Pseudomonas putida KT2440-induced RBM47 regulates non-small cell lung cancer stem cell properties and T cell-mediated antitumor activity

Introduction

Lung cancer (LC) remains the leading cause of cancer-related mortality globally (1). Among all LCs, approximately 85% of cases are non-small cell LC (NSCLC) (2). Although advances in early detection, targeted therapies, and immunotherapy have significantly improved outcomes and prolonged survival, the majority of patients present with advanced, incurable disease at diagnosis, and NSCLC remains largely incurable. A major challenge is the persistence of cancer stem cells (CSCs) and tumor-initiating cells (T-ICs), which possess self-renewal and indefinite proliferative abilities (3). The literature suggests that CSCs regulate tumor initiation, proliferation, metastasis, recurrence, and chemoresistance (4). Furthermore, CSCs are the crucial factors that modulate tumor metastasis and recurrence in several cancers including NSCLC (5,6). The molecular mechanisms governing the maintenance and expansion of CSCs, however, remain poorly understood.

RNA-binding proteins (RBPs) bind to specific RNA molecules and modulate posttranscriptional gene expression (7). Various RBPs have been associated with malignant tumor progression and poor prognosis (8). One of these is the RNA-binding motif protein 47 (RBM47) linked to NSCLC. The role of RBM47 is still debated, as some studies report an association of RBM47 overexpression with worse prognosis, while others suggest a favorable prognostic impact in NSCLC (9-11). The human lungs have a diverse microbiota comprising various organisms. Research indicates the presence of over nine bacterial genera in the lungs (12). The makeup of pulmonary microbiota has been implicated in several lung conditions (13), including LC (14). Several factors affect bronchopulmonary microbiome- for example, older age is linked with decreased microbiome diversity (15). The microbiome is also significantly affected by gender, with males and females exhibiting distinct microbiomes due to the gender-specific hormones (16).

The association between bronchopulmonary microbiome and NSCLC remains largely unexplored (17), with only a few studies reported in the literature evaluating this association. The microbiota is believed to influence the occurrence, development, and prognosis of NSCLC, although the molecular mechanisms underlying this interaction remain largely unclear (18).

In this study, we aimed to investigate how Pseudomonas putida KT2440 influences non-small cell lung CSC (NSCL-CSC) properties and T cell-mediated antitumor activity. We focused on the influence of tumor microbiome on RBM47, which has emerged as a major regulator of immune responses. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1945/rc).

Methods

Sample collection

A total of 30 paired tumor and adjacent non-tumor tissues from NSCLC patients were obtained from Longyan First Affiliated Hospital of Fujian Medical University from 2018 to 2022. Samples were collected in accordance with the institutional protocol. Immediately after collection, tissue samples were rapidly frozen in liquid nitrogen and stored at −80 ℃ for further use. Informed consent was obtained from all the subjects prior to tissue procurement. The Ethics Committee of Longyan First Affiliated Hospital of Fujian Medical University approved the study protocol (No. 2020LYF17042). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Cell culture and RNA interference

The NSCLC cell lineages (NCI-H1299 and NCI-H1650) were propagated at 37 ℃ in a 5% CO₂ environment with Dulbecco’s modified Eagle medium (DMEM) augmented with L-glutamine (2 mM), 10% fetal bovine serum (FBS), and gentamicin (25 µg/mL). Both the cell lines were subjected to RBM47 knockdown (KD), overexpression, and their control lentiviruses (LVs; Ribobio, Guangzhou, China). The infection stability was assessed using puromycin. The LV-control, LV-RBM47, control-KD, RBM47-KD, small interference RNAs (siRNAs), and negative control (NC) SOX4 were acquired from Ribobio.

