What this is
- , an m6A demethylase, is upregulated in non-small cell lung cancer (NSCLC).
- It influences tumor progression and response to anti-PD-L1 immunotherapy by modulating .
- The study investigates the mechanisms of in the tumor microenvironment and its potential as a therapeutic target.
Essence
- promotes NSCLC progression by enhancing interactions between tumor cells and macrophages, impacting the efficacy of anti-PD-L1 therapy.
Key takeaways
- expression correlates with PD-L1 levels and macrophage infiltration in NSCLC tissues, suggesting its role in creating an immunosuppressive microenvironment.
- regulates JAK2 expression through , activating the JAK2/p-STAT3 pathway, which promotes tumor growth and PD-L1 expression.
- Knockdown of in mouse models reduced tumor growth and enhanced the effectiveness of anti-PD-L1 therapy, indicating its potential as a therapeutic target.
Caveats
- The study primarily focuses on correlations and mechanisms without direct clinical trial data to support the efficacy of targeting .
- Further research is needed to validate as a biomarker for predicting immunotherapy response in diverse patient populations.
Definitions
- ALKBH5: An m6A demethylase involved in RNA modification, influencing gene expression and tumor behavior.
- m6A modification: A common RNA modification that affects RNA stability, translation, and interactions with proteins, impacting cancer progression.
- tumor-associated macrophages (TAMs): Macrophages located in the tumor microenvironment that can promote tumor growth and suppress anti-tumor immunity.
AI simplified
Background
The high mortality rate of lung cancer, with non-small cell lung cancer (NSCLC) being the most common pathological type, remains a critical concern [1]. Established tumors have an immunosuppressive microenvironment owing to the interaction between tumor and immune cells and related signaling molecules [2, 3]. An in-depth understanding of the tumor microenvironment (TME) has driven advances in immunotherapy. Among cancer immunotherapies, immune checkpoint inhibitors, including programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors, are the most effective and widely used immunotherapeutic agents [4]. Although they represent a promising therapeutic approach, the resistance of cancer cells to immune checkpoint inhibitors limits their efficacy [5, 6]. Only approximately 20% of NSCLC patients achieve remission [7]. Recent research suggests that resistance to immune checkpoint inhibitors is closely related to changes in the TME [8–10]. Understanding the mechanisms that mediate the interaction between cancer cells and the TME could provide insights into immune evasion, informing the development of more effective immunotherapeutic strategies for NSCLC.
Numerous types of chemical modifications exist in cellular RNAs, including 5-methylcytosine, N6-methyladenosine (m6A), and N7-methylguanosine; such modifications increase the complexity of RNA biogenesis, structure, localization, and function [11]. Research on m6A—the most common mRNA modification—is increasing each year. The regulation of m6A involves methyl writers (METTL3/14/16 [methyltransferase-like 3/14/16] and WTAP [Wilms’ tumor 1-associated protein]), erasers (ALKBH5 [alkB homolog 5] and FTO [fat mass and obesity-related protein]), and readers (YTHDF1/2/3 [YTH domain family 1/2/3]) [12]. Better understanding of these regulatory molecules has highlighted the importance of m6A methylation in disease progression. Notably, m6A RNA modifications are reportedly involved in tumorigenesis and metastasis, and increasing evidence suggests a role for m6A RNA modifications in modulating the TME [13]. m6A RNA modification affects tumor immune escape to different degrees by reshaping the TME under hypoxic conditions, regulating acidity and metabolic reprogramming, inducing epithelial–mesenchymal transition, and influencing immune cells infiltration and immune checkpoint expression [14–16]. Immune cells are an essential component of the TME. Immune cell abnormal activation, differentiation, and expansion can cause immune failure and inhibit immune cell functions in the TME, leading to tumor escape. m6A RNA modification in immune cells is involved in the activation of T-lymphocytes, dendritic cells, and natural killer cells, as well as macrophage reprogramming [17]; thus, reshaping antitumor immunity and regulating immune escape. However, the mechanism by which m6A methylation remodels the TME and affects tumor immune monitoring has remained unknown.
In the present study, we investigated the role of ALKBH5 in regulating tumor-associated macrophages (TAMs), the phenotypes of NSCLC cells, and any interactions between the two that promoted NSCLC progression and influenced the efficacy of anti PD-L1 immunotherapy, in addition to elucidating the underlying mechanisms. Our results suggest that ALKBH5, as a biomarker for predicting anti PD-L1 immunotherapy outcomes in NSCLC patients, is a potential target for addressing anti PD-L1 immunotherapy resistance in NSCLC.
