Regulation of Epidermal Growth Factor Receptor Signaling and Erlotinib Sensitivity in Head and Neck Cancer Cells by miR-7

<div><p>Elevated expression and activity of the epidermal growth factor receptor (EGFR)/protein kinase B (Akt) signaling pathway is associated with development, progression and treatment resistance of head and neck cancer (HNC). Several studies have demonstrated that microRNA-7 (miR-7) regulates EGFR expression and Akt activity in a range of cancer cell types via its specific interaction with the EGFR mRNA 3′-untranslated region (3′-UTR). In the present study, we found that miR-7 regulated EGFR expression and Akt activity in HNC cell lines, and that this was associated with reduced growth <em>in vitro</em> and <em>in vivo</em> of cells (HN5) that were sensitive to the EGFR tyrosine kinase inhibitor (TKI) erlotinib (Tarceva). miR-7 acted synergistically with erlotinib to inhibit growth of erlotinib-resistant FaDu cells, an effect associated with increased inhibition of Akt activity. Microarray analysis of HN5 and FaDu cell lines transfected with miR-7 identified a common set of downregulated miR-7 target genes, providing insight into the tumor suppressor function of miR-7. Furthermore, we identified several target miR-7 mRNAs with a putative role in the sensitization of FaDu cells to erlotinib. Together, these data support the coordinate regulation of Akt signaling by miR-7 in HNC cells and suggest the therapeutic potential of miR-7 alone or in combination with EGFR TKIs in this disease.</p> </div

miR-7 regulates EGFR expression and Akt signaling in HNC cell lines.

<p>(A) Western blotting analysis of EGFR, P-EGFR, Akt and P-Akt levels in HN5 (left panel) and FaDu (center panel) and SCC-25 (right panel) cells 3 d after transfection with miR-7 or miR-NC precursor molecules or vehicle (LF2000) only. β-actin is included as a loading control. (B) RT-qPCR analysis of EGFR mRNA expression in HN5 cells 24 h after transfection with miR-7 or miR-NC precursor molecules. Data was normalized to GAPDH mRNA expression and expressed relative to miR-NC-transfected cells. (C) Luciferase reporter assay with FaDu cells co-transfected with miR-7 or miR-NC precursor molecules, a firefly luciferase full-length EGFR mRNA 3′-UTR reporter plasmid, and a <i>Renilla</i> luciferase reporter plasmid. Firefly luciferase activity was assessed 24 h post-transfection, normalized to <i>Renilla</i> luciferase measurements, and data was expressed relative to vehicle (LF2000) only-transfected cells. Error bars represent standard deviations. All data are representative of three independent experiments. *, p<0.001, miR-7 vs miR-NC.</p

Coordinate regulation of EGFR/Akt signaling by miR-7 in HNC cells.

<p>Schematic representation of molecules in the EGFR/Akt signaling pathway that are inhibited by miR-7 in HNC. IPA software was used to map common miR-7-downregulated genes (shown in green) in FaDu and HN5 cells onto the canonical PI3K/Akt pathway. The density of shading represents the fold-change downregulation of a gene by miR-7. Blue circles indicate that a gene is a predicted or validated target of miR-7 by IPA analysis. Several genes belonging to the PI3K/Akt pathway that were downregulated by miR-7 in HN5 cells only are shaded in light blue.</p

Synergistic inhibition of cell growth and EGFR/Akt signaling in erlotinib-resistant FaDu cells by miR-7 and erlotinib.

<p>(A) Cell titre analysis of FaDu cells that were transfected with vehicle only (LF2000), miR-7, or miR-NC for 3 d, and then treated with erlotinib (7.5 µM) or vehicle (DMSO) for a further 4 d. Data is expressed relative to vehicle-transfected, vehicle-treated FaDu cells (LF2000 minus erlotinib, first column). (B) Western blotting analysis of EGFR, P-EGFR, Akt and P-Akt levels in FaDu cells that were transfected with vehicle only (LF2000), or miR-7 or miR-NC for 3 d and then treated ± erlotinib (7.5 µM) for 24 h. β-actin is included as a loading control. (C) Densitometry analysis of P-Akt levels from western blotting between FaDu cells transfected with miR-NC or miR-7 and then treated with erlotinib (7.5 µM) for 24 h. Data is shown relative to miR-NC-transfected cells. Error bars represent standard deviations. All data are representative of three independent experiments. *, p<0.05, miR-7 minus erlotinib vs miR-NC minus erlotinib, and LF2000 plus erlotinib vs LF2000 minus erlotinib; **, p<0.01, miR-7 plus erlotinib vs miR-NC plus erlotinib. † indicates synergy between miR-7 and erlotinib as defined by the Bliss additivism model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047067#pone.0047067-Bliss1" target="_blank">[34]</a>.</p

Microarray analysis of miR-7-downregulated genes in HN5 and FaDu cells.

