Inhibition of amyloid precursor protein processing enhances gemcitabine-mediated cytotoxicity in pancreatic cancer cells

Abstract

Pancreatic adenocarcinoma or pancreatic cancer is often diagnosed at a very late stage at which point treatment options are minimal. Current chemotherapeutic interventions prolong survival marginally, thereby emphasizing the acute need for better treatment options to effectively manage this disease. Studies from different laboratories have shown that the Alzheimer disease-associated amyloid precursor protein (APP) is overexpressed in various cancers but its significance is not known. Here we sought to determine the role of APP in pancreatic cancer cell survival and proliferation. Our results show that pancreatic cancer cells secrete high levels of sAPPα, the α-secretase cleaved ectodomain fragment of APP, as compared with normal non-cancerous cells. Treatment of cells with batimastat or GI254023X, inhibitors of the α-secretase ADAM10, prevented sAPPα generation and reduced cell survival. Additionally, inhibition of sAPPα significantly reduced anchorage independent growth of the cancer cells. The effect of batimastat on cell survival and colony formation was enhanced when sAPPα downregulation was combined with gemcitabine treatment. Moreover, treatment of batimastat-treated cells with recombinant sAPPα reversed the inhibitory effect of the drug thereby indicating that sAPPα can indeed induce proliferation of cancer cells. Down-regulation of APP and ADAM10 brought about similar results, as did batimastat treatment, thereby confirming that APP processing is important for growth and proliferation of these cells. These results suggest that inhibition of sAPPα generation might enhance the effectiveness of the existing chemotherapeutic regimen for a better outcome.

Keywords: ADAM ADAMTS; Amyloid Precursor Protein; Pancreatic Cancer; Protein Secretion; Secretases.

FIGURE 1.

Pancreatic cancer cells show enhanced APP expression and processing. A, schematic showing APP processing by β- and γ-secretases, which follows the amyloidogenic pathway to produce Aβ, sAPPβ (secreted β-secretase cleaved APP), and AICD (APP intracellular domain) or the non-amyloidogenic processing by α and γ-secretases to generate sAPPα, P3 (small fragment generated by α and γ-secretase cleavage of APP), and AICD. B, pancreatic cancer cell lines AsPC1, CD18, MiaPaCa2, and Panc1 were compared with normal pancreatic epithelial cells, HPDE6E7, for expression and secretion of sAPPα and expression of ADAM10 in the presence or absence of batimastat. C, analysis of epithelial and mesenchymal markers in different pancreatic cancer cell lines. Vimentin is shown in panel 2 and E-cadherin in panel 3. Actin was used as loading control.

FIGURE 2.

Batimastat inhibits ADAM10 and down-regulates sAPPα generation. A, MiaPaCa2 and Panc1 cells were treated with 5 μm batimastat or 25 μm GM6001. Tissue culture supernatant was blotted for sAPPα using 6E10 antibody, and cell extract was analyzed for ADAM10 levels (light and dark exposures of blot are shown). Actin was used as loading control. B, zymography was performed on a 10% gelatin zymogram with tissue culture supernatant of untreated MiaPaCa2 cells or those treated with either 5 μm batimastat or 25 μm GM6001. Cell lysates from the corresponding samples used for zymography were blotted for actin to ensure equal loading. C, MiaPaCa2 cells were treated with 5 μm batimastat or 5 μm GI254023X. Tissue culture supernatant was blotted for sAPPα using 6E10 antibody (top panel), and cell lysate was analyzed for ADAM10 (panel 2) and ADAM17 (bottom panel) levels. Actin was used as loading control (panel 3). D, MiaPaCa2 cells were treated with 5 μm batimastat, 100 ng/ml gemcitabine or both, and ADAM10 levels were analyzed by Western blotting. An antibody directed against the C terminus of ADAM10 was used to detect the different forms of ADAM10. Actin was used as loading control. E, phoenix cells were transfected with either untagged pcDNA3-ADAM10 or C-terminally Myc-tagged pRK5M-ADAM10 and cell lysates were used to detect the different forms of ADAM10 (light and dark exposures are shown). Myc antibody was used to detect the expression of Myc-tagged ADAM10 (myc, panel 4), and actin was used as loading control (actin, panel 3). Tissue culture supernatant was used to detect the levels of secreted sAPPα using 6E10 antibody (panel 5, sAPPα). F, sAPPα levels were quantified using ImageJ software from three independent experiments and normalized to actin. G, MiaPaCa2 and myc-ADAM10 transfected Phoenix cells were treated with or without 5 μm batimastat, samples were prepared in RIPA lysis buffer and deglycosylated following the manufacturer's protocol. Western blot analysis of samples was performed with ADAM10 C-terminal antibody. Actin was used as a loading control. H, ADAM10 levels in total cell lysate of Phoenix cells transfected with pcDNA3 or Myc-tagged pRK5 ADAM10 before immunoprecipitation with NeutrAvidin was detected by ADAM10 antibody. Actin was used as loading control. I, phoenix cells transfected with pcDNA3 or myc-tagged pRK5M-ADAM10 were analyzed using anti-Myc antibody for membrane bound ADAM10 fragments in the presence or absence of batimastat using biotinylation-based cell surface protein isolation (top and second panels). Na/K ATPase was used as loading control (third panel). 6E10 antibody was used to detect membrane-associated APP levels (bottom panel). J, to confirm that the ∼80 kDa band we observed in the pancreatic cancer cells is a form of ADAM10, we transfected MiaPaCa2 cells with a control or ADAM10 siRNA, performed biotinylation-based membrane isolation and analyzed the samples by Western blot using C-terminal ADAM10 antibody. Na/K ATPase was used as loading control (lower panel).

