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.

considered to be targeted by a given miRNA when the miRNA-mRNA relationship held true in at least 4 different target prediction algorithms. From the above datasets, we identified mRNAs that were commonly upregulated in prostate cancer compared to normal prostate and mRNAs upregulated in metastasis compared to primary cancer. The list of miRNAs targeting seven or more of the genes upregulated (5-fold increase) in prostate cancer was intersected with a list of miRNAs targeting genes upregulated (10fold increase) in metastasis. Concurrently, the miRNAs that were downregulated in PCa from literature search were generated. MiRNAs that target genes that are upregulated in cancer or cancer metastasis and at the same time downregulated in cancer were included for further analysis. We also downloaded publically available data from the TCGA (https://tcga-data.nci.nih.gov/tcga/) for patients with prostate adenocarcinoma (PRAD).
These analyses were performed in an R statistical environment (version 3.0.1) (http:///www.r-project.org/). The tests were two-sided and considered statistically significant when p< 0.05. The expression of miR-34a was obtained from the Illumina mirnaseq Level_3 data set. We derived from the "isoform_quantification" file the "reads_per_million_miRNA_mapped" values for miR-34a. To find the relationship between miR-34a expression and T staging (pathologic T), and the relationship between miR-34a and the Gleason score, we first employed a Shapiro-Wilk test to verify if the data followed a normal distribution. Accordingly, an ANOVA test, and the nonparametric Kruskal-Wallis test were applied to assess the relationship between miR-34a and the pathologic T, and between miR-34a and the Gleason score respectively. A box-and-whisker plot (Box plot represents first (lower bound) and third (upper bound) quartiles, whiskers represent 1.5 times the interquartile range) was used to visualize the data (log2 (x+1)).

Migration and invasion assay:
Migration and invasion assays were performed using migration (catalog #354578) and invasion (catalog #354480) assay inserts (BD Biosciences, Bedford, MA). The number of migrated or invaded cells for 5 fields for each insert was counted under a bright-field microscope and plotted as the number of cells migrated or invaded per field. Each experiment was performed in triplicate.

Inhibition of autophagy and apoptosis
Hydroxychloroquine (HCQ) was purchased from Sigma and used at concentration of 1nM. PC3 cells were pre-treated with HCQ for 24h prior to miR-34a transfection and cell lysates were prepared. NS3694 was purchased from Sigma and 24h before N.C. or miRNA transfection, 100 nM of NS3694 was added to PC3 cells and following 72 hours of transfection, cell lysates were prepared for western blots. Proliferation of cells pretreated with NS3694 and then transfected with N.C. or miR-34a was performed as described above. with the concentrated viral titer in growth media containing 8 μg/ml polybrene and GFP+ cells were sorted by BD FACS Aria II following which they were transfected with either N.C. or miR-34a mimics for 72 hours.

Autophagic flux assay
pGFP-RFP-LC3 plasmid was transfected into PC3 cells, and then cells were transfected with N.C. or miR-34a and plated on a 24-well plate. The GFP and RFP positive cells were visualized with a fluorescent microscope (Nikon) for time-lapse analysis. Each experiment was repeated at least three times, and representative images from control (N.C) or miR-34a transfected PC3 cells were identified and combined in Image J software to generate autophagy flux videos.

Confocal Imaging
PC3 cells grown in 4-well chamber slide were transfected with N.C. or miR-34a for 72 hours and incubated with 1ug/ml acridine orange for 30 min in the dark. The cells were then fixed with 4% paraformaldehyde and sucrose solution and mounted. Fixed cells were then imaged under a confocal microscope at 20X magnification.

Immunofluorescence (IF) and in situ hybridization (ISH)
For TUNEL staining, DeadEnd colorimetric TUN-EL system (Promega) was used according to manufacturer's instructions. Briefly, formalin fixed paraffin embedded (FFPE) slides were deparaffinized and antigen retrieval was performed with 1X Dako Antigen Retrieval buffer. Slides were then washed with PBS, blocked with 3% H 2 O 2 in methanol for peroxidase blocking, and in 4% fish gelatin for protein blocking. The slides were incubated with 4% paraformaldehyde in PBS for 10 minutes at room temperature (RT), washed with PBS, incubated in 0.2% TritonX-100 in PBS for 15 min at RT, washed with PBS and incubated with Equilibration buffer (Promega) for 10 min at RT. TUNEL incubation buffer (Promega) was added to each slide for 1 hour at 37°C in the dark. Slides were washed in 2X SSC (Promega) and counterstained with Hoechst mounting media (Life Technologies) and visualized under fluorescence microscope.
DIG-labeled probes for miR-34a and U6 endogenous control were purchased from Exiqon (Woburn, MA) and in situ hybridization (ISH) was performed on tumor sections according to the manufacturer's instructions by the Center for RNA Interference and noncoding RNA at UTMDACC. IF and ISH images were quantified by ImageJ and NIS software.

Cy5.5 delivery
Chitosan (CH) nanoparticles complexed with control or Cy5.5-siRNA were prepared as described previously [57]. Briefly, a CH solution was obtained by dissolving in 0.25% acetic acid and nanoparticles were spontaneously generated by the addition of TPP (0.25% w/v) and either control or Cy5.5-siRNA at a concentration of 1μg/μL to CH solution under constant stirring at room temperature. One million PC3MM2-LG cells were injected in the femur of the mice. Ten days after cell injection, mice received control or Cy5.5-CH nanoparticles (5μg/100μL of nanoparticles) via tail-vein injection.
Three days after nanoparticle injection, mice were sacrificed; femurs were harvested and imaged with IVIS 200.