Introduction

Lung cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related mortality worldwide [1]. Non-small cell lung cancer (NSCLC) represents approximately 85% of lung cancers and is the most common subtype of lung cancer [2]. Despite rapid advances in the clinical treatment of NSCLC in recent years, the prognosis of NSCLC patients is still poor due to recurrence and distant metastasis [3]. Cisplatin-based chemotherapy is the first-line treatment for the majority of metastatic NSCLC cases, but drug resistance develops rapidly [4, 5]. However, the mechanisms of cisplatin resistance and NSCLC metastasis are still poorly understood.

Phosphodiesterases (PDEs) are a class of enzymes that hydrolyse cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), reducing the signaling of these important intracellular second messengers [6]. PDEs consist of 11 family members, and each family member contains multiple subtypes [7]. PDEs are being pursued as therapeutic targets in multiple diseases, including the cardiovascular system, metabolism, pulmonary system, nervous system, immunity, and cancers [8]. For example, PDE3 inhibitors are used to treat heart failure and peripheral artery disease, and PDE4 inhibitors are approved to treat inflammatory diseases [9]. Multiple studies have shown that PDEs, such as PDE4 and PDE5, play a vital role in the progression of tumors and are regarded as potential therapeutic targets for cancer treatment [9, 10]. The PDE1 family member has three subtypes, including PDE1A, PDE1B and PDE1C, with different affinities for cAMP and cGMP [11]. The effect and mechanism of PDE1 in regulating cancer progression remain elusive.

N⁶-methyladenosine (m⁶A) is the most abundant RNA modification, and the process of m⁶A modification is reversible: m6A is installed by “writers”, removed by “erasers”, and recognized by “readers” [12]. YT521-B homology domain family member 2 (YTHDF2), belonging to the YTH domain protein family, has been validated as m⁶A “reader” and regulates the stability of messenger RNAs (mRNAs) [13]. YTHDF2 promotes the progression of lung adenocarcinoma by recognizing m6A modification and influencing mRNA fate [14]. Furthermore, YTHDF2 orchestrates the reprogramming of tumor- associated macrophages in the tumor microenvironment (TME), and YTHDF2 is an effective target to enhance cancer immunotherapy [15]. In this study, we first demonstrated that PDE1A promoted NSCLC metastasis and cisplatin resistance via interacting with YTHDF2.

Materials and methods

Materials

Vinpocetine (V107535) was purchased from Aladdin (Shanghai, China). Cisplatin (S1166), SH-4-54 (S7337), SQ22536 (S8283), and GW4869 (S7609) were obtained from Selleck Chemicals (Houston, TX, USA). Antibodies against β-actin (SC-1616), GAPDH (SC-25778), MET (SC-514148), and caspase-3 (SC-7148) were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against N-cadherin (14215), STAT3 (9139), p- STAT3 (Y-705) (9145), p-Met (Tyr1234) (3077), cleaved-PARP (9541), Vimentin (5741), JAK2 (3230S), and NRF2 (12721) were obtained from Cell Signaling Technology (Danvers, MA, USA). An antibody against α-tubulin (AT7819) was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against PDE1A (12442-2-AP), YTHDF2 (24744-1-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Cell lines and cell culture

Human NSCLC cell lines (A549, NCI-H1299, and NCI-H460), a bronchial epithelial cell line (HELF), and umbilical vein endothelial cells (HUVECs) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. All the cell lines were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China), cultured at 37 °C with 5% CO₂ and confirmed to be mycoplasma-free.

siRNA transfection

Scramble siRNA, siPDE1A, siPDE1B, siPDE1C, siSTAT3, and siYTHDF2 were synthesized by GenePharma (Suzhou, China). Then, NSCLC cells were transfected with siRNA (40 nM) using PolyPlus-transfection reagent in accordance with the instructions. The sequences of siRNAs are summarized in Table S1.

Sulforhodamine B (SRB) assay

NSCLC cells were treated with the indicated compounds, and subsequently fixed with ice-cold TCA and stained with 0.4% SRB (w/v) solution. Cell proliferation was determined by SRB assay according to previously reported methods [16].

