Targeted inhibition of SIRT6 via engineered exosomes impairs tumorigenesis and metastasis in prostate cancer

Abstract

The treatment for metastatic castration-resistant prostate cancer patients remains a great challenge in the clinic and continuously demands discoveries of new targets and therapies. Here, we assess the function and therapeutic value of SIRT6 in metastatic castration-resistant prostate cancer. Methods: The expression of SIRT6 was examined in prostate cancer tissue microarray by immunohistochemistry staining. The functions of SIRT6 and underlying mechanisms were elucidated by in vitro and in vivo experiments. We also developed an efficient method to silence SIRT6 by aptamer-modified exosomes carrying small interfering RNA and tested the therapeutic effect in the xenograft mice models. Results: SIRT6 expression is positively correlated with prostate cancer progression. Loss of SIRT6 significantly suppressed proliferation and metastasis of prostate cancer cell lines both in vitro and in vivo. SIRT6-driven prostate cancer displays activation of multiple cancer-related signaling pathways, especially the Notch pathway. Silencing SIRT6 by siRNA delivered through engineered exosomes inhibited tumor growth and metastasis. Conclusions: SIRT6 is identified as a driver and therapeutic target for metastatic prostate cancer in our findings, and inhibition of SIRT6 by engineered exosomes can serve as a promising therapeutic tool for clinical application.

Keywords: Notch pathway; SIRT6; castration-resistant prostate cancer; engineered exosomes; therapy.

Figure 1

SIRT6 expression is positively correlated with prostate cancer progression. (A) Copy number of SIRT6 in normal prostate tissue, primary prostate cancer, and metastasis prostate cancer samples from selected Oncomine database (Grasso Prostate cohort, n=122; Vanaja Prostate cohort, n=40). (B) Relative mRNA expression of SIRT6 in GEO database (GDS2546, n=167). (C) Enrichment plots of Prostate cancer related gene signatures according to SIRT6 mRNA levels and Enrichment plots of cancer-related pathways dependent on SIRT6 expression in TCGA (PanCancer Atlas) dataset. (D) Immunohistochemical staining (IHC) of SIRT6 on prostate cancer biopsy samples grouped by Gleason score. Scale bars, 500 µm. (E) Representative IHC of SIRT6 expression in a prostate cancer tissue array. Scale bars, 500 µm and enlarged scale bars, 50 µm (Data are expressed as means ± SEM, Student's t-test, *P<0.05, ***P<0.001, ****P<0.0001).

Figure 2

SIRT6 is essential for the growth of prostate cancer cells. (A) Enrichment plot of Cyclin D1 according to SIRT6 expression in Metastatic Prostate Cancer (SU2C/PCF Dream Team, PNAS 2019) dataset. (B) Proliferation assay in SIRT6 stable overexpression (SIRT6-OE) and control (SIRT6-EV) cancer cells. (C)-(D) Colony formation in different prostate cancer cells (DU145 and PC3) with SIRT6 being either up- or down-regulated. (E) Proliferation of prostate cancer cells stably transfected with scramble sequence or shSIRT6. (F) Soft agar assay in C42B cells stably transfected with scramble sequence or shSIRT6. (G) IF staining of Ki67 (red) and DAPI (blue, nucleus) in C42B cells stably transfected with scramble sequence or shSIRT6. Scale bars, 50 µm. The data were represented as mean and SEM from three independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

Figure 3

SIRT6 induces a metastasis-promoting phenotype in prostate cancer cells. (A) Wound healing assay and (B) migration assay of prostate cancer cell groups (stably overexpressing SIRT6, SIRT6-OE vs. empty vector control, SIRT6-EV). Scale bars, 100 µm. (C) Western blot of EMT related protein levels in SIRT6-EV cells and SIRT6-OE cells. (D) Migration assay of prostate cancer cell lines stably transfected with either empty vector (scramble) or shSIRT6. Scale bars, 100 µm. (E-F) The expression of EMT-related markers in these two groups of cells was analyzed by qPCR and Western blot. (G) Immunofluorescence of Vimentin in SIRT6 knockdown C42B cells (Vimentin, red; DAPI, blue nucleus; Scale bars, 50 µm).

