Coordinated targeting of the EGFR signaling axis by microRNA-27a*

Abstract

Epidermal growth factor receptor (EGFR) has been characterized as a critical factor in the development and progression of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). However, monotherapy with EGFR-specific agents has not been as dramatic as preclinical studies have suggested. Since complex regulation of the EGFR signaling axis might confound current attempts to inhibit EGFR directly, we searched for microRNAs (miRNAs) that may target the EGFR signaling axis. We identified miR-27a (miR-27a-3p) and its complementary or star (*) strand, miR-27a* (miR-27a-5p), as novel miRNAs targeting EGFR, which were significantly downregulated in multiple HNSCC cell lines. Analysis of human specimens demonstrated that miR-27a* is significantly underexpressed in HNSCC as compared to normal mucosa. Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a. Analysis for potential targets of miR-27a* led to the identification of AKT1 (protein kinase B) and mTOR (mammalian target of rapamycin) within the EGFR signaling axis. Treatment with miR-27a* led to coordinated downregulation of EGFR, AKT1 and mTOR. Overexpression of EGFR signaling pathway components decreased the overall effect of miR-27a* on HNSCC cell viability. Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth. Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo. These findings identify miR-27a* as a functional star sequence that exhibits novel coordinated regulation of the EGFR pathway in solid tumors and potentially represents a novel therapeutic option.

Figure 1. miR-27a* has putative binding sites in EGFR mRNA and shows decreased expression in HNSCC cell lines and human tumor tissues

(A) Identification of specific miR-27a(), −27a*(), −27b(), −27b*(#), −7(), −128() candidate binding sites within EGFR mRNA using in silico screening methods; (B) Hairpin representation of the pre-miR-27a with the sequences of miR-27a* (green) and miR-27a (magenta) highlighted; (C) Decreased expression of mature miR-27a and −27a* by qRT-PCR in 10 HNSCC cell lines and normal oral keratinocytes (OKF-6 and HOK16B). Values normalized to OKF-6, p<0.005; (D) Analysis of miR-27a* RNA in human HNSCC and normal mucosal specimens by qRT-PCR revealed an overall decrease in miR-27a* expression levels in HNSCC, p<0.0001; (E) Comparison of miR-27a* levels in matched normal/HNSCC tissue pairs demonstrated decreased expression in the tumors as compared to matched normal tissue, p<0.01.

Figure 2. miR-27a* transfection decreases cell viability in HNSCC cells and other solid tumor types

(A) Cell viability is decreased at in three HNSCC cell lines after transfection of miR-27a* compared to miR-27a, −7, and -Control, *p<0.001; (B) Photomicrograph details cell number 72 hrs after transfection of miR-27a* mimic into wells containing 20,000 cells each; (C) Cell viability is decreased in other solid tumor cell lines (PC3 prostate, HEC1A endometrial, MIA PaCa pancreas, and MDA-468 breast) after transfection of miR-27a* compared to miR-27a, −7, and -Control, *p<0.001.

Figure 3. miR-27a* coordinately downregulates the EGFR signaling axis via independent direct interactions with EGFR, AKT1, and mTOR

(A) Further in silico screening of downstream members of the EGFR signaling axis, identified 5 putative binding sites for miR-27a* (black bars) on AKT1 and 11 binding sites for miR-27a* on mTOR; (B) Immunoblot shows decreased EGFR expression after the transfection of miR-27a*, −27a and −7 precursors. Downstream AKT1 and mTOR are also decreased when transfected with miR-27a*, but not miR-27a or −7; (C) Densitometry analysis quantifies the differences observed in the immunoblots, *p<0.01; (D) Detailed map of reporter plasmid constructs depicting the EGFR sequence placed downstream of luciferase in pGL3; (E) Luciferase assay after transfection of EGFR reporter plasmids and miR-27a* in HNSCC cells demonstrates E2145-pGL3 and E3908-pGL3 have functional binding sites for miR-27a* and that E160-pGL3 is a non-functional site, *p<0.05 and **p<0.005; (F) Luciferase assay after transfection of AKT1 and mTOR reporter plasmids plus miR-27a* in HNSCC cells demonstrates the 3'UTR of both signaling mediators have functional binding sites for miR-27a*, *p<0.001.

Figure 4. Overexpression of EGFR axis signaling components reverses the loss of HNSCC cell viability mediated by miR-27a*, which increases apoptosis and reduces migration

(A) The effect of miR-27a* on cell viability is decreased in 22A and 22B cells by overexpression of EGFR, AKT1 and mTOR 48 hrs prior to miR-27a* expression as compared to control vector, *p<0.01; (B) Overexpression following transfection with EGFR, AKT1 and mTOR (EAmT) vectors was confirmed by immunoblot analysis. (C) Annexin V assay shows increased apoptosis in HNSCC cells after transfection with miR-27a*, *p<0.005; (D) Transfection with miR-27a* leads to increased PARP cleavage compared to miR-27a and -Control; (E) Wound healing assay demonstrates decreased migration distance after 24 hrs after transfection of miR-27a* compared to miR-Control, −27a, and −7, *p<0.001.

Figure 5. miR-27a* expression reduces tumor growth in vivo and direct intratumoral injection reduces tumor growth

(A) Orthotopic xenografts of 22B cells expressing miR-27a* (pSuper-27a*) show reduced growth compared to control vector (pSuper); * at day 16 indicates point at which differences in tumor volume became statistically significant, p<0.05; (B) Tumor growth curve of 22B cells with inducible miR-27a* expression. One group of mice was treated with doxycycline (Doxy) to induce miR-27a* expression after tumors developed (day 0). Tumor volumes were significantly decreased in mice who received doxycycline; * marks day 18 when differences in tumor volumes became statistically significant, p<0.05; (C) Scatter plot depicting a statistically significant increase in miR-27a* RNA collected from murine tumor tissue in the doxycycline group compared to the non-doxycycline group, p<0.01; (D) Direct intratumoral injection significantly reduces growth of 17B tumors in mice treated with pSuper-27a* compared to control and pSuper; * marks day 18 when differences in tumor volumes became statistically significant (p<0.05) for pSuper-27a* compared to the control groups.