Wound healing assay

NSCLC cells were seeded on a 24-well plate and cultured as a monolayer to 90% confluence. The monolayer was scratched with a 10 µL pipette tip, and then the cells were cultured with FBS-free culture medium for 24 h. Images of the wounded cell monolayer were taken using a microscope (Olympus, Japan) at 0 h and 24 h. The wound closure rate was calculated as follows: (G₀-G₂₄) / G₀ × 100%, where G₀ and G₂₄ represent the gap areas at 0 and 24 h, respectively.

Migration and invasion assays using transwells

Migration and invasion assays were performed using a 24-well transwell chamber system (pore size: 8 µm, Corning, USA). A total of 5 × 10⁴ cells were seeded in the upper chamber of an insert with 0.4 ml serum-free culture media in 24-well plates. Then, 0.6 ml culture medium with 20% FBS was added to the lower chamber. For invasion assays, the upper transwell chamber of the insert was coated with Matrigel (BD Biosciences, Bedford, MA, USA) before plating cells. After incubation for 24 h, migratory or invasive cells were stained with 0.5% crystal violet and analyzed under a light microscope.

Protein extraction and western blotting analysis

Briefly, cells were washed three times with cold PBS and pelleted. The pellet was resuspended in lysis buffer, incubated on ice with frequent vortex for 30 min, and the lysate was obtained by centrifugation at 12000 rpm for 30 min. Proteins were fractionated by SDS-PAGE, transferred onto PVDF membranes, blocked in 5% nonfat milk in PBS/Tween-20, and then blotted with specific primary antibody overnight at 4 °C, followed by incubation with secondary antibody for 1 h at room temperature. Bands were detected by the Odyssey CLX Image Studio system (version 5.0.21, LiCor Odyssey, LI-COR Biosciences).

Immunoprecipitation and LC-MS/MS

For coimmunoprecipitation, the cells were lysed with IP buffer and centrifuged at 10000 × g for 30 min at 4 °C. The cell lysates were treated with protein G magnetic beads at 4 °C for 1 h. Subsequent immunoprecipitation reactions were set up with equal quantities of the lysates. Primary antibody was added to the lysate, and the mixture was incubated overnight with slow shaking at 4 °C, and then incubated with protein G magnetic beads at 4 °C for 1 h. Subsequently, the lysates were centrifuged at 3000 g at 4 °C for 5 min. We aspirated the supernatant, washed the protein G magnetic beads with lysis buffer 3-4 times, and detected by SDS-PAGE or LC-MS/MS (Micrometer Biotech Company, Hangzhou, China).

Extraction of RNA and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from cells by utilizing TRIzol™ Reagent (Invitrogen, Thermo Fisher Scientific). RNA was reverse-transcribed into cDNA using an iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA). qRT-PCR was performed using the PerfectStart Green qPCR SuperMix kit (TransGen Biotech, Beijing, China). The levels of mRNA were analyzed by the Bio-Rad CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA). GAPDH was used as an endogenous control for mRNA qualification and the 2-ΔΔCt method was applied to calculate the relative expression. The primers used are listed in Table S2.

RNA binding protein immunoprecipitation (RIP)

RIP assays were performed using a Magna RIP Kit (17-701, Millipore, MA, USA) following the manufacturer’s instructions. In brief, magnetic beads precoated with 5 mg normal antibodies against PDE1A/YTHDF2 or rabbit IgG (Millipore) were incubated with cell lysates at 4 °C overnight. The beads containing immunoprecipitated RNA-protein complexs were treated with proteinase K to remove proteins. Then RNAs of interest were purified with TRIzol and measured by qRT-PCR.

Plasmid and shRNA transfection and infection

Two micrograms of overexpressing plasmid or shRNA of the indicated genes was transfected into cells using PolyPlus-transfection reagent. For shRNA used in lentivirus-mediated interference, complementary sense and antisense oligonucleotides encoding shRNAs targeting PDE1A were synthesized, annealed, and cloned into pLKO.TRC vector (Addgene, 10878). The PDE1A- FLAG overexpression plasmid was synthesized by GenScript (Nanjing, Jiangsu, China). YTHDF2-HA expression plasmids were synthesized via cloning YTHDF2 with an HA tag into the pcDNA3.1(-) vector.

