The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

Abstract

Background: Using novel small-molecular inhibitors, we explored the feasibility of the class I PI3K/Akt/mTORC1 signaling pathway as a therapeutic target in canine oncology either by using pathway inhibitors alone, in combination or combined with conventional chemotherapeutic drugs in vitro.

Results: We demonstrate that growth and survival of the cell lines tested are predominantly dependent on class I PI3K/Akt signaling rather than mTORC1 signaling. In addition, the newly developed inhibitors ZSTK474 and KP372-1 which selectively target pan-class I PI3K and Akt, respectively, and Rapamycin which has been well-established as highly specific mTOR inhibitor, decrease viability of canine cancer cell lines. All inhibitors demonstrated inhibition of phosphorylation of pathway members. Annexin V staining demonstrated that KP372-1 is a potent inducer of apoptosis whereas ZSTK474 and Rapamycin are weaker inducers of apoptosis. Simultaneous inhibition of class I PI3K and mTORC1 by ZSTK474 combined with Rapamycin additively or synergistically reduced cell viability whereas responses to the PI3K pathway inhibitors in combination with conventional drug Doxorubicin were cell line-dependent.

Conclusion: This study highlighted the importance of class I PI3K/Akt axis signaling in canine tumour cells and identifies it as a promising therapeutic target.

Figure 1

Schematic diagram of the class I PI3K/Akt/mTOR axis pathway. The targets of the inhibitors (ZSTK474, Wortmannin, KP372-1 and Rapamycin) used in this study are indicated.

Figure 2

Western blot analysis of components of the class I PI3K and ERK pathways in human and canine cancer cells. Whole cell lysates (comprising 50 μg total protein) were subjected to western blotting analysis with β-actin as a loading control.

Figure 3

Sensitivity of canine and human cancer cells to inhibitors targeting class I PI3K/Akt/mTOR pathway. Cells were treated with a range of doses of the pan-class I PI3K inhibitor ZSTK474 for 3 days (A), Akt inhibitor KP372-1 for 2 days (B), or mTOR inhibitor Rapamycin for 3 days (C). After drug treatment, the number of viable cells was determined by using CellTiter-Glo® Luminescent Cell Viability Assay. Results were expressed as mean (±SD) counts of quadruplicate wells. The values of the viability rates of the drug-treated cells were compared with the vehicle (DMSO)-treated cells on the same culture plates.

Figure 4

Effects of the inhibitors on class I PI3K/Akt/mTOR axis signaling in canine and human cancer cells. Cells were seeded at a density of 20,000 cells per ml overnight, followed by treatment with 1 μM ZSTK474 (A), 400 nM KP372-1 (B), or 100 nM Rapamycin (C) for 5 hrs. Whole cell lysates (comprising 50 μg total protein) were subjected to western blot with the indicated antibodies. β-actin was used as a loading control.

Figure 5

Effects of the inhibitors on class I PI3K/Akt/mTOR axis signaling in canine C2 cells. Cells were treated with pan-class I PI3K inhibitor Wortmannin (W) at 1 μM and ZSTK474 (Z) at 1 μM, mTOR inhibitor Rapamycin (R) at 100 nM (A) and Akt inhibitor KP372-1 at 0, 150, 200 and 400 nM (B) for the indicated period of time. Whole cell lysates (comprising 50 μg total protein) were subjected to western blot with the indicated antibodies. β-actin was used as a loading control. N/A indicates data unavailable due to induction of apoptosis in all cells.

Figure 6

Effects of ZSTK474, KP372-1 and Rapamycin on induction of apoptosis. Cells were treated with 20 μM ZSTK474 for 2 days, 400 nM KP372-1 for 1 day, 20 μM Rapamycin for 2 days, or vehicle control as described in Materials and Methods. Induction of apoptosis was determined by annexin V/propidium iodide (PI) staining and flow cytometry analysis. Scatter pots with percentages of live, early apoptosis and late apoptosis were indicated at lower left, lower right and upper right quadrants, respectively in A while B demonstrates this data in a bar chart format. This data is representative of two independent experiments.

Figure 7

Effects of ZSTK474 in combination with Rapamycin on cells. Cells were treated with the indicated doses of ZSTK4747, Rapamycin, the combination of the former two inhibitors or vehicle control for 3 days. Additive, synergistic, or antagonistic inhibitory effects of the drug combination on cell viability were determined when the experiment values (cell viability percentages) of the drug combination were overlapped with, lower than, or higher than Bliss theoretical values respectively (A). Western blot analysis was performed to determine the inhibitory effects of ZSTK474 in combination with Rapamycin on class I PI3K activity (B). Cells were seeded at a density of 20,000 cells per ml overnight, followed by treatment with 5 μM ZSTK474 (10 μM ZSTK474 in REM cells), 100 nM Rapamycin, the combination of the former two inhibitors or vehicle control for 18 hrs. Whole cell lysates (comprising 50 μg total protein) were subjected to western blot with the indicated antibodies. β-actin was used as a loading control.

Figure 8

Effects of the combination of the class I PI3K/Akt/mTOR pathway inhibitors and Doxorubicin on cell viability. SB (A) and REM (B) cells were treated with the indicated does of the class I PI3K/Akt/mTOR pathway inhibitors, Doxorubicin, the combination of the former two drugs or vehicle control for 3 days (2 days for KP372-1). Additive, synergistic or antagonistic inhibitory effects of the drug combination on cell viability were determined when the experiment values (cell viability percentages) of the drug combination were overlapped with, lower than, or higher than Bliss theoretical values respectively.