Fig 2: Targeting PDE4D suppresses in vivo tumor development of pancreatic cancer.(A and B) PDE4D inhibition suppresses tumor growth. MIA PaCa-2 cells stably expressing shGFP (control) or shPDE4D were subcutaneously injected into mice for tumor xenograft experiments. In addition, MIA PaCa-2 cells were subcutaneously injected into mice for tumor xenograft experiments and injected with either roflumilast (5 mg/kg) or GEBR-7b (0.3 mg/kg) intraperitoneally once every other day. Tumor growth in A was measured every week after injecting cancer cells into mice. Tumor weight in B was measured after resection of xenograft tumor from mice. Control n = 13, shPDE4D n = 12, roflumilast n = 13, and GEBR-7b n = 13. (C and D) PDE4D inhibition suppresses mTORC1 activity in pancreatic cancer. The xenograft tumor samples were assessed by immunoblotting (C) for mTORC1 activity via pS6K1. S6K1 and ß-actin are controls. The xenograft tumor samples were assessed by immunohistochemistry (D) for pS6 (Ser235/236). Quantification of immunohistochemical staining was obtained using ImageJ. Scale bars: 50 µm. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Dunnett’s multiple-comparison test (A) or 1-way ANOVA with Dunnett’s multiple-comparison test (B and C).

Fig 3: PDE4D forms a complex with mTORC1 and promotes mTORC1 activity.(A) Resveratrol inhibits mTORC1 activity. HEK293A cells were treated for 2 hours with resveratrol (0–100 µM). mTORC1 activity was analyzed by immunoblotting for pULK1 (Ser758), pS6K1 (Thr389), and p4EBP1 (Thr37/46). (B) Depletion of PDE4D inhibits mTORC1 activity. HEK293A cells were stably generated with 3 shRNAs targeting PDE4D. mTORC1 activity was analyzed as in A. (C) mTORC1 activity is rescued in PDE4D-depleted cells by reintroducing FLAG-tagged PDE4D. Wild-type or an inactive PDE4D mutant (D620A) was expressed in shPDE4D cells. mTORC1 activity was analyzed as in A. s.e., short exposure; l.e., long exposure. (D) Elevated PDE4D levels increase mTORC1 activity. FLAG-tagged PDE4D4 was overexpressed in cells for 48 hours. mTORC1 activity was analyzed as in A. (E) Increased PDE4D levels promote mTORC1 activity. FLAG-tagged PDE4D4 (0.5–2 µg) was transfected into HEK293A cells for 48 hours, and then stimulated with forskolin (10 µM) for 1 hour. mTORC1 activity was analyzed as in A. The quantification of pS6K1 was analyzed using ImageJ and normalized to S6K1. The data represent mean ± SD of triplicate experiments. NS, not significant. **P < 0.01, ****P < 0.0001 by 1-way ANOVA with Dunnett’s test for multiple comparisons. (F) PDE4D binds to mTORC1 and AKAP13. Cells were transfected with FLAG-tagged PDE4D4 for 48 hours and then lysates immunoprecipitated (IP) with anti-FLAG antibody (PDE4D4). mTORC1 (mTOR, Raptor, mLST8), AKAP13, and FLAG (PDE4D4) were probed for. WCL, whole-cell lysates. (G) PDE4D interacts with PKA catalytic subunits. FLAG-tagged PDE4D4 was overexpressed in cells and cells were treated with forskolin (10 µM) for 1 hour. Lysates were immunoprecipitated with anti-FLAG antibody (PDE4D4). Raptor, PKA RIa, PKA RIIa, PKA Cata, pCREB (Ser133), CREB, FLAG (PDE4D4), and ß-actin were probed for. Immunoblots probed for ULK1, S6K1, 4EBP1, ß-actin, pCREB (PKA activation), CREB, PDE4D, and FLAG are controls.

