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

(A) The expression of PDE1A was detected in lung cancer and normal lung tissue. The data were obtained from The Human Protein Atlas (https://www.proteinatlas.org). (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 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). Single 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. (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). (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. (G) The Pearson correlation coefficient between the expression of YTHDF2 and STAT3 was identified using UALCAN.

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. (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.

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

(A) NSCLC cells were transfected with control siRNA or siPDE1A for 48 h, and the expression of indicated proteins were determined. (B) NSCLC cells were transfected with empty vector or PDE1A overexpressing plasmid for 48 h, and the expression of indicated proteins were determined. (C) NSCLC cells were treated with vinpocetine for 24 h, and the expression of indicated proteins were determined. (D) NSCLC cells were transfected with control siRNA or siPDE1A for 24 h, and then cells were treated with cisplatin for 72 h, SRB assay was performed to evaluate cell proliferation. (E) NSCLC cells were transfected with empty vector or PDE1A overexpressing plasmid for 24 h, and then cells were treated with cisplatin for 72 h, SRB assay was performed to evaluate cell proliferation. (F) NSCLC cells were treated with vinpocetine and/or cisplatin for 72 h, and SRB assay was performed to evaluate cell proliferation. (G) NSCLC cells were treated with vinpocetine and/or cisplatin for 48 h, and the expression of indicate proteins were detected. (H) NSCLC cells were transfected with control siRNA or siPDE1A for 48 h, and the mRNA level of indicated proteins were determined. (I) NSCLC cells overexpressing PDE1A were transfected with control siRNA and YTHDF2 siRNA for 48 h, and the expression of the indicated proteins was detected.