<p>(A) Volcano plots representing array probes between HN5 (left) and FaDu (right) cells 24 h after transfection with miR-7 or miR-NC precursor molecules. Assigning a cut off of ±1.5-fold change (miR-7 vs miR-NC) and p<0.05, significantly downregulated probes are in green and significantly upregulated probes are in red. (B) Cluster analysis of miR-7-downregulated genes in HN5 and FaDu cells, where green and red shading corresponds to downregulated and upregulated genes, respectively. (C) Venn diagram of miR-7-downregulated genes in HN5 and FaDu cells. (D) Scatter plot of miR-7-downregulated genes common to HN5 and FaDu cells (R² = 0.435, p<0.001).</p

A functional miR-7 target signature in HNC cells.

<p>miR-7-downregulated genes common to both HN5 and FaDu cells (103) were assigned to annotated cancer-associated processes using IPA software. These included “cell cycle”, “cell movement”, “cell proliferation”, “cell development”, “tumorigenesis”, “protein synthesis”, “angiogenesis” and “cell death”. Official gene symbols are used for each miR-7-downregulated gene and a blue circle indicates that a gene is a predicted or validated target of miR-7 by IPA analysis.</p

miR-7 inhibits HNC cell growth <i>in vitro</i>.

<p>(A) Colorimetric cell titre assay of HN5 cells 5 d after transfection in 96-well plates with miR-7 or miR-NC precursor molecules, or vehicle (LF2000) only. (B) Graphical representation of cell titre assay from (A). Data represents the relative number of viable HN5 cells normalized to LF2000-treated HN5 cells. (C) TaqMan RT-qPCR analysis of miR-7 expression in HN5 clones with stable expression of miR-7 (clone 39) or miR-NC (clone 2). Data was normalized to U44 snRNA expression and expressed relative to HN5 miR-NC clone 2. (D) Western blotting analysis of EGFR, Akt and P-Akt levels in HN5 clones with stable expression of miR-7 (clone 39) or miR-NC (clone 2). β-actin is included as a loading control. (E) Manual cell counting of HN5 cells with stable expression of miR-7 (clone 39) or miR-NC (clone 2). Data is expressed relative to miR-NC. (F) Clonogenicity assay of HN5 cells with stable expression of miR-7 (clone 39) or miR-NC (clone 2) 10 d after cells were seeded in 10 cm dishes. Error bars represent standard deviations. All data are representative of three independent experiments. *, p<0.01, miR-7 vs miR-NC; **, p<0.005, miR-7 vs miR-NC.</p

miR-7 inhibits HNC xenograft growth <i>in vivo</i>.

<p>(A) HN5 tumor xenograft growth following subcutaneous injection of stable HN5 miR-NC clone 2 (red) or miR-7 clone 39 (blue) cells into nude mice. Mean tumor volume (mm³) is plotted over time (d). (B) Representative photographs of tumor xenografts for cells with stable miR-NC expression (clone 2, left) and stable miR-7 expression (clone 39, right). (C) TaqMan RT-qPCR analysis of miR-7 expression in HN5/miR-7 and HN5/miR-NC stable tumor xenografts at experimental endpoint. Data was normalized to U44 snRNA expression and miR-7 levels are shown relative to HN5/miR-NC clone 2 tumors. (D) Western blotting analysis of Akt and P-Akt levels between HN5/miR-7 and HN5/miR-NC stable tumor xenografts. β-actin and Akt are included as loading controls. (E) Densitometry analysis of P-Akt levels between HN5/miR-7 (clone 39) and HN5/miR-NC (clone 2) tumor xenografts by western blotting. P-Akt levels were normalized to total Akt expression. Error bars represent standard deviations. *, p<8.0×10⁻⁵, miR-7 vs miR-NC; **, p<1.0×10⁻⁸, miR-7 vs miR-NC; ***, p = 0.06, miR-7 vs miR-NC.</p

Characterization of erlotinib sensitivity of HN5, FaDu and SCC-25 HNC cells.

<p>HN5 (red), FaDu (blue) and SCC-25 (green) cells were seeded in 96-well plates and treated with the EGFR inhibitor erlotinib (final concentration 0–100 µM). The half maximal effective concentration (EC₅₀) of erlotinib was determined for each cell line after measurement of the relative number of viable cells by cell titre assay 3 d after the addition of erlotinib. Data are normalized to the lowest concentration of erlotinib. Error bars represent standard deviations. Data are representative of three independent experiments.</p

910 metagenome-assembled genomes from the phytobiomes of three urban-farmed leafy Asian greens

10.1038/s41597-020-00617-9Scientific Data7127