Recombinant MDA-7/IL24 Suppresses Prostate Cancer Bone Metastasis through Downregulation of the Akt/Mcl-1 Pathway

Abstract

Prostate cancer is a principal cause of cancer-associated morbidity in men. Although 5-year survival of patients with localized prostate cancer approaches 100%, survival decreases precipitously after metastasis. Bone is the preferred site for disseminated prostate cancer cell colonization, altering the equilibrium of bone homeostasis resulting in weak and fragile bones. Currently, no curative options are available for prostate cancer bone metastasis. Melanoma differentiation associated gene-7 (MDA-7)/IL24 is a well-studied cytokine established as a therapeutic in a wide array of cancers upon delivery as a gene therapy. In this study, we explored the potential anticancer properties of MDA-7/IL24 delivered as a recombinant protein. Using bone metastasis experimental models, animals treated with recombinant MDA-7/IL24 had significantly less metastatic lesions in their femurs as compared with controls. The inhibitory effects of MDA-7/IL24 on bone metastasis resulted from prostate cancer-selective killing and inhibition of osteoclast differentiation, which is necessary for bone resorption. Gain- and loss-of-function genetic approaches document that prosurvival Akt and Mcl-1 pathways are critically important in the antibone metastatic activity of MDA-7/IL24. Our previous findings showed that MDA-7/IL24 gene therapy plus Mcl-1 inhibitors cooperate synergistically. Similarly, an Mcl-1 small-molecule inhibitor synergized with MDA-7/IL24 and induced robust antibone metastatic activity. These results expand the potential applications of MDA-7/IL24 as an anticancer molecule and demonstrate that purified recombinant protein is nontoxic in preclinical animal models and has profound inhibitory effects on bone metastasis, which can be enhanced further when combined with an Mcl-1 inhibitory small molecule. Mol Cancer Ther; 17(9); 1951-60. ©2018 AACR.

Figure 1:

Production and characterization of His-Tagged recombinant MDA-7/IL-24 protein. A. Schematic diagram of production of His-tagged recombinant MDA-7/IL-24 protein. B. Confirmation of MDA-7/IL-24 protein by Western blotting. C. MTT assays examined the activity of MDA-7/IL-24 on PC3-ML cells. Data showed a significant inhibition in cell proliferation. D. Expression of downstream MDA-7/IL-24 signaling cascade molecules including p27, Beclin-1, and BiP/GRP78 were confirmed using Western blotting, which are upregulated in MDA-7/IL-24-treated cells. EF1α was used as a loading control. E. Colony formation (clonal) assays were performed with different PC cells in triplicates. Approximately, 200 cells were plated, treated with His-MDA-7/IL-24, and 2 weeks after treatment they were stained with crystal violet. Numbers of colonies were counted and the data was plotted. Data represents mean ± S.D. of two independent experiments; **, P< 0.01; ***, P< 0.001 versus control.

Figure 2:

Anti-metastatic activity and inhibition of osteoclast differentiation by MDA-7/IL-24 protein. A. In vivo bone metastasis assay evaluating the effect of His-MDA-7 on bone metastasis development (5 mg/kg, 2 X a week for 3 weeks). n = 5 in each group. B. Luciferase intensity was quantified and bar graph showing the significant inhibition in luciferase intensity in MDA-7/IL-24-treated animals. C. Survival plot showing the role of MDA-7/IL-24 in the enhancement of survival of animals. Data represents mean ± S.D. of two independent experiments: **, P<0.01 versus control. Bone marrow cells were collected at the end of the study and osteoclast differentiation was induced. Mature osteoclasts were stained using a TRAP staining kit and osteoclast activity was measured by TRACP enzymatic assay kit as described in Materials and methods. Number of osteoclasts (D) and osteoclast activities (E) in control and MDA-7/IL-24-treated samples are as shown in the graphs. Five replicates were done for each group. Data represents mean ± S.D. of two independent experiments; *, P< 0.05; ***, P< 0.001 versus control.

Figure 3:

Effect of MDA-7/IL-24 on signaling cascades in RAW 264.7 cells. Cells were treated with the indicated reagents/constructs and Western blotting was performed to investigate the signaling cascades. A. Cells were treated with RANKL (100 ng/ml) and His-MDA-7 (10 μg/ml). After 5 days, cells were lysed and Western blotting was done. Akt phosphorylation decreased with MDA-7/IL-24 treatment, which was reversed upon RANKL treatment. Mcl-1, NFATc1, and phosphor-GSK3β, which are downstream signaling molecules, followed a similar pattern. B. Cells were treated with a PI3 kinase inhibitor LY294002 (10 μM) and His-MDA-7 (10μg/ml). Treatment with an Akt inhibitor or MDA-7/IL-24 attenuated the level of Phosphor-Akt. Combined treatment with an Akt inhibitor and MDA-7/IL-24 further downregulated Phosphor-Akt and downstream molecules. C. Cells were transfected with Myr-Akt (constitutively active Akt) and treated with His-MDA-7 (10 μg/ml). Western blotting was performed to check the expression of the indicated proteins. Phosphor-GSK3β, NFATc1, and Mcl-1 expression increased with the over expression of a constitutively active Akt, which were inhibited by treatment with His-MDA-7. EF1α was used as a loading control in all the experiments. The densitometric quantification of p-Akt/Akt, p-GSK3β/GSK3β, NFATc1/EF1α, and Mcl-1/EF1α under different experimental conditions in RAW 264.7 cells is shown in supplemental figure 5A, B and C.

Figure 4:

Combinatorial effect of His-MDA-7 and BI-97D6 on metastasis of PC in bone and osteoclast differentiation. A. In vivo bone metastasis assays evaluated the effects of His-MDA-7 and BI-97D6. Experimental treatment protocol is described in Materials and methods. B. Luciferase intensity was quantified in triplicates and the bar graph shows the significant inhibition in luciferase intensity in MDA-7/IL-24-treated animals. Addition of BI-97D6 further enhanced the inhibitory effects of MDA-7/IL-24 on bone metastasis development. Osteoclasts were stained with a TRAP staining kit and osteoclastic activity was measured using a TRACP enzymatic assay kit. Number of osteoclasts (C) and osteoclastic activity was measured (D), which is represented graphically. Four replicates were taken for each group. Data represents mean ± S.D. of two independent experiments; *, P< 0.05; **, P< 0.01; ***, P< 0.001 versus control.

Figure 5:

Stable PC cells overexpressing Akt and effect of Akt expression on PC bone metastasis, response to MDA-7/IL-24 and osteoclast differentiation. A. Phosphor-Akt expression in control PC3-ML and stable Akt overexpressing PC3-ML^Akt clones. B. In vivo bone metastasis study using PC3-ML cells and PC3-ML cells overexpressing CA-Akt (PC3-ML^Akt). Constitutive activation of Akt diminished the inhibitory effects of MDA-7/IL-24 on PC-induced bone metastasis. C. Luciferase intensities are as represented. Effect of constitutive Akt expression on osteoclast activity (D) and number of osteoclasts (E) were measured in triplicates and are represented graphically. Bone marrow cells from mice (described in B) were collected and 5 X 10⁵ cells were induced for osteoclast differentiation. Data represents mean ± S.D. of two independent experiments; *, P< 0.05; ***, P< 0.001 versus control.

Figure 6:

Schematic representation of MDA-7/IL-24-mediated inhibition in progression of PC-induced bone metastasis through modulation of the bone microenvironment.