.0 (http://timer.cistrome.org/) [20] was used to assess the expression differences in of TNFRSF12A mRNA across various cancer-affected tissues compared to their corresponding adjacent tissues within the TCGA dataset. Immunohistochemistry (IHC) data from HPA (https://www.proteinatlas.org/) were used to compare the protein expression levels of TNFRSF12A in normal and cancerous tissues. KMPlotter website (https://kmplot.com/analysis/) [21] was used to analyze the correlation between TNFRSF12A mRNA levels and overall survival (OS) in multiple cancer types. ChIP-Atlas (https://chip-atlas.org/) [22] and Cistrome (http://cistrome.org/db/) [23] were employed to explore potential regulatory relationships between genes of interest and relevant transcription factors. Furthermore, the STRING database (https://string-db.org/) [24] was used to predict protein interconnections among the genes of interest. Additional information is available in S2 Table.
3. Cell culture
Colorectal cancer cell lines (human: DLD-1, RKO, and HT-29; mouse: CT26) and HEK293T were obtained from the American Type Culture Collection (ATCC). These cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, Grand Island, NY) or RPMI-1640 medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (ExCell Bio, Shanghai, China) at 37℃ and 5% CO2.
4. Plasmid construction and transfection
The short hairpin RNAs (shRNAs) targeting TNFRSF12A and BIRC3 were ordered from the Beijing Genomics Institute (BGI, Beijing, China). The corresponding sequences are listed in S3 Table. The annealed shRNA oligonucleotides were ligated into the pLKO-Tet-puro vector (#21915, Addgene, Cambridge, MA). The TNFRSF12A overexpression plasmid was constructed from pLenti-TRE-EF1-rtTA3-IRES-Puro H125 (Obio Technology, Shanghai, China). For stable transfection, the prepared plasmids were packaged with lentivirus package system, psPAX2 and pMD2.G, within HEK293T cells. Subsequently, the virus-containing supernatant was collected, filtered, and added to the targeted cells. The transfected cells were selected using puromycin (MeilunBio, Dalian, China), and then were induced using doxycycline (Dox, MeilunBio). For the transient transfection, the prepared plasmids were directly added into cells with the transfection reagent. The transfection efficiency was assessed using quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, or fluorescence microscopy.
5. Cell counting kit-8 assay
Cells were seeded in 96-well plates at a density of 5×103 cells per well. The cell viability at different post-treatment time points was assessed using cell counting kit-8 (CCK-8) reagent (APExBIO, Houston, TX). Cells under examination were incubated in medium containing 10% CCK-8 reagent at 37℃ for 90 minutes. Subsequently, the absorbance at 450 nm was measured to determine the relative cell viability.
6. Colony formation assay
Cells were seeded in 12-well plates at a density of 1×103 cells per well. Different groups of cells were cultured with their respective treatments. After approximately 7 days, when single-cell colonies reached a size of nearly 50 cells, they were rinsed with phosphate-buffered saline, fixed with 4% paraformaldehyde, and then stained with 0.1% crystal violet. Pictures of wells were captured by a camera.
7. Wound healing assay
Wounds were made using 4-well culture inserts (ibidi, Gräfelfing, Germany) placed in 12-well plates. Cells were cultured in each well with inserts at a density of 8×104 cells, and the inserts were removed once the cells had attached. Wound images at various time points were captured using an inverted microscope (Nikon, Tokyo, Japan). ImageJ software was employed for image analysis.
8. Quantitative real-time PCR
The total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA) and the reverse transcription was carried out with the EasyScript First-Strand cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China) according to the provided protocols. RT-qPCR was performed using the PerfectStart Green qPCR SuperMix Kit (TransGen Biotech). The relative expression levels of the genes of interest were calculated with the 2–ΔΔCt method. β-Actin or Gapdh mRNA was used as an internal reference. All primer sequences are listed in S3 Table.