FIGURE 3.

Inhibition of sAPPα enhances the cytotoxic effect of gemcitabine on pancreatic cancer cells. A, 10,000 CD18 cells were plated in 24-well culture dishes and treated with 5 μm batimastat, 100 ng/ml gemcitabine, or a combination of both for 24 h. Cytotoxicity assay using MTT reagent was performed and samples analyzed at A₅₄₀. B, 10,000 MiaPaCa2 cells were plated in 24-well culture dishes and treated with the indicated drugs, and the extent of cytotoxicity was determined using MTT reagent at A₅₄₀. C, CD18 cells were plated in 24-well plates and treated with the indicated drugs for 24 h and cytotoxicity was measured using the MTT reagent at A₅₄₀. Each experiment was performed more than three times and (*) indicates significant difference with p value less than 0.05 from an unpaired two-tailed Student's t test. D, 10,000 CD18 cells were plated in 24-well plates and treated with 5 μm batimastat, 5 μm GI254023X, 100 ng/ml gemcitabine, or combinations for 24 h. MTT reagent was used to determine cytotoxicity at A₅₄₀. E, 10,000 MiaPaCa2 cells were plated in 24-well plates and treated with 5 μm batimastat, 5 μm GI254023X, 100 ng/ml gemcitabine or combinations for 24 h. MTT reagent was used to determine cytotoxicity at A₅₄₀. F, CD18 and MiaPaCa2 cells were treated with 5 μm batimastat, 100 ng/ml gemcitabine or a combination of the drugs for 24 h, and cell lysates collected for analysis of PARP cleavage. The same blot was reprobed for actin, which served as a loading control. G, cleavage of PARP was quantified from three independent experiments and normalized to actin using the ImageJ software as represented by the bar graph. Each of the cytotoxicity and quantification experiments were performed more than three times, and (*)(#)(●)(■) indicate significant difference with p value less than 0.05 from an unpaired two-tailed Student's t test.

FIGURE 4.

Cytotoxic effect of batimastat is brought about by inhibition of sAPPα generation. A, CD18 cells were transfected with siRNA to APP or ADAM10 and Western blot analysis was performed using 6E10 and ADAM10 antibodies to confirm knockdown of the respective genes. B, APP or ADAM10 were knocked down in MiaPaCa2 cells and Western blot was used to confirm knockdown. The effect of ADAM10 and APP knockdown in CD18 (C) and MiaPaCa2 (D) cells was compared with batimastat treatment in a cytotoxicity assay using MTT reagent at A₅₄₀. CD18 (E) and MiaPaCa2 (F) cells were treated with 100 nm recombinant sAPPα alone or in combination with 5 μm batimastat for 24 h and changes in cell death was analyzed with the MTT reagent at A₅₄₀. Each experiment was performed three times, and (*)(#)(**) indicates significant difference with p value less than 0.05 from an unpaired two-tailed Student's t test.

FIGURE 5.

ADAM10, and not ADAM17, plays a role in sAPPα-mediated pancreatic cancer cell proliferation. A, MiaPaCa2 cells were transfected with ADAM17 siRNA at different concentration starting from 20 nm to 60 nm and Western blot of cell lysates were performed to determine extent of ADAM17 knockdown (top panel). Actin was used as loading control (bottom panel). Cell supernatant was used to detect sAPPα level using 6E10 antibody. B, MiaPaCa2 cells transfected with control siRNA, ADAM17 siRNA or ADAM10 siRNA were treated with or without 5 μm batimastat for 24 h, and cell death was analyzed with the MTT reagent at A₅₄₀. Each experiment was performed three times, and (*)(#) indicates significant difference with p value less than 0.05 from an unpaired two-tailed Student's t test. C, sAPPα levels in ADAM10 or ADAM17 knockdown cells were detected using 6E10 antibody. D, Western blot analysis was performed to determine levels of ADAM10 (left) or ADAM17 (right) in MiaPaCa2 cells transfected with ADAM10 or ADAM17 siRNA. Actin was used as loading control.

FIGURE 6.

Effect of sAPPα inhibition on colony formation by pancreatic cancer cells. A, 5,000 CD18 cells were seeded in soft agar and treated with 1 μm batimastat, 100 ng/ml gemcitabine or combination of both drugs (left panel in A) to study their effect on the ability of cells to form colonies over a period of 2 weeks. Soft agar assay performed with CD18 cells using 5 μm batimastat, 100 ng/ml gemcitabine or a combination of both drugs (right panel in A). Similarly, 5,000 MiaPaCa2 (B) or Panc1 (C) cells were seeded in soft agar and treated with 10 μm batimastat, 100 ng/ml gemcitabine, or a combination of the drugs to study their effect on colony formation. Treatment with each dose was performed in triplicate, and each experiment was performed three times.