Chitosan nanoparticle-mediated delivery of miRNA-34a decreases prostate tumor growth in the bone and its expression induces non-canonical autophagy

While several new therapies are FDA-approved for bone-metastatic prostate cancer (PCa), patient survival has only improved marginally. Here, we report that chitosan nanoparticle-mediated delivery of miR-34a, a tumor suppressive microRNA that downregulates multiple gene products involved in PCa progression and metastasis, inhibited prostate tumor growth and preserved bone integrity in a xenograft model representative of established PCa bone metastasis. Expression of miR-34a induced apoptosis in PCa cells, and, in accord with downregulation of targets associated with PCa growth, including MET and Axl and c-Myc, also induced a form of non-canonical autophagy that is independent of Beclin-1, ATG4, ATG5 and ATG7. MiR-34a-induced autophagy is anti-proliferative in prostate cancer cells, as blocking apoptosis still resulted in growth inhibition of tumor cells. Thus, combined effects of autophagy and apoptosis are responsible for miR-34a-mediated prostate tumor growth inhibition, and have translational impact, as this non-canonical form of autophagy is tumor inhibitory. Together, these results provide a new understanding of the biological effects of miR-34a and highlight the clinical potential for miR-34a delivery as a treatment for bone metastatic prostate cancer

Dasatinib and BMS-754807 have different effects on tumorigenic properties of PCa cells.

<p>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.</p

Dasatinib (DSA) and BMS-754807 (BMS-807) inhibit tumor growth after subcutaneous implantation of xenograft MDA PCa 133 cells in nude mice.

<p>(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. *<i>P</i><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.</p

Modulation of IGF-1–induced Akt1 and Akt2 phosphorylation in PC-3 cells by dasatinib (DSA) and BMS-754807 (BMS-807).

<p>(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.</p

Dual inhibition of IR/IGF-1R and SFK decreases tumor growth <i>in vitro</i> more effectively than single pathway inhibition does.

<p>(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. *<i>P</i><0.05; N.S. not significant.</p