Synergistic effect of antagonists to KRas4B/PDE6 molecular complex in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among all human cancers as it is highly resistant to chemotherapy. K-Ras mutations usually trigger the development and progression of PDAC. We hypothesized that compounds stabilizing the KRas4B/PDE6δ complex could serve as PDAC treatments. Using in silico approaches, we identified the small molecules C14 and P8 that reduced K-Ras activation in primary PDAC cells. Importantly, C14 and P8 significantly prevented tumor growth in patient-derived xenotransplants. Combined treatment with C14 and P8 strongly increased cytotoxicity in PDAC cell lines and primary cultures and showed strong synergistic antineoplastic effects in preclinical murine PDAC models that were superior to conventional therapeutics without causing side effects. Mechanistically, C14 and P8 reduced tumor growth by inhibiting AKT and ERK signaling downstream of K-RAS leading to apoptosis, specifically in PDAC cells. Thus, combined treatment with C14 and P8 may be a superior pharmaceutical strategy to improve the outcome of PDAC.

Figure 1.. C14 stabilizes the K-Ras4B/PDEδ complex and inhibits the growth of human pancreatic cancer cell lines.

(A) Interaction of compound C14 (red) with the K-Ras4B^wt (purple) PDE6δ (aqua) complex. (B) Interaction of compound C14 with the K-Ras4B^G12D/PBDE6δ complex. (C) Interaction of compound C14 with the K-Ras4B^G12C/PBDE6δ complex. (D) Representative bright-field images of the cell lines ARPE-19, hTERT-HPNE, PANC-1, and MIA PaCa-2 treated with 100 μM C14, DMSO as vehicle and untreated control cells. Bar = 20 μm. (E) Dose response of MIA PaCa-2, PanC-1 pancreatic ductal adenocarcinoma (PDAC) cells and the normal pancreatic cell line hTERT-HPNE treated with different concentrations of C14 compound for 72 h and compared with relative control DMSO. (F) Percentage of PDAC cell lines viability at 72 h treated with 150 μM of the C14 compound. (G, H) Relative cell viability curves after treatment of the PDAC cell line MIA PaCa-2 (G) and the normal pancreatic cell line hTERT-HPNE (H) with different compounds known to target the K-Ras4B/PBDE6δ complex were compared with negative control DMSO. (n = 5), with a significance of P < 0.001.

Figure S1.. Evaluation and identification of analogs of compound C14.

(A, B) Evaluation of cell viability after treatment with P1, P2, P3, P4, and P5 analogs of C14 at 90.18 μM in MIA PaCa-2 (A) and hTERT-HPNE (B) cells. (C, D) Evaluation of cell viability after treatment with P6, P7, P8, P9, and P10 analogs of C14 at 90.18 μM in MIA PaCa-2 (C) and hTERT-HPNE (D) cells. (E, F) Evaluation of cell viability after treatment with P11, P12, P13, P14, and P15 analogs of C14 at 90.18 μM in MIA PaCa-2 (E) and hTERT-HPNE (F). (E, F, G, H) Evaluation of cell viability after treatment with P16, P17, P18, P19, and P20 analogs of C14 at 90.18 μM in MIA PaCa-2 (G) and hTERT-HPNE (H) n = 6; ***P < 0.001.

Figure 2.. P8 analog stabilizes the K-Ras4B/PDEδ complex and inhibits growth of pancreatic ductal adenocarcinoma (PDAC) cell lines and induced apoptosis better than C14 compound.