Animal models

All in vivo procedures were authorized by the Animal Experimentation Ethics Committee of Longyan First Affiliated Hospital of Fujian Medical University (No. 2020LYFDW0003), and in compliance with Longyan First Affiliated Hospital of Fujian Medical University guidelines for the care and use of animals. A protocol was prepared before the study without registration. For animal experiments, 4-week-old BALB/c female nude mice were purchased from Shanghai Experimental Animal Center of Chinese Academic of Sciences (Shanghai, China) and were vaccinated with KT2440 or broth control, as described previously (19). Six weeks into the experiment, the mice were sacrificed, and their lung tissues were dissected for gene expression analyses.

Spheroid assay

The cells (300/well) were propagated for 1 week on ultralow attachment 96-well plates (Corning, Corning, NY, USA) in DMEM/F12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) augmented with 1% FBS, basic fibroblast growth factor (bFGF) (20 ng/mL), and epidermal growth factor (EGF) (20 ng/mL). The spheroids were counted from three representative views. The protocol was repeated three times.

In vitro limiting dilution analysis

Cells at concentrations of 2-, 4-, 8-, 16-, 32-, and 64-well (eight wells for each concentration) were propagated for 1 week in ultralow attachment 96-well plates in DMEM/F12 augmented with 1% FBS, bFGF (20 ng/mL), and EGF (20 ng/mL). The CSCs were quantified via ELDA software (http://bioinf.wehi.edu.au/software/elda/index.html) (20).

RiboTrap RNA-protein binding assay

RNA immunoprecipitation (RIP) was performed using the EZ-Magna RIP RBP Immunoprecipitation Kit (Merck Millipore, Burlington, MA, USA). In brief, cells were cross-linked with 1% formaldehyde and lysed with protease and RNase inhibitors. Magnetic beads preincubated with IgG or indicated antibody were incubated with lysates at 4 ℃ overnight. Eluted RNAs were purified and detected with quantitative polymerase chain reaction (qPCR). Total RNA was regarded as the input control.

Flow cytometry

For sorting CD90⁺ and CD133⁺ cells from NSCLC individuals and cell lines, primary CD133 (BioLegend, Inc., San Diego, CA, USA) and CD90 antibodies (BioLegend, Inc.) were incubated with the cells for 30 min at ambient temperature, and then subjected to flow cytometry via a MoFlo XDP cell sorter (Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions. The data from three separate experiments were used for real-time polymerase chain reaction (RT-PCR) assessment.

RT-PCR analysis

Whole NSCLC cellular and tissue RNA was separated via TRIzol (Invitrogen, Thermo Fisher Scientific); its purity was detected via a ultraviolet (UV) spectrophotometer (NanoDrop ND-1000, Thermo Fisher Scientific), while the integrity was confirmed via agarose gel electrophoresis. The RNA was then reverse transcribed into complementary DNA (cDNA) with M-MLV RTase cDNA Synthesis Kit (Promega, Madison, WI, USA). The RNA was then subjected to RT-PCR via SYBR Green PCR Kit (Roche, Basel, Switzerland) and the Light Cycler 480 System (Roche) with initial denaturation (1 cycle at 95 ℃ for 5 min), denaturation (40 cycles at 95 ℃ for 15 s), annealing (60 ℃ for 30 s), and extension (72 ℃ for 30 s). Melting curves were used to confirm the primer specificity after the reaction. For each sample, experiments were performed in triplicate.

Western blotting assay

For specimen acquisition, a cell lysis buffer was used as per a previously described protocol (20). Briefly, proteins (25 µg) were isolated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a nitrocellulose membrane, blocked with 5% nonfat milk, and labeled with primary antibody and then with IRDye 800CW-conjugated secondary antibody. Finally, the proteins were assessed for fluorescein intensity with the LI-COR imaging system (LI-COR Biosciences, Lincoln, NE, USA).

RIP assay

For RIP assay, a Magna RIP RBP Immunoprecipitation Kit (#17-701; MilliporeSigma, Burlington, MA, USA) was employed as described previously (20).