Methods
Reagents
Actinomycin D (M4881) was purchased from AbMole Bioscience (Houston, TX). AG490 (HY-12000) was purchased from MedChemExpress (Monmouth Junction, NJ). Matrigel matrix (356,234) was purchased from Corning (Corning, NY). Phorbol 12-myristate 13-acetate (PMA; FMS-FZ207) was purchased from Fcmacs Biotech (Nanjing, Jiangsu, China). Recombinant human CCL2 (300–04), CXCL10 (300–12), and IL-6 (200–06) were purchased from PeproTech (Cranbury, NJ). Puromycin (ST551) was purchased from Beyotime Biotechnology (Haimen, Jiangsu, China). Anti-mouse PD-L1 antibody (BE0101) was purchased from BioXcell (Lebanon, NH).
Tissue specimens
Fresh NSCLC tissues and corresponding paracancerous tissues were collected from the Department of Thoracic Surgery of Jinling Hospital between 2020 and 2023. Paraffin-embedded NSCLC tissues were collected from the Department of Pathology of Jinling Hospital.
Cell culture
All cell lines were obtained between 2020 and 2023. The culture medium used for human bronchial epithelial, human lung cancer and THP-1 cells was RPMI 1640 supplemented with 10% FBS (Gibco, Billings, MT). The culture medium used for Lewis lung cancer (LLC) cells and human umbilical vein endothelial cells (HUVECs) was DMEM supplemented with 10% FBS (Gibco). All cells were cultured at 37 °C with 5% CO2.
Constructs and transfection
Lipofectamine 3000 (Invitrogen, Carlsbad, CA) was used to transfect cells. siRNAs targeting either ALKBH5 (si-ALKBH5) or YTHDF2 (si-YTHDF2) and a negative control siRNA (si-NC) were synthesized by RiboBio (Guangzhou, Guangdong, China). shRNA targeting ALKBH5 (sh-ALKBH5) and a negative control shRNA (sh-NC) were synthesized by Hanheng Biotechnology (Shanghai, China). Recombinant plasmids overexpressing wild-type ALKBH5 (OE-ALKBH5), the catalytic mutant ALKBH5 H204A (OE-H204A), or control (OE-NC) were constructed by Jinruisi (Nanjing, Jiangsu, China). LLC cells with stable overexpression or knockdown of ALKBH5 were infected with the lentivirus synthesized by Hanheng and selected based on puromycin (2 μg/mL) screening. The sequences used are listed in Table S1.
Co-culture experiments
Co-culture experiments were performed using 0.4-μm Transwell chambers (Corning). The upper chamber was filled with the cancer cell suspension, and the lower chamber was filled with 100 ng/mL of PMA-stimulated THP-1 cell suspension. Following 72 h co-culture at 37 °C with 5% CO2, the THP-1 cells were collected.
Cell proliferation assays
For the Cell Counting Kit-8 (CCK-8) assay, cancer cells (2,000 cells/well) were seeded in 96-well plates. CCK-8 solution (Meilunbio, Dalian, Liaoning, China) was added and incubated for 2 h before measuring the absorbance at 450 nm (BioTek, Winooski, VT). For the colony formation assay, cancer cells (1,000 cells/well) were seeded in 6-well places, cultured for 10–14 days, and then fixed and stained. Cells were counted using ImageJ (version 1.46; NIH).
Cell migration assay
Eight-micrometer Transwell chambers (Corning) were used for cell migration assays. The cancer (4 × 104 cells/well) or PMA-stimulated THP-1 (20 × 104 cells/well) cell suspension was added to the upper chamber. Transfected NSCLC cells were incubated in serum-free medium for 24 h, after which the culture supernatants were collected and centrifuged at 1500 rpm for 10 min to remove the debris. The cell supernatant was used as conditioned medium. RPMI 1640 medium or the serum-free conditioned medium from the transfected NSCLC cells was supplemented with 20% FBS and added to the lower chamber. After 48 h culture, the migrated cells were fixed and stained. Cells were counted using ImageJ.
Tube formation assay
HUVECs (1.5 × 104 cells/well) were resuspended in the supernatant of NSCLC cells and seeded in Matrigel-coated 96-well plates. After 24 h incubation at 37 ºC with 5% CO2, tube formation was examined using an inverted microscope (MVX10; Olympus, Tokyo, Japan). Images were analyzed using ImageJ.
RNA extraction, quantitative real-time PCR (qRT-PCR), and RNA sequencing
Total RNA was extracted using TRIzol reagent (Vazyme, Nanjing, Jiangsu, China) and reverse transcribed into cDNA. qRT-PCR was performed using a SYBR Green PCR Kit (Vazyme). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control. The primer sequences are listed in Table S2. RNA sequencing was performed by Novogene (Beijing, China). Differentially expressed genes and enriched pathways were identified using the “edgeR” package in R (version 3.12.1; R Foundation for Statistical Computing).