In vivo animal experiment

Female nude mice (BALB/c, 4-6 weeks old) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. A total of 2 × 10⁶ NCI-H1299 cells transfected with shPDE1A/control shRNA or PDE1A overexpressing plasmid/empty vector were suspended in 0.1 ml of PBS and injected into mice via the tail vein. After 60 days, the mice were sacrificed, and the lung tissues were collected to observe pulmonary nodules. The lung, liver, kidney, pancreas, and other tissues were separated, fixed in 4% paraformaldehyde, and stained using H&E. All procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University City College (No. 22001).

mRNA stability assay

NSCLC cells were seeded in six-well plates and grown to approximately 30% confluence followed by siRNA transfection and incubation for 24 h. Then, cells were incubated with actinomycin D (5 μg/ml) for 0, 3 or 6 h followed by RNA extraction. The half-life of mRNA was analyzed by qRT-PCR. The mRNA expression for each group at the indicated time was calculated and normalized to GAPDH.

Statistical analysis

Data are presented as the mean ± SD from 3 independent experiments. Two- tailed Student’s t test was used to compare 2 groups. P values < 0.05 were considered significant. *P < 0.05; **P < 0.01; ***P < 0.001.

Results

PDE1A overexpression predicts a poor prognosis in lung cancer patients

First, we found that PDE1A was highly expressed in lung cancer compared with normal lung tissue using immunohistochemistry (Figure 1A and Figure S1) [17, 18]. As shown in Figure 1B, overexpression of PDE1A was also observed in three NSCLC cell lines compared with normal human lung fibroblasts (HELF cells). Additionally, the overexpression of PDE1A was also observed in lung cancer from high-risk patients compared with low-risk patients (p<0.0001, Figure 1C), and lung cancer patients in the high-risk group had shorter survival times than those in the low-risk group (Figure 1D) [19, 20]. Furthermore, lung cancer patients with high levels of PDE1A in their tumors had shorter overall survival than those with low PDE1A expression, indicating that PDE1A overexpression was correlated with a poor prognosis in lung cancer patients (Figure 1E) [21, 22]. Thus, PDE1A might be a novel prognostic predictor in lung cancer treatment and contribute to lung cancer progression.

Figure 1.

PDE1A promotes the metastasis and EMT of NSCLC cells both in vitro and in vivo

To investigate the biological function of PDE1A in lung cancer development, gene set enrichment analysis (GSEA) and overrepresentation enrichment analysis (ORA) were performed to analyze the biological process of PDE1A in NSCLC using LinkedOmics [23]. As shown in Figure S2A, PDE1A might be involved in the adhesion, migration, and motility of NSCLC cells, which are critical parameters in the metastatic dissemination of cancer cells. Tumor angiogenesis, the recruitment of new blood vessels, enables a pre-existing tumor to grow and metastasize [24]. PDE1A might also participate in mesenchyme development, angiogenesis, vasculature development, cellular response to VEGF stimulus, blood vessel morphogenesis and development (Figure S2A-C). Thus, we hypothesized that PDE1A might promote the metastatic potential of NSCLC cells.

First, PDE1A silencing did not cause a significant decrease in the proliferation of NSCLC cells relative to that in the control siRNA group (Figure S3A). Moreover, PDE1A overexpression had no significant effect on the proliferation of NSCLC cells (Figure S3B). As bioinformatics analysis demonstrated that PDE1A might promote the metastatic potential of NSCLC cells, wound healing and transwell assays were used to investigate the migration and invasion capacity of PDE1 family members. Knockdown of PDE1 family members suppressed the migratory ability of NCI-H1299 cells, and siPDE1A exerted a stronger suppression effect on the migration of NCI-H1299 cells than siPDE1B and siPDE1C transfection (Figure S4A). Meanwhile, siPDE1A resulted in more profound suppression of EMT progression in NCI-H1299 cells than siPDE1B and siPDE1C transfection (Figure S4B). Thus, PDE1 family members, particularly PDE1A, might be involved in the metastatic behavior of NSCLC cells.

