Regulation of heparin-binding EGF-like growth factor by miR-212 and acquired cetuximab-resistance in head and neck squamous cell carcinoma

Abstract

Background: We hypothesized that chronic inhibition of epidermal growth factor receptor (EGFR) by cetuximab, a monoclonal anti-EGFR antibody, induces up-regulation of its ligands resulting in resistance and that microRNAs (miRs) play an important role in the ligand regulation in head and neck squamous cell carcinoma (HNSCC).

Methodology/principal findings: Genome-wide changes in gene and miR expression were determined in cetuximab-sensitive cell line, SCC1, and its resistant derivative 1Cc8 using DNA microarrays and RT-PCR. The effects of differentially expressed EGFR ligands and miRs were examined by MTS, colony formation, ELISA, and western blot assays. Heparin-binding EGF-like growth factor (HB-EGF) and its regulator, miR-212, were differentially expressed with statistical significance when SCC1 and 1Cc8 were compared for gene and miR expression. Stimulation with HB-EGF induced cetuximab resistance in sensitive cell lines. Inhibition of HB-EGF and the addition of miR-212 mimic induced cetuximab sensitivity in resistant cell lines. MicroRNA-212 and HB-EGF expression were inversely correlated in an additional 33 HNSCC and keratinocyte cell lines. Six tumors and 46 plasma samples from HNSCC patients were examined for HB-EGF levels. HB-EGF plasma levels were lower in newly diagnosed HNSCC patients when compared to patients with recurrent disease.

Conclusions/significance: Increased expression of HB-EGF due to down-regulation of miR-212 is a possible mechanism of cetuximab resistance. The combination of EGFR ligand inhibitors or miR modulators with cetuximab may improve the clinical outcome of cetuximab therapy in HNSCC.

Figure 1. Determination of cetuximab sensitivity.

Cetuximab response determination of SCC1 and 1Cc8 HNSCC cell lines by; A) MTS assay, B) colony formation assay, C) graphical presentation of the colony formation assay, A. U.- Arbitrary Unit, and D) growth inhibition in mouse xenografts. CTX-cetuximab. Ctrl-control.

Figure 2. Determination of the EGFR ligand expression and receptor kinase activation in SCC1 and 1Cc8 cell lines.

A) Western blot analyses of receptor tyrosine kinases and their downstream proteins with/without HB-EGF stimulation. B) EGFR and pro-HB-EGF protein expression levels in mouse-xenograft tumors generated from SCC1 and 1Cc8. C) EGFR ligand levels in conditioned media of SCC1 and 1Cc8 with/without cetuximab treatment determined by ELISA assays. P-values were generated comparing SCC1 versus 1Cc8 for control or cetuximab treatment (* P<0.05, ** P<0.01, ***P<0.001). D) Western blot analyses for pro-HB-EGF, pro-TGFA and TACE/ADAM17 levels with/without cetuximab treatment in cell lysates from SCC1 and 1Cc8. AREG- amphiregulin, EGF- epidermal growth factor, HB-EGF- heparin-binding EGF-like growth factor, TGFA- transforming growth factor alpha.

Figure 3. Effects of EGFR ligands in cetuximab sensitivity.

A) Western blot analyses of HER family receptor kinases and their downstream proteins with/without EGFR ligand stimulation in the presence of cetuximab. B) Induction of cetuximab resistance by exogenous EGFR ligands in three cetuximab sensitive HNSCC cell lines determined by MTS assay. C) Induction of cetuximab resistance by exogenous HB-EGF in SCC1 determined by colony formation assay. D) Pro-HB-EGF level in the cell lysate of 1Cc8 after knockdown of HB-EGF shown in Western blot. Growth inhibition rate of 1Cc8 cells transfected with shRNA HB-EGF and an empty vector measured by MTS assay.

Figure 4. Identification of microRNA-212 as a potential regulator of HB-EGF expression.

A) Top 20 genes putatively targeted by miR-212. The numbers next to the gene symbols are p-values. Red: higher gene expression, Green: lower gene expression. B) Relative expression of top 10 miRs targeting HB-EGF in SCC1 and 1Cc8. C) Relative expression levels of miR-212 and HB-EGF in 34 HNSCC cell lines and a keratinocyte cell line. HB-EGF expression data were obtained from DNA microarray analyses and miR-212 expression data were obtained from TLDA microRNA arrays.

Figure 5. MicroRNA-212 regulates HB-EGF expression in cetuximab resistant cells.

A) Pro-HB-EGF expression following the transfection with miR-212 mimics in three cetuximab resistant cell lines. B) Growth inhibition rate of 1Cc8 cells with exogenous miR-212 mimics, cetuximab or a combination of miR-212 mimics and cetuximab measured by colony formation assay. C) MicroRNA-212 directly regulates HB-EGF by binding to the 3′ untranslated region of HB-EGF in cetuximab resistant 1Cc8 cells while it was not significant in cetuximab sensitive SCC1 cells (*, p = 0.47; **, p = 0.038). RLU – Raw Light Units. D) Growth rate comparison of SCC1 cells in the presence of cetuximab, exogenous miR-212 inhibitor, or a combination of miR-212 inhibitor and cetuximab measured by colony formation assay.

Figure 6. The HB-EGF levels in patients with HNSCC.

A) Relative expression levels of five ligands in one normal oral mucosa and six tumors from patients with HNSCC. B) HB-EGF protein levels in plasma from patients with HNSCC. “Primary” plasma samples were taken at the time of diagnosis. “Recurrent” samples were taken at the time of recurrence.