9. Western blot
Cells were lysed using a cold lysis buffer containing 10% protease inhibitor (Roche, Basel, Switzerland), and the lysates were then centrifuged at 4℃, 12,000 rpm for 30 minutes. The resulting protein supernatant was collected and quantified using the BCA protein quantitative Kit (BestBio, Shanghai, China). Subsequently, 15 μg protein per lane was loaded and separated on an sodium dodecyl sulfate polyacrylamide gel electrophoresis gel, followed by transfer onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked with 5% skim milk and then washed with Tris-buffered saline containing 0.1% Tween-20 (TBST). Next, the membrane was incubated with primary antibodies at 4℃ overnight, followed by secondary antibodies for 2 hours at room temperature. Specific blots were detected using ECL on a ChemiDoc imaging system (Bio-Rad, Hercules, CA). Semi-quantitative analysis was performed by measuring the band intensity using ImageJ software. β-Actin protein was used as an internal reference protein. The primary antibodies used were as follows: β-actin (sc-47778, 1:2,000, Santa Cruz Biotechnology, Santa Cruz, CA), TNFRSF12A (ab109365, 1:1,000, Abcam, Cambridge, MA), BIRC3 (24304-1-AP, 1:2,000, Proteintech, Wuhan, China), RELA (8242S, 1:1,000, CST, Danvers, MA), and p-RELA (Ser536, 310013, 1:1,000, ZENBIO, China). The secondary antibodies used were as follows: goat anti-Rabbit IgG HRP (31460, 1:10,000, Invitrogen) and goat anti-Mouse IgG HRP (31430, 1:10,000, Invitrogen). JSH-23 (J863496, 5 μM, Macklin, Shanghai, China) was used for the rescue experiment. A certain protein relative expression value is derived through measuring band intensity by ImageJ software and calculating the ratio of the protein to β-actin (for RELA activation, which is the ratio of p-RELA to RELA).
10. RNA-sequencing
The RNA samples of the cells under investigation were extracted and sent to BGI Genomics for subsequent library construction and sequencing. The raw data were filtered, aligned, and quantified to determine the gene counts in each sample. Rstudio software (2023.09.0+463) was used for some analysis or plotting. The “DESeq2” [25] R package was used for differential gene analysis, while the “pheatmap” [26] and “ggplot2” [27] R packages were employed for heat mapping and volcano mapping. Additionally, the “clusterProfiler” [28] R package was used for gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The enrichment results can be found in S4 Table. All original data were deposited in the GEO database (accession No. GSE261177).
11. Homograft mouse model
This study was approved by the Animal Ethical and Welfare Committee of the Sixth Affiliated Hospital, Sun Yat-sen University (IACUC-2022081602). BALB/c mice aged 4 to 6 weeks were obtained from Guangdong GemPharmatech (China). To induce subcutaneous tumorigenesis, 5×106 cells per mouse were administered. The mice were then randomly divided into the doxycycline-treatment group and the corresponding control group. Mice in the doxycycline-treatment group were fed with water containing 2 mg/mL Dox. The subcutaneous tumors were measured every 3 days and the tumor volume was calculated using the formula (length×width2)/2. The mice were humanely euthanized via CO2 suffocation when the subcutaneous tumor volume reached 1.5 cm3.
12. Statistical analysis
All experiments were conducted with a minimum of three biological replicates. Statistical analysis was carried out using GraphPad Prism 8 software, and the data were presented as mean±standard deviation. The student’s t test was used to compare means between two groups, while the one-way ANOVA was employed to compare means among multiple groups. Significance is defined as p < 0.05.
Results
1. TNFRSF12A is highly expressed in multiple cancer types and associated with poor prognosis
We performed pan-cancer expression analysis of TNFRSF12A using the TIMER2.0 database and the HPA database. According to the TIMER2.0 database, TNFRSF12A mRNA levels were significantly elevated in tumor tissues of most cancer types (Fig. 1A). Furthermore, TNFRSF12A protein level was notably elevated in tumor tissues from the IHC results in the HPA database (Fig. 1B). Besides, investigation of the correlation of TNFRSF12A expression with patient prognosis by KMPlotter website analysis revealed that elevated TNFRSF12A mRNA levels were associated with shorter survival in different cancers (Fig. 1C, S5 Fig.). These findings suggest that TNFRSF12A may potentially function as a cancer-promoting factor and serve as an indicator of poor prognosis in cancer patients.
