ADAM17 inhibition enhances platinum efficiency in ovarian cancer

Abstract

Chemotherapeutic resistance evolves in about 70 % of ovarian cancer patients and is a major cause of death in this tumor entity. Novel approaches to overcome these therapeutic limitations are therefore highly warranted. A disintegrin and metalloprotease 17 (ADAM17) is highly expressed in ovarian cancer and required for releasing epidermal growth factor receptor (EGFR) ligands like amphiregulin (AREG). This factor has recently been detected in ascites of advanced stage ovarian cancer patients. However, it is not well understood, whether and how ADAM17 might contribute to chemo resistance of ovarian cancer. In this study, we identified ADAM17 as an essential upstream regulator of AREG release under chemotherapeutic treatment in ovarian cancer cell lines and patient derived cells. In the majority of ovarian cancer cells cisplatin treatment resulted in enhanced ADAM17 activity, as shown by an increased shedding of AREG. Moreover, both mRNA and the protein content of AREG were dose-dependently increased by cisplatin exposure. Consequently, cisplatin strongly induced phosphorylation of ADAM17-downstream mediators, the EGFR and extracellular signal-regulated kinases (ERK). Phorbol 12-myristate 13-acetate (PMA), similarly to cisplatin, mediated AREG shedding and membrane fading of surface ADAM17. Inhibition of ADAM17 with either GW280264X or the anti-ADAM17 antibody D1 (A12) as well as silencing of ADAM17 by siRNA selectively reduced AREG release. Thus, ADAM17 inhibition sensitized cancer cells to cisplatin-induced apoptosis, and significantly reduced cell viability. Based on these findings, we propose that targeting of ADAM17 in parallel to chemotherapeutic treatment suppresses survival pathways and potentially diminish evolving secondary chemo resistance mechanisms.

Keywords: ADAM17; amphiregulin; chemo resistance; ovarian cancer; tumor therapy.

Figure 1. Cisplatin increases ADAM17-dependent AREG release in ovarian carcinoma cell lines

After 48 h of cisplatin treatment with the indicated concentrations, cells were trypsinized, counted and lyzed. Optical densities (ODs) of ADAM17 and AREG levels in lysates and AREG amounts in supernatants were measured by sandwich ELISA, and the calculated concentration values were normalized to the total cell number. Cisplatin treatment increased ADAM17-protein amounts (left panel) and AREG levels (center panel) in cell lysates and AREG release into culture supernatants (right panel) in Igrov-1 cells and A2780 cells. Skov-3 cells did not respond to cisplatin treatment. Data from three to five independent experiments per cell line are presented as mean + SEM. Stars indicate significant differences compared to untreated cells (0 μM). Friedman test; P = significance, ^* (p<0.05); ^** (p<0.01).

Figure 2. Modulation of ADAM17 on tumor cell surface after cisplatin treatment

ADAM17 expression was analyzed on the cell surface of Igrov-1 cells without treatment (control, Ctrl; thin line) or after treatment with the indicated concentrations of Cisplatin (cis) or Phorbolester (PMA) (bold lines) after 48 h. Cells were stained by anti-human TACE (ADAM 17) mAb and by appropriate isotype controls for untreated (Ctrl, dotted line) and treated (dashed lines) samples. Histograms of one representative of five independent experiments are shown (A). The indicated numbers present the mean fluorescence intensity (MFI); shown as the difference of IgG-MFI to ADAM17-MFI. The MFI of control cells [Ctrl (NaCl)] was set to 100 % for comparison of surface ADAM17 expression to cisplatin or PMA treated cells. Mean + SEM of the MFI of five experiments were calculated and are presented as percentage of Ctrl (NaCl). Stars indicate significant differences to the corresponding Ctrl. ANOVA; P = significance, ^* (p<0.05); ^** (p<0.01) (B).

