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Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers.

Thiel KW, Hernandez LI, Dassie JP, Thiel WH, Liu X, Stockdale KR, Rothman AM, Hernandez FJ, McNamara JO, Giangrande PH - Nucleic Acids Res. (2012)

Bottom Line: RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2.We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression.The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2(+)-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating 'cell-type specific', 'cell-internalizing RNA ligands (aptamers)' capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2(+)-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

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Chimera-mediated death of HER2+-mammary carcinoma cells. (A) Predicted secondary structures of the HER2 aptamers-Bcl-2 siRNA chimeras using RNAStructure. Red nucleotides indicate the Bcl-2 siRNA guide strand sequence. Chimeras were generated by annealing the Bcl-2 guide strand to the complementary passenger strand sequence covalently linked to the 3′-end of each aptamer. (B) Cell-type specific internalization of the aptamer-siRNA chimeras was compared to that of the aptamers alone and analyzed by qRT-PCR as in Figure 1C. (C) Internalization of chimeras in N202.1A(HER2+) cells versus normal mouse mammary carcinoma cells (NMuMG) (B). (D) Silencing of Bcl-2 at the mRNA level was determined by qRT-PCR after incubation of N202.1A(HER2+) cells with chimeras for 38 h (top panel) or 96 h (bottom panel). Bcl-2 mRNA levels were normalized to GAPDH mRNA levels for each sample. (E) 5′-Rapid amplification of cDNA ends (5′-RACE) PCR analysis to assess siRNA mediated cleavage of Bcl2 mRNA in cells treated with the various HER2-Bcl2 chimeras (A1-Bcl2, B1-Bcl2, C3-Bcl2, D1-Bcl2, E1-Bcl2). A non-internalizing chimera (SCR1-Bcl2) was used as a control in these assays. (F) N202.1A(HER2+) cells were treated with either aptamer-Bcl-2 siRNA chimeras (Bcl2-chimeras; top panels) or aptamer-control siRNA chimeras (Con-chimeras; bottom panels) for 72 h, then with media containing chimeras (solid gray) and low-dose cisplatin (20 µM) (red line) for an additional 24 h. Following cisplatin treatment, cells were stained with an antibody to active cleaved caspase-3 and processed by flow cytometry to assess % apoptosis.
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gks294-F5: Chimera-mediated death of HER2+-mammary carcinoma cells. (A) Predicted secondary structures of the HER2 aptamers-Bcl-2 siRNA chimeras using RNAStructure. Red nucleotides indicate the Bcl-2 siRNA guide strand sequence. Chimeras were generated by annealing the Bcl-2 guide strand to the complementary passenger strand sequence covalently linked to the 3′-end of each aptamer. (B) Cell-type specific internalization of the aptamer-siRNA chimeras was compared to that of the aptamers alone and analyzed by qRT-PCR as in Figure 1C. (C) Internalization of chimeras in N202.1A(HER2+) cells versus normal mouse mammary carcinoma cells (NMuMG) (B). (D) Silencing of Bcl-2 at the mRNA level was determined by qRT-PCR after incubation of N202.1A(HER2+) cells with chimeras for 38 h (top panel) or 96 h (bottom panel). Bcl-2 mRNA levels were normalized to GAPDH mRNA levels for each sample. (E) 5′-Rapid amplification of cDNA ends (5′-RACE) PCR analysis to assess siRNA mediated cleavage of Bcl2 mRNA in cells treated with the various HER2-Bcl2 chimeras (A1-Bcl2, B1-Bcl2, C3-Bcl2, D1-Bcl2, E1-Bcl2). A non-internalizing chimera (SCR1-Bcl2) was used as a control in these assays. (F) N202.1A(HER2+) cells were treated with either aptamer-Bcl-2 siRNA chimeras (Bcl2-chimeras; top panels) or aptamer-control siRNA chimeras (Con-chimeras; bottom panels) for 72 h, then with media containing chimeras (solid gray) and low-dose cisplatin (20 µM) (red line) for an additional 24 h. Following cisplatin treatment, cells were stained with an antibody to active cleaved caspase-3 and processed by flow cytometry to assess % apoptosis.

Mentions: To rule out the possibility that the RNA aptamers are remaining on the cell surface following the stringent salt wash step, we performed fluorescence microscopy (Figure 2F). In this assay, chemically synthesized C1 and E1 RNA aptamers were conjugated to FAM and incubated with either HER2+- (N202.1A) or HER2−- (N202.1E) cells for 1 h prior to imaging. Cells were washed with the stringent salt wash and remaining RNAs visualized by fluorescence microscopy. As shown in Figure 2F, aptamers C1 and E1 internalize specifically into HER2+-cells but not HER2−-cells. We observe perinuclear and cytoplasmic punctuated fluorescence. Furthermore, a negative control RNA aptamer (SCR) does not internalize under these conditions. These data are in support of the Bcl2 silencing data (Figure 5D and E) and confirm that the selected RNA aptamers internalize into HER2-expressing cells.


Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers.

