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. 2015 Oct;14(10):2313-20.
doi: 10.1158/1535-7163.MCT-14-1071. Epub 2015 Jul 30.

miR-124 Regulates the Epithelial-Restricted with Serine Box/Epidermal Growth Factor Receptor Signaling Axis in Head and Neck Squamous Cell Carcinoma

Manchao Zhang  1 , Longzhu Piao  1 , Jharna Datta  1 , James C Lang  1 , Xiujie Xie  1 , Theodoros N Teknos  1 , Anna K Mapp  2 , Quintin Pan  3
Affiliations

miR-124 Regulates the Epithelial-Restricted with Serine Box/Epidermal Growth Factor Receptor Signaling Axis in Head and Neck Squamous Cell Carcinoma

Manchao Zhang et al. Mol Cancer Ther. 2015 Oct.

Abstract

Epithelial-restricted with serine box (ESX), a member of the ETS transcription factor family, is elevated and regulates EGFR in head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanisms that contribute to ESX dysregulation remain to be elucidated. In this study, in silico analysis of the 3'-untranslated region (UTR) of ESX predicted two miR-124-binding sites. Delivery of miR-124 inhibited the 3'UTR ESX-driven reporter activity by 50% (P < 0.05) confirming ESX as a direct target of miR-124. Loss of miR-124 was found to be a frequent event in HNSCC. miR-124 expression was significantly depleted in the primary tumor compared with matched normal tissue in 100% (12/12) of HNSCC patients; relative mean miR-124 expression of 0.01197 and 0.00118 (P < 0.001, n = 12) in matched normal adjacent tissue and primary HNSCC tumor, respectively. Overexpression of miR-124 decreased ESX and EGFR levels in miR-124(low)/ESX(high)/EGFR(high) SCC15 HNSCC cells and reduced cell invasion, migration, proliferation, and colony formation. SCC15 cells with miR-124 restoration were less tumorigenic in vivo than miR-control SCC15 cells (70% inhibition, P < 0.01). Restoration of miR-124 in SCC15 cells enhanced the antiproliferative efficacy of the EGFR/Her2 tyrosine kinase inhibitors. Furthermore, recapitulation of EGFR in miR-124-overexpressing SCC15 cells was sufficient to completely block the antiproliferative effects of lapatinib and afatinib. Taken together, our work provides intriguing evidence that miR-124 is a novel therapeutic approach to reduce ESX/EGFR, and may be a tractable strategy to enhance the response rate of HNSCC patients to current anti-EGFR/Her2 therapies.

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Conflict of interest statement

Authors declare no competing financial interests in relation to the work described.