As shown in Figure 2A and 2B, suppression of PDE1A markedly reduced the migratory and invasive capacity of NSCLC cell lines, and the wound healing assay also showed that NSCLC cells with PDE1A knockdown had a slower wound closure rate than control siRNA-transfected cells (Figure 2C and 2D). Furthermore, PDE1A knockdown increased E-cadherin expression and reduced N-cadherin expression, indicating that PDE1A silencing suppressed EMT progression in NSCLC cells (Figure 2E). Meanwhile, the PDE1 inhibitor vinpocetine significantly suppressed the migration and EMT of NSCLC cells (Figure 2F - H). To determine the effects of PDE1A on NSCLC cell migration and invasion in vivo, nude mouse models were established using PDE1A- shRNA- and control-shRNA-treated NCI-H1299 cells. As shown in Figure 2I and Figure S4C, the number of pulmonary metastatic nodules was decreased in the PDE1A-shRNA group compared with the control-shRNA group in nude mice.

Figure 2:

In contrast, PDE1A overexpression significantly enhanced the migratory and invasive capacities of NSCLC cells (Figure 3A and 3B). In addition, NSCLC cells with high PDE1A expression had a higher wound closure rate than those transfected with empty vector (Figure 3C). Meanwhile, PDE1A overexpression decreased E-cadherin expression and elevated N-cadherin expression, indicating that PDE1A promoted EMT progression of NSCLC cells (Figure 3D). To compare the expression of PDE1A in NSCLC cells with high and low invasive potential, we established series of NSCLC cell lines with differential invasive abilities through multiple selections using a transwell chamber assay (Figure 3E, Figure S5A and S5B). The protein and mRNA levels of PDE1A were higher in highly invasive NSCLC cells than in NSCLC cells with low invasive potential (Figure 3F and 3G). In an in vivo nude mouse experiment, NSCLC cells overexpressing PDE1A produced more pulmonary metastatic nodules than the parental NSCLC cells (Figure 3H and Figure S5C).

Figure 3:

NSCLC cells overexpressing PDE1A promote angiogenesis in the TME

GSEA demonstrated that PDE1A expression was positively correlated with angiogenesis in lung cancer (Figure 4A). Thus, we constructed a tumor microenvironment by coculturing NSCLC cells with vascular endothelial cells (Figure 4B). NSCLC cells overexpressing PDE1A promoted the migration of HUVECs, and NSCLC cells with low levels of PDE1A suppressed the migration of HUVECs (Figure 4C and 4D). Next, we treated NSCLC cells with GW4869 to reduce exosome release and found that GW4869 suppressed the enhancement of the migratory ability of HUVECs induced by NSCLC cells overexpressing PDE1A (Figure 4E). Meanwhile, compared with negative control, shPDE1A significantly suppressed tumor angiogenesis of NSCLC in vivo (Figure 4F). Thus, NSCLC cells overexpressing PDE1A promote angiogenesis in the TME.

Figure 4:

PDE1A promotes the metastasis of NSCLC cells via the STAT3 signaling pathway

Then, we investigated whether PDE1A-enhanced metastasis was dependent on cAMP metabolism activity. As shown in Figure S6A, the cAMP inhibitor SQ22536 failed to rescue the migrative ability suppressed by siPDE1A in NSCLC cells, indicating that the basic molecular function might not be involved in the metastasis of PDE1A. To better explore the mechanism of PDE1A in NSCLC progression, we performed bioinformatic analysis of PDE1A coexpressed genes, which revealed that PDE1A might be involved in the JAK/STAT3, Hedgehog, and TGF-β pathways in NSCLC (Figure 5A). Meanwhile, GSEA enrichment analysis demonstrated that PDE1A might participate in IL-6 production (Figure 5B), thus we hypothesized that IL- 6/JAK/STAT3 signaling might be involved in PDE1A-promoted metastasis in NSCLC. As shown in Figure 5C, PDE1A overexpression increased the phosphorylation level of STAT3 in NSCLC cells. In contrast, PDE1A knockdown or the PDE1 inhibitor vinpocetine suppressed the phosphorylation of STAT3 in NSCLC cells (Figure 5D and 5E). Moreover, STAT3 suppression by siRNA or SH-4-54 significantly reversed the enhancement of NSCLC cell migration induced by PDE1A overexpression (Figure 5F and 5G). In addition, the suppression of STAT3 inhibited PDE1A-induced EMT progression in NSCLC cells (Figure 5H and 5I). Thus, PDE1A promoted the metastasis of NSCLC cells via activating the STAT3 signaling pathway, but the direct interaction between PDE1A and STAT3 could not be observed in NSCLC cells (Figure 5J). Moreover, PDE1A was mainly overexpressed in the cytoplasm in NSCLC cells (Figure 5K), and we next further explored the mechanism by which PDE1A in promotes the STAT3 signaling pathway in the cytoplasm.