Programmed death-ligand 1 (PD-L1) and RBM47 degradation rate

The PD-L1 and RBM47 messenger RNA (mRNA) degradation rate was measured with the transcription inhibitor actinomycin D (20).

Cell Counting Kit-8 (CCK-8) assay

Relevant cells were seeded in 96-well plates at a density of 1×10³ cells/well. Cell viability was determined by the CCK-8 assay kit, following manufacturer’s instructions at indicated timepoints after seeding.

Luciferase reporter assay

Luciferase reporter assay was performed as described in the literature (20).

Statistical analysis

All statistical measurements were carried out with GraphPad Prism (Dotmatics, Boston, MA, USA) and are expressed as the mean ± standard deviation (SD) of three experimental replicates. Two-tailed Student t-test, Fisher exact, Pearson Chi-squared, Wilcoxon signed-rank, Pearson correlation, and nonparametric Mann-Whitney tests were carried out as appropriate. P<0.05 was set as the significance threshold.

Results

KT2440 induced the N⁶-methyladenosine (m⁶A) modification of RBM47 to reduce its mRNA expression

The mRNA expression of RBM47 was decreased in mouse lung tissues infected with KT2440 for 6 weeks as compared to that in control lungs (Figure 1A), whereas the m⁶A of RBM47 mRNA was increased (Figure 1B), suggesting that KT2440 induced m⁶A modification in RBM47 to decrease its mRNA expression. The SRAMP (http://www.cuilab.cn/sramp/) database was searched to identify the three most frequent m⁶A methylation loci in RBM47 (Figure 1C). KD of different m6A demethylases or methylases indicated that inhibiting methyltransferase-like 3 (METTL3) methyltransferase but not METTL14, fat mass- and obesity-associated protein (FTO), Wilms tumor 1-associated protein (WTAP), or alkB homolog 5 (ALKBH5) significantly decreased RBM47 expression (Figure 1D,1E). Furthermore, m⁶A qPCR confirmed that METTL3 mediates hypermethylation of RBM47 mRNA in the NSCLC cell line (Figure 1F). To further evaluate the impact of m6A modification on RBM47, mutagenesis and luciferase reporter analyses were carried out. These revealed that METTL3 KD reduced the luciferase function of wild-type but not mutant mice, further supporting the importance of m⁶A in RBM47 expression (Figure 1G). The stability of mRNA and translation controlled by reader proteins are impacted by the RNA m⁶A modification (21). In our study, only the inhibition of YTHDF1, and not of IGF2BP1, YTHDF2, IGF2BP3, IGF2BP2, or YTHDF3, substantially upregulated RBM47 mRNA expression in NSCLC cells (Figure 1H,1I). Moreover, the RIP assay indicated the binding of YTHDF1 to RBM47 mRNA in NSCLC cells (Figure 1J). Additionally, METTL3 overexpression shortened RBM47 mRNA half-life while YTDF1 partially rescued this effect (Figure 1K).