Protein extraction, western blotting, and enzyme-linked immunosorbent assay (ELISA)
Cells and tissues were lysed in RIPA buffer (Servicebio, Wuhan, China). Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Burlington, MA). The membrane was blocked and incubated with specific antibodies. Proteins were visualized using an ECL detection system (Millipore). Protein bands were quantified using ImageJ, and GAPDH was used as an internal reference for relative quantitative analysis. The antibodies used are listed in Table S3. Specific secretory proteins were detected using a human or mouse ELISA Kit (AiFang Biological, Changsha, Hunan, China), according to the manufacturer’s instructions.
RNA stability assay
Transfected NSCLC cells were incubated with actinomycin D (5 μg/mL) for 0, 4, and 8 h. Total RNA was then extracted from each group and analyzed using qRT-PCR.
Ribonucleoprotein immunoprecipitation (RIP) assay
The RIP assay was performed using a Magna RIP Kit (Millipore) (10% of the lysate was used as the input control). After washing and purification, RNA was analyzed using qRT-PCR. The antibodies used for RIP assay are listed in Table S3.
Immunohistochemistry
Immunohistochemistry was performed using paraffin-embedded tissue sections. After antigen retrieval and blocking, the sections were incubated with primary and secondary antibodies and stained with DAB and hematoxylin. Immunohistochemical scores were calculated as described previously [18]. The antibodies used are listed in Table S3.
Flow cytometry
PMA-stimulated THP-1 cells were collected after co-culture with cancer cells. The cells were incubated with specific antibodies for 30 min at 4ºC. Flow cytometry was performed using a flow cytometer (BD FACSCanto; BD, Franklin Lakes, NJ). The antibodies used are listed in Table S3.
Immunofluorescence
Transfected NSCLC cells or PMA-stimulated THP-1 cells, after co-culture with cancer cells, were seeded on coverslips in 24-well plates. After fixation and permeabilization, the cells were incubated with primary and secondary antibodies. The cells were counterstained with DAPI and observed by fluorescence microscopy. The antibodies used are listed in Table S3.
m6A RNA methylation
The m6A content in total RNA was quantified using an m6A RNA Methylation Assay Kit (Abcam). Total RNA (250 ng) and the m6A standard were incubated with the binding solution followed by incubation with diluted capture and detection antibodies. The m6A content was determined by measuring the absorbance at 450 nm (BioTek).
Methylated RNA immunoprecipitation (MeRIP)–qRT-PCR
MeRIP–qRT-PCR was performed using a MeRIP m6A Kit (RiboBio). Total RNA (100 µg) was cut into 100 to 150 bp fragments. The fragmented RNA and magnetic beads coated with m6A or IgG antibodies were co-incubated for 2 h at 4 °C (10% of the total RNA was used as the input control). After elution and purification, RNA was analyzed using qRT-PCR. Potential m6A modification sites on JAK2 were predicted using the online software SRAMP (https://www.cuilab.cn/sramp↗) and RMBase v3.0 (https://rna.sysu.edu.cn/rmbase3/index.php↗). Two pairs of specific primers were designed based on the predicted modification sites. The primer sequences are listed in Table S2.
Dual luciferase reporter assay
The wild type JAK2 sequence and the corresponding mutated sequence (replaced adenosine [A] with thymidine [T] in the potential m6A motifs) were inserted into the pmirGLO reporter vector (keyGEN BioTECH, Nanjing, Jiangsu, China) to obtain the JAK2-WT and JAK2-MUT luciferase reporter plasmids, respectively. NSCLC cells were co-transfected with the reporter plasmids and si-NC or si-ALKBH5 in 12-well plates for 48 h. Finally, using the Dual Luciferase Reporter Gene Assay kit (Yeasen Biotechnology, Shanghai, China), the cell lysate was collected, and both firefly and Renilla luciferase activities were detected.
Animal experiments
C57BL/6 J mice (aged 4–6 weeks) were assigned randomly to each group. LLC cells (approximately 1 × 106) with stable overexpression or knockdown of ALKBH5 were suspended in PBS, mixed with an equal volume of matrix, and injected into the axilla. When tumors reached an appropriate size, mice in the treatment group were treated with anti-PD-L1 antibody (200 µg intraperitoneally three times a week). The tumors were excised, weighed, and preserved. Tumor volume was calculated as length × width2 × 0.5. Tumor inhibition rate (%) was calculated using the following formula: [(tumor weight of control group—tumor weight of treatment group)/tumor weight of the control group] × 100% [19].
Statistical analysis
Data are expressed as the mean ± SEM. Student’s t-test or two-way ANOVA was used to compare the differences between groups. Pearson’s correlation coefficient was determined to evaluate the correlation between two genes. All experiments were performed independently at least three times. Statistical analysis was conducted using GraphPad Prism (version 9; GraphPad Software). *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.