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.
2. TNFRSF12A promotes the growth, colony formation, and migration of human colorectal cancer cells in vitro
The pan-cancer analysis of TNFRSF12A indicates that the observations in colorectal cancer closely mirror those seen in the majority of cancer types. Given the substantial prevalence and mortality rates associated with colorectal cancer, the investigation into the function of TNFRSF12A in this context is deemed highly important.
To delve into the role of TNFRSF12A in colorectal cancer, we selected two human colorectal cancer cell lines, DLD-1 and RKO, which have moderate to high levels of TNFRSF12A, as well as CT26 mouse colorectal cancer cells, to establish stable knockdown and overexpression TNFRSF12A cell lines. All vectors utilized were of the doxycycline-inducible expression type. Both TNFRSF12A shRNA (sh#1 and sh#2) effectively reduced the levels of mRNA and protein of TNFRSF12A, while the overexpression plasmid (oeT) increased those levels (Fig. 2A and B).
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.
Next, we employed the CCK-8 reagent to evaluate cell viability at 0, 24, 48, 72, and 96 hours in cell culture. The results showed a deceleration in the growth of colorectal cancer cells post-NFRSF12A knockdown, while an acceleration was observed upon overexpression (Fig. 2C). Analysis of colony formation revealed a significant decrease in both the number and size of colonies upon TNFRSF12A knockdown, whereas overexpression of TNFRSF12A led to larger and more abundant cell colonies (Fig. 2D). Additionally, the results from the wound healing assay demonstrated a decrease in the closure rate of colorectal cancer cells after TNFRSF12A knockdown and an increase after TNFRSF12A overexpression (Fig. 2E). It is noteworthy that DLD1 belongs to consensus molecular subtype I (CMS1) and RKO belongs to CMS4 of colorectal cancer. Both cell lines exhibit microsatellite instability. Hence, to corroborate the findings, we conducted similar experiments in the HT-29 cell line (CMS3, microsatellite stability), demonstrating that knockdown of TNFRSF12A inhibited cell proliferation, while overexpression of TNFRSF12A promoted cell proliferation (S6 Fig.). In summary, TNFRSF12A is implicated in the regulation of growth, colony formation, and migration of colorectal cancer cells in vitro.
3. TNFRSF12A promotes colorectal cancer growth in mouse models
Following the investigation into the function of TNFRSF12A in human colorectal cancer cells in vitro, we proceeded to evaluate its impact in mouse models. As anticipated, the knockdown of TNFRSF12A in mouse colorectal cells CT26 resulted in reduced cell growth, colony formation, and migration. Conversely, overexpression of TNFRSF12A in CT26 cells led to an increase in cell growth, colony formation, and migration (Fig. 3A-E). Next, we utilized BALB/c mice to investigate the in vivo effect of TNFRSF12A. By utilizing lentivirus to establish stable TNFRSF12A knockdown and overexpression CT26 cells, we assessed the influence of TNFRSF12A on tumor growth through subcutaneous tumorigenesis assay. Although the time frame of tumorigenesis varied between the TNFRSF12A knockdown (shCtl vs. sh#1) and overexpression experiments (oeCtl vs. oeT) for ethical reasons, it was evident that the TNFRSF12A knockdown group (sh#1) exhibited reduced tumor growth, resulting in smaller tumor volume and weight compared to the control knockdown group (shCtl). Conversely, TNFRSF12A overexpression had the opposite effect (Fig. 3F-H). In conclusion, TNFRSF12A promotes the growth of colorectal cancer in vivo.
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.
