NNMT orchestrates the proangiogenic phenotype of cancer-associated fibroblasts via epigenetically regulating ETS2 in oral squamous cell carcinoma

doi:10.21203/rs.3.rs-3642085/v1

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NNMT orchestrates the proangiogenic phenotype of cancer-associated fibroblasts via epigenetically regulating ETS2 in oral squamous cell carcinoma

https://doi.org/10.21203/rs.3.rs-3642085/v1

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Tumor angiogenesis is pivotal in tumorigenesis and progression. While cancer-associated fibroblasts (CAFs) are known to promote angiogenesis in oral squamous cell carcinoma (OSCC), the underlying mechanisms by which CAFs facilitate angiogenesis within the tumor microenvironment remain elusive. Nicotinamide N'-methyltransferase (NNMT), a member of the N-methyltransferase family, is found to be highly expressed and is identified as a key molecule in the activation of CAFs and the progression of OSCC. Here, combined with our newly established assembled organoid model and fibroblast-endothelial cell (EC) co-culture model, we discovered that stromal NNMT contributed to angiogenesis and tumor growth. Intriguingly, knocking down NNMT in CAFs in OSCC reduced VEGFA expression both in vivo and in vitro. At the molecular level, high expression of NNMT in CAFs promotes ETS2 expression by regulating H3K27 methylation level through mediating methylation deposition. Moreover, ETS2 was furtherly verified to be an activating transcription factor of VEGFA in this study. Collectively, our findings delineated a molecular network in which stromal NNMT initiated an epigenetic reprogramming-ETS2-VEGFA signaling axis, thereby modulating angiogenesis in OSCC.

Angiogenesis

NNMT

organoid

histone methylation

OSCC

Oral squamous cell carcinoma (OSCC) remains a predominant malignancy of the lips and oral cavity [1]. The tumor microenvironment (TME) of OSCC is intricately heterogeneous, comprised largely of stromal components such as fibroblasts, immune cells, blood vessels, and extracellular matrix (ECM), which are instrumental in tumor progression [2]. CAFs are the paramount and functionally significant entities within the OSCC stroma and play a central role in the TME. Accumulating evidence has revealed that CAFs could help tumor cells to promote angiogenesis [3–4].

Tumor angiogenesis, which refers to the formation of new blood vessels supplying tumors with essential nutrients and oxygen, is crucial for tumor growth, invasion, and metastasis [5]. Importantly, CAFs significantly influence tumor angiogenesis by regulating the secretion of proangiogenic factors, notably vascular endothelial growth factor A (VEGFA) [6]. Despite the extensive utilization of VEGF antagonists (e.g., monoclonal antibodies and TKIs) in cancer therapy, resistance to anti-angiogenic agents remains a great challenge [7–8]. Hence, a deeper understanding of the mechanisms underlying tumor angiogenesis and the factors modulating VEGFA expression is imperative for refining therapeutic strategies. Within this paradigm, we identified NNMT as a prominent regulator of angiogenesis in OSCC progression.

NNMT, a member of N-methyltransferase family, facilitates the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide, consuming SAM in the process [9]. This activity results in a "methyl sink," leading to DNA/histone hypomethylation, thereby modulating cellular proliferation and migration from an epigenetic perspective [10–14]. Previous studies confirmed the high NNMT protein expression in the OSCC stroma, and particularly, NNMT in fibroblasts was found to regulate collagen synthesis and ECM remodeling [15, 16]. Our current research highlights that ETS2, an ETS family transcription factor known to facilitate endothelial cell branching and tube formation [17, 18], is transcriptionally modulated by NNMT. With ETS2 expression found to be upregulated in breast cancer mesenchymal cells and its ablation in fibroblasts shown to thwart tumor angiogenesis [19], we postulate a NNMT-ETS2-VEGFA regulatory axis in CAFs that drives angiogenesis in OSCC.