Results
ALKBH5 is upregulated in NSCLC and is associated with immune response
Kaplan–Meier analysis showed that increased ALKBH5 expression was associated with shorter overall survival in patients with lung cancer (Fig. 1D). Conversely, increased ALKBH5 expression was associated with longer overall survival in patients who received immunotherapy (Fig. 1E). These contradictory results suggest that ALKBH5 may play a role in modulating the TME.
To determine whether ALKBH5 expression in NSCLC tissues correlates with immunotherapy response, we collected paraffin-embedded tissue samples from 55 patients with advanced NSCLC who received immunotherapy. Patients were divided into two groups (with and without durable clinical benefit), according to the Response Evaluation Criteria in Solid Tumors (version 1.1). Statistical analysis showed that the ALKBH5 immunohistochemical score was higher in patients with durable clinical benefit (DCB) than in those with no durable clinical benefit (NDB) (Fig. 1F).
Based on ALKBH5 immunohistochemical scores, the patients were divided into high (6–12) and low (0–5) ALKBH5 subgroups. We also evaluated the relationship between ALKBH5 expression, PD-L1 (a classic immunosuppressive molecule), and immune cell markers, which play important roles in the TME. Increased ALKBH5 expression was associated with higher PD-L1, CD68, and CD206 scores (Fig. 1G). However, no correlation was observed between ALKBH5 expression and CD8 score (Fig. 1G). The positive relationship between ALKBH5 and PD-L1 expression was confirmed in 40 cases of NSCLC by performing Pearson correlation coefficient analysis (Fig. 1H). Collectively, the results show that ALKBH5 is highly expressed in NSCLC tissues and cell lines and is associated with an immunosuppressive microenvironment and immunotherapy response.
ALKBH5 is upregulated in NSCLC and is associated with the immune response.qRT-PCR analysis of m6A regulators (ALKBH5, FTO, METTL3/14/16, and WTAP) in 40 pairs of NSCLC tissues and adjacent normal tissues.qRT-PCR analysis of ALKBH5 expression in A549, BEAS-2B, H460, H1299, H1975, HCC-827, PC-9, and SPC-A1 cells.Western blot analysis of ALKBH5 expression in A549, BEAS-2B, H460, H1299, H1975, HCC-827, PC-9, and SPC-A1 cells. GAPDH was used as the loading control.Kaplan–Meier analysis of the relationship between ALKBH5 expression and overall survival in patients with lung cancer.Kaplan–Meier analysis of the relationship between ALKBH5 expression and overall survival in patients with cancer treated with immunotherapy.Representative immunohistochemical images of ALKBH5 in DCB (n = 30) and NDB (= 25) groups (left panel; scale bar = 20 µm) and immunohistochemistry correlation analysis (right panel).Representative immunohistochemical images of CD8, CD68, CD206, and PD-L1 in the low (= 28) and high ALKBH5 (= 27) expression groups (upper panel; scale bar = 20 µm) and immunohistochemistry correlation analysis (lower panel).Pearson correlation coefficient analysis of ALKBH5 and PD-L1 expression in 40 cases of NSCLC. *< 0.05; **< 0.01; ***< 0.001; NS, not significant A B C D E F G H n n n P P P
ALKBH5 upregulates JAK2 expression to activate the JAK2/p-STAT3 pathway
ALKBH5 upregulates JAK2 expression to activate the JAK2/p-STAT3 pathway.qRT-PCR analysis of the efficiency of siRNA-mediated knockdown of ALKBH5 in A549 and H1299 cells.qRT-PCR analysis of JAK2 expression in A549 and H1299 cells transfected with ALKBH5 siRNA.Western blot analysis of ALKBH5 and JAK2 expression in A549 and H1299 cells transfected with ALKBH5 siRNA (loading control = GAPDH).The mRNA stability of JAK2 in ALKBH5-knockdown A549 and H1299 cells treated with actinomycin D (5 µg/mL).RIP–qRT-PCR analysis of the binding affinity of ALKBH5 to JAK2 in A549 and H1299 cells (control = IgG).Pearson correlation coefficient analysis of ALKBH5 and JAK2 mRNA expression in 40 cases of NSCLC.Western blot analysis of ALKBH5, JAK2, and p-STAT3 expression in A549 and H1299 cells with or without ALKBH5 knockdown (loading control = GAPDH).Immunofluorescence analysis of p-STAT3 expression in A549 and H1299 cells with or without ALKBH5 knockdown (scale bar = 20 μm). **< 0.01; ***< 0.001; NS, not significant A B C D E F G H P P
ALKBH5 regulates JAK2 expression in an m6A-YTHDF2-dependent manner
Given that the ALKBH5 overexpression-induced decrease in m6A modification leads to JAK2 upregulation, we hypothesized that the opposite regulatory pattern may be due to the involvement of YTHDF2, which promotes mRNA degradation by recognizing m6A modifications [21]. To explore the potential relationship between YTHDF2 and JAK2, we transfected A549 and H1299 cells with siRNA to inhibit YTHDF2 expression. The transfection efficiency was confirmed by qRT-PCR (Fig. 3G). YTHDF2 knockdown increased JAK2 mRNA and protein expression (Fig. 3H and I). RIP–qRT-PCR confirmed the binding of YTHDF2 to JAK2 mRNA in NSCLC cells (Fig. 3J). The RNA stability assay showed that the decay of JAK2 mRNA was significantly slowed down in NSCLC cells subjected to YTHDF2 knockdown (Fig. 3K). Moreover, the ALKBH5 knockdown-mediated downregulation of JAK2 was reversed by silencing YTHDF2 in NSCLC cells (Fig. 3L). Thus, the ALKBH5-mediated regulation of JAK2 is mediated by the recognition of m6A modifications on JAK2 mRNA by YTHDF2.
