CD70-Targeted Micelles Enhance HIF2α siRNA Delivery and Inhibit Oncogenic Functions in Patient-Derived Clear Cell Renal Carcinoma Cells

Abstract

The majority of clear cell renal cell carcinomas (ccRCCs) are characterized by mutations in the Von Hippel−Lindau (VHL) tumor suppressor gene, which leads to the stabilization and accumulation of the HIF2α transcription factor that upregulates key oncogenic pathways that promote glucose metabolism, cell cycle progression, angiogenesis, and cell migration. Although FDA-approved HIF2α inhibitors for treating VHL disease-related ccRCC are available, these therapies are associated with significant toxicities such as anemia and hypoxia. To improve ccRCC-specific drug delivery, peptide amphiphile micelles (PAMs) were synthesized incorporating peptides targeted to the CD70 marker expressed by ccRCs and anti-HIF2α siRNA, and the ability of HIF2α-CD27 PAMs to modulate HIF2α and its downstream targets was evaluated in human ccRCC patient-derived cells. Cell cultures were derived from eight human ccRCC tumors and the baseline mRNA expression of HIF2A and CD70, as well as the HIF2α target genes SLC2A1, CCND1, VEGFA, CXCR4, and CXCL12 were first determined. As expected, each gene was overexpressed by at least 63% of all samples compared to normal kidney proximal tubule cells. Upon incubation with HIF2α-CD27 PAMs, a 50% increase in ccRCC-binding was observed upon incorporation of a CD70-targeting peptide into the PAMs, and gel shift assays demonstrated the rapid release of siRNA (>80% in 1 h) under intracellular glutathione concentrations, which contributed to ~70% gene knockdown of HIF2α and its downstream genes. Further studies demonstrated that knockdown of the HIF2α target genes SLC2A1, CCND1, VEGFA, CXCR4, and CXCL12 led to inhibition of their oncogenic functions of glucose transport, cell proliferation, angiogenic factor release, and cell migration by 50−80%. Herein, the development of a nanotherapeutic strategy for ccRCC-specific siRNA delivery and its potential to interfere with key oncogenic pathways is presented.

Keywords: CD70; HIF2α; clear cell renal cell carcinoma; micelle; nanoparticle; siRNA.

Figure 1

Physicochemical characterization of HIF2α-CD27 PAMs. (A) Schematic of HIF2α-CD27 PAM self-assembly. (B) Transmission electron micrograph of HIF2α-CD27 PAMs. Scale bar = 200 nm. (C) Zeta potential of CD27 PAMs before and after HIF2α siRNA incorporation. N = 3. (D) Gel electrophoresis assay demonstrating incorporation of siRNA into PAMs at 1 mol% and protection of siRNA from RNAse-mediated degradation after 1 h. Lanes: (i) HIF2α-CD27 PAMs, (ii) HIF2α siRNA, (iii) HIF2α-CD27 PAMs incubated with RNAse-treated FBS for 1 h, (iv) HIF2α siRNA incubated with RNAse-treated FBS for 1 h. (E) Gel electrophoresis assay with PAMs up to 24 h after GSH treatment demonstrate rapid siRNA release following exposure to intracellular GSH levels (1 mM) and minimal siRNA release (<25%) after extracellular GSH exposure (1 μM). N = 3.

Figure 2

Baseline mRNA expression in patient-derived ccRCC cell cultures. Patient-derived ccRCC cells have increased (A) HIF2A, (B) CD70, (C) SLC2A1, (D) CCND1, (E) VEGFA, (F) CXCR4, (G) CXCL12 mRNA expression compared to HK-2 kidney epithelial cells. N = 8. * p < 0.05, ** p < 0.001, **** p < 0.0001.

Figure 3

In vitro binding to patient-derived ccRCC cells and to tumor tissue sections. Confocal microscopy images of patient-derived ccRCC cells that were immunostained for CD70 (green), counterstained with DAPI (blue), and incubated for 30 min with 50 μM FITC-labeled micelles (red). (A) CD27 PAMs have increased binding to ccRCC cells in vitro compared to (B) scrCD27 PAMs. (C) CD27 PAM binding is reduced after pre-incubation with anti-CD70 antibodies, confirming specificity of CD27 PAMs for CD70. (D) CD27 PAMs have significantly increased binding compared to scrCD27 PAMs, but not if target cells are pre-treated with anti-CD70. N = 6. * p < 0.05. (E) ccRCC tumor tissue sections incubated for 1 h with 10 μM CD27 micelles have increased nanoparticle signal compared to (F) scrCD27 micelles. Scale bar = 50 μm. (G) Representative H&E-stained ccRCC tumor tissue section. Scale bar = 100 μm.

Figure 4

mRNA expression of HIF2A (left) and CD70 (right) following 48 h treatment with HIF2α-CD27 PAMs. The 48 h HIF2α-CD27 PAM treatment (500 nM siRNA) significantly reduced HIF2A and CD70 mRNA expression compared to non-targeted PAM and free siRNA controls in patient-derived ccRCC cells. N = 5. * p < 0.05 relative to PBS. ^# p < 0.05 relative to HIF2α-CD27 PAMs. ^& p < 0.05 relative to HIF2α-scrCD27 PAMs.

Figure 5

Gene knockdown and functional effects of HIF2α-CD27 PAM treatment. 48 h HIF2α-CD27 PAM treatment (500 nM siRNA) reduces (A) SLC2A1 mRNA expression and (B) glucose transport relative to non-targeted PAMs, free siRNA, and PBS in patient-derived ccRCC cells. HIF2α-CD27 PAMs also reduce (C) CCND1 mRNA expression and (D) ccRCC proliferation 5 d following treatment. N = 4–6. * p < 0.05 relative to PBS. ^# p < 0.05 relative to HIF2α-CD27 PAMs. ^& p < 0.05 relative to HIF2α-scrCD27 PAMs.

Figure 6

48 h HIF2α-CD27 PAM treatment (500 nM siRNA) reduces (A) VEGFA mRNA expression in patient-derived ccRCC. (B) HUVECs cultured with HIF2α-CD27 PAM-conditioned culture medium have reduced proliferation relative to PBS-conditioned culture medium. N = 5. * p < 0.05 relative to PBS. ^# p < 0.05 relative to HIF2α-CD27 PAMs. ^& p < 0.05 relative to HIF2α-scrCD27 PAMs.

Figure 7

Effect of HIF2α-CD27 PAMs on ccRCC migration. (A) The 48 h HIF2α-CD27 PAM treatment (500 nM siRNA) slows wound closure of ccRCC cells over 24 h compared to PBS and free siRNA treatment. (B) The 48 h HIF2α-CD27 PAM treatment (500 nM) reduces mRNA expression of CXCR4 (left) and CXCL12 (right). N = 4 or 5. * p < 0.05 relative to PBS. ^# p < 0.05 relative to HIF2α-CD27 PAMs.