Figure 1 The KT2440-induced m⁶A modification of RBM47 to decrease its mRNA expression. (A) RBM47 levels in mice lung tissues infected with KT2440 for 40 weeks as compared to those in control lungs were assessed via qPCR assay. (B) m⁶A-RBM47 levels in mice lung tissues infected with KT2440 for 40 weeks as compared with those in control lungs were assessed via m⁶A-qPCR assay. (C) Assessment of specific RBM47 m⁶A methylation loci via the SRAMP website. (D) qPCR analysis of NSCLC cells infected with LV-ALKBH5, LV-METTL14, LV-FTO, LV-WTAP, LV-METTL3, or LV-NC. (E) qPCR analysis for assessing the expression of RBM47 in NSCLC cells infected with siRNA (si-ALKBH5, si-METTL14, si-FTO, si-WTAP, si-METTL3, or si-NC). (F) m⁶A-site levels in RBM47 were elucidated by transfecting m⁶A-qPCR in NSCLC cells with LV-METTL3 or LV-NC. (G) Relative luciferase function of pMIR-REPORT-RBM47 in mutant (A-to-T mutation) or wild-type m⁶A sites in NSCLC cells co-transfected with LV-METTL3, or LV-NC, respectively. The luciferase activity of Renilla firefly was used for normalization. (H) NSCLC cells transfected with siRNA (si-YTHDF1/2/3, si-IGF2BP1/2/3, or si-control) were examined via qPCR assay. (I) RBM47 levels in NSCLC cells infected with siRNA (si-YTHDF1/2/3, si-IGF2BP1/2/3, or si-control) were examined via qPCR analysis. (J) RBM47 binding to the YTHDF1 protein in NSCLC was verified via RIP-qPCR. (K) qRT-PCR assessment of RBM47 expression relative to actin in actinomycin D (10 µM)-treated NSCLC cells. *, P<0.05. ALKBH5, alkB homolog 5; CMV, cytomegalovirus; FTO, fat mass- and obesity-associated protein; IgG, immunoglobulin G; LV, lentivirus; m⁶A, N⁶-methyladenosine; METTL, methyltransferase-like; mRNA, messenger; NC, negative control; NSCLC, non-small cell lung cancer; qPCR, quantitative polymerase chain reaction; qRT-PCR, quantitative real-time polymerase chain reaction; RBM47, RNA-binding motif protein 47; RIP, RNA immunoprecipitation; siRNA, small interference RNA; WTAP, Wilms tumor 1-associated protein.

RBM47 was downregulated in NSCL-CSCs

CD133 and CD90 are well-known CSC markers (22). According to Pearson correlation analysis, the levels of RBM47 were negatively associated with the expression of primary NSCLC tissue-isolated CD133 and CD90 (Figure 2A,2B). The NSCL-CSCs were subjected to the sphere formation or flow cytometry sorting assays, which revealed the marked downregulation of RBM47 in isolated CD133⁺ or CD90⁺ NSCLC cells as compared with CD133⁻ or CD90⁻ NSCLC cells (Figure 2C,2D). Similarly, RBM47 expression was downregulated in NSCLC spheroids as compared to adherent NSCLC cells (Figure 2E), and its expression further declined with serial spheroid passaging (Figure 2F).

Figure 2 RBM47 was downregulated in NSCL-CSCs. (A) The association of RBM47 levels with CD133 in primary NSCLC cells (n=30) was examined via RT-PCR. (B) The association of RBM47 levels with CD90 in primary NSCLC cells (n=30) was examined via RT-PCR. (C) RBM47 levels in CD133⁺ NSCLC and CD133⁻ NSCLC cells were analyzed via RT-PCR assay. (D) RBM47 levels in CD90⁺ NSCLC cells and CD90⁻ NSCLC cells were compared via RT-PCR analysis. (E) The RBM47 level in NSCLC spheroids and adherent cells was determined via RT-PCR assay. (F) The levels of RBM47 in serial NSCLC spheroids passages were examined via RT-PCR. *, P<0.05. CT, cycle threshold; mRNA, messenger RNA; NSCL-CSC, non-small cell lung cancer stem cell; NSCLC, non-small cell lung cancer; RBM47, RNA-binding motif protein 47; RT-PCR, real-time polymerase chain reaction.

RBM47 inhibited NSCL-CSC self-renewal and tumorigenesis

The NSCLC cells were transfected with the LV-RBM47 virus to assess its potential biological activity in NSCL-CSCs via RT-PCR (Figure 3A). Compared to controls, RBM47-overexpressing cells showed reduced expression of stemness markers (Figure 3B,3C), lower self-renewal capacity (Figure 3D), and fewer CSCs in limiting dilution assays (Figure 3E). In vivo, these cells also showed reduced tumorigenic potential (Figure 3F).