Figure 5:

PDE1A physically interacts with YTHDF2 and promotes the metastasis of NSCLC cells

To investigate the mechanism by which PDE1A promotes NSCLC metastasis and activates the STAT3 pathway, the proteins interacting with PDE1A in NSCLC were determined using immunoprecipitation followed by mass spectrometry analysis (Table S3). To identify the key proteins involved in PDE1A-mediated STAT3 activation, we generated a Venn diagram, which showed that there were 9 overlapping genes among those coexpressed with STAT3 in NSCLC clinical samples, PDE1A-interacting proteins, and overexpressed in NSCLC compared with normal tissues (Figure 6A). Meanwhile, GO enrichment analysis of PDE1A interacting genes was used to predict the molecular function of PDE1A, and PDE1A might participate in m6A-containing RNA binding in NSCLC progression (Figure 6B). Thus, we hypothesized that PDE1A might interact with YTHDF2 and participate in m6A- containing RNA binding during NSCLC progression. The physical binding between PDE1A and YTHDF2 was confirmed by silver staining and immunoprecipitation (Figure 6C and 6D). Furthermore, YTHDF2 knockdown reversed the enhancement of NSCLC migration induced by PDE1A overexpression, indicating that PDE1A might interact with YTHDF2 and promote the metastasis of NSCLC (Figure 6E and Figure S6B and S6C). The mRNA and protein levels of YTHDF2 were upregulated in NSCLC compared with normal lung tissues (Figure S7A and S7B) [25]. In addition, YTHDF2 overexpression predicted poor outcomes in lung cancer patients (Figure S7C) [21, 22]. Meanwhile, the activation of STAT3 by PDE1A could be reversed by YTHDF2 knockdown in NSCLC cells (Figure 6F). Furthermore, PDE1A was positively correlated with YTHDF2 expression with a Pearson correlation coefficient above 0.3 in NSCLC tissues (Figure 6G) [25]. Thus, PDE1A might regulate the STAT3 signaling pathway via interacting with YTHDF2.

Figure 6:

PDE1A interacts with YTHDF2 to regulate the SOCS2/STAT3 signaling pathway

To gain mechanistic insights regarding the key target mRNA by which the PDE1A/YTHDF2 complex regulates the STAT3 signaling pathway, we generated a Venn diagram, which showed that there were 33 overlapping genes among RNAs that physically interact with YTHDF2, YTHDF2- coexpressed genes, and STAT3-coexpressed genes in lung cancer (Figure 7A and Table S4) [26–29]. Then, the interactions between the YTHDF2 protein and the mRNAs of 33 overlapping genes were predicted by RNA-Protein Interaction Prediction online tool. There were three predicted targets of YTHDF2 with high scores and highly correlated with STAT3 signaling as previously reported, including NRF2, SOCS2, and MET (Table S5) [30]. SOCS family members are cytokine-inducible negative regulators of the JAK/STAT pathway, and SOCS2 suppresses the binding of JAK2 and STAT3, the activity of JAK, and STAT3 activation [31]. Thus, we hypothesized that PDE1A might interact with YTHDF2, influence the fate of SOCS2 mRNA, and regulate the STAT3 signaling pathway in NSCLC cells. As shown in Figure 7B, the interaction between YTHDF2 protein and SOCS2 mRNA was verified by RIP, and we also proved the binding between PDE1A protein and SOCS2 mRNA using RIP. Meanwhile, siPDE1A significantly enhanced the stability of SOCS2 mRNA in NSCLC cells (Figure 7C). Furthermore, YTHDF2 or PDE1A negatively regulated the expression of SOCS2 mRNA in NSCLC cells, and YTHDF2 overexpression successfully reversed the siPDE1A-induced SOCS2 mRNA accumulation. In contrast, siYTHDF2 enhanced siPDE1A-induced SOCS2 mRNA accumulation (Figure 7D). These data indicated that YTHDF2 might negatively regulate the expression of SOCS2 mRNA via cooperating with PDE1A.

