Modification of HER2 pre-mRNA alternative splicing and its effects on breast cancer cells - PubMed (original) (raw)

Modification of HER2 pre-mRNA alternative splicing and its effects on breast cancer cells

Jing Wan et al. Int J Cancer. 2009.

Abstract

The oncogene HER2 is overexpressed in a variety of human tumors, providing a target for anti-cancer molecular therapies. Here, we employed a 2'-O-methoxyethyl (MOE) splice switching oligonucleotide, SSO111, to induce skipping of exon 15 in HER2 pre-mRNA, leading to significant downregulation of full-length HER2 mRNA, and simultaneous upregulation of Delta15HER2 mRNA. SSO111 treatment of SK-BR-3 cells, which highly overexpress HER2, led to inhibition of cell proliferation and induction of apoptosis. The novel Delta15HER2 mRNA encodes a soluble, secreted form of the receptor. Treating SK-BR-3 cells with exogenous Delta15HER2 protein reduced membrane-bound HER2 and decreased HER3 transphosphorylation. Delta15HER2 protein thus has similar activity to an autoinhibitory, natural splice variant of HER2, Herstatin, and to the breast cancer drug Herceptin. Both SSO111 and Delta15HER2 may be potential candidates for the development of novel HER2-targeted cancer therapeutics.

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Conflict of interest statement

Financial disclosure: RK declares a potential conflict of interest as an employee of AVI BioPharma, Inc.

Figures

Figure 1

Figure 1

Skipping of exon 15 in HER2 pre-mRNA. A) SSOs were designed to target the 5’ splice site of exon 15. Skipping of exon 15 induced by SSOs generated a downstream stop codon, and results in a truncated HER2 ECD. Thick bar: SSO. Part of HER2 pre-mRNA (3’ to 5’, with intron underlined), SSO111, and SSO111m (with mismatches underlined and highlighted in bold) were shown in B). B) and C) Results from the analysis of total cellular RNA by RT-PCR are shown: B) Dose-dependence of SK-BR-3 cells treated with SSO111 at designated concentrations for 24 hours; C) Time-course of Sk-BR-3 cells treated with SSO111 for the designated time at 100 nM. The lengths of the PCR products (in base pairs) are indicated. Lower panels in B) and C): quantitative analysis of the results. Gray bars: SSO111 transfected cells; white bars: 111m-transfected cells.

Figure 2

Figure 2

Western blot analysis of HER2 protein from SK-BR-3 cells transfected with SSO111. A) dose dependence (at 48 hours). B) time course (at 100 nM). Top panels: HER2 protein; lower panels: β-actin as a loading control. See “Methods and Materials” for details.

Figure 3

Figure 3

Growth inhibition of SK-BR-3 cells by SSO111 treatment. A) Western blot demonstrating relative HER2 expression in MCF7 and SK-BR-3 cell lines (20 µg protein/lane). B) MTS assay of SK-BR-3 (black bar) and MCF7 (gray bar) cells after 72 hours incubation with 100 nM SSO111. Shown are the mean ± standard deviation of triplicates.

Figure 4

Figure 4

PARP cleavage as a marker for apoptosis after SSO111 treatment. In SK-BR-3 cells: A) dose dependence (at 48 hours); B) time course (at 100 nM). In MCF7 cells: C) time course (at 100 nM). Full-length PARP: 116 kDa; cleavage product: 85 kDa.

Figure 5

Figure 5

Effects of Δ15HER2-His protein on HER2/HER3 receptors. Western blot analysis of (A) SK-BR-3 and (B) MCF7 cell lysates after treatment with purified Δ15HER2-His protein at the designated concentrations. For analysis of phosphorylated Akt (p-Akt) in A) and phosphorylated HER3 (p-HER3) protein in B), cells were stimulated with heregulin (15 min, 20 ng/ml). C) Growth inhibition of SK-BR-3 cells by Δ15HER2-His protein treatment after 72 hours incubation analyzed by MTS assay. Shown are the mean ± standard deviation of triplicates.

Figure 6

Figure 6

Interaction between Δ15HER2-His protein and HER2/HER3. A) SK-BR-3 and B) MCF7 cell lysates were collected 48 hours after transfection with Δ15HER2-His plasmid, the protein was immunoprecipitated with anti-His tag antibody and blotted with anti-HER2 antibody and anti-HER3 antibody, respectively.

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