(A) Interaction of compound P8 (blue) with K-Ras4B^wt (pink)/PDE6δ (aqua complex). (B) Interaction of P8 with the K-Ras4B^G12D/PBDE6δ complex. (C) Interaction of P8 with the K-Ras4B^G12C/PBDE6δ complex. (D) Interaction of P8 with the K-Ras4B^G12V/PBDE6δ complex. (E, F, G, H) Relative cell viability of the PDAC cells PANC-1, MIA PaCa-2, and Capan-1 and the normal pancreatic cell line hTERT-HPNE treated with different concentrations of P8 for 72 h (n = 5). (I, J, K) Clonogenic assays of the PDAC cell lines PANC-1, MIA PaCa-2, and Capan-1 treated with the IC₅₀ of P8, C14, gemcitabine, and deltarasin (n = 5). (L, M) Cell death analyses by flow cytometry in hTERT-HPNE, PANC-1, MIA PaCa-2, and Capan-1 cells treated with the IC₅₀ of P8 and C14, DMSO as vehicle or medium alone after staining with Anexin-V, 7-AAD, and CytoCalcein Violet. (L, M) Quantification of the plots shown in (L) n = 5; ***P < 0.001.

Figure 3.. P8 and C14 decrease the activation of K-Ras and phosphorylation of AKT and ERK in pancreatic ductal adenocarcinoma cell lines with K-Ras mutation.

(A, B, C, D) K-Ras-GTP, K-Ras-GDP expression determined by Western blot in hTERT-HPNE (A), PANC-1 (B), MIA PaCa-2 (C), and Capan-1 (D) cell lines treated with the IC₅₀ of P8, C14, gemcitabine, and deltarasin for 3 h. Total protein extracts were precipitated using RAF-RBD beads. Total RAS (Ras-T) and GAPDH are shown as loading controls. Pixel intensities of K-Ras GTP were normalized to total RAS and GAPDH. (E, F, G) pAKT, AKT, pERK, ERK expression determined by Western blot in PANC-1 (E), MIA PaCa-2 (F), and Capan-1 (G) cell lines treated with the IC₅₀ of P8, C14, gemcitabine, and deltarasin or vehicle. The intensity of pAKT, AKT, pERK, ERK relative to GAPDH was determined by densitometric analysis. GAPDH was used as a loading control. Quantification of pixel intensities of pERK and pAKT relative to total ERK and AKT levels, respectively, are shown in the graphs to the right. Data are shown as SDM; n = 5; ***P < 0.001. Source data are available for this figure.

Figure S2.. Characterization of pancreatic ductal adenocarcinoma (PDAC) tissues and primary cells.

(A) Expression of CK19 in MGKRAS003, MGKRAS004, and MGKRAS005 PDAC tissues and primary cells by confocal immunofluorescence microscopy (Leica SP8, Barcelona, Spain). Bar = 50 µm; n = 3. (B) Expression of MUC1 in MGKRAS003, MGKRAS004, and MGKRAS005 PDAC tissues and primary cells by confocal immunofluorescence microscopy. Bar = 50 µm; n = 3. (C) Nucleotide sequences and histograms of sequencing of exon 2 containing KRAS in MGKRAS003, MGKRAS004, and MGKRAS005 tissues and primary cells. Source data are available for this figure.

Figure S3.. Characterization of skin-derived primary cell cultures.

(A) Expression of IB-10 in PBD033 and JGC028 primary skin-derived cultures by confocal immunofluorescence microscopy (Leica SP8, Barcelona, Spain). Bar = 50 µm; n = 3. (B, C) Expression of CD90, CD105, CD73, CD34, CD45, and HLADR in PBD033 and JGC028 primary skin-derived cultures as analyzed by flow cytometry after correction for autofluorescence.

Figure 4.. P8 and C14 inhibit the growth of and induce apoptosis in primary pancreatic ductal adenocarcinoma (PDAC) cell cultures.

(A, B, C, D, E) Effects of P8 and C14 at various concentrations (5, 10, 30, 50, 100, 150, and 200 μM) for 72 h in the primary noncancerous cultures PBD033 and JGC028, and the primary PDAC cultures MGKRAS003, MGKRAS004, and MGKRAS005. (F, G, H) Clonogenic assays of the primary PDAC cultures MGKRAS003, MGKRAS004, and MGKRAS005 treated with the IC₅₀ of P8, C14, gemcitabine, and deltarasin. (I, J) Cell death analyses of PBD033, JGC028 MGKRAS003, MGKRAS004, and MGKRAS005 were determined by flow cytometry after staining with annexin-V, 7-AAD, and CytoCalcein Violet. (I, J) Quantification of the plots is shown in (I). Data are shown as SDM; n = 5; ***P < 0.001.

