Dual inhibition of PI3K and mTOR signaling pathways decreases human pancreatic neuroendocrine tumor metastatic progression

Abstract

Objectives: Patients with advanced pancreatic neuroendocrine tumors have limited therapeutic options. Everolimus (RAD001), an inhibitor of the mammalian target of rapamycin (mTOR) pathway, has been shown to increase progression-free survival, but not overall survival, indicating a need to identify additional therapeutic targets. Inhibition of mTOR complex 1 by RAD001 may induce upstream AKT upregulation. We hypothesized that dual inhibition of AKT along with mTOR will overcome the limited activity of RAD001 alone.

Methods: The BON cell line has been used as a model to study pancreatic neuroendocrine tumor cell biology. Western blots and cell growth assays were performed with mTOR inhibitor RAD001 (50 nM), mitogen-activated protein kinase inhibitor PD0325901 (50 nM), PI3K (phosphatidylinositol 3-kinase) inhibitor LY294002 (25 μM), or vehicle control. Nude mice were treated daily for 6 weeks with RAD001 (oral gavage) and with LY29400 (subcutaneous) 1 week after intrasplenic injection of BON cells.

Results: Cellular proliferation was most attenuated with the combination therapy of LY29400 and RAD001. Similarly, the volume of liver metastasis was lowest in the group treated with both LY29400 (100 mg/kg per week, subcutaneous) and RAD001 (2.5 mg/kg per day) compared with that in the vehicle group (P = 0.04).

Conclusion: The combination therapy of LY29400 and RAD001 decreased the cell growth in vitro and progression of liver metastasis in vivo compared with vehicle or with single-drug therapy.

Figure 1

Effect of mTOR inhibitor RAD001 (RAD), MEK inhibitor PD0325901 (PD) and PI3K inhibitor LY294002 (LY), as single agents and in dual combination, on MAPK and AKT signal transduction. (A) BON cells were treated with the indicated inhibitors, for 30 minutes. Cell lysates were subjected to electrophoresis, followed by Western blotting using the indicated phosphor-specific antibodies. Blots were then stripped and reprobed with βactin to confirm equal protein loading. Representative blots are shown. (B, C, D). Graphical representation of protein expression levels (pAKT, pp70S6K, and pERK1,2, respectively) relative to vehicle control treated BON cells from 3 independent experiments. T-test was used to compare vehicle vs. treatment (*p<0.05, **p<0.01).

Figure 2

Effect of mTOR inhibitor RAD001, MEK inhibitor PD0325901, and PI3K inhibitor LY294002, as single agents and in dual combination, compared to vehicle treatment on BON cell growth in vitro over time.

Figure 3

Effect of vehicle treatment (A), mTOR inhibitor RAD001 (B), PI3K inhibitor LY294002 (C) as single agents, and in dual combination (D), in an in vivo model of hepatic metastases from BON cells (representative experiment shown). For example, bulky tumor (>50% of the liver parencyma by gross inspection) was seen in 4/6 livers. See Table 1 for Summary of 3 combined experiments.