To substantiate this hypothesis, we introduced a novel angiogenesis assay and devised an assembled organoid system incorporating endothelial cells, simulating the direct interplay between distinct cell types. Our findings revealed that NNMT in CAFs could regulate ETS2 expression by reducing histone H3K27 methylation. Furthermore, ETS2 acts as an activating transcription factor for VEGFA in CAFs, amplifying VEGFA transcription and thus promoting angiogenesis. This underscores the significance of NNMT in tumor angiogenesis, positioning it as a prospective therapeutic target against tumor angiogenesis.

Human tissue samples

Primary OSCC specimens and adjacent normal tissue from patients who didn’t receive radiotherapy and chemotherapy were acquired from the School and Hospital of Stomatology, Wuhan University, which was approved by the Ethics Committee of School and Hospital of Stomatology, Wuhan University.

Isolation and collection of primary fibroblasts and OSCC cells

Paracancerous fibroblasts (PFs) and CAFs were isolated from OSCC tissues as previously described [20], and primary OSCC cells were collected as described [21], which both were in accordance with the Ethics Committee of School and Hospital of Stomatology at Wuhan University.

Lentiviral infection

Lentivirus targeting knockdown and overexpression of NNMT were purchased from Hanbio Technology (Shanghai, China). Lentivirus targeting knockdown and overexpression of ETS2 was purchased from GeneChem (Shanghai, China). An empty vector lentivirus with red and green fluorescent was purchased from GeneChem (Shanghai, China). CAFs and PFs were infected with lentivirus for 72 hours, after which, the infected cells were cultured with 4 µg/ml puromycin for 24 hours.

Animals experiment

All Animal studies were approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University (Approval Number: S07921100A). 4-to 6-week-old BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. For the xenograft experiment, 2×10⁵ primary oral cancer cells, 2×10⁵ CAFs or PFs treated differently, and 4×10⁴ human umbilical vein endothelial cells (HUVEC) were mixed in 50 µl PBS added with 50 µl matrigel and injected subcutaneously into mice. Tumor volumes were determined every two days by caliper measurements, tumor volume = (width² × length)/2. After indicated days, the mice were euthanized, and the tumor loads were removed for further analysis.

RNA isolation and quantitative PCR

Total RNA was extracted using RNAiso Plus (Takara, Tokyo, Japan) and reverse-transcribed to cDNA (Takara) [21]. The primer sequences are provided in Table 1.

Enzyme-linked immunosorbent (ELISA) assay

Conditioned medium was acquired from PFs or CAFs cultured in FBS-free DMEM/HG medium for 24 hours. Afterward, ELISA kits (4 A Biotech Co, Ltd, China) were applied to determine the concentration of VEGF-A.

Tube-formation assay

1×10⁴ HUVEC were seeded into 48-well plates precoated with 300 µl Matrigel ( Cultrex Basement Membrane Extract, BME, R&D Systems, 432-010-01), and cultivated in the conditioned medium of PF or CAFs accepting different treatments. Six hours later, representative photos of tube-like structures were taken.

Generation of fibroblasts-HUVEC clusters

PFs or CAFs infected with green fluorescent empty vector lentivirus and HUVEC infected with red fluorescent empty vector lentivirus were mixed at the indicated ratio of 5:1 to construct fibroblasts-EC clusters. Cells were resuspended in DMEM/HG (Thermo Fisher Scientific, Inc.) and ECM (Cell Science) in an ultra-low attached plate (Corning, 3471). 24 hours later, The cluster with diameter > 75µm was defined as a cluster. The length of tube-like structures in each cluster were counted.

Constitution of primary oral squamous cell carcinoma assembled organoids

The fibroblasts, OSCC cells, and HUVEC were mixed at the indicated ratio of 5:1:1 to construct fibroblast-EC-OSCC cell clusters and then resuspended in DMEM/F12 (Thermo Fisher Scientific, Inc.), DMEM/HG (Thermo Fisher Scientific, Inc.), and ECM (Cell Science) in an ultra-low attached plate (Corning, 3471), and after 48 hours, the spheroid with diameter > 75µm was defined as a cluster. To construct assembled organoids, fibroblasts - ECs - OSCC cell clusters were collected and mixed with matrigel (200 clusters/300 matrigel), then embedded into preheated 24-well plates and incubated in a mixed medium of ECM (Sciencecell, #1001) and organoid culture medium.