Figure 7:

PDE1A suppression enhances the anti-NSCLC activity of cisplatin via regulating MET and NRF2 expression

As the PDE1A/ YTHDF2 complex might also regulate MET and NRF2, the activation of MET or NRF2 triggers NSCLC resistance to chemotherapy, such as cisplatin treatment [32, 33]. As shown in Figure 8A and 8B, siPDE1A significantly suppressed the expression of MET and NRF2 compared with control siRNA, and PDE1A overexpression enhanced the expression of MET and NRF2 in NSCLC cells. Furthermore, vinpocetine restrained the expression of MET and NRF2 in NSCLC cells (Figure 8C). These data suggest that PDE1A could positively regulate MET and NRF2; thus, we hypothesized that PDE1A might be involved in cisplatin resistance in NSCLC cells. Indeed, PDE1A knockdown significantly enhanced the cytotoxicity of cisplatin, and PDE1A overexpression decreased cisplatin-induced cytotoxicity in NSCLC cells (Figure 8D and 8E). Meanwhile, vinpocetine plus cisplatin synergistically suppressed the proliferation of NSCLC cells compared with single agent treatment alone (Figure 8F). Furthermore, vinpocetine plus cisplatin enhanced apoptosis induction in NSCLC cells compared with single agent alone (Figure 8G). In addition, knockdown of PDE1A significantly suppresses the mRNA level of MET and NRF2, and siYTHDF2 reversed the activation of MET and NRF2 induced by PDE1A overexpression (Figure 8H and 8I). These data indicate that PDE1A positively regulates MET and NRF2 via interacting with YTHDF2, and that PDE1A suppression enhances the anti- NSCLC activity of cisplatin.

Figure 8:

Discussion

NSCLC is becoming a leading cause of death globally due to its fast progression and metastatic potential, and effective therapeutic targets are urgently needed to block NSCLC metastasis [34]. PDEs are regarded as therapeutic targets for multiple diseases, but the feasibility of targeting PDEs to treat NSCLC metastasis may need further investigation [35]. In this study, we showed for the first time that PDE1 promoted metastasis and EMT progression in NSCLC cells. Furthermore, the expression of PDE1A was closely correlated with the disease progression of NSCLC. These data first indicated with PDE1A might be an efficacious therapeutic target for patients with metastatic NSCLC.

The stimulation of the PDEs requires physiological concentrations of Ca²⁺ and calmodulin, and Ca²⁺/calmodulin-dependent cyclic nucleotide PDE1 is involved in the communication between the cyclic nucleotide and Ca²⁺ second messenger systems [36]. The increasing of Ca²⁺ in cancer cells stimulates exosome biogenesis and release under both physiological and pathological conditions [37, 38]. In this context, we assumed that PDE1A might be involved in exosome biogenesis and release in NSCLC cells and play a vital role in message communication in the TME. GSEA showed that PDE1A might be involved in angiogenesis, vasculature development, and blood vessel development. Indeed, NSCLC cells overexpressing PDE1A promoted angiogenesis in the TME, and PDE1A knockdown significantly suppressed angiogenesis of NSCLC in vivo. Furthermore, an exosome release inhibitor successfully reversed the angiogenesis promoted by NSCLC cells overexpressing PDE1A. Thus, this study first illustrated that PDEs might play an important role in angiogenesis and the TME via regulating exosome biogenesis and release in cancer cells.