Figure 3 Overexpression of RBM47 inhibited NSCL-CSC self-renewal and tumorigenesis ability. (A) NSCLC cells were infected with LV-NC/LV-RBM47 and examined via RT-PCR analysis. (B,C) RT-PCR results of the CD133 and CD90 in LV-NC/LV-RBM47 NSCLC cells. (D) Spheroid-formation assay of LV-NC/LV-RBM47 NSCLC cells and representative sphere images. Magnification ×200. (E) The in vitro and (F) in vivo limiting dilution analysis of the proportion of CSCs in LV-NC/LV-RBM47 NSCLC cells. Tumors were observed over 2 months; for each group n=5. *, P<0.05. CSC, cancer stem cell; LV, lentivirus; mRNA, messenger RNA; NC, negative control; NSCL-CSC, non-small cell lung cancer stem cell; NSCLC, non-small cell lung cancer; RBM47, RNA-binding motif protein 47; RT-PCR, real-time polymerase chain reaction.

Furthermore, the NSCLC cells were transfected with the RBM47-KD virus to evaluate its potential biological activity in NSCL-CSCs via RT-PCR (Figure 4A). Compared with the control NSCLC cells, RBM47-KD cells showed an increased abundance of stemness-associated markers (Figure 4B,4C). Moreover, RBM47-KD cells had an increased self-renewal capacity (Figure 4D). The in vitro limiting dilution analysis revealed a notably increased CSC proportion (Figure 4E), whereas the in vivo limiting analysis indicated a markedly enhanced tumorigenesis capacity (Figure 4F) in NSCLC cells with RBM47 KD. These data suggest that RBM47 inhibits NSCL-CSCs self-renewal and tumorigenesis activity.

Figure 4 KD of RBM47 enhanced NSCL-CSC’s self-renewal and tumorigenesis ability. (A) NSCLC cells were infected with control-KD/RBM47-KD and examined via RT-PCR analysis. (B,C) RT-PCR results of the CD133 and CD90 in control-KD/RBM47-KD NSCLC cells. (D) Spheroids-formation assay of control-KD/RBM47-KD NSCLC cells and representative sphere images. Magnification ×200. (E) The in vitro and (F) in vivo limiting dilution analysis of the proportion of CSCs in control-KD/RBM47-KD NSCLC cells. Tumors were observed over 2 months; for each group n=5. *, P<0.05. CSC, cancer stem cell; KD, knockdown; mRNA, messenger RNA; NC, negative control; NSCL-CSC, non-small cell lung cancer stem cell; NSCLC, non-small cell lung cancer; RBM47, RNA-binding motif protein 47; RT-PCR, real-time polymerase chain reaction.

RBM47 suppressed liver CSC self-renewal and tumorigenesis activity via the Wnt pathway

A previous study indicated that RBM47 suppresses the metastasis of NSCLC by modulating the Wnt/β-catenin signaling and AXIN1 mRNA stability (23). The Wnt/β-catenin pathways’ hyperactivation has been reported as the most common event occurring in CSCs (24). To further confirm the functional connection between RBM47 and Wnt, a specific Wnt inhibitor (pyrvinium pamoate) was added to RBM47-KD and control NSCLC cells, which abolished the differences in the self-renewal ability (Figure 5A), proportion (Figure 5B), and tumorigenesis of CSCs (Figure 5C) between the RBM47-KD and control NSCLC cells. This indicates that RBM47 suppresses tumorigenesis and CSCs’ self-renewal properties via the Wnt pathway.

Figure 5 RBM47 suppressed NSCL-CSC’s tumorigenesis and self-renewal ability via the Wnt pathway. (A) Control-KD and RBM47-KD cells were inoculated with pyrvinium pamoate or DMSO, and their spheroid-formation ability was assessed. Magnification ×200. (B) Control-KD and RBM47-KD cells were inoculated with pyrvinium pamoate or DMSO, after which in vitro limiting dilution analysis was performed. (C) Control-KD and RBM47-KD cells were inoculated with pyrvinium pamoate or DMSO, after which in vivo limiting dilution analysis was performed. NS, not significant (P>0.05); *, P<0.05. CSC, cancer stem cell; DMSO, dimethyl sulfoxide; KD, knockdown; NSCL-CSC, non-small cell lung cancer stem cell; RBM47, RNA-binding motif protein 47.

