Dual targeting of phosphoinositide 3-kinase and mammalian target of rapamycin using NVP-BEZ235 as a novel therapeutic approach in human ovarian carcinoma

Abstract

Purpose: This study evaluates the effect of dual PI3K and mTOR inhibition using NVP-BEZ235 in preclinical models of ovarian cancer as a potential novel therapeutic strategy.

Experimental design: Inhibition of PI3K/Akt/mTOR signaling by NVP-BEZ235 was demonstrated by immunoblotting. The effect on cell proliferation was assessed in 18 ovarian cancer cell lines, including four pairs of syngeneic cisplatin-sensitive and cisplatin-resistant cell lines. The in vivo effects of NVP-BEZ235 on established tumor growth were evaluated using an immunocompetent, transgenic murine ovarian cancer model (LSL-K-ras(G12D/+)Pten(loxP/loxP)).

Results: NVP-BEZ235 decreased cell proliferation in all ovarian cancer cell lines assayed and sensitized cisplatin-resistant cells to the cytotoxic effects of cisplatin. Cell lines with PI3K-activating mutations or Pten deletions were significantly more sensitive to the effect of NVP-BEZ235 than cell lines without these mutations (P < 0.05). A statistically significant correlation was found between relative levels of p4E-BP1 and the IC(50) for NVP-BEZ235. In LSL-K-ras(G12D/+)Pten(loxP/loxP) mice with established intraperitoneal tumor disease, oral administration of NVP-BEZ235 decreased pAkt, p4E-BP1 and Ki67 in tumor tissue, and resulted in significantly longer survival compared to control animals (P < 0.05). NVP-BEZ235 also induced cell cycle arrest, caspase 3 activity, and reduced cell migration.

Conclusions: Targeting PI3K and mTOR simultaneously using NVP-BEZ235 effectively inhibits ovarian cancer cell growth even in the presence of platinum resistance and prolongs survival of mice with intra-abdominal ovarian tumor disease. We propose that dual PI3K and mTOR inhibition using NVP-BEZ235 may be an effective novel therapeutic approach in patients with ovarian cancer.

Figure 1. NVP-BEZ235 blocks phosphorylation of Akt, S6 and 4E-BP1

A and B. IGROV1, SKOV3 and OVCAR5 cells were serum-starved overnight, then treated with NVP-BEZ235 (A) or RAD001 (B) in culture medium for 24h. Total cell extracts were analyzed by immunoblotting for pAkt, pS6 or p4E-BP1. Total Akt, S6 and β-actin levels are shown as loading controls.

Figure 2. NVP-BEZ235 decreases ovarian cancer cell proliferation. Cells mutated in the PI3K/Akt pathway or with high p4E-BP1 levels demonstrate increased sensitivity

A. Ovarian cancer cells were treated with NVP-BEZ235 for 72h. Treatment with RAD001 is shown in comparison. Results for cell proliferation are expressed as a percentage of no treatment control (% maximum) ± the standard error. B. The IC₅₀ for the effect of NVP-BEZ235 on cell growth was determined from the data in Figure 2A. Cell lines with PI3K activating mutations or Pten deletions are shown as gray columns. Insert, Average IC₅₀ for cells lines with (gray columns) and without (white columns) PI3K mutations or Pten deletions. C. Correlation of basal p4E-BP1, pS6 or pAkt protein levels with the IC₅₀ for NVP-BEZ235. A two-tailed Pearson’s correlation analysis showed a significant correlation between NVP-BEZ235 IC₅₀ and relative levels of p4E-BP1 protein, but not pS6 or pAkt.

Figure 2. NVP-BEZ235 decreases ovarian cancer cell proliferation. Cells mutated in the PI3K/Akt pathway or with high p4E-BP1 levels demonstrate increased sensitivity

A. Ovarian cancer cells were treated with NVP-BEZ235 for 72h. Treatment with RAD001 is shown in comparison. Results for cell proliferation are expressed as a percentage of no treatment control (% maximum) ± the standard error. B. The IC₅₀ for the effect of NVP-BEZ235 on cell growth was determined from the data in Figure 2A. Cell lines with PI3K activating mutations or Pten deletions are shown as gray columns. Insert, Average IC₅₀ for cells lines with (gray columns) and without (white columns) PI3K mutations or Pten deletions. C. Correlation of basal p4E-BP1, pS6 or pAkt protein levels with the IC₅₀ for NVP-BEZ235. A two-tailed Pearson’s correlation analysis showed a significant correlation between NVP-BEZ235 IC₅₀ and relative levels of p4E-BP1 protein, but not pS6 or pAkt.

