Post-Transcriptional Regulation of the GASC1 Oncogene with Active Tumor-Targeted siRNA-Nanoparticles

Abstract

Basal-like breast cancer (BLBC) accounts for the most aggressive types of breast cancer, marked by high rates of relapse and poor prognoses and with no effective clinical therapy yet. Therefore, investigation of new targets and treatment strategies is more than necessary. Here, we identified a receptor that can be targeted in BLBC for efficient and specific siRNA mediated gene knockdown of therapeutically relevant genes such as the histone demethylase GASC1, which is involved in multiple signaling pathways leading to tumorigenesis. Breast cancer and healthy breast cell lines were compared regarding transferrin receptor (TfR) expression via flow cytometry and transferrin binding assays. Nanobioconjugates made of low molecular weight polyethylenimine (LMW-PEI) and transferrin (Tf) were synthesized to contain a bioreducible disulfide bond. siRNA complexation was characterized by condensation assays and dynamic light scattering. Cytotoxicity, transfection efficiency, and the targeting specificity of the conjugates were investigated in TfR positive and negative healthy breast and breast cancer cell lines by flow cytometry, confocal microscopy, RT-PCR, and Western blot. Breast cancer cell lines revealed a significantly higher TfR expression than healthy breast cells. The conjugates efficiently condensed siRNA into particles with 45 nm size at low polymer concentrations, showed no apparent toxicity on different breast cancer cell lines, and had significantly greater transfection and gene knockdown activity on mRNA and protein levels than PEI/siRNA leading to targeted and therapeutic growth inhibition post GASC1 knockdown. The synthesized nanobioconjugates improved the efficiency of gene transfer and targeting specificity in transferrin receptor positive cells but not in cells with basal receptor expression. Therefore, these materials in combination with our newly identified siRNA sequences are promising candidates for therapeutic targeting of hard-to-treat BLBC and are currently further investigated regarding in vivo targeting efficacy and biocompatibility.

Keywords: GASC1; artificial virus; breast cancer; nanoparticle; oncogene; polyethylenimine (PEI); siRNA; targeted therapy; targeting; transferrin; transferrin receptor.

Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1

Screening of TfR expression levels in different cancer cell lines compared to MCF 10A cells. Using flow cytometry, TfR overexpression on the breast cancer cell line HCC1954 was verified, which also shows a comparably high GASC1 amplification. The transferrin receptor expression (CD71, TfR) was compared to the MCF-10A cell line, an immortalized, nontransformed epithelial cell line, showing no GASC1 amplification. The expression was verified by using a PE-labeled anti-CD71 antibody, and nonspecific binding of the antibody was precluded based on results with an isotype control. Data are expressed as the mean ± SD (n = 3); ns, nonsignificant; ****p < 0.0001.

Figure 2

Comparison of FITC–transferrin or FITC–albumin internalization in HCC1954 (lower left bar graph) and MCF10A (lower right bar graph) cell lines at different concentrations of transferrin and albumin via flow cytometry. Data are expressed as the mean ± SD (n = 3); ns, nonsignificant; ****p < 0.0001.

Figure 3

Schematic illustration of transferrin cross-linking with PEI by SPDP.

Figure 4

Hydrodynamic diameter (nm) and zeta potential (mV) of siRNA polyplexes made with PEI, Tf–PEI, and Tf-PEG-PEI at N/P ratio of 7 at room temperature. Data are expressed as the mean ± SD (n = 3).

Figure 5

AFM topography of siRNA polyplexes made with PEI (left) and Tf–PEI (right) at N/P ratio of 7 and imaged after drying at room temperature.

Figure 6

Condensation efficiency of Tf–PEI and Tf-PEG-PEI complexes with siRNA measured by SYBR Gold assay at different N/P ratios (1–20) compared to polyethylenimine (PEI 5 kDa). Results are given as the average of three measurements ± SD (n = 3).