Immunohistochemical and immunofluorescence assays

Human tumor tissues and xenograft tumors embedded in paraffin and cut into 6 um were performed immunohistochemical (IHC) and immunofluorescence (IF) staining as previously described [21]. The following antibodies were applied to conduct immunohistochemical staining. Anti-NNMT antibody (dilution 1:200, #E6N2Z, CST); anti-CD31 antibody (dilution 1:200, GB11063–3, Servicebio). The following antibodies were used for immunofluorescence. Anti-NNMT antibody (dilution 1:200, #E6N2Z, CST); anti-CD31 antibody (dilution 1:200, GB11063–3, Servicebio); anti-VEGFA, antibody (dilution 1:200, #A12303, Abclonal).

Western blot assay

Immunoblotting was performed as previously described [21]. The following antibodies were used for immunoblotting. Anti-NNMT antibody (dilution 1:1000, #E6N2Z, CST); anti-VEGFA antibody (dilution 1:1000, #A12303, Abclonal); anti-ETS2 antibody (dilution 1:1000, #12280, Proteintech); anti-H3K27me3 antibody (dilution 1:1000, #9733, CST);anti-Histone H3 antibody (dilution 1: 2000, #9733, CST ).

Luciferase reporter assay

The 293T cells were cultured in 48-well plates and transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The pGL3-basic luciferase plasmid, pGL3-VEGFA construct luciferase plasmid, and the pRL-TK plasmid were purchased from GeneChem, (Shanghai, China). 0.25 µg of each VEGFA construct promoter was transfected and 0.04 ug pRL‐TK was used as an internal control per well. In co-transfection assays, 0.25 µg of each VEGFA promoter construct was co-transfected into cells with or without 0.25 µg pCMV‐ETS2 expression vector and 0.04 ug pRL‐TK vectors. Luciferase activities were measured 48 hours after transfection using the dual‐luciferase reporter assay system (Promega, Madison, MI, USA) following the manufacturer's protocol.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (CHIP) assays were performed according to the manufacturer's instructions of the kit (Thermo Fisher Scientific, Rockford, USA). The primer sequences for ETS2, and promoter sequence of VEGFA are listed in Table 1.

Statistical analysis

All experimental results were repeated a minimum of three times and presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using SPSS and GraphPad software. For statistical analysis, either one-way ANOVA or the Student’s t-test was employed. The variance within each group was comparable. Statistical significance was defined as p < 0.05.

High expression of NNMT in stromal CAFs is closely associated with tumor angiogenesis in OSCC tissues

In our previous study, we proved the high stromal expression pattern of NNMT in CAFs and its correlation with poor prognosis in OSCC patients [15]. To further determine the correlation between high stromal NNMT expression and tumor angiogenesis, we first analyzed the existing TCGA database using the Gene Expression Profiling Interactive Analysis (GEPIA) web tool. The results showed that, in patients with head and neck squamous cell carcinoma (HNSCC), the expression of NNMT was positively correlated with EC markers, PECAM1, and CD34 (Fig. 1a-b,). Additionally, we performed gene set enrichment analysis (GSEA) from TCGA-HNSC datasets, and the results showed that the upregulation of angiogenesis signatures (the gene set: HALLMARK) was strongly correlated with NNMT expression (Fig. 1c). Next, to calculate the microvessel density, we performed immunohistochemistry (IHC) to evaluate CD31 expression level in 54 samples from patients with stage I–IV oral cancer, Angio tool software was used to determine the MVD in the tissues (Fig. 1d-e, FigS1a). Next, IHC and the multiplex immunofluorescence staining assay were performed to analyze the NNMT protein levels and MVD in the OSCC tissue microarray. The results showed that NNMT expression was highly restricted to the stromal component of OSCC samples, and higher NNMT protein levels were significantly correlated with higher MVD (Fig. 1f-g, Fig.S1a). Overall, these results indicated that stromal NNMT expression was significantly correlated with tumor angiogenesis in OSCC.