ALKBH5 regulates JAK2 expression in an m6A-YTHDF2-dependent manner.qRT-PCR analysis of the efficiency of ALKBH5 overexpression in H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, and OE-H204A.Overall m6A deposited RNAs in H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, and OE-H204A detected using the colorimetric m6A assay.Western blot analysis of ALKBH5, JAK2, and p-STAT3 expression in H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, or OE-H204A (loading control = GAPDH).MeRIP–qRT-PCR analysis of m6A modification sequences of JAK2 transcript in ALKBH5 overexpression and knockdown H1299 cells.Schematic showing the sequence of the wild type and m6A motif mutation (A to T) plasmids containing luciferase reporter gene vectors.Dual luciferase reporter analysis of wild type and m6A motif mutation reporter vector plasmids in A549 and H1299 cells with or without ALKBH5 knockdown.qRT-PCR analysis of the efficiency of YTHDF2 knockdown in siRNA-transfected A549 and H1299 cells.qRT-PCR analysis of JAK2 mRNA expression in A549 and H1299 cells transfected with YTHDF2 siRNA.Western blot analysis of JAK2 and YTHDF2 expression in A549 and H1299 cells with or without YTHDF2 knockdown (loading control = GAPDH).RIP–qRT-PCR analysis of the binding affinity of YTHDF2 to JAK2 in A549 and H1299 cells (control = IgG).The mRNA stability of JAK2 in YTHDF2-knockdown A549 and H1299 cells treated with actinomycin D (5 µg/mL).Western blot analysis of JAK2 expression in ALKBH5-knockdown A549 and H1299 cells with or without YTHDF2 deficiency (loading control = GAPDH). *< 0.05; **< 0.01; ***< 0.001; NS, not significant A B C D E F G H I J K L P P P
ALKBH5 induces PD-L1 expression and lungcell proliferation, migration, and angiogenesis via the JAK2/p-STAT3 pathway cancer
The JAK2/p-STAT3 pathway is an important pathway involved in the endogenous expression of PD-L1 (in tumor and immune cells) that promotes cancer progression [22–24]. The previous results show that ALKBH5 expression is positively correlated with PD-L1 expression in NSCLC tissues, and that ALKBH5 positively regulates PD-L1 expression in NSCLC cell lines (Figure S4). Therefore, we next investigated whether ALKBH5 induces PD-L1 expression and promotes NSCLC progression through the JAK2/p-STAT3 pathway.
