Synergistic Targeting HER2 and EGFR with Bivalent Aptamer-siRNA Chimera Efficiently Inhibits HER2-Positive Tumor Growth

Abstract

HER2 overexpression is identified on 20-30% breast cancer and other cancers at different levels. Although HER2 targeted monoclonal antibody combined with chemical drugs has shown improved outcomes in HER2 expressing patients, drug resistance and toxicity have limited their efficacy. To overcome drug resistance, cotargeting multiple HER receptors was proven to be effective. EGFR/HER2 dimerization can active PI3K/AKT pathway, and resistance to HER2-targeted drugs is associated with upregulation of EGFR. Here, we developed a novel HER2/EGFR targeted nucleic acid therapeutic to address current drug limits. The new therapeutic is constructed by fusing HER2 aptamer-EGFR siRNA sense strand with HER2 aptamer-EGFR siRNA antisense strand into one molecule: a bivalent HER2 aptamer-EGFR siRNA aptamer chimera (HEH). In breast cancer cell lines, HEH can be selectively taken up into HER2 expressing cells and successfully silence EGFR gene and down regulate HER2 expression. In breast cancer xenograft models, HEH is capable of triggering cell apoptosis, decreasing HER2 and EGFR expression, and suppressing tumor growth. The therapeutic efficacy of HEH is superior to HER2 aptamer only, which suggests that HEH has synergistic effect by targeting HER2 and EGFR. This study demonstrated that HEH has great potential as a new HER2 targeted drug to address toxicity and resistance of current drugs and may provide a cure for many HER2 positive cancers.

Keywords: HER2; aptamer; bivalent; siRNA; siRNA delivery; synergistic treatment; targeted therapeutic.

Figure 1

Construction and characterization of HEH. (A) Schematic illustration of HER aptamer-EGFR siRNA-HER2 aptamer chimera (HEH). (B) Detection of HER2 and EGFR expression in breast cancer cell lines by Western blot. (C) Evaluation of the cytotoxicity of H2EH3. (D) Dose-dependent cytotoxicity assay of HEH on different breast cancer cell lines. Data are the mean ± SE from three independent experiments. (E) HEH stability in cell culture medium.

Figure 2

Detection of cell apoptosis and death by flow cytometry. BT474 and SKBR3 cells were treated with HEH, HScH, and HER2 aptamer for 48 h and 72 h, and then cells were stained with Alexa Fluor 488 Annexin V-Propidium Iodide and analyzed by flow cytometry.

Figure 3

Detection of HEH internalization by Z-stack confocal microscopy. Cy5-labeled HEH, muHEH, HER2 aptamer, or EGFR siRNA was added into BT474 cells for 12 h at 37 °C. Lysotracker Green and DAPI were added into cells at the same time as the chimeras. LysoTracker Green was used to show lysosomes and endosomes. DAPI (blue) was used to display nucleus. Confocal laser scanning microscopy with z-stack was performed to show cell binding and internalization.

Figure 4

Evaluation of protein levels by Western blot. (A) Detection of EGFR and Cleaved Caspase 3 in SKBR3 cells after treatment with  HER2 aptamer and HEH for 48 h and 72 h. (B) Detection of EGFR and Cleaved Caspase 3 inBT474 cells after treament  with HEH and HER2 aptamer for 72 h. (C) Detection of HER2 in SKBR3 cells after treatment with HER2 aptamer and HEH for 72 h (D) Detection of HER2 in BT474 cells after treatment with HER2 aptamer and HEH for 72 h  Quantification of protein levels normalized by GPDH using ImageJ. The results are the mean ± SEM from three independent experiments. ∗P < 0.05, ∗∗P < 0.01.

Figure 5

Cell binding specificity and biodistribution of HEH. (A) Evaluation of cell binding specificity by flow cytometry. HER2 positive and HER2 negative breast cancer cell lines were incubated with Cy5 labeled HEH or muHEH for 1 h at 37 °C, and detected with flow cytometry. Black line, cell only; green line, muHEH; light purple, HEH. (B) Biodistribution assay. Athymic female mice were implanted with BT474 cells. After 4 weeks, tumor bearing mice were i.v. injected with Cy5-HEH or Cy5-muHEH. Cy5 fluorescence of whole body was captured at the time points of 0.5 h, 3 h, 12 h, and 24 h using Xenogen IVIS100.

Figure 6

HEH inhibits tumor growth in BT474 breast cancer xenografts. Mice with subcutaneous tumors were i.p. injected with HEH and controls (PBS, HER2 apt) for 4 week. (A) Tumor growth curve. Tumor sizes were measured twice a week with digital calipers (n = 4). (B) Dissected tumors after treatment. (C) Quantitation of dissected tumor size from (B) (n = 4). (D) Body weight were measured and averaged (n = 4). ∗p < 0.05; ∗∗p < 0.005. Data represent the mean ± SEM.

Figure 7

Histology analysis of tumor and detection of biomarkers by immunohistochemistry. Formalin-fixed paraffin-embedded sections of xenograft tumors were analyzed with HE staining for detection of morphologic changes and IHC staining for detection of protein levels of EGFR, HER2, and Cleaved Caspase-3. Scale bar, 50 μm.

Figure 8

Assessment of systemic toxicity of HEH. (A) Histological examination of organ damage after HEH treatment with HE staining. Detection of mouse serum (B) IFNα and (C) IL-6 with ELISA assay. The results are the mean ± SEM (N = 4).