NNMT regulates the pro-angiogenic phenotypes of fibroblasts in OSCC

To verify the contribution of NNMT to regulating the expression of pro-angiogenetic markers, immunoblotting assays were performed in CAFs stably infected with sh-Ctrl and sh-NNMT (Fig.S2a). The result demonstrated that the expression of VEGFA was significantly decreased in CAFs when NNMT was knocked down (Fig. 2a). Next, ELISA assays showed that the secretion of VEGFA was decreased in the culture supernatant of NNMT knockdown CAFs (Fig. 2b). To explore whether NNMT in CAFs could contribute to promoting angiogenesis in vitro, HUVEC was used. And matrigel tube formation assays indicated that the number of meshes formed by HUVEC were significantly decreased when incubated with the conditioned medium (CM) from NNMT knockdown CAFs (Fig. 2c-d). As in our previous study, we have verified that PFs, the potential precursor cells of CAFs, could be activated by NNMT overexpression [15]. To identify whether NNMT overexpression in PFs could contribute to angiogenesis, we conducted several experiments. The results showed that NNMT overexpression in PFs could help to upregulate VEGFA protein expression and VEGFA secretion in PFs (Fig. 2e-f). Matrigel tube formation assays indicated that the number of meshes formed by HUVEC were significantly increased when incubated with the CM from NNMT overexpression PFs (Fig. 2g-h).

In addition, to further clarify the function of stromal NNMT in promoting angiogenesis, we established a three-dimensional (3D) co-culture system of fibroblasts with HUVECs (Fig. 2i). (see methods for more details). In this 3D co-culture system, fibroblasts co-cultured with endothelial cells (ECs) could help ECs form lumen-like structure (Fig. 2i), which was further verified in the confocal scanning 3D model (Fig. 2j). Interestingly, the lumen formation ability of HUVEC was significantly decreased when co-cultured with NNMT knockdown CAFs (Fig. 2k, Fig.S2b). In addition, IF results showed knockdown of NNMT could decrease the number of ECs in the co-culture system (Fig. 2l-m). Taken together, these results indicated that stomal NNMT could promote the pro-angiogenic phenotypes of fibroblasts in OSCC in vitro.

Stromal NNMT accelerates angiogenesis by increasing VEGFA expression in vivo and in vitro

To clarify whether NNMT in fibroblasts contributes to tumor angiogenesis, an assembled organoid model and a co-implanted xenograft model, both integrating the patient-derived tumor organoids (PDOs) and corresponding fibroblasts, as well as HUVEC, were established for further experiments (Fig. 3a). To establish the assembled organoid model, we mixed the endothelial cells, fibroblasts, and organoid at a ratio of 1:5:5 to generate clusters, 24 hours later, clusters were mixed with matrigel and seeded into 24-well plates (Fig. 3b). As a result, the volume of assembled organoids integrated with NNMT overexpression PFs was significantly increased (Fig. 3c-d). In addition, the number of endothelial cells in assembled organoids integrated with NNMT overexpression PFs was significantly increased (Fig. 3e). More importantly, IF staining for CD31 expression in assembled organoids further confirmed these results (Fig. 3f, Fig.S3a). Next, a co-implanted xenograft model was established (Fig. 3a). We found that xenografts integrated with NNMT knockdown CAFs possessed worse tumor formation ability, while xenografts integrated with NNMT overexpression PFs exhibited enhanced tumor formation ability (Fig. 3g). Both the volume and weight of tumors integrated with NNMT knockdown CAFs were lower than those in the negative control group (Fig. 3h-i). As expected, the volume and weight of tumors of integrated with PFs with NNMT overexpression were higher than those in the negative control group (Fig. 3j-k). In addition, tumors with NNMT overexpression PFs displayed increased expression of CD31 and VEGFA. In contrast, expression levels of CD31 and VEGFA in tumors with NNMT knockdown CAFs were greatly suppressed (Fig.S3b-d). Overall, these results demonstrated that stromal NNMT accelerated angiogenesis by increasing VEGFA expression in vivo and in vitro, thus promoting tumor progression.