Figure 3. Inhibition of ADAM17 reduces cell viability and enhances apoptosis in cisplatin sensitive cells

Cells were treated with 6 μM cisplatin (cis; diluted in NaCl) or NaCl (control) for 24 h for measurement of apoptosis (A, B, C) and for 48 h to measure cell viability (D). GI and GW (each diluted in DMSO) were used at a concentration of 3 μM to block ADAM10 and ADAM10/ADAM17, respectively. As a control, cells were treated with DMSO. For comparison of three to five independent experiments the caspase activity of control cells was set to one (A) or the number (no.) of viable cells was set to 100 % of control cells (NaCl and DMSO) (D). Data are presented as mean + SEM. For FACS-analyses, cells were washed and stained with Annexin V-FITC (An V) and Propidium iodide (PI). All analyses were measured on a FACS Calibur (BD Biosciences) (B, C). (B) One representative experiment out of five independent ones is shown with the dot plot analysis and the appropriate percentage of dead cells. (C) The mean distribution + SEM of alive (An V/PI-negative), early apoptotic (An V-positive, PI-negative), late apoptotic/necrotic (An V/PI-positive) or necrotic (An V-negative, PI-positive) tumor cells of five independent experiments are presented. Stars above the black bars indicate significant differences between cisplatin treatment and the corresponding NaCl treatment (left). ANOVA; P = significance, ^* (p<0.05); ^** (p<0.01); ^*** (p<0.001); ^**** (p<0.0001).

Figure 4. Cisplatin-induced AREG release is selectively ADAM17-dependent and cisplatin increases EGFR and ERK phosphorylation

Cells were treated with 6 μM cisplatin or the equivalent volume of NaCl (A) 100 nM PMA (solved in DMSO) or DMSO (B) and 3 μM of the ADAM10 inhibitor GI or the combined ADAM10/ADAM17 inhibitor GW or DMSO for 24 h, as indicated (A, B). Additionally, ADAM17 was blocked by 200 nM of the anti-ADAM17 IgG antibody D1 (A12) or an equivalent amount of normal human IgG was used as a control (C). AREG amounts in supernatants were investigated by AREG-ELISA and data were normalized to total protein amount of the cell lysates. For comparison of three independent experiments, the AREG-level of cells treated with NaCl and DMSO (A) or NaCl, Ctrl.IgG (C) was set to one. Means + SEM from three independent experiments are presented. Stars above black bars indicate differences between cisplatin treatment and the corresponding NaCl treatment (left). ANOVA; P = significance, ^* (p<0.05); ^** (p<0.01); ^*** (p<0.001). For western blot analyses (D), cells were treated for 48 h with the indicated amounts of cisplatin or NaCl as a solvent. Proteins were analyzed with the indicated primary antibodies. β-actin was used as a loading control. One of three representative blots is shown.

Figure 5. Blockage of ADAM17 effectively sensitizes patient-derived ovarian cancer cells to cisplatin

Primary cancer cells were isolated from ascites of five ovarian cancer patients (Pat.As.1, 2, 3, 4, 5) and from ovarian cancer-tissue (Pat. T1). Patients are identified by different symbols. Following 6 days of cell treatment with 9 μM cisplatin or the equivalent amount of NaCl, cells were harvested. ADAM17 amounts in lysates and AREG levels in supernatants and lysates were determined by ELISA. The calculated concentrations were normalized to total cell number. Data are presented as mean + SEM of the patient collective (A). For caspase and apoptosis measurement, cells were grown with and without addition of cisplatin [10 μM] or the equivalent amount of NaCl as a solvent for 48 h. To block ADAM10 and ADAM10/ADAM17, the inhibitors GI and GW were used at a concentration of 3 μM. Experiments were performed as three biological replicates for Pat.As.4 and are depicted as mean + SEM for this patient and mean + SD for Pat.As.5 and Pat.T1, where due to material restriction only technical replicates could be performed (B). For FACS analyses 0.5×10⁶ Pat.As.4 cells were cultured for 48 h with NaCl (control) or with the indicated concentration of cisplatin (cis; diluted in NaCl) either in the presence of DMSO (control) or with GI or GW (each diluted in DMSO). After incubation, cells were washed and stained with Annexin V-FITC (An V) and PI. All analyses were measured on a FACS Calibur (BD Biosciences). (C) One representative experiment out of five independent ones is shown with the dot plot analysis. (D) The mean distribution + SEM of alive (An V/PI-negative), apoptotic (An V-positive, PI-negative), late apoptotic/necrotic (An V/PI-positive) or necrotic (An V-negative, PI-positive) Pat.As.4 cells of five independent experiments are presented. Stars above black bars indicate differences between cisplatin treatment and the corresponding NaCl treatment (left). Wilkox test (A) ANOVA (B, D); P = significance, ^* (p<0.05); ^** (p<0.01); ^*** (p<0.001); ^**** (p<0.0001).