Thiel KW, Hernandez LI, Dassie JP, Thiel WH, Liu X, Stockdale KR, Rothman AM, Hernandez FJ, McNamara JO, Giangrande PH - Nucleic Acids Res. (2012)

Chimera-mediated death of HER2+-mammary carcinoma cells. (A) Predicted secondary structures of the HER2 aptamers-Bcl-2 siRNA chimeras using RNAStructure. Red nucleotides indicate the Bcl-2 siRNA guide strand sequence. Chimeras were generated by annealing the Bcl-2 guide strand to the complementary passenger strand sequence covalently linked to the 3′-end of each aptamer. (B) Cell-type specific internalization of the aptamer-siRNA chimeras was compared to that of the aptamers alone and analyzed by qRT-PCR as in Figure 1C. (C) Internalization of chimeras in N202.1A(HER2+) cells versus normal mouse mammary carcinoma cells (NMuMG) (B). (D) Silencing of Bcl-2 at the mRNA level was determined by qRT-PCR after incubation of N202.1A(HER2+) cells with chimeras for 38 h (top panel) or 96 h (bottom panel). Bcl-2 mRNA levels were normalized to GAPDH mRNA levels for each sample. (E) 5′-Rapid amplification of cDNA ends (5′-RACE) PCR analysis to assess siRNA mediated cleavage of Bcl2 mRNA in cells treated with the various HER2-Bcl2 chimeras (A1-Bcl2, B1-Bcl2, C3-Bcl2, D1-Bcl2, E1-Bcl2). A non-internalizing chimera (SCR1-Bcl2) was used as a control in these assays. (F) N202.1A(HER2+) cells were treated with either aptamer-Bcl-2 siRNA chimeras (Bcl2-chimeras; top panels) or aptamer-control siRNA chimeras (Con-chimeras; bottom panels) for 72 h, then with media containing chimeras (solid gray) and low-dose cisplatin (20 µM) (red line) for an additional 24 h. Following cisplatin treatment, cells were stained with an antibody to active cleaved caspase-3 and processed by flow cytometry to assess % apoptosis.
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gks294-F5: Chimera-mediated death of HER2+-mammary carcinoma cells. (A) Predicted secondary structures of the HER2 aptamers-Bcl-2 siRNA chimeras using RNAStructure. Red nucleotides indicate the Bcl-2 siRNA guide strand sequence. Chimeras were generated by annealing the Bcl-2 guide strand to the complementary passenger strand sequence covalently linked to the 3′-end of each aptamer. (B) Cell-type specific internalization of the aptamer-siRNA chimeras was compared to that of the aptamers alone and analyzed by qRT-PCR as in Figure 1C. (C) Internalization of chimeras in N202.1A(HER2+) cells versus normal mouse mammary carcinoma cells (NMuMG) (B). (D) Silencing of Bcl-2 at the mRNA level was determined by qRT-PCR after incubation of N202.1A(HER2+) cells with chimeras for 38 h (top panel) or 96 h (bottom panel). Bcl-2 mRNA levels were normalized to GAPDH mRNA levels for each sample. (E) 5′-Rapid amplification of cDNA ends (5′-RACE) PCR analysis to assess siRNA mediated cleavage of Bcl2 mRNA in cells treated with the various HER2-Bcl2 chimeras (A1-Bcl2, B1-Bcl2, C3-Bcl2, D1-Bcl2, E1-Bcl2). A non-internalizing chimera (SCR1-Bcl2) was used as a control in these assays. (F) N202.1A(HER2+) cells were treated with either aptamer-Bcl-2 siRNA chimeras (Bcl2-chimeras; top panels) or aptamer-control siRNA chimeras (Con-chimeras; bottom panels) for 72 h, then with media containing chimeras (solid gray) and low-dose cisplatin (20 µM) (red line) for an additional 24 h. Following cisplatin treatment, cells were stained with an antibody to active cleaved caspase-3 and processed by flow cytometry to assess % apoptosis.
Mentions: To rule out the possibility that the RNA aptamers are remaining on the cell surface following the stringent salt wash step, we performed fluorescence microscopy (Figure 2F). In this assay, chemically synthesized C1 and E1 RNA aptamers were conjugated to FAM and incubated with either HER2+- (N202.1A) or HER2−- (N202.1E) cells for 1 h prior to imaging. Cells were washed with the stringent salt wash and remaining RNAs visualized by fluorescence microscopy. As shown in Figure 2F, aptamers C1 and E1 internalize specifically into HER2+-cells but not HER2−-cells. We observe perinuclear and cytoplasmic punctuated fluorescence. Furthermore, a negative control RNA aptamer (SCR) does not internalize under these conditions. These data are in support of the Bcl2 silencing data (Figure 5D and E) and confirm that the selected RNA aptamers internalize into HER2-expressing cells.

Bottom Line: RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2.We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression.The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2(+)-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating 'cell-type specific', 'cell-internalizing RNA ligands (aptamers)' capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2(+)-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

Show MeSH
Related in: MedlinePlus