ALKBH5 induces PD-L1 expression and the malignant phenotype of NSCLC by regulating the JAK2/p-STAT3 pathway.qRT-PCR analysis of PD-L1 expression in ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM).Western blot analysis of PD-L1 and p-STAT3 expression in ALKBH5-overexpressing H1299 and H1975 cells treated with AG490 (50 μM; loading control = GAPDH).CCK-8 assay of the proliferation of ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM).Colony formation assay of ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM).Transwell assay of the migration of ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM).Quantification of tube formation in HUVECs treated with the supernatant of ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM; magnification, × 100).qRT-PCR analysis of VEGFA expression in ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM).ELISA of VEGFA secreted into the supernatant of ALKBH5-overexpressing H1299 and H1975 cells treated with or without AG490 (50 μM). *< 0.05; **< 0.01; ***< 0.001 A B C D E F G H P P P
CCL2 and CXCL10 are targets of ALKBH5
![RNA sequencing identifies CCL2 and CXCL10 as targets of ALKBH5.Volcano plot of up- (red) and down-regulated (green) genes (log2[Fold Change] > 1 and< 0.05) in H1299 cells transfected in triplicate with si-ALKBH5 or si-NC.Gene Ontology analysis of differentially expressed genes after ALKBH5 knockdown.qRT-PCR analysis of the expression of six selected chemokines in A549 and H1299 cells transfected with ALKBH5 siRNA.ELISA of CCL2 and CXCL10 secreted into the supernatant of ALKBH5-knockdown A549 and H1299 cells.qRT-PCR analysis of CCL2 and CXCL10 expression in H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, and OE-H204A.ELISA of CCL2 and CXCL10 secreted into the supernatant of H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, and OE-H204A. *< 0.05; **< 0.01; ***< 0.001; NS, not significant A B C D E F P P P P](https://europepmc.org/articles/PMC11177518/bin/13046_2024_3073_Fig5_HTML.jpg.jpg "Click to view full size")
RNA sequencing identifies CCL2 and CXCL10 as targets of ALKBH5.Volcano plot of up- (red) and down-regulated (green) genes (log2[Fold Change] > 1 and< 0.05) in H1299 cells transfected in triplicate with si-ALKBH5 or si-NC.Gene Ontology analysis of differentially expressed genes after ALKBH5 knockdown.qRT-PCR analysis of the expression of six selected chemokines in A549 and H1299 cells transfected with ALKBH5 siRNA.ELISA of CCL2 and CXCL10 secreted into the supernatant of ALKBH5-knockdown A549 and H1299 cells.qRT-PCR analysis of CCL2 and CXCL10 expression in H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, and OE-H204A.ELISA of CCL2 and CXCL10 secreted into the supernatant of H1299 and H1975 cells transfected with OE-NC, OE-ALKBH5, and OE-H204A. *< 0.05; **< 0.01; ***< 0.001; NS, not significant A B C D E F P P P P
ALKBH5 recruits PD-L1TAMs and promotes M2 macrophage polarization by inducing CCL2 and CXCL10 secretion +
ALKBH5 recruits PD-L1TAMs and promotes M2 macrophage polarization.Transwell assay of the migration of PMA-stimulated THP-1 cells co-cultured with conditioned medium from ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins.qRT-PCR analysis of PD-L1 expression in PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins.Western blot analysis of PD-L1 expression in PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins (loading control = GAPDH).Flow cytometry analysis of the mean fluorescence intensity (MFI) of PD-L1 in CD206TAMs co-cultured with ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins.Immunofluorescence analysis of CD206PD-L1TAMs in PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins (blue = DAPI; green = CD206; red = PD-L1; scale bar = 20 µm).Flow cytometry analysis of the percentage of CD11bCD206TAMs in PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown A549 and H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins. *< 0.05; **< 0.01; ***< 0.001 + + + + + + A B C D E F P P P
TAM-secreted IL-6 and ALKBH5 synergistically to activate the JAK2/p-STAT3 pathway
ALKBH5 has a synergistic effect with IL-6 on the activation of the JAK2/p-STAT3 pathway.Schematic of the co-culture model (created with BioRender.com).qRT-PCR analysis of IL-6 expression in A549 or H1299 cells, THP-1 macrophages, and PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown or control A549 and H1299 cells.ELISA of IL-6 secreted from A549 or H1299 cells, THP-1 macrophages, and PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown or control A549 and H1299 cells.qRT-PCR analysis of ALKBH5 expression in H1299 and H1975 cells treated with or without IL-6 (20 ng/mL).Western blot analysis of ALKBH5, p-JAK2, and p-STAT3 expression in H1299 and H1975 cells treated with or without IL-6 (20 ng/mL; loading control = GAPDH).Western blot analysis of p-STAT3 expression in ALKBH5-overexpressing H1299 and H1975 cells treated with or without IL-6 (20 ng/mL; loading control = GAPDH). *< 0.05; **< 0.01; ***< 0.001; NS, not significant A B C D E F P P P
ALKBH5 promotes tumor growth in vivo and is sensitive to anti PD-L1 therapy
To clarify the relationship between ALKBH5 expression and immunotherapy response, ALKBH5-knockdown LLC cells were used to establish a subcutaneous mouse model of NSCLC. The knockdown efficiency was confirmed by qRT-PCR and western blotting (Fig. 8G and H). The mice were divided into four groups according to ALKBH5 expression and anti-PD-L1 treatment. The process of drug administration experiment was shown in Fig. 8I. The results showed that ALKBH5 knockdown and anti-PD-L1 treatment inhibited tumor growth and reduced tumor burden (Fig. 8J). The inhibitory effect of anti-PD-L1 therapy on tumor growth was attenuated in ALKBH5-knockdown mice, suggesting that lung cancer cells with high ALKBH5 expression are more sensitive to anti-PD-L1 therapy (Fig. 8K). Collectively, the in vivo studies showed that ALKBH5 promotes cancer progression by facilitating tumor growth and maintaining an immunosuppressive microenvironment, whereas anti-PD-L1 therapy is more effective in tumors with high ALKBH5 expression.