Figures

Figure 1
Figure 1. miR-124 is depleted and regulates ESX in HNSCC
(a) Two putative miR-124 binding sites in the 3′-UTR of ESX. Wildtype miR-124 binding sites are highlighted in red and mutant miR-124 binding sites are highlighted in blue. (b) miR-124 directly regulates ESX. HEK293T cells were co-transfected with wildtype or mutant ESX-3′UTR and pre-miR-control or pre-miR-124. ESX-3′UTR Mut 1 contains mutations located at the miR-124 binding site 1. ESX-3′UTR Mut 2 contains mutations located at the miR-124 binding site 2. ESX-3′UTR Mut1, 2 contains mutations located at the miR-124 binding sites 1 and 2. Renilla and Firefly luciferase activities were measured using the Dual-Luciferase Reporter assay system. Renilla luciferase is normalized to Firefly luciferase and data are presented as mean ± SEM. *p<0.01, n=3. (c and d) miR-124 is depleted in HNSCC cell lines and primary HNSCC tumors. Total RNA was extracted from established cell lines and primary HNSCC tumors and paired adjacent normal epithelium from HNSCC patients. miR-124 expression was assessed by qPCR using a validated TaqMan primer set. Data are presented as mean ± SEM. (e) Restoration of miR-124 reduces ESX levels in HNSCC. Stable polyclonal SCC15/miR-control and SCC15/miR-124 cells were generated by antibiotic selection. Total RNA was extracted and measured for miR-124 expression using qPCR. Data is presented and mean ± SEM. **p<0.01, n=3. Whole cell lysates were extracted and immunoblot analyses were performed for ESX, EGFR and Her2.
Figure 2
Figure 2. Restoration of miR-124 promotes a global anti-tumor effect in HNSCC in vitro
(a) Cell proliferation. Cell proliferation was measured using the CCK-8 reagent to detect metabolic active cells. Data are from three independent experiments and presented as mean ± SEM. *p<0.01, n=3. (b) Colony formation. Colonies were stained with crystal violet and counted. Data are from three independent experiments and presented as mean ± SEM. *p<0.05, n=3. (c) Cell invasion. Cell invasion was assessed using the Modified Boyden chamber invasion assay with Matrigel basement membrane. Invasive cells were counted, normalized to SCC15/miR-control and presented as mean ± SEM from three independent experiments. *p<0.01, n=3. (d) Cell migration. Cell migration was determined using the wound healing assay. % of filled area is calculated, normalized to SCC15/miR-control and presented as mean ± SEM from three independent experiments. *p<0.05, n=3. (e) Cell adhesion. Cells were plated on fibronectin-coated plates and subsequently washed with PBS. The remaining attached cells were stained with 0.5% crystal violet and counted. Data are from three independent experiments and presented as mean ± SEM. *p<0.01, n=3.
Figure 3
Figure 3. Restoration of miR-124 suppresses the tumorigenicity of HNSCC in vivo
(a) Tumor growth. SCC15/miR-control or SCC15/miR-124 cells were injected subcutaneously to the flanks of the nude mice. Tumors were measured using a caliper and tumor volumes were calculated. Data is presented as mean ± SEM. *p<0.01, n=7. (b) Intratumoral miR-124 expression. SCC15/miR-control and SCC15/miR-124 tumors were resected and total RNA was isolated. miR-124 expression was measured using qPCR. Data is presented as mean ± SEM. *p<0.01. (c) Intratumoral ESX, EGFR and Her2 levels. ESX, EGFR and Her2 levels was assessed by standard immunohistochemistry. A representative image is shown for the SCC15/miR-control and SCC15/miR-124 tumors.
Figure 4
Figure 4. miR-124 modulates the ESX/EGFR axis to potentiate the anti-tumor efficacy of EGFR/Her2 TKIs
(a) Restoration of miR-124 potentiates the activity of afatinib and lapatinib. SCC15/miR-control and SCC15/miR-124 cells were treated with lapatinib or afatinib at the IC50 dose. Colonies were stained with crystal violet. Data is normalized to vehicle-treated SCC15/miR-control cells and presented as mean ± SEM. *p<0.01, SCC15/miR-control vs. SCC15/miR-124 for vehicle, lapatinib, or afatinib, n=3. **p<0.01 SCC15/miR-124/vehicle, SCC15/miR-control/lapatinib or SCC15/miR-control/afatinib vs. SCC15/miR-124/lapatinib or SCC15/miR-124/afatinib, n=3. (b) Ectopic over-expression of EGFR in SCC15/miR-124 cells. Stable polyclonal SCC15/miR-124/vector and SCC15/miR-124/EGFR cells were generated by antibiotic selection. Whole cell lysates were extracted and immunoblot analyses were performed for EGFR, STAT3 and pSTAT3. (c) Recapitulation of EGFR in SCC15/miR-124 cells promotes resistance to EGFR/Her2 TKIs. SCC15/miR-control, SCC15/miR-124/vector and SCC15/miR-124/EGFR cells were treated with lapatinib or afatinib at the IC50 dose. Colonies were stained with crystal violet. Data is normalized to vehicle-treated SCC15/miR-control cells and presented as mean ± SEM. *p<0.01, SCC15/miR-124/vector vs. SCC15/miR-124/EGFR for vehicle, lapatinib, or afatinib, n=3.
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