Fig 4: Pharmacologic PDE4D inhibition impairs mTORC1 signaling and pancreatic cancer cell growth.(A) High PDE4D levels in patients with pancreatic adenocarcinoma from GEPIA (74). *P < 0.05. (B) High PDE4D has low survival rate in patients with pancreatic adenocarcinoma (74). Log-rank test was performed for statistical analysis (A and B). (C) High PDE4D expression in patients with pancreatic ductal adenocarcinoma. Pancreatic ductal adenocarcinoma (PDAC) tissue sections paired with benign tissues. T, tumor; NLD, normal duct. Scale bar: 100 µm. Significance was assessed by 2-tailed Student’s t test. (D) PDE4D levels correlate with mTORC1 activity in pancreatic cancer cells. (E and F) PDE4D inhibition blocks mTORC1 activity. (E) MIA PaCa-2 cells were treated with roflumilast or GEBR-7b for 2 hours. mTORC1 activity was analyzed. (F) MIA PaCa-2 cells stably expressing shPDE4D have low mTORC1 activity. (G) PDE4D inhibition reduces pancreatic cancer cell size. Cell size was determined for shPDE4D MIA PaCa-2 cells, or MIA PaCa-2 cells treated with roflumilast or GEBR-7b for 48 hours. One-way ANOVA with Dunnett’s test was performed. P < 0.0001 for control vs. shPDE4D #1. P < 0.05 for control vs. shPDE4D #2 and control vs. GEBR-7b. P < 0.001 for control vs. roflumilast. (H) Pharmacological inhibition of PDE4D reduces proliferation of pancreatic cancer cells. MIA PaCa-2 cells with shPDE4D, or MIA PaCa-2 cells treated with roflumilast or GEBR-7b were counted. Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-way ANOVA with Dunnett’s multiple-comparison test. (I and J) Pharmacological inhibition of PDE4D reduces colony formation. (I) Colony formation assays in MIA PaCa-2 cells 2 weeks after seeding cells (1 × 103). (J) Colonies in I were counted using ImageJ for the quantification. Bar graph represents mean ± SD. ***P < 0.001, ****P < 0.0001 by 1-way ANOVA with Dunnett’s multiple-comparison test. Immunoblots probed for PDE4D, S6K1, 4EBP1, pCREB (Ser133) (measure of PKA activation), CREB, and ß-actin are controls.

Fig 5: PDE4D promotes mTORC1 lysosomal localization.(A) Depletion of PDE4D inhibits amino acid–induced mTORC1 activation. HEK293A cells stably expressing shGFP (control) or shRNA targeting PDE4D (shPDE4D) were generated. Cells were starved in amino acid–free media for 2 hours, pretreated with or without forskolin (10 μM) for 1 hour, and then stimulated with amino acids for 1 hour. mTORC1 activity with pS6K1 (Thr389) was analyzed. S6K1, pCREB (Ser133) (measure of PKA activation), CREB, and β-actin are controls. (B–D) Depletion of PDE4D blocks mTORC1 lysosomal localization. (B) HEK293A cell lines stably expressing shGFP (control) or shPDE4D were starved in amino acid–free media for 2 hours, pretreated with or without forskolin (10 μM) for 1 hour, and then stimulated with amino acids for 1 hour. Immunofluorescence experiments were performed with anti-mTOR (green) and -LAMP2 (lysosome marker, red) antibodies. Representative images were obtained under a Zeiss LSM 900 confocal microscope with 100× objective. (C) Staining intensity profiles across the 3.6-μm distance of the green (mTOR) and red (LAMP2) channels in the magnified pictures. (D) Ten immunofluorescence images per group were quantified using Squassh in ImageJ. The data represent mean ± SD. ****P < 0.0001 by 2-way ANOVA with Tukey’s test for multiple comparisons. (E) PDE4D localizes in the cytoplasm. Cells were fractionated after forskolin (10 μM) treatment for 1 hour and then analyzed by immunoblotting for PDE4D. LAMP2 (lysosome marker), lamin A/C (nuclear marker), and α-tubulin (cytoplasm marker) are controls. Arrow indicates PDE4D. (F) PDE4D does not localize to the lysosome. HEK293A were starved in amino acid–free media for 2 hours, pretreated with or without forskolin (10 μM) for 1 hour, and then stimulated with amino acids for 1 hour. Immunofluorescence experiments were performed with anti-PDE4D (green) and -LAMP2 (lysosome marker, red) antibodies. Representative images were obtained under an LSM 900 confocal microscope with 100× objective. AA, amino acid.