4. TNFRSF12A overexpression enhances NF-κB signaling pathway in colorectal cancer cells
Given its high expression in tumors, TNFRSF12A is likely involved in self-activation. To delve deeper into how TNFRSF12A influences tumor behavior, we conducted RNA-seq analysis on TNFRSF12A-overexpressed (oeT) DLD-1 cells and their corresponding control cell (oeCtl). The analysis of differentially expressed genes revealed 470 down-regulated genes and 171 upregulated genes in the TNFRSF12A overexpression group (Fig. 4A). Notably, TNF receptor-related factor 1 (TRAF1), NFKB2 and BIRC3 were highly upregulated upon TNFRSF12A overexpression (Fig. 4B). Moreover, GO analysis demonstrated enhanced TNF signaling, NF-κB signaling, cytokine-receptor binding and chronic inflammation in TNFRSF12A overexpressed DLD-1 cells (Fig. 4C). Additionally, KEGG analysis highlighted the TNF pathway, NF-κB pathway, cytokine-receptor interactions, and transcriptional dysregulation in TNFRSF12A overexpressed DLD-1 cells (Fig. 4D). Our investigation also revealed the upregulation of multiple genes associated with the NF-κB signaling pathway (Fig. 4E). Importantly, mRNA levels of several potential RELA targets were significantly increased upon TNFRSF12A overexpression, while mRNA levels of potential targets of RELB, NFKB1, and NFKB2 did not show significant changes (Fig. 4F). This observation indicated the activation of canonical NF-κB signaling in TNFRSF12A-overexpressed DLD-1 cells.
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.
5. TNFRSF12A overexpression upregulates RELA’s potential target BIRC3
Based on the results of our RNA-seq data analysis, the NF-κB signaling pathway is activated after TNFRSF12A overexpression. This is consistent with previous studies that TNFRSF12A involves in activating NF-κB signaling. Additionally, the RNA-seq analysis showed a significant upregulation of BIRC3, an inhibitor of apoptosis known to be activated by NF-κB signaling in splenic marginal zone lymphoma (PMID: 21881048), upon TNFRSF12A overexpression. This finding led up to hypothesize that TNFRSF12A might positively regulate BIRC3 through NF-κB signaling pathway in colorectal cancer cells. To validate this hypothesis, we employed RT-qPCR to assess the expression of BIRC3 in TNFRSF12A knockdown and overexpression DLD-1 and RKO cells. As expected, the expression of BIRC3 was decreased upon TNFRSF12A knockdown and increased following TNFRSF12A overexpression (Fig. 5A). Moreover, the RELA ChIP-seq assay using chromatin from LoVo colorectal cancer cells (SRX359916) revealed that RELA binds to the promoter of BIRC3, suggesting a potential transcriptional regulation of BIRC3 by RELA (Fig. 5B). Further analysis using the STRING database uncovered a strong correlation among TNFRSF12A, BIRC3 and RELA (Fig. 5C). These combined results indicate that TNFRSF12A positively regulate BIRC3 through the mediation of RELA.
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.
6. TNFRSF12A overexpression affects colorectal cancer cell behavior via the TNFRSF12A/RELA/BIRC3 axis
To ascertain whether BIRC3 serves as a key downstream factor influencing colorectal cancer cells during TNFRSF12A overexpression, we developed BIRC3 knockdown plasmids (shBIRC3#1 and shBIRC3#2) and verified the transient knockdown efficiency by RT-qPCR (Fig. 6A). Subsequently, we conducted rescue experiments by transiently transfecting the BIRC3 knockdown plasmid into TNFRSF12A overexpressed colorectal cancer cell lines. As previously noted, the growth rate, colony formation, and migration capacity of the colorectal cancer cells increased upon TNFRSF12A overexpression. However, the pro-tumor phenotype either disappeared or diminished after BIRC3 knockdown in TNFRSF12A overexpressed cells (Fig. 6B-D). Moreover, upregulated BIRC3 level and an increased p-RELA/RELA ratio were observed both in vivo and in vitro through TNFRSF12A overexpression (Fig. 6E and F). While JSH-23, a RELA activation inhibitor, when supplemented to TNFRSF12A overexpression cancer cells inhibited TNFRSF12A promoted BIRC3-upregulation (Fig. 6F). Collectively, these results strongly suggest that BIRC3 is a pivot downstream factor of TNFRSF12A in colorectal cancer cells. In summary, TNFRSF12A influences the growth, colony formation, and migration of colorectal cancer cells through the TNFRSF12A/RELA/BIRC3 axis.