NNMT increases VEGFA expression through epigenetically regulating ETS2 expression

The above results indicated that NNMT could increase VEGFA expression, and VEGFA plays a crucial role in NNMT-induced tumor angiogenesis. To investigate the exact mechanism of NNMT increasing VEGFA expression, we bioinformatically analyzed the potential transcription factors that could bind to the promoter region of VEGFA (Fig. 4a-b). Then we evaluated the mRNA expression of these transcription factors in NNMT knockdown CAFs and their scramble nonsense transfectants. The results showed that only ETS2 showed low mRNA expression in NNMT knockdown cells, while others showed no significant differences (Fig. 4c). Consistently, ETS2 protein expression was significantly downregulated in NNMT knockdown CAFs, while significantly upregulated in NNMT overexpression CAFs (Fig. 4e). These results suggested that ETS2 might be involved in the regulation of VEGFA in fibroblasts by NNMT. To further confirm whether ETS2 directly regulates VEGFA, we analyzed the existing TCGA database using the GEPIA web tool. The results showed that the expression of ETS2 was positively correlated with VEGFA expression in HNSCC tissues (Fig.S4a). CAFs and PFs were then infected with ETS2 knockdown and overexpression lentiviruses and their scramble nonsense transfectants, respectively. And we found that VEGFA protein expression was significantly decreased in ETS2 knockdown CAFs, while increased in ETS2 overexpression PFs (Fig. 4f-g).

To further confirm that ETS2 serves as a transcription factor of VEGFA, we searched for the putative binding motif of the transcription factor ETS2 (Fig. 4h) and found two putative binding sites of ETS2 in the promoter region (between − 2000 and 0) of VEGFA from JASPAR website (http://jaspar.genereg.net) (Fig. 4i) [22]. To identify exact regulatory elements in the VEGFA promoter for ETS2, a series of deletions of the potential VEGFA promoter sequences were cloned into pGL3-basic vectors. The reduced promoter activity implies that the truncated region might encompass crucial binding sites for transcription factors. Transient transfections in 293T cells revealed the significance of the − 1159 to 0 bp 5' flanking sequence in conferring VEGFA promoter activity (Fig. 4j).

To further explore the effect of ETS2 on VEGFA promoter activation, we conducted transfections in 293T cells using a luciferase reporter system driven by the VEGFA promoter, with a mutation in the E1 binding region. The VEGFA promoter activity exhibited a significant increase when the wild-type construct was co-transfected with ETS2. In contrast, co-transfection of the mutated E1 construct did not result in a substantial increase in promoter activity. Therefore, overexpression of ETS2 upregulates VEGFA‐luciferase activity (Fig. 4k). Moreover, the ChIP assay showed that ETS2 could directly bind to the VEGFA promoter and the E1 site was significantly enriched in comparison with the E2 site. (Fig. 4l). These results demonstrate that ETS2 does act as a transcription factor of the VEGFA promoter. Moreover, ELISA assays showed that secretion of VEGFA was decreased in the culture supernatant of ETS2 knockdown CAFs (Fig. 4m). Matrigel tube formation assays indicated the number of meshes formed by HUVEC was significantly decreased when incubated with the CM from ETS2 knockdown CAFs (Fig. 4n, Fig.S4b). Interestingly, in the 3D co-culture model, the lumen formation ability of HUVEC co-cultured with ETS2 knockdown CAFs was significantly decreased compared to control (Fig.S4c-d). Collectively, These results demonstrated that ETS2 serves as a transcription factor of VEGFA and participates in NNMT-mediated tumor angiogenesis.