Figure 4

SIRT6 promotes the proliferation and metastasis of prostate cancer cells in vivo. (A) PC3M-luc cells were stably transfected with control pCDH empty vector (SIRT6-EV) or pCDH SIRT6-overexpression vector (SIRT6-OE), and orthotopically implanted to BALC/c nude mice. The growth of orthotopically implanted tumor was monitored by bioluminescence, and quantification of luminescence was summarized in (B). (C) Representative images of HE staining (left panel) and metastasis in liver (right panel) of the SIRT6-overexpression orthotopic prostate cancer models compared with the control group. Scale bars, 100 µm. (D) IHC analysis of SIRT6 (left panel) and Ki67 (right panel) expression in tumors. Scale bars, 100 µm. (E) Survival of above-described two groups of mice: SIRT6-EV (n=6) vs. SIRT6-OE (n=6). (F) Bioluminescence imaging of orthotropic models from each group implanted with PC3M-luc cells stably transfected either with empty vector (scramble sequence) (n=6) or shSIRT6 vector (n=6). (G) Quantification and (H) area of luminescence in orthotropic models from each group. (I) Representative image of HE staining (left panel) and metastasis in liver (right panel) of the SIRT6 knockdown orthotopic prostate cancer model compared with the scramble group. Scale bars, 100 µm. (J) IHC analysis of SIRT6 (left panel) and Survivin (right panel) expression in tumors. Scale bars, 100 µm.

Figure 5

The tumor-promoting effect of SIRT6 involves multiple cancer-related signaling pathways, mainly the Notch pathway. (A) Migration assay and (B) colony formation of prostate cancer cells treated with SIRT6 deacetylation inhibitor OSS_128167. Scale bars, 100 µm. (C) Migration assay and (D) proliferation assay in SIRT6 knock-down prostate cancer cells transfected with SIRT6 WT or mutants (G60A and R65A). Scale bars, 100 µm. (E) Top 8 pathways significantly enriched in SIRT6 high expression group compared with low levels of SIRT6 group in Metastatic Prostate Cancer (SU2C/PCF Dream Team, cell 2015) dataset. (F) Enrichment plot of Notch pathway according to SIRT6 expression level in the same prostate cancer cohort. (G) The receptors and ligands expression of Notch pathway in stable SIRT6-OE cells and SIRT-EV cells determined by RT-PCR. (H) 4×CBF1 luciferase activity and (I) the expression of NICD in prostate cancer cells transfected with combination of NICD and SIRT6. (J) Enrichment plot of mTOR pathway according to SIRT6 expression level. (K) Overexpression of SIRT6 increases the levels of p-S6, p-S6K, downstream of mTOR pathway. (L) Silencing SIRT6 inhibits the expression of p-S6, p-S6K.

Figure 6

Preparation and characterization of E3 aptamer-modified exosomes. (A) Schematic illustration of the procedure for the E3 aptamer-modified siRNA loaded exosomes. (B) Gel electrophoresis of the Apt-PEG-Chol (Lane 5) compared to free E3 Apt (Lane 4). (C) FAM-labeled siRNA was loaded into PKH27-marked exosomes and applied to C42B cells. After 12 h incubation, cells were fixed and visualized by confocal microscopy (FAM-siRNA, green; PKH27-Exos, red; DAPI, blue for nucleus; Scale bars, 10 µm). (D) Flow cytometry analyses of the three cell lines after incubation with the E3 Apt-modified, DIO-labeled exosomes. (E) Confocal images of aptamer (FAM labeled)-modified exosomes (marked by PKH27) absorbed by three cell lines (FAM-Apt, green; PKH-27-Exos, red; DAPI, blue nucleus, scale bars, 20 µm).

Figure 7

Delivery of therapeutic SIRT6 siRNA by aptamer-modified exosomes suppresses prostate tumor proliferation and metastasis. (A) Overall fluorescence imaging of tumor- bearing (subcutaneous implantation of PC3 cells) or no tumor-bearing mice after the injection of aptamer-modified, DIR-labeled exosomes. (B) Ex vivo fluorescence images of tumor and other major organs. (C) Representative image of the subcutaneous tumors in mice after treatment with aptamer-modified siRNA-loaded exosomes (n=4) compared with the control group (n=4). (D) Tumor volume changes of the subcutaneous mice models after treatment. (E) Representative IHC staining of SIRT6 in two groups of tumor tissues. Scale bars, 100 µm. (F) Representative IHC staining of Ki67 in two groups of tumor tissues. Scale bars, 100 µm. (G) The weight of tumor in orthotopically implanted mouse model after treatment with aptamer-modified siRNA-loaded exosomes (n=4) compared with the control group (n=4). (H) Full-scale HE staining of livers from the orthotopic mouse model. Metastatic nodules in livers were dissected counted. (I) Representative IHC staining of Vimentin and cleaved Caspase-3 in two groups of tumor tissues. Scale bars, 100 µm.