During our attempt to reveal the downstream signaling pathway of PDE1A, we found that the IL-6/JAK/STAT3 signaling pathway was highly enriched in PDE1A coexpressed genes from NSCLC. Furthermore, PDE1A promoted the metastasis of NSCLC cells via the STAT3 signaling pathway. Targeting the IL- 6/JAK/STAT3 signaling pathway is considered as a promising therapeutic strategy for the management of NSCLC [39]. However, the direct interaction between PDE1A and STAT3 could not be observed in NSCLC cells. Thus, we next explored the mechanism by which PDE1A promotes the STAT3 signaling pathway. In our study, we demonstrated that PDE1A interacted with YTHDF2 and participated in NSCLC progression, and our data first verified the interaction between YTHDF2 and PDE1A in NSCLC cells. Meanwhile, YTHDF2 might act as an m6A RNA “reader” by interacting with PDE1A, but the mechanism might need further investigation. YTHDF2 destabilizes mRNAs via degrading target transcripts, but it also stabilizes important oncogenic drivers, such as MYC and VEGFA transcripts, in an m6A- dependent manner [40]. We demonstrated that YTHDF2 destabilized SOCS2 mRNA and stabilized MET and NRF2 mRNA via interacting with PDE1A, but the mechanism by which YTHDF2 sorts mRNA might need further investigation.

Treatment with cisplatin frequently develops chemoresistance leading to therapeutic failure, and the exploitation of chemosensitization strategies have significant clinical implications [41]. In this study, we demonstrated that PDE1A positively regulated the expression of MET and NRF2 in NSCLC cells, and the expression of PDE1A was closely correlated with the anti-NSCLC activity of cisplatin. Our findings are the first to indicate that the expression of PDEs might be involved in NSCLC resistance to cisplatin treatment. Vinpocetine has been clinically used to treat stroke, senile dementia, and memory disturbances; no significant side effects or toxicity of vinpocetine have been reported at therapeutic doses, and it is safe for long-term use [42]. Vinpocetine is also regarded as a PDE1 inhibitor and increases the levels of cAMP and cGMP [43]. This study first revealed that vinpocetine sensitized cells to cisplatin treatment via regulating MET and NRF2. Thus, vinpocetine plus cisplatin might be a novel therapeutic regimen for treating NSCLC patients with cisplatin resistance. Our finding was supported by the observations that cAMP signaling increases histone deacetylase 8 expression and augments cisplatin-induced apoptosis [44].

Collectively, the results of this study suggest that PDE1A promotes metastasis and cisplatin resistance in NSCLC cells. Furthermore, PDE1A overexpression is correlated with angiogenesis and poor outcomes of NSCLC patients. In addition, PDE1A interacts with YTHDF2 and regulates the JAK/STAT3 signaling pathway via degrading SOCS2 mRNA. Meanwhile, PDE1A regulates cisplatin resistance via stabilizing MET and NRF2. Therefore, this study reveals the effect and mechanism of PDE1A in promoting HCC metastasis and cisplatin resistance, this work not only uncovers a novel PDE1A/YTHDF2/STAT3 signaling pathway in NSCLC progression but also provides novel therapeutic strategies to treat NSCLC patients with metastasis or cisplatin resistance.

Acknowledgements

This study was funded by Huadong Medicine Joint Funds of the Zhejiang Provincial Natural Science Foundation of China (LHDMY22H160001), Natural Science Foundation of Ningbo City (2022J206), National Natural Science Foundation of China (82273352), Public-service Technology Research Plan of Zhejiang Province (LGF21H310002), Zhejiang Provincial Natural Science Foundation (LY21H160017).

Additional information

Authors’ contributions

Chong Zhang: Conception and design of the work, drafting the work, writing- original draft, writing-review and editing; Yangling Li: Conception and design of the work, supervision, funding acquisition; Zuoyan Zhang: acquisition, analysis, or interpretation of data for the work; Yuchen Wu: acquisition, analysis, or interpretation of data for the work; Jing Cheng: acquisition, analysis, or interpretation of data for the work; Kaizhi Luo: acquisition, analysis, or interpretation of data for the work; Zhidi Li: acquisition, analysis, or interpretation of data for the work; Manman Zhang: acquisition, analysis, or interpretation of data for the work; Jian Wang: funding acquisition. All authors have approved the final article.

Conflict of Interest

The authors have declared that no competing interest exists.

Ethics approval and consent to participate

The experiments were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Availability of data and material

The data presented in the study are included in the article and additional material.