RBM47 enhanced antitumor T-cell immunity

PD-L1 in tumor cells interacts with programmed death receptor-1 (PD-1) on stimulated T cells, causing exhaustion and T-cell apoptosis, a mechanism central to the immune escape of tumor cells (25). To determine whether RBM47 modulates T-cell antitumor immunity via PD-L1, an in vitro T-cell killing assay was performed by coculturing activated human peripheral blood mononuclear cells (HPBMCs) with human NSCLC cell lines. For activating and propagating HPBMCs, interleukin-2 (IL-2) and anti-CD3/CD28 antibodies were employed. The lymphocytes employed were 60% composed of CD3⁺ T cells. NSCLC cells transfected with LV-RBM47 demonstrated increased susceptibility to T-cell killing (Figure 6A). In HPBMCs, a coculture with the LV-RBM47 cells led to enhanced CD3⁺ cell expression as compared to a coculture with LV-NC cells (Figure 6B). Furthermore, the mRNA levels of granzyme B (GZMB), perforin 1 (PRF1), interferon-γ (IFN-γ), and granulysin (GNLY) increased in HPBMCs cocultured with LV-RBM47 cells (Figure 6C). Overall, these results indicate that in tumor cells, RBM47 overexpression increases T cell-induced antitumor activity.

Figure 6 RBM47 enhanced antitumor T-cell immunity. (A) Activated HPBMCs’ tumor-killing activity was assessed via CCK-8 assay. Briefly, the coculturing of NSCLC cells was carried out in the absence or presence of HPBMCs for 24 h. Normalization of the results was carried out using corresponding controls without HPBMCs. (B) Stimulated HPBMCs and LV-NC/LV-RBM47 were cocultured for 3 days at a ratio of 4:1. The CD3 percentage in HPBMC was examined by FCM. Representative plots of CD3+ T cells. A control group was employed for results normalization. (C) Quantitative RT-PCR was carried out to assess GNLY, PRF1, IFNG, and GZMB in stimulated HPBMCs cocultured with LV-NC/LV-RBM47 or NSCLC cells for 48 h. The ratio of tumor cells to HPBMC was 1:4. *, P<0.05. CCK-8, Cell Counting Kit-8; FCM, flow cytometry; FITC-H, fluorescein isothiocyanate-height; GNLY, granulysin; GZMB, granzyme B; HPBMC, human peripheral blood mononuclear cell; IFNG, interferon-γ; KD, knockdown; LV, lentivirus; mRNA, messenger RNA; NC, negative control; NSCLC, non-small cell lung cancer; PRF1, perforin 1; RBM47, RNA-binding motif protein 47; RT-PCR, real-time polymerase chain reaction; SSC-A, side scatter-area.

RBM47 destabilized PD-L1 mRNA via 3'-untranslated region (3'-UTR) binding

As an RBP, RBM47 binds the 3'-UTR of target mRNAs to modulate their relative stability. In this study, we confirmed that RBM47 interacted with the PD-L1 3'-UTR in NSCLC cell lysates (Figure 7A). Furthermore, the mRNA synthesis in cells was blocked by α-amanitin, and subsequently, the relative stability of PD-L1 and β-actin mRNAs and 18S ribosomal RNA was observed over 24 hours. It was determined that RBM47 overexpression substantially reduced the PD-L1 degradation half-life (Figure 7B). These data indicate that RBM47 can destabilize PD-L1 mRNA by directly interacting with its 3’-UTR.