ALKBH5 promotes tumor growth in vivo and is sensitive to anti PD-L1 therapy.qRT-PCR analysis of the efficiency of ALKBH5 overexpression in LLC cells.Western blot analysis of the efficiency of ALKBH5 overexpression in LLC cells (loading control = GAPDH).Left panel: ALKBH5 overexpression promotes tumor growth in C57BL/6 J mice (= 4). Middle panel: Tumor volumes measured by growth curve every three days from day 6 to day 21 after cell transplantation. Right panel: Tumor weight determined immediately after removal from C57BL/6 J mice.qRT-PCR analysis of tumor mRNA expression of CCL2, CXCL10, JAK2, and PD-L1.ELISA of CCL2 and CXCL10 in plasma.Representative immunohistochemical images of ALKBH5, CD8, CD31, CD206, F4/80, JAK2, Ki-67, and PD-L1 expression in ALKBH5-overexpressing and control mice (upper panel; scale bar = 20 µm) and immunohistochemistry correlation analysis (lower panel).qRT-PCR analysis of the efficiency of ALKBH5 knockdown in LLC cells.Western blot analysis of the efficiency of ALKBH5 knockdown in LLC cells (loading control = GAPDH).Schematic of the cell transplantation and drug administration experiment (created with BioRender.com).Left panel: ALKBH5 knockdown and anti-PD-L1 therapy attenuated tumor growth in C57BL/6 J mice (= 4). Middle panel: Tumor volumes measured by growth curve every three days from days 6 to 21 after cell transplantation. Right panel: Tumor weight determined immediately after removal from C57BL/6 J mice.Tumor inhibition rate of ALKBH5-knockdown and control mice treated with anti-PD-L1 therapy. *< 0.05; **< 0.01; ***< 0.001; NS, not significant A B C D E F G H I J K n n P P P
Discussion
Immune cells, including macrophages, granulocytes, lymphocytes, and dendritic cells can create a microenvironment that regulates tumor progression [31–33]. Additionally, tumor cells can recruit immune cells, or affect the activity of immune cells, by secreting various inflammatory cytokines and chemokines [34–36]. This interaction maintains an immunosuppressive microenvironment. TAMs (an important component of the TME) have an M2-like phenotype, as well as immunosuppressive and pro-tumor functions [37]. Examining the interaction between tumor cells and TAMs and understanding the drivers of this interaction may provide novel avenues for NSCLC treatment.
PD-L1 upregulation is an important mechanism of tumor immune escape. Tumor cell-expressed PD-L1 binds to the PD-1 receptor on CD8+ T lymphocytes, thereby inhibiting the activity of effector T cells, which weakens the anti-tumor activity of the immune system [38]. TAMs are an important source of PD-L1 in the TME. On the one hand, PD-L1 expression in TAMs is induced by cytokines and chemokines, while on the other hand, it is regulated by intrinsic carcinogenic pathways, with the former playing a more important role [39, 40]. However, the relationship between PD-L1+ TAMs and tumor progression remains controversial, and the relevance of PD-L1+ TAMs to immunotherapy outcomes is unclear. Most studies [39–41] support the role of PD-L1+ TAMs in negatively regulating T cell activity and promoting immune escape. However, some studies [42, 43] also suggest that PD-L1+ TAMs may contribute to immune activation in the TME, which is beneficial to the prognosis of patients with cancer.
According to previous studies, m6A modification plays an important role in regulating PD-L1 expression. ALKBH5, FTO, METTL3, and METTL16 directly modify PD-L1 mRNA by m6A methylation [44–48]. ALKBH5 may also indirectly regulate PD-L1 expression via the m6A modification of other mRNAs in cancer cells [16, 49]. In addition, ALKBH5 can promote PD-L1 expression in TAMs in liver cancer [50]. However, few studies have looked into the regulatory role of m6A methylation in PD-L1 expression and immune surveillance in lung cancer. Yu et al. found that METTL3 directly affects the stability of PD-L1 mRNA and the efficacy of anti-PD-1 in an m6A-dependent manner [51]. Liu et al. and Sun et al. found that METTL3 regulates the ubiquitination-mediated degradation of PD-L1 protein via methylation modification of some non-coding RNAs, thus affecting the infiltration and activation of CD8+T cells [52, 53]. In addition, Xu et al. showed that the low expression of the METTL3/IL-18 axis in cancer-associated fibroblasts promotes PD-L1 expression in NSCLC cells, thus inhibiting the immune activity of CD8+T and aggravating the PD-L1 mediated immunosuppression of NSCLC [54]. Based on this information, we explored the effect of the demethylating enzyme ALKBH5 on the anti-PD-L1 efficacy in NSCLC through the cross-talk of tumor cells and TAMs, hoping to shed further light on the role of m6A modification in immunotherapy. We found, on the one hand, that ALKBH5 regulates JAK2 expression through m6A methylation, activating the JAK2/p-STAT3 pathway to increase PD-L1 expression and promote NSCLC progression. On the other hand, CCL2 and CXCL10 are transcriptionally activated by ALKBH5, further recruiting PD-L1+ TAMs and promoting M2 macrophage polarization. Increased ALKBH5 expression confers increased sensitivity to anti-PD-L1 therapy in lung cancer cells. This conclusion is also supported by Liu et al. [55], who found that among nearly 500 patients with NSCLC, those harboring macrophages expressing PD-L1 had better overall survival when treated with anti-PD-1/PD-L1 therapy.
