Article Figures & Data
Figures
- Figure 1.
A PDX model for enzalutamide-resistant CRPC. A, Development of patient-derived LTL-313B and LTL-313BR tumor tissue xenograft lines by serial subrenal capsule transplantation. The castration-resistant LTL-313BR subline was derived from the hormone-naïve LTL-313B line. B, LTL-313BR showed primary enzalutamide resistance. NS, not significant. Mice bearing LTL-313B or LTL-313BR tumors under the renal capsules were treated for 4 weeks with 10 mg/kg enzalutamide or vehicle (Veh). Tumor volume was measured at the end of the treatment. The LTL-313BR subline was resistant to enzalutamide treatment without prior exposure to the drug, whereas the parental LTL-313B remained sensitive.
- Figure 2.
Level of IAP member, BIRC6, is elevated in models showing acquired and primary enzalutamide resistance. A, The mRNA expressions of the IAP family members were determined in PDX models (LTL-313BR vs. LTL-313B) and LNCaP cell line–based xenografts models (enzalutamide-sensitive CRPC V16D vs. enzalutamide-resistant MR42D and MR49F) by transcriptomic sequencing. MR42D and MR49F are enzalutamide-resistant sublines developed after in vivo exposure to enzalutamide of parental V16D xenografts, that is, showing acquired enzalutamide resistance. B, BIRC6 protein expression determined by IHC in LTL-313B and LTL-313BR xenografts. Scale bar, 10 μm. Magnification, ×400. IHC scores 0, 1, 2, and 3 refer to negative, weak, mild, and strong staining intensities, respectively. ENZ-R, enzalutamide resistant.
- Figure 3.
BIRC6-targeting ASO-6w2 suppressed growth of enzalutamide-resistant LTL-313BR xenografts and induced apoptosis. A, BIRC6-targeting ASO (ASO-6w2) effectively suppressed proliferation of enzalutamide-resistant MR49F cells in vitro in a dose-dependent manner. Decreased BIRC6 protein expression after ASO-6w2 treatment was confirmed by Western blotting. B, In vivo therapeutic potential of ASO-6w2 was examined in the enzalutamide-resistant PDX model LTL-313BR. Groups of mice bearing LTL-313BR xenografts were treated with Scrb ASO or ASO-6w2 at 30 mg/kg on the first day, followed by 15 mg/kg for 20 days. Tumors were harvested and sera obtained for analysis 1 week after the end of the treatment. C, The ASO-6w2–treated group showed significantly smaller tumor volumes than the Scrb-treated group. D, The increase in average serum PSA levels of the ASO-6w2–treated mice was also significantly lower than that of the Scrb control. Whisker, median/mean ± interquartile range. E, Representative images of cells stained by IHC for cleaved caspase-3. The ASO-6w2–treated group showed a significant increase in apoptosis, with a 2-fold increase in the number of cleaved caspase-3–positive cells. The numbers of positively stained cells were quantified in 3 to 5 fields per sample (magnification, ×400). Scale bar, 50 μm. Error bars, mean ± SD. F, Suppression of BIRC6 expression by ASO-6w2 was confirmed by immunohistochemical staining. Scale bar, 10 μm. ENZ-R, enzalutamide resistant.
- Figure 4.
ASO-6w2 suppressed the expression of prosurvival genes upregulated in enzalutamide-resistant tumors. The LTL-313BR enzalutamide-resistant CRPC xenograft line showed upregulated expression of prosurvival genes in AR, TMPRSS2 (AR pathway), IGFBP5 (IGF signaling), BCL2, TNFRSF11A, and NRP1 (NFκB pathway) compared with the parental, enzalutamide-sensitive LTL-313B line. Results are presented as mRNA reads relative to those of the LTL-313B line from transcriptomic sequencing expression data shown in stripped histograms. The expression of these genes in LTL-313BR were shown to be reduced upon treatment with ASO-6w2, including significant downregulations of TMPRSS2, IGFBP5, BCL2, TNFRSF11A, and a reduced expression trend for AR and NRP1. Relative mRNA expressions were determined by qRT-PCR; n = 7 per group. Error bars, mean ± SD. ns, not significant.
Tables
- Table 1.
Top enriched gene sets in MSigDB all canonical pathways and GO biological process in ASO-6w2 versus Scrb-treated LTL-313BR tumors
Functions ASO-6w2 vs. Scrb - Top enriched gene sets Up/down FDR GPCR signaling Reactome signaling by GPCR ↓ 8.06E−19 Reactome GPCR ligand binding ↓ 3.63E−14 Reactome G alpha I signalling events ↓ 4.96E−11 Matrisome (extracellular matrix) Naba matrisome ↑↓ 1.33E−15 Naba matrisome associated ↑↓ 3.08E−13 Receptor activity ↓ 7.00E−11 Transmembrane transport Establishment of localization ↑ 3.08E−16 Transport ↑ 1.35E−15 Reactome transmembrane transport of small molecules ↑ 2.07E−12 Reactome SLC-mediated transmembrane transport ↑ 1.34E−08 Response to external stimulus Response to external stimulus ↑ 4.10E−15 Response to wounding ↑ 3.05E−13 Response to stress ↑ 1.23E−10 NOTE: Analysis was based on gene expression profiling in ASO-6w2- and Scrb-treated LTL-313BR xenografts (n = 4 per group; fold change > 1.5).
Supplementary Data
- Supplementary figure S1 - BIRC6 mRNA levels in MR49F cells treated with ASO-6w2
- Supplementary figure S2 - MTS proliferation assay of MR42D ENZ-resistant CRPC cells treated with ASO-6w2
- Supplementary figure S3 - MTS Proliferation assay of MR49F cells treated with siControl or siXIAP at 20 nM. Results presented as growth rate relative to Mock at 96-hour after transfection
- Supplementary figure S4 - Serum PSA data of individual mice before and after treatment with ASO
- Supplementary figure S5 - Weights of mice during treatment of LTL-313BR xenografts with ASO-6w2
- Supplementary figure S6 - Hoechst 33258 staining of LTL-313BR tumours in Scrb or ASO-6w2 treated mice. White arrows indicate nuclei of mouse fibroblasts
- Supplementary figure S7 - Serum PSA (ng/ml) per unit tumour volume (mm3) in individual mice and in Scrb and ASO-6w2 groups
- Supplementary Table S1 - The qPCR primer sequences used
- Supplementary Table S2 - Gene expression changes in GPCR signalling genesets associated with ASO-6w2 treatment of LTL-313BR xenografts
- Supplementary Table S3 - Gene expression changes in matrisome genesets associated with ASO-6w2 treatment of LTL-313BR xenografts
- Supplementary figure legends - Supplementary figure legends
Volume 23, Issue 6