Figure 7 RBM47 destabilized PD-L1 mRNA via 3'-UTR binding. (A) Analysis of RBM47s’ ability to bind PD-L1 mRNA 3'-UTR in NSCLC lysates via BrdU-labeled mRNA and protein G beads. After proteins were eluted, RBM47 levels were assessed via Western blotting. (B) The PD-L1 and actin mRNA stability was elucidated via qRT-PCR in α-amanitin (50 µM)-treated samples to further block RNA synthesis. 18S rRNA was used as a normalizing control as α-amanitin does not affect it. (C) A schematic of KT24400-induced m6A modification of the RBP RBM47 modulating NSCL-CSC properties and T cell-mediated antitumor function. ATP, adenosine triphosphate; CTP, cytidine triphosphate; dNTP, deoxy-ribonucleoside triphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; LV, lentivirus; m⁶A, N⁶-methyladenosine; METTL, methyltransferase-like; mRNA, messenger RNA; NC, negative control; NSCL-CSC, non-small cell lung cancer stem cell; NSCLC, non-small cell lung cancer; PD-L1, programmed death-ligand 1; qRT-PCR, quantitative real-time polymerase chain reaction; RBM47, RNA-binding motif protein 47; RBP, RNA-binding protein; UTP, uridine triphosphate; UTR, untranslated region; WB, Western blotting.

Discussion

Research suggests that the microbiome is an essential regulator of cancer and may promote its progression (26). Although most cancer research has primarily focused on the gut microbiome, a few studies have sought to elucidate the key microbial species causing cancer in other organs, such as the colon, skin, liver, and lungs (26). Furthermore, an association of the lung microbiome with the survival of patients with LC has been established, with LC progression being potentially regulated via specific immune pathways (26). In this study, we identified Pseudomonas putida KT2440 as a lung-resident microbe capable of modulating host gene expression through epitranscriptomic mechanisms. Specifically, we found that KT2440 mediated the m6A modification of RBM47, reducing its mRNA expression to enhance LC progression. This represents the first evidence linking a lung microbiome species to RNA methylation-mediated regulation of a tumor suppressor gene in NSCLC.

RNA m⁶A modification is the most frequent type of mRNA modification and is widely found in mammal cells, specifically in the last mRNA exon (27). Its methylation is modulated by reader, eraser, and writer proteins (28). Moreover, the m⁶A methyltransferase complex primarily comprises METTL14, METTL3, and WTAP, which act as writers, catalyzing methylation; meanwhile, FTO and ALKBH5 are demethylases (erasers). Ultimately, the reader (YTHDF1, YTHDF2, and YTHDF3), that recognizes m⁶A methylated mRNA and influences its translation, splicing, stability, and nuclear transportation, determines its fate (29). Various m⁶A targets have been associated with stem cells’ self-renewal and differentiation, dendritic cell antigen presentation, cell tissue development, T-cell homeostasis, UV-induced DNA damage response, circadian rhythm modulation, postnatal mouse cerebellum development, mouse fertility, and innate immune response (30). RBPs have been indicated to be critically involved in the regulation of cell migration, survival, and death (31). Furthermore, these RBPs are strongly associated with the progression of various tumor types (31), with RBM47 mediating NSCLC progression. Our study is the first to elucidate a novel m⁶A modification of RBM47 in NSCLC. It was found that RBM47 was modulated by its mRNA m⁶A modification, and METTL3 was the primary m⁶A enzyme regulating RBM47 mRNA levels. m⁶A modification influences mRNA stability, nuclear export, splicing, and its targets’ translation, based on their distinct recognizing proteins (YTHDF1, YTHDF2, and YTHDF3) (29). In this study, it was found that RBM47 mRNA levels reduced by METTL3 were due to the accelerated RBM47 mRNA degradation induced by YTHDF1, an m⁶A reader protein. These results suggest that microbial-induced m⁶A modification may represent a previously unrecognized mechanism of tumor suppressor silencing in LC.