NNMT modulates the expression of ETS2 by suppressing the tri-methylation of H3K27

As NNMT drives gene expression changes in CAFs through attenuation of the S-adenosyl methionine (SAM): S-adenosyl homocysteine(SAH) ratio, which regulates the methylation of histones [16]. We performed targeted immunoblotting to detect relative levels of histone lysine methylation, and the results confirmed that H3K27 tri-methylation was upregulated upon knockdown of NNMT in CAFs of OSCC. In addition, H3K27 tri-methylation was downregulated upon NNMT overexpression in PFs. Treatment of NNMT knockdown CAFs with the EZH2 histone methyltransferase inhibitor DZNep [16] restored H3K27me3 expression (Fig. 5a). In addition, treatment of NNMT overexpression PFs with GSK-J1, an inhibitor of H3K27me3 demethylases [23], restored H3K27me3 expression (Fig. 5b). To Further explore whether NNMT regulates ETS2 expression through H3K27 trimethylation, we performed CHIP assays and found that NNMT activity reduced H3K27me3 occupancy at the promoter site of ETS2 (Fig. 5e-f). Therefore, these results demonstrated that NNMT modulates ETS2 expression by suppressing the tri-methylation of H3K27me3.

NNMT-ETS2-VEGFA signal axis orchestrates the pro-angiogenic properties of CAFs to promote tumor angiogenesis

As ETS2, which is under epigenetic regulation by NNMT, serves as a transcription factor of VEGFA, we speculated that ETS2 is involved in high NNMT expression-mediated tumor angiogenesis in CAFs of OSCC. To address this question, we used CAFs stably expressing sh-NNMT plus ETS2 overexpression. Immunoblotting results showed that knockdown of NNMT significantly decreased VEGFA expression, while overexpression of ETS2 dramatically increased VEGFA expression (Fig. 6a). ELISA assays also showed that the VEGFA secretion was decreased in the culture supernatant of CAFs with NNMT knockdown, While overexpression of ETS2 rescued VEGFA secretion (Fig. 6b). Moreover, the angiogenic effect of CMs from CAFs with sh-NNMT was attenuated, while the angiogenic function of sh-NNMT was reversed by overexpression of ETS2 (Fig. 6c-d). Consistently, HUVECs co-cultured with CAFs with NNMT knockdown exhibited a worse lumen formation ability, and this effect was recovered by ETS2 overexpression (Fig. 6e-f). In addition, IF staining showed knockdown of NNMT markedly decreased the number of ECs, while these results were reversed by ETS2 overexpression (Fig. 6g-h,). Interestingly, the volume of assembled organoids and the number of endothelial cells were significantly decreased when integrated with NNMT knockdown CAFs, whereas this effect was significantly rescued by overexpression of ETS2 (Fig. 6i, Fig.S5a-b). To assess the effect of ETS2 involved in high NNMT expression of CAFs mediated tumor angiogenesis in vivo, a co-implanted xenograft model integrated with CAFs, HUVEC, and organoid was applied, and tumor masses were harvested on day 28. The results showed knockdown of NNMT in CAFs strongly suppressed tumor volume and weight of xenograft, while overexpression of ETS2 abolished the inhibition of tumor formation ability of sh-NNMT (Fig. 6j-l). Furthermore, IF staining of tumor tissues showed that VEGFA and CD31 expression was decreased with sh-NNMT, but these results were reversed by ETS2 overexpression (Fig. 6m-o). Collectively, these data demonstrated that stromal NNMT could regulate VEGFA through epigenetically regulating the transcription of ETS2, thus promoting tumor angiogenesis of OSCC (Fig. 7).

