High expression of PDE1A predicts a poor prognosis of lung cancer patients.

(A) The expression of PDE1A was detected in NSCLC and normal lung tissue. The data were obtained from The Human Protein Atlas (https://www.proteinatlas.org). IOD/area means Integral optical density/area. T-test was used to compare the difference between lung cancer and normal lung groups. (B) The expression of PDE1A in HELF, A549, NCI-H1299, and NCI-H460 cells was detected by western blot. (C and D) Box plot analysis of the PDE1A mRNA levels in clinical lung cancer tissue samples. The data were collected and statistical analyzed from SurvExpress (http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp). Gene: PDE1A; Access database Numbers: Chitale Lung (n=185); Censored: Recurrence Months or Survival months. (E) The data was collected from PROGgene V2 Prognostic Database (http://www.progtools.net/gene/index.php). Survival analysis is done using backend R script which employs R library ‘survival’ to perform Cox proportional hazards analysis (function ‘coxph’) and to plot prognostic plots (function ‘survfit’). TSingle user input genes: PDE1A; Cancer type: LUNG; Survival measure: Death; Bifurcate gene expression at: Median; GSE30219-Off-context gene expression in lung cancer identifies a group of metastatic-prone tumors.

PDE1A knockdown suppresses the metastasis of NSCLC cells.

(A-B) NSCLC cells were transfected with control siRNA and PDE1A siRNA for 24 h, cells were transferred to Transwell chambers without (A) or with (B) a Matrigel coating on the insert membrane, and the cell migrative and invasive abilities were determined, respectively. (C-D) NSCLC cells were transfected with control siRNA and PDE1A siRNA for 24 h, and the wound healing assay was established in NSCLC cells. (E) NSCLC cells were transfected with control siRNA and PDE1A siRNA for 48 h, and the expression of indicated proteins were detected. (F-G) NSCLC cells were treated with DMSO or vinpocetine (5, 10, 20 µM) for 24 h, and the migrative ability of treated NSCLC cells was determined using Transwell assay for 24 h. (H) NSCLC cells were treated with DMSO or vinpocetine (5, 10, 20 µM) for 24 h, and the expression of indicated proteins was determined. (I) The pulmonary metastatic nodules were stained using H&E staining and counted in nude mice harboring NCI-H1299 cells transfected with PDE1A shRNA and control shRNA.

PDE1A promotes the metastasis and EMT of NSCLC cells.

(A-B) NSCLC cells were transfected with PDE1A plasmid and empty vector for 24 h, cells were transferred to Transwell chambers without (A) or with (B) a Matrigel coating on the insert membrane, and the cell migrative and invasive abilities were determined, respectively. (C) NSCLC cells were transfected with PDE1A plasmid and empty vector for 24 h, and the wound-healing assay was established in NSCLC cells. (D) NSCLC cells were transfected with PDE1A plasmid and empty vector for 48 h, and the expression of indicated proteins were detected. (E) The highly invasive NSCLC cells were separated using transwell chamber assay, and P3 cells were obtained from P0 cells after three generations. (F-G) The protein (F) and mRNA (G) levels of indicated genes were determined in P3 and P0 NSCLC cells. (H) The pulmonary metastatic nodules were stained using H&E and Bouin’s solution and counted in nude mice harboring NCI-H1299 cells transfected with PDE1A plasmid and empty vector.

NSCLC cells overexpressing PDE1A promote angiogenesis in the TME.

(A) Data were collected from LinkedOmics. Statistical tests in LinkFinder include Pearson’s correlation coefficient, Spearman’s rank correlation, Student’s t-test, Wilcoxon test, Analysis of Variance, Kruskal– Wallis analysis, Fisher’s exact test, Chi-Squared test, Jonckheere’s trend test and Cox’s regression analysis. Multiple-test correction is performed using the Benjamini and Hochberg method to generate the False Discovery Rate. (B-D) NSCLC cells were transfected with empty vector / PDE1A overexpressing plasmid or control siRNA / siPDE1A for 48 h, and then NSCLC cells were placed on the upper panel of transwell with 0.4 μm insert, HUVECs were placed on the lower panel of transwell, and wound healing assay was performed to determine the migrative abilities of HUVECs. (E) NSCLC cells with PDE1A overexpression were treated with 10 µM GW4869, and wound healing assay was performed to determine the migrative abilities of HUVECs. (F) NSCLC cells were transfected with empty vector or shPDE1A, then cells were transplanted into nude mice via subcutaneous injection, the blood vessels were counted after 60 days.