Since Wnt/β-catenin signaling pathway hyperactivation is a frequent event in CSCs (12), the activation of this pathway affects stabilization and β-catenin nuclear translocation and ultimately upregulates the target genes’ transcription (32). This pathway is also linked with cancer progression and is activated in various tumors, including NSCLC. However, the function and associated mechanisms of the Wnt pathways in NSCLC remain undetermined. Our RNA-sequencing and functional assays revealed that RBM47 suppresses NSCL-CSC tumorigenesis and self-renewal by inhibiting Wnt/β-catenin signaling, identifying a novel RBM47-Wnt axis in CSC regulation. These findings provide mechanistic insight into how RBM47 loss contributes to CSC maintenance and tumor progression.

The PD-L1 (also known as B7-H1) is a crucial immune checkpoint inhibitor that promotes tumor immune escape by interacting with PD-1 on T cells, inducing T-lymphocyte anergy, functional exhaustion, and apoptosis (33). Anti-PD-1/PD-L1 immunotherapy has been employed to inhibit this interaction and activate T-cell immunity in various cancers (26). The PD-1-PD-L1 has a complex function as it is regulated by an array of processes, such as posttranscriptional and posttranslational modifications, gene transcription, and exosome transport (34). Posttranslational modifications (e.g., phosphorylation, acetylation, palmitoylation, SUMOylation, ubiquitination, and glycosylation) are crucial for regulating protein stabilization and their interaction with other proteins in the PD-1-PD-L1 axis (35). In our study, we identified a tumor-intrinsic RBM47-PD-L1 regulatory axis, in which RBM47 binds to the 3'-UTR of PD-L1 mRNA and promotes its degradation, thereby enhancing T cell-mediated cytotoxicity. This suggests that RBM47 not only regulates tumor cell-intrinsic pathways but also modulates the tumor immune microenvironment.

There were still existed several limitations in the study, such as lacking of exploring the specific molecular mechanism of KT2440 regulating m⁶A modification and explaining how RBM47 simultaneously affects CSC characteristics and PD-L1 expression, and also lacking of discussing the differences between in vitro experiments and in vivo clinical scenarios, and proposing future research directions. Further study also needed to display the detection results of Wnt pathway activation, such as Western blot band plots and grayscale quantification of β-catenin protein expression. Besides, the study also needs to supplement the known mechanisms by which tumor microbiota regulates the occurrence of LC, and explain the basic function of RBM47 in RNA metabolism and its potential association with LC stem cells and immune microenvironment, clarifying the rationality of the correlation between various elements.

Conclusions

We identified a novel mechanism in which KT2440 induces m⁶A modification of RBM47, which in turn modulates NSCL-CSC properties and T cell-mediated antitumor activity (Figure 7C). These data may inform the identification of targets for the diagnosis, treatment, and prevention of LC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1945/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1945/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1945/prf

Funding: This study was funded by the Observation on the Therapeutic Effect of Probiotic Intervention Combined with Immune Checkpoint Inhibitors on Non-Small Cell Lung Cancer (No. 2020LYF17042).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1945/coif). A.I. receives honoraria for lectures from Amgen, AstraZeneca, Merck Sharp & Dohme, Novartis, Roche, medical writing grant from Merck Serono, travel support from Amgen, AstraZeneca, Roche, Sanofi, and serves on the Advisory Board for Amgen, AstraZeneca, Merck Sharp & Dohme, Novartis, Roche. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments, and approved by the Ethics Committee of Longyan First Affiliated Hospital of Fujian Medical University (No. 2020LYF17042). All participants provided informed consent. Animal experiments were performed under a project license granted by the Ethics Committee of Longyan First Affiliated Hospital of Fujian Medical University (No. 2020LYFDW0003), and in compliance with Longyan First Affiliated Hospital of Fujian Medical University guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Gray)