Herein, we also showed that ALKBH5-recruited TAMs secrete IL-6 into the TME of NSCLC cells. IL-6 activated the JAK2/p-STAT3 pathway in NSCLC cells, which exerts a synergistic effect with ALKBH5, thus maintaining an immunosuppressive microenvironment that drives tumorigenesis. These findings further our understanding of the interaction between tumor cells and TAMs in the TME.
Illustrative model of the proposed mechanism.ALKBH5 promotes NSCLC progression and susceptibility to anti-PD-L1 therapy by modulating the interactions between tumor and macrophages (created with BioRender.com)
Conclusions
The findings of the present study imply that ALKBH5 is a potential biomarker to predict the response of patients with NSCLC to anti PD-L1 immunotherapy, and that targeting ALKBH5 may be a promising strategy to enhance anti-tumor immunity. They also contribute to our understanding of the mechanism underlying m6A modifications in the TME of NSCLC. Nevertheless, further studies are needed to target m6A regulators to enhance both the anti-tumor immune response and the efficacy of combining targeted m6A modification with immunotherapy.
Supplementary Information
Additional file 1: Table S1. siRNA sequences.Additional file 2: Table S2. qRT-PCR primer sequences.Additional file 3: Table S3. Antibody sources and dilutions.Additional file 4: Figure S1. SRAMP predicted m6A modification sites on JAK2. A m6A modification sites predicted on the main JAK2 transcript based on online software SRAMP. Additional file 5: Figure S2. Basal expression level of JAK2 in normal bronchial epithelial cells and common NSCLC cells. A qRT-PCR analysis of JAK2 expression in A549, BEAS-2B, H460, H1299, H1975, HCC-827, PC-9, and SPC-A1 cells. B Western blot analysis of JAK2 expression in A549, BEAS-2B, H460, H1299, H1975, HCC-827, PC-9, and SPC-A1 cells. GAPDH was used as the loading control. *P < 0.05; **P < 0.01; ***P < 0.001.Additional file 6: Figure S3. Construction of specific primers based on potential m6A modification sites on JAK2 transcript. A The online software SRAMP and RMBase V3.0 predicted 7 and 49 potential m6A modification sites on JAK2 transcript, respectively. B Three overlapping potential m6A modification sites on the JAK2 transcript were located at 1730 bp, 1759 bp, and 3605 bp on the JAK2 transcript from the 5’ end. Two pairs of specific primers were constructed to amplify the fragments near the sites (sequence 1 and sequence 2).Additional file 7: Figure S4. ALKBH5 induces PD-L1 expression in NSCLC cells. A qRT-PCR analysis of PD-L1 expression in A549 and H1299 cells transfected with ALKBH5 siRNA. B Western blot analysis of PD-L1 expression in A549 and H1299 cells transfected with ALKBH5 siRNA (loading control = GAPDH). **P < 0.01; ***P < 0.001.Additional file 8: Figure S5. SRAMP predicted m6A modification sites on CCL2 and CXCL10. A m6A modification sites predicted on CCL2 based on online software SRAMP. B m6A modification sites predicted on CXCL10 based on online software SRAMP.Additional file 9: Figure S6 ALKBH5 promotes M2 macrophage polarization in NSCLC. A qRT-PCR analysis of the expression of M1 (CD86, IL-1β, and TNF-α) and M2 (CD163, CD206, and IL-10) polarization markers in PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown H1299 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins. B qRT-PCR analysis of the expression of M1 (CD86, IL-1β, and TNF-α) and M2 (CD163, CD206, and IL-10) polarization markers in PMA-stimulated THP-1 cells co-cultured with ALKBH5-knockdown A549 cells treated with or without CCL2 (200 ng/mL) and CXCL10 (50 ng/mL) recombinant proteins. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.