Figure 7

Release profile of siRNA from transferrin modified polyplexes (N/P 7; 50 pmol of siRNA/well) compared to polyethylenimine (PEI 5 kDa) as a function of time (0–185 min) with heparin concentration of 0.2 IU per well. Results are shown as relative mean fluorescence ± SD (n = 3).

Figure 8

Comparison of siRNA uptake with different formulations (Blank (untreated cells), PEI (positive control), Tf–PEI, Tf-PEG-PEI) in GACS1 positive (HCC1954 and COLO824) and GASC1 negative (MCF10A and SUM102) cell line models after fluorescence quenching of membrane bound nanoparticles with trypan blue (asterisks indicate cell samples treated with trypan blue). Data are expressed as the mean ± SD (n = 3); ns, nonsignificant.

Figure 9

Confocal microscopy images of cellular interaction of different complexes with the HCC1954 cell line after 4 h incubation with siRNA polyplexes. HCC1954 cells were seeded with 25,000 cells per chamber and transfected with 25 pmol of siRNA (TYE 563 labeled, indicated by red color) for 4 h. Cells were fixed and the nuclei stained with DAPI (indicated by blue color). Representative pictures are shown (100× magnification): (A) blank; siRNA polyplexes with (B) Lipofectamine; (C) Tf–PEI (N/P 7); and (D) PEI (N/P 7).

Figure 10

Cytotoxicity profile of siRNA polyplexes made with Tf–PEI or Tf-PEG-PEI measured by the CellTiter-Blue method compared to PEI/siRNA polyplexes. HCC1954, COLO824, SUM102, and MCF10A cell lines were treated with polyplexes for 4 h, and the viability of cells was measured after 24 h. Cell toxicity is presented as impaired cell viability as a percentage relative to control cells. Data are expressed as the mean ± SD (n = 5). *P < 0.05.

Figure 11

GASC1 gene knockdown optimization with different siRNA sequences (A–D sequences from Qiagen, and siRNA sequences E–H and mixed from Dharmacon). Per well, 500,000 HCC1954 cells were transfected with Lipofectamine 2000 (LF) for 24 h with 100 pmol of different siRNA sequences against GASC1. RT-PCR was performed with specific GASC1 primers; normalization was carried out against the housekeeping gene PUM1. NC = scrambled siRNA; error bars reflect SD, n = 3. *P < 0.05 and **P < 0.01.

Figure 12

GASC1 gene knockdown optimization with siRNA sequence A in HCC1954 cells at different time points (A), or different siRNA concentration 24 h post transfection (B). Lipofectamine 2000 (LF) was used as transfection agent, and RT-PCR was performed with specific GASC1 primers; normalization was carried out against the housekeeping gene PUM1. NC = scrambled siRNA; error bars reflect SD, n = 3; ns, nonsignificant; *P < 0.05, ***P < 0.001, and ****p < 0.0001.

Figure 13

(A) GASC1 knockdown of Tf–PEI with siRNA sequence V7 shows that the conjugate was able to knock down mRNA levels of GASC1 more efficiently than the commercially available siRNA sequences in HCC1954 cells. V4, V6, and V7 are self-designed GASC1 siRNA sequences. NC = scrambled siRNA; PC = positive control siRNA (hGAPDH), error bars reflect SD, n = 3; ns, nonsignificant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****p < 0.0001. (B) GASC1 protein knockdown in HCC1954 cells with Tf–PEI compared to PEI alone was confirmed by Western blot. Alpha-tubulin was used as the loading control. Top row: A, PEI/V7GASC1 siRNA; B, PEI/NC siRNA; C, Tf–PEI/V7GASC1; D, Tf–PEI/NC siRNA.

Figure 14

Therapeutic effects of GASC1 knockdown with Tf–PEI polyplexes on days 2 (D2) and 10 (D10) after seeding measured as inhibition of proliferation of HCC1954 cells. V7 is a self-designed GASC1 siRNA sequence; NC = scrambled siRNA; error bars reflect SD, n = 3.