Figure 3. NVP-BEZ235 blocks pAkt and p4E-BP1, reduces Ki67 expression, and prolongs survival in LSL-K-ras^G12D/+Pten^loxP/loxP mice bearing ovarian tumors

A. Ovarian tumor formation in the right ovarian bursa of LSL-K-ras^G12D/+Pten^loxP/loxP mice injected with Cre expressing recombinant adenovirus (AdCre). The left ovary was injected with a control, GFP expressing adenovirus (AdGFP) and lacked formation of tumor. B. Mice with established ovarian tumors were treated with 40 mg/kg NVP-BEZ235 3 times every 12 hours. Tumors were harvested and immunohistochemistry was performed for the indicated proteins. Images were taken at 200 × magnification. C. Mice with established tumor disease were treated daily for 4 weeks with 40 mg/kg NVP-BEZ235 (red line, ), and survival compared to control animals (black line, ●).

Figure 4. NVP-BEZ235 decreases cell proliferation in cisplatin-resistant ovarian cancer cells and sensitizes ovarian cancer cells to cisplatin

A. Cisplatin sensitivity of SKOV3, OVCAR5, A2780 and OV2008 cells and their corresponding isogenic cisplatin-resistant cells, SKOV3-CisR, OVCAR5-CisR, CP70 and C13*, respectively. B. Effect of NVP-BEZ235 on proliferation of cisplatin-resistant ovarian cancer cells. C. NVP-BEZ235 sensitizes cells to cisplatin in cisplatin-resistant (upper panel) and cisplatin-sensitive (lower panel) cells treated as indicated with cisplatin with or without NVP-BEZ235.

Figure 4. NVP-BEZ235 decreases cell proliferation in cisplatin-resistant ovarian cancer cells and sensitizes ovarian cancer cells to cisplatin

A. Cisplatin sensitivity of SKOV3, OVCAR5, A2780 and OV2008 cells and their corresponding isogenic cisplatin-resistant cells, SKOV3-CisR, OVCAR5-CisR, CP70 and C13*, respectively. B. Effect of NVP-BEZ235 on proliferation of cisplatin-resistant ovarian cancer cells. C. NVP-BEZ235 sensitizes cells to cisplatin in cisplatin-resistant (upper panel) and cisplatin-sensitive (lower panel) cells treated as indicated with cisplatin with or without NVP-BEZ235.

Figure 5. NVP-BEZ235 blocks cell cycle progression, induces caspase 3 activity and decreases cell migration

A. Cisplatin-resistant (SKOV3-CisR, OVCAR5-CisR, CP70, C13*) (upper panel) and cisplatin-sensitive (SKOV3, OVCAR5, A2780, OV2008, IGROV1) (lower panel) cells accumulate in G₀/G₁ phase after 24h treatment with 50 nM NVP-BEZ235. B. Induction of caspase 3 activity in cisplatin-resistant (upper panel) and cisplatin-sensitive (lower panel) cells treated with NVP-BEZ235. Cells were treated with cisplatin as a positive control. C. NVP-BEZ235 reduces migration of cisplatin-resistant (upper panel) and cisplatin-sensitive (lower panel) cells in a Boyden transwell dual chamber system.

Figure 5. NVP-BEZ235 blocks cell cycle progression, induces caspase 3 activity and decreases cell migration

A. Cisplatin-resistant (SKOV3-CisR, OVCAR5-CisR, CP70, C13*) (upper panel) and cisplatin-sensitive (SKOV3, OVCAR5, A2780, OV2008, IGROV1) (lower panel) cells accumulate in G₀/G₁ phase after 24h treatment with 50 nM NVP-BEZ235. B. Induction of caspase 3 activity in cisplatin-resistant (upper panel) and cisplatin-sensitive (lower panel) cells treated with NVP-BEZ235. Cells were treated with cisplatin as a positive control. C. NVP-BEZ235 reduces migration of cisplatin-resistant (upper panel) and cisplatin-sensitive (lower panel) cells in a Boyden transwell dual chamber system.

Figure 5. NVP-BEZ235 blocks cell cycle progression, induces caspase 3 activity and decreases cell migration

A. Cisplatin-resistant (SKOV3-CisR, OVCAR5-CisR, CP70, C13*) (upper panel) and cisplatin-sensitive (SKOV3, OVCAR5, A2780, OV2008, IGROV1) (lower panel) cells accumulate in G₀/G₁ phase after 24h treatment with 50 nM NVP-BEZ235. B. Induction of caspase 3 activity in cisplatin-resistant (upper panel) and cisplatin-sensitive (lower panel) cells treated with NVP-BEZ235. Cells were treated with cisplatin as a positive control. C. NVP-BEZ235 reduces migration of cisplatin-resistant (upper panel) and cisplatin-sensitive (lower panel) cells in a Boyden transwell dual chamber system.