Figure S4.. Evaluation of viability of primary cultures treated with gemcitabine and deltarasin.

(A) Effect of gemcitabine on cell viability at various concentrations (0–8,000 nM) after treatment for 72 h in the primary pancreatic ductal adenocarcinoma cultures MGKRAS003 WT, MGKRAS004G12V, and MGKRAS005G12C. (B) Effect of deltarasin at various concentrations (0–8,000 nM) after treatment for 72 h on cell viability in the primary pancreatic ductal adenocarcinoma cultures MGKRAS003 WT; MGKRAS004G12V and MGKRAS005G12C (n = 3); ***P < 0.001.

Figure 5.. P8 and C14 decrease the activation of K-Ras and phosphorylation of AKT and ERK in primary pancreatic ductal adenocarcinoma cell cultures with K-Ras mutation.

(A, B, C, D, E) K-Ras-GTP, K-Ras-GDP expression determined by Western blot in PBD033 (A), JGC028 (B), MGKRAS003 (C), MGKRAS004 (D), and MGKRAS005 (E) cells treated with the IC₅₀ of P8, C14, gemcitabine, and deltarasin for 3 h. Total protein extracts were precipitated using RAF-RBD beads. Total RAS (Ras-T) and GAPDH are shown as loading controls. Pixel intensities of K-Ras GTP were normalized to the controls. (F, G, H) pAKT, AKT, pERK, ERK expression determined by Western blot in MGKRAS003 (F), MGKRAS004 (G), and MGKRAS005 (H) cells treated with the IC₅₀ of P8, C14 compounds, and compared with negative control DMSO (vehicle) and positive controls gemcitabine and deltarasin. The intensity of pAKT, AKT, pERK, ERK relative to GAPDH was determined by densitometric analysis. GAPDH was used as a loading control. Quantification of pixel intensities of pERK and pAKT relative to total ERK and AKT levels, respectively, are shown in the graphs to the right. Data are shown as SDM; n = 5; ***P < 0.001. Source data are available for this figure.

Figure 6.. Synergistic effect of P8 and C14 on cell lines and primary pancreatic ductal adenocarcinoma cultures.

(A) Synergistic interaction of P8 (blue), C14 (red) with the K-Ras4B^wt (pink and yellow)/PDE6δ (aqua complex). (B) Synergistic interaction of P8/C14 with the K-Ras4B^G12D/PBDE6δ complex. (C) Synergistic interaction of P8/C14 with the K-Ras4B^G12C/PBDE6δ complex. (D) Synergistic interaction of P8/C14 with the K-Ras4B^G12V/PBDE6δ complex. (E) Isobologram of the IC₅₀ of compounds P8 and C14. (F, G, H, I, J) Dose response on cell viability of MIA PaCa-2 (F) and PANC-1 (G) cell lines and MGKRAS003 (H), MGKRAS004 (I) and MGKRAS005 (J) primary pancreatic ductal adenocarcinoma cultures of compounds P8/C14 at various concentrations of each (5, 10, 30, 50, 100, 150, and 200 μM). (K, L, M, N, O) Clonogenic assays of MIA PaCa-2 (K), PANC-1 (L), MGKRAS003 (M), MGKRAS004 (N), and MGKRAS005 (O) treated with the IC₅₀ concentration of P8 and C14 compounds. (P) Cell death analyses of PANC-1, MIA PaCa-2, MGKRAS003, MGKRAS004, and MGKRAS005 by flow cytometry after staining with annexin-V, 7-AAD, and CytoCalcein Violet. Data are shown as SDM; n = 5; ***P < 0.001.

Figure S5.. Evaluation of cytotoxicity and genotoxicity of P8 and C14.