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

(A) A Venn diagram was generated using LinkedOmics, ORA was performed to analysis the molecular pathway regulated by PDE1A in NSCLC. Sample cohort: TCGA_NSCLC; Institute: UNC; Data type: RNAseq; Platform: HiSeq RNA; Attribute: PDE1A; Statistical methods: Spearman Correlation test; Patients: 515; Tools: ORA; Gene Ontology analysis: Wikipathway and Panther Pathway; Select Rank Criteria: FDR; Select Sign: Positively correlated; Significance Level: 0.05; TOP40 was selected to generate Venn diagram. (B) GSEA was performed to analyze the biological process of PDE1A in NSCLC. (C) NSCLC cells were transfected with PDE1A plasmid and empty vector for 48 h, and the expression of indicated proteins were detected. (D) NSCLC cells were transfected with control siRNA and PDE1A siRNA for 48 h, and the expression of indicated proteins were detected. (E) NSCLC cells were treated with DMSO or vinpocetine (5, 10, 20 µM) for 24 h, and the expression of indicated proteins was determined. (F) NSCLC cells overexpressing PDE1A were transfected with control siRNA and STAT3 siRNA for 48 h, the migrative abilities of NSCLC cells were determined by Transwell assay. (G) NSCLC cells overexpressing PDE1A were treated with STAT3 inhibitor SH-4-54 (5 µM) for 24 h, the migrative abilities of NSCLC cells were determined by Transwell assay. (H) NSCLC cells overexpressing PDE1A were transfected with control siRNA and STAT3 siRNA for 48 h, and the expression of indicated protein was detected by western blot. (I) NSCLC cells overexpressing PDE1A were treated with STAT3 inhibitor SH-4-54 (5 µM) for 24 h, and the expression of indicated protein was detected by western blot. (J) The interaction between PDE1A and STAT3 was determined by immunoprecipitation. (K) NCI-H1299 cells were transfected with empty vector and PDE1A overexpressing plasmid for 48 h, and the contribution of PDE1A in the cytoplasm and nucleus was determined.

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

(A) Venn diagram showing the overlap among PDE1A interacting proteins (The data was collected from mass spectrometry analysis in NSCLC cells), STAT3 coexpressed genes (collected from gene correlation using UALCAN), upregulated proteins in NSCLC compared with normal tissues (analyzed by UALCAN based on CPTAC database), and upregulated genes in NSCLC compared with normal tissues (analyzed by UALCAN based on TCGA database). Pearson correlation analysis of UALCAN was used to evaluate gene correlation analyses, and Welch’s T-test was estimated to detect the significance of differences in expression levels between two groups. (B) GO enrichment analysis of PDE1A interacting genes. (C) Immunoprecipitation followed by silver staining was performed to identify protein and protein interaction using A549 cell lysate with the anti-PDE1A antibody. (D) Immunoprecipitation was used to confirm protein and protein interaction in NCI-H1299 cells. (E) NSCLC cells overexpressing PDE1A were transfected with control siRNA and YTHDF2 siRNA for 48 h, the migrative abilities of NSCLC cells were determined by Transwell assay. (F) NSCLC cells overexpressing PDE1A were transfected with control siRNA and YTHDF2 siRNA for 48 h, and the expression of indicated protein was detected by western blot.