In this study, we confirmed that NNMT expression was positively associated with microvessel density in cancerous tissues of OSCC patients. Subsequently, combined with the traditional matrigel tube formation assay and our novel fibroblasts-ECs co-culture model, we verified the role of stromal NNMT in regulating angiogenesis. In addition, assembled organoid model integrated with fibroblasts, HUVEC, and organoid was established and authenticated the role of stromal NNMT in enhancing tumor angiogenesis and promoting tumor growth in vivo and in vitro. Furthermore, we identified the potential pro-angiogenetic transcription factor ETS2 as an activating transcription factor of VEGFA. In addition, ETS2 could be epigenetically regulated by NNMT histone tri-methylation of H3K27. In summary, we proposed that the NNMT-ETS2-VEGFA signal axis orchestrates the pro-angiogenic properties of fibroblasts to promote tumor angiogenesis, and NNMT-mediated angiogenesis is a potential target for patients with OSCC.

Tumor angiogenesis is a multifaceted process instigated by augmented production and bioavailability of various soluble growth factors [24]. Although first-generation angiogenesis inhibitors and pro-angiogenic drugs have shown only moderate success, therapeutic strategies targeting angiogenesis remain an avenue with significant promise [25]. The diminished efficacy was primarily attributed to the emergence of intrinsic resistance mechanisms and reduced drug responsiveness stemming from interactions with the microenvironment [26]. Traditional angiogenesis models can only simulate indirect intercellular interactions, including the effects of cytokine secretion, so novel preclinical models that mimic the increased complexity present in vitro are needed. The concept of EC–fibroblast cocultures were first described by Bishop et al [27]. Daniela and colleagues then developed an improved human fibroblast-EC coculture assay that mimics sprouting angiogenesis from EC-coated microbeads, simulating existing endothelial structures [28]. In our study, the new fibroblast-EC coculture model was established, which could simulate direct interactions between fibroblasts and ECs, as well as, help ECs form lumen structure. More importantly, our fibroblast-EC coculture model provided a novel mechanism study model for matrix cell-promoted tumor angiogenesis.

Organoids have emerged as pivotal tools for probing tumorigenesis and the cancer evolution in vitro, demonstrating significant promise for translational research [29–35]. Currently, organoids originating from diverse human tumor types have been effectively characterized [33, 36–37]. However, many of these models tend to overlook the intricate stromal, vascular, and immune milieu of tumors, which is pivotal for devising next-generation cancer therapies that target desmoplasia, angiogenesis, and immune responses [38]. Significant strides have been taken to emulate the tumor vasculature within organoid cultures, fostering a deeper comprehension of angiogenic signaling trajectories and paving the way for potent therapeutic approaches [39–40]. Tumoroids showcasing vascular features were adeptly developed by co-culturing tumor cells with endothelial colony-forming cells originating from ECs and lung fibroblasts [41]. In our study, we established OSCC patient-derived organoids integrated with respective PFs or CAFs and HUVEC. This model is corroborated to mimic the in vivo intercellular communication observed in OSCC. Undoubtedly, the inception and utilization of this model offer enhanced avenues for mechanistic exploration and targeted therapeutic interventions.

Historically, anti-angiogenic therapies, particularly those targeting VEGFA, have been hailed as breakthroughs in oncology. However, resistance against these treatments showcases a crucial drawback in their efficacy [42]. NNMT is a metabolic enzyme responsible for the methylation of nicotinamide (NAM) using S-adenosyl methionine (SAM) as the primary methyl donor [43]. This enzymatic action directly connects one-carbon metabolism with cellular methylation balance and the concentration of nicotinamide adenine dinucleotide (NAD+) [44]. In addition, The expression of NNMT influences alterations in gene expression and induces changes in cell phenotype. This is achieved through the hypomethylation of DNA, RNA, or histones, a process associated with the modulation of the SAM: SAH ratio, which indicates methylation capacity of cells [16]. Since the enzyme plays a fundamental role in methylation processes and its role in regulating cell metabolism, its dysregulation could be a commonality among various malignancies, positioning it as a potential pan-cancer target. Previous studies have reported high expression of NNMT in various cancers and it may be a potent target for anticancer therapy [45–46]. Our study elucidates the epigenetic modifications driven by NNMT in the transcription of ETS2, which regulates the key angiogenic factor VEGFA. The combination of traditional anti-angiogenic agents with these novel approaches might address the aforementioned resistance and thus herald a new phase in cancer therapy.

