Efficient Epidermal Growth Factor Receptor Targeting Oligonucleotide as a Potential Molecule for Targeted Cancer Therapy

Abstract

Epidermal growth factor receptor (EGFR) is associated with the progression of a wide range of cancers including breast, glioma, lung, and liver cancer. The observation that EGFR inhibition can limit the growth of EGFR positive cancers has led to the development of various EGFR inhibitors including monoclonal antibodies and small-molecule inhibitors. However, the reported toxicity and drug resistance greatly compromised the clinical outcome of such inhibitors. As a type of chemical antibodies, nucleic acid aptamer provides an opportunity to overcome the obstacles faced by current EGFR inhibitors. In this study, we have developed and investigated the therapeutic potential of a 27mer aptamer CL-4RNV616 containing 2'-O-Methyl RNA and DNA nucleotides. Our results showed that CL-4RNV616 not only displayed enhanced stability in human serum, but also effectively recognized and inhibited the proliferation of EGFR positive Huh-7 liver cancer, MDA-MB-231 breast cancer, and U87MG glioblastoma cells, with an IC50 value of 258.9 nM, 413.7 nM, and 567.9 nM, respectively. Furthermore, TUNEL apoptosis assay revealed that CL-4RNV616 efficiently induced apoptosis of cancer cells. In addition, clinical breast cancer biopsy-based immunostaining assay demonstrated that CL-4RNV616 had a comparable detection efficacy for EGFR positive breast cancer with commonly used commercial antibodies. Based on the results, we firmly believe that CL-4RNV616 could be useful in the development of targeted cancer therapeutics and diagnostics.

Keywords: Chemically-modified oligonucleotides; aptamers; nucleic acids.

Figure 1

Relative binding capacity of different modified EGFR aptamer sequences and their specificity analysis using a negative target of the lipoprotein receptor protein (LDL-R) protein. EGFR protein was incubated with 100 nM of biotin labelled aptamers CL-4RNV615 to CL-4RNV618 for 1 h, and the binding capacity was analysed by ELONA assay. * p < 0.05.

Figure 2

Determination of binding affinity of CL-4 and CL-4RNV616 aptamers to EGFR protein. Equilibrium dissociation constants (K_d) was determined by incubating EGFR protein at varying concentrations of aptamer (0–200 nM) using LDL-R protein as a negative control. K_d was derived using the GraphPad Prism program 8.0.

Figure 3

Evaluation of the stability of aptamers in 90% human serum. After incubation, samples were separated on a 20% Urea-PAGE denaturing gel, with a 30nt RNA sequence as the loading control.

Figure 4

Determination of specific binding of CL-4RNV616 aptamer to EGFR positive cells via FACS assay. (A) Quantification of the binding of CL-4RNV616 aptamer to EGFR-positive cell lines and to the EGFR-negative HEK-293 cells via flow cytometric analysis. FAM labelled CL-4RNV616 aptamer was incubated at a concentration of 200 nM with EGFR positive or negative cell lines for 30 min at 37 °C. (B) Determination of equilibrium dissociation constants (K_d) of CL-4RNV616 aptamer to EGFR positive cells via flow cytometry by incubating cells at varying concentrations of aptamer (0–500 nM) using an EGFR negative HEK293 cell line as a negative control. K_d was derived using the GraphPad Prism program 8.0.

Figure 5

Specific binding of CL-4RNV616 aptamer to EGFR-positive cells. CL-4RNV616 aptamer specifically binds to EGFR-positive Huh-7, U87MG, and MDA-MB-231 cells but not to EGFR-negative HEK293 cells. Cells were imaged via fluorescent microscopy after incubation with 200 nM of aptamer at 37 °C for 30 min. Green, FAM (aptamer), and blue, Hoechst 33342 (nuclei).

Figure 6

Evaluation of cytotoxicity and IC50 of CL-4RNV616 using EGFR positive Huh-7, MDA-MB-231, and U87MG cells.

Figure 7

Evaluation of apoptotic induction effect of CL-4RNV616 aptamer on EGFR positive cancer cells. Representative micrograph of TUNEL assays on indicated cells after 72 h of treatment (at 500 nM). Blue, Hoechst 33342 (nuclei) and red, Rhodamine for apopTag-positive nuclei. The relative quantification was determined via the Image J program. * p < 0.05.

Figure 8

Tissue immunostaining of both EGFR positive and negative breast cancer by CL-4RNV616 aptamer and EGFR antibody. H&E stain was conducted for morphological confirmation.