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

(A) YTHDF2-RNA complexes were identified by LC-MS/MS and collected from reference; YTHDF2 corelated genes were collected from TNMplot (https://tnmplot.com/analysis/), Gene: YTHDF2, Gene vs. all genes correlation: Genechip data, Tissue: Lung; STAT3 corelated genes were collected from cBioPortal (http://www.cbioportal.org/), Lung cancer (SMC, cancer research 2016); n=22; Gene: STAT3. The interaction between YTHDF2 protein and the mRNA of 33 overlapping genes were predicted by RNA-Protein Interaction Prediction (http://pridb.gdcb.iastate.edu/RPISeq/index.html), and the values of RF classifier and SVM classifier above 0.5 were considered positive. Comparison of the normal and the tumorous samples was performed by the Mann– Whitney U test, and Normal, tumorous and metastatic tissue gene comparison can be analyzed using Kruskal–Wallis test. (B) The interactions between protein and mRNAs were verified by RIP experiments. (C) NSCLC cells were transfected with control siRNA and siPDE1A for 48 h, and the stability of mRNA was determined by qRT-PCR. (D) NSCLC cells were transfected with control siRNA and siPDE1A for 48 h, and the expression of SOCS2 mRNA was determined by qRT-PCR.

The expression of PDE1A in normal lung (A) and lung cancer (B).

The data were obtained from The Human Protein Atlas (https://www.proteinatlas.org).

The biological processes related to PDE1A in NSCLC is predicted using the LinkedOmics.

Data collected from LinkedOmics (www.linkedomics.org/admin.php) are shown. ORA (A) and GSEA (B) were performed to analyze the biological processes related to PDE1A in NSCLC. Sample cohort: TCGA_NSCLC; Institute: UNC; Data type: RNAseq; Platform: HiSeq RNA; Attribute: PDE1A; Statistical methods: Spearman Correlation test; Patients: 515; Tools: ORA and GSEA; Gene Ontology analysis: biological process. (C) GSEA demonstrated that PDE1A participated in mesenchyme development and the cellular response to VEGF stimulus.

The expression of PDE1A does not affect the proliferation of NSCLC cells.

(A-B) NSCLC cells were transfected with PDE1A siRNA or control siRNA (A) and empty vector or PDE1A plasmid (B) for 24 h in 6-well plates, then transferred to 96-well plated for the indicated times, and finally subjected to SRB assay.

PDE1A silence suppresses the metastasis of NSCLC cells.

(A) NSCLC cells were transfected with control siRNA, siPDE1A, siPDE1B, and siPDE1C for 24 h, and cells were transferred to Transwell chambers without a Matrigel coating on the insert membrane, and the cell migration were determined after 24 h. (B) NSCLC cells were transfected with indicated siRNA for 48 h, and the expression of the indicated proteins were detected. (C) The lungs of nude mice were separated and stained with Bouin’s solution and the pulmonary metastatic nodules were counted. (D) The relative body weight of each group in the in vivo metastasis studies in figure 2I.

PDE1A promotes the metastasis of NSCLC cells.

(A) The migrative abilities of P3 and P0 NSCLC cells were determined. (B) The mRNA levels of indicated genes were determined in P3 and P0 NSCLC cells using qRT-PCR. (C) Before the NSCLC cells were injected into the nude mice, the migrative abilities of NSCLC cells were determined using Transwell assay. (D)The relative body weight of each group in the in vivo metastasis studies in figure 3H.

PDE1A promotes the metastatic potential in a cAMP-independent manner.

(A) NSCLC cells were transfected with control siRNA and PDE1A siRNA, after 24 h cells were collected for Transwell assay, cells were treated with DMSO or 5 µM SQ22536 for 24 h, and the migrative abilities of NSCLC cells were determined. (B-C) NSCLC cells were transfected with control siRNA and YTHDF2 siRNA for 48 h, and the knockdown efficiency of YTHDF2 was confirmed by western blot (B) and qRT-PCR (C).

Overexpression of YTHDF2 predicts poor outcomes for lung cancer patients.

(A-B) The mRNA and protein levels of PDE1A in NSCLC and normal lung tissues are shown. Data collected from UALCAN was shown. Gene: YTHDF2; TCGA dataset: Lung adenocarcinoma (A); CPTAC dataset: Lung adenocarcinoma (B). (C) The prognostics value of YTHDF2 in NSCLC patients was identified using PROGgeneV2 online tool (www.progtools.net/gene).