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.
Discussion
TNFRSF12A has been recognized for its involvement in promoting cancer progression in various cancer types, influencing key aspects such as proliferation [14], metastasis [12], and tumor microenvironment [13], although some studies have suggested its potential for cancer suppression. As TNFRSF12A is a tumor-associated cell membrane receptor, Li et al. [29] developed TNFRSF12A× CD3 BiTE and TNFRSF12A-specific CAR-T cells. These innovations exhibited promising anti-tumor efficacy both in vitro and in vivo, providing substantial potential for the clinical advancement of TNFRSF12A. Therefore, performing comprehensive pan-cancer research on TNFRSF12A could significantly enhance its clinical applicability.
After conducting pan-cancer bioinformatics analysis of the TNFRSF12A using diverse data sources such as TCGA, GEO, and HPA, we identified that TNFRSF12A is high expressed across most cancer types and is associated with poor prognosis. Considering the significant impact of colorectal cancer with the spectrum of cancer types, we have prioritized investigating the function of TNFRSF12A in this specific cancer. Our findings revealed that knockdown of TNFRSF12A inhibited colorectal cancer cell growth, clonogenic capacity, and migration, while its overexpression led to the opposite effect. Notably, based on RNA-seq and transcription factor databases, we hypothesized the existence of the TNFRSF12A/RELA/BIRC3 regulatory axis. Subsequently, rescue experiments corroborated the vital role of BIRC3 in TNFRSF12A-promoted growth, colony formation, and migration of colorectal cancer cells (Fig. 7), suggesting BIRC3 is a key downstream factor of TNFRSF12A. But the precise mechanism remains to be further explored. Furthermore, the mechanism by which TNFRSF12A regulates colorectal cancer requires further study in other types of cancer to determine whether it is a common mechanism in pan-cancer and whether it could be used as a universal target for anti-cancer therapy.
Scheme of inferred tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) function in colorectal cancer. BIRC3, baculoviral inhibitor of apoptosis protein repeat containing 3.
In summary, our study demonstrates that TNFRSF12A has the potential to enhance the growth, colony formation, and migration of colorectal cancer cells. These effects are likely mediated through the TNFRSF12A/RELA/BIRC3 axis, indicating its potential as a therapeutic target for cancer.
Electronic Supplementary Material
Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).
Notes
Ethical Statement
The animal experiment in this study was approved by the Animal Ethical and Welfare Committee of the Sixth Affiliated Hospital, Sun Yat-sen University (IACUC-2022081602).
Author Contributions
Conceived and designed the analysis: Wang C, Zhao Y, Yuan P.
Collected the data: Wang C, Chen Y.
Contributed data or analysis tools: Wang C, Zhao Y, Yang Z, Wu W.
Performed the analysis: Wang C, Chen Y, Shi Y.
Wrote the paper: Wang C, Zhao Y, Ma R, Sun Y, Yuan P.
Funding acquisition: Wang B, Yuan P.
Conflicts of Interest
Conflict of interest relevant to this article was not reported.
Acknowledgements
This study was funded in part by the National Natural Science Foundation of China (Grant No. 31970674 to PY); the key project funding in the Sixth Affiliated Hospital, Sun Yat-sen University (Grant No. 2022JBGS13 to PY); Guangdong Yiyang Healthcare Charity Foundation (Grant No. JZ2023010 to PY); Beijing Bethune Charitable Foundation (Grant No: BQE-TY-SSPC(5)-S-01 to BW); Guangdong Provincial Clinical Research Center for Digestive Diseases (Grant No. 2020B1111170004).
The bioinformatics analysis results in this study were partly based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga and GEO database, directly or indirectly.