Our study underscores the pivotal role of stromal NNMT in regulating tumor angiogenesis in OSCC and emphasizes the significance of the NNMT-ETS2-VEGFA axis in the tumor-stromal context. This insight may benefit future targeted therapies for OSCC that modulate angiogenesis via NNMT.

HNSCC: head and neck squamous cell carcinoma; CAFs: Cancer-associated fibroblasts; OSCC: Oral squamous cell carcinoma; NNMT: Nicotinamide N'-methyltransferase; ECs: Endothelial cells; TME: Tumor microenvironment; ECM: Extracellular matrix; VEGFA: vascular endothelial growth factor A; SAM: S-adenosylmethionine; PFs: Paracancerous fibroblasts; HUVEC: Human umbilical vein endothelial cell; IHC: immunohistochemical; IF: immunofluorescence; Elisa: Enzyme-linked immunosorbent assay; CHIP: Chromatin immunoprecipitation; GEPIA: Gene expression profiling interactive analysis; GSEA: Gene set enrichment analysis; MVD: microvessel density; CM: Conditioned medium; 3D: three dimentional; PDO: Patient-derived tumor organoid; TF: Transcription factor

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This study was supported by the National Natural Science Foundation of China (No.82273306 to SZJ and No.82373093 to SZ).

Acknowledgements

We would like to thank Dr. Xiazhou Fu (Wuhan University) for his expert guidance. This study was supported by the National Natural Science Foundation of China (No.82273306 to SZJ and No.82373093 to SZ).

Ethics approval

All protocols were approved by the Ethics Committee of School and Hospital of Stomatology, Wuhan University, and informed consent was obtained from all patients. All Animal studies were approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University (Approval Number: S07921100A).

Authors’ contributions

Conceptualization, WXM, and ZH; Literature Search, WXM and LXY; Data Collection, WXM and SCY; Formal Analysis, WXM; Validation, WXM, ZH, LXY, CY, SCY, WYF, and BJQ; Investigation and Visualization, WXM; Methodology, WXM and LXY; Writing Original Draft, WXM; Project Administration, SZ and SZJ; Review & Editing, SZ and SZJ; Supervision, SZJ. All authors read and approved the final version of the manuscript.

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No competing interests reported.

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NNMT orchestrates the proangiogenic phenotype of cancer-associated fibroblasts via epigenetically regulating ETS2 in oral squamous cell carcinoma

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Abstract

Figures

Background

Materials and Methods

Human tissue samples

Isolation and collection of primary fibroblasts and OSCC cells

Lentiviral infection

Animals experiment

RNA isolation and quantitative PCR

Enzyme-linked immunosorbent (ELISA) assay

Tube-formation assay

Generation of fibroblasts-HUVEC clusters

Constitution of primary oral squamous cell carcinoma assembled organoids

Immunohistochemical and immunofluorescence assays

Western blot assay

Luciferase reporter assay

Chromatin immunoprecipitation

Statistical analysis

Results

NNMT regulates the pro-angiogenic phenotypes of fibroblasts in OSCC

Stromal NNMT accelerates angiogenesis by increasing VEGFA expression in vivo and in vitro

NNMT increases VEGFA expression through epigenetically regulating ETS2 expression

NNMT modulates the expression of ETS2 by suppressing the tri-methylation of H3K27

NNMT-ETS2-VEGFA signal axis orchestrates the pro-angiogenic properties of CAFs to promote tumor angiogenesis

Discussion

Conclusion

Abbreviations

Statements and Declarations

References

Additional Declarations

Supplementary Files

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