(A, B) Effects of P8 and C14 at different concentrations (5, 10, 30, 50, 100, 150, and 200 μM) on cell viability of stimulated and non-stimulated H-PBMC’s (n = 5). (C) Frequency of micronuclei in polychromatic erythrocytes isolated from bone marrow of BALB/c mice treated with 60 μM P8, C14, and 40 μM gemcitabine for 24 h. (D, E, F, G) Evaluation of protein presence (D), pH (E), bilirubin (F), and glucose (G) in the urine of BALB/c mice after treatment with 60 μM P8, C14, and gemcitabine for 24 h. (H, I, J, K) Evaluation of protein presence (H), pH (I), bilirubin (J), and glucose (K) in the urine of BALB/c mice after treatment with 60 μM P8, C14, and gemcitabine for 16 d; (n = 6); ***P < 0.001.

Figure 7.. The combination of P8 and C14 reduces tumor growth in subcutaneous and orthotopic xenograft models.

(A) Effects of P8, C14, and C14/P8 at different concentrations (5, 10, 30, and 60 mg/kg, and the combination of 30 mg/kg) in a subcutaneous xenograft model of injection of MIA PaCa-2 cells in the skin of the back of male Nu/Nu mice (n = 6). (B) Quantification of tumor volume every day after treatment with P8, C14, and C14/P8 at different concentrations (n = 6). Below the graph are images of representative tumors of each of the treatments performed. (C) Body weight was measured daily during treatment with P8, C14, and C14/P8. (D) Representative WB of MIA PaCa-2 tumor lysates treated with P8, C14, gemcitabine shows complete inhibition of AKT and ERK phosphorylation using total AKT, ERK, and GAPDH as loading controls. The relative quantification of the Western blot results is shown in the graphs. (E) Effects of P8, C14, and C14/P8 at different concentrations (30 and 60 mg/kg, and the combination of 30 mg/kg) in an orthotopic xenograft model of injection of MIA PaCa-2 cells in the pancreas of mice NU/NU male (n = 6). (F) Body weight was measured daily during treatment with P8, C14, and C14/P8. (G) Representative images of the effect of treatment with P8, C14, P8/C14, and gemcitabine. (H) Survival graph during treatment with P8, C14, P8/C14, and gemcitabine. Data represent mean±SDM of at least six independent experiments; ***P < 0.001. Source data are available for this figure.

Figure 8.. Evaluation of the malignancy markers CK19, CA125, and Ki-67 in tumors derived from subcutaneous xenografts, by means of immunohistochemistry.

(A) Representative hematoxylin–eosin and immunohistochemistry images of the tumor sections derived from mice treated with P8, C14, P8/C14, gemcitabine or vehicle. (B, C, D) Quantification of signal intensities of CK19 (B), CA125 (C), and Ki-67 (D). Tumor sections were counted at 100 cells per field with six fields per section: ***P < 0.001. Source data are available for this figure.

Figure 9.. Combined treatment with P8 and C14 reduces tumor growth in primary culture xenograft models.

(A) Tumor growth effects of P8, C14, and C14/P8 at different concentrations (5, 10, 30, and 60 mg/kg, and a combination of 30 mg/kg each) in a subcutaneous xenograft model using MGKRAS004 cells. (B) Effect of tumor growth after treatment with P8, C14, and C14/P8 at different concentrations on the MGKRAS004 xenograft. (C) Body weight was measured daily during treatment with P8, C14, and C14/P8. (D) Representative images of MGKRAS004 tumors obtained from each group. (E) Effects of P8, C14, and C14/P8 at different concentrations (5, 10, 30, and 60 mg/kg, and a combination of 30 mg/kg each) in a subcutaneous xenograft model (n = 6) using MGKRAS005 cells. (F) Effect of tumor growth after treatment with P8, C14, and C14/P8 at different concentrations on the MGKRAS005 xenograft. (G) Body weight was measured daily during treatment with P8, C14, and C14/P8. (H) Mice representative images of MGKRAS005 tumor development obtained from each group of treatments with C14, P8 compounds, and gemcitabine. Data represent mean±SDM of at least six independent experiments; ***P < 0.001.