Figure 1.
Dual inhibition of IR/IGF-1R and SFK decreases tumor growth in vitro more effectively than single pathway inhibition does.
(A) PC-3 or LNCaP cells were cultured for 96 hours with and without dasatinib (DSA; 100 nM) and BMS-754807 (BMS-807; 2 µM for PC-3 and 0.5 µM for LNCaP cells), alone and in combination, and cell numbers were determined as described in the methods. (B) PC-3 and LNCaP cells were incubated for 24 hours with and without DSA (100 nM) and BMS-754807 (5 µM for PC-3 and 1 µM for LNCaP cells), alone and in combination, and cell cycle staining after 24 hours was performed using propidium iodide. (C) PC-3 cells were incubated for 48 hours with and without DSA (100 nM) and BMS-754807 (2 µM), alone and in combination, and the percentage of apoptotic cells was determined by flow cytometric analysis of annexin-V staining. (D)Left: cell cycle staining with propidium iodide in PC-3–shIGF-1R cells. Right: PC-3–shIGF-1R and control cells were incubated for 48 hours with and without DSA (100 nM), and the percentage of apoptotic cells was determined by flow cytometric analysis of annexin-V staining. All experiments were performed in triplicate. *P<0.05; N.S. not significant.
Figure 2.
Modulation of IGF-1–induced Akt1 and Akt2 phosphorylation in PC-3 cells by dasatinib (DSA) and BMS-754807 (BMS-807).
(A) PC-3 cells were serum starved for 72 hours and then pre-incubated for 2 hours with BMS-754807 at either 2 µM or 5 µM, with dasatinib at 100 nM, or with both agents. After 2 hours, the cells were stimulated with 50 ng/mL recombinant human IGF-1 (rhIGF-1) for 3 minutes. Next, protein was harvested and the (phospho)-proteins IGF-1R, Src, and Akt were determined by western blot (WB). Vinculin was used as the loading control. (B and C) PC-3 cells were stimulated with DSA (100 nM) and BMS-754807 (5 µM) as above, and then the cells were harvested and immunoprecipitated for Akt1 (B) or Akt2 (C), followed by immunoblotting for phospho-Akt. (D) PC-3 cells were treated as in (A), and western blot was run for S6 and phospho-S6.
Figure 3.
Dasatinib (DSA) and BMS-754807 (BMS-807) inhibit tumor growth after subcutaneous implantation of xenograft MDA PCa 133 cells in nude mice.
(A) MDA PCa 133 (derived from bone metastases) cells were implanted subcutaneously in nude mice as described in Methods. Once the tumors reached a volume of 3 mm3, the mice were treated with dasatinib and BMS-754807 alone and in combination (n = 4 for each group). Tumors were measured twice a week. Shown is the relative increase in tumor size over time in each group. *P<0.05. (B) The mice were euthanized 16 days after treatment initiation, the tumors were harvested, and western blots were done for the indicated (total and phospho)-proteins. Shown are representative results from a tumor in each treatment group. (C) Quantification of TUNEL staining to detect apoptosis. Shown is the mean(± SEM) number of apoptotic cells per high-power field (HPF) in each group.
Figure 4.
Dasatinib (DSA) and BMS-754807 (BMS-807) decrease intratibial bone destruction and tumor formation and modulate bone turnover markers.
Bone-destructive PC3-MM2 cells were injected intratibially in nude mice. Following treatment with dasatinib, BMS-754807 alone and combined, the mice were euthanized, and x-rays and computed tomographic (CT) scanning of the long bones were done (control, n = 10; dasatinib, n = 9; BMS-754807, n = 9; combination, n = 8). Blood was collected for serum bone turnover markers. (A) The degree of bone destruction was blindly graded in a semiquantitative fashion based on x-rays from all mice. The graph displays the proportion of high-grade lesions (i.e., 2 or 3) in each treatment group. * P<0.05 combination vs. dasatinib only. (B) Representative CT scans (top) and x-rays (bottom) from the mice in each group. Arrows and numbers indicate the lesion site and the grade of bone destruction. (C and D) Serum levels of murine alkaline phospatase (C) and N-telopeptide (D) were determined by ELISA. Bars indicates SEM. * P<0.05.
Figure 5.
Dasatinib and BMS-754807 have different effects on tumorigenic properties of PCa cells.
Circulating IGF-1 from multiple sources (bone, tumor cells, adipocytes, etc.) binds to IGF-1R, which leads to downstream activation of Akt and suppression of apoptosis. BMS-754807 inhibits IGF-1R phosphorylation and thus leads to partial inhibition of Akt1 and Akt2. While Akt2 phosphorylation is Src independent, Akt1 phosphorylation is also Src mediated, so full blockade is dependent on dual inhibition of Src and IGF-1R. Activation of IGF-1R, but also of many other cell surface receptors (integrins, vascular endothelial growth factor-receptor [VEGF-R], Axl, c-Met, etc.), involves Src activation, which increases PCa cell motility, migration, and invasion. Dasatinib inhibits Src phosphorylation, thereby decreasing invasive/migratory properties of PCa cells. Since Akt is activated by both IGF-1R and Src, blockade of either pathway alone will only partially inhibit Akt phosphorylation, whereas the dual blockade almost completely abrogates Akt phosphorylation. FAK: focal adhesion kinase.