MicroRNA-608 and MicroRNA-34a Regulate Chordoma Malignancy by Targeting EGFR, Bcl-xL and MET

<div><p>Chordomas are rare malignant tumors that originate from the notochord remnants and occur in the skull base, spine and sacrum. Due to a very limited understanding of the molecular pathogenesis of chordoma, there are no adjuvant and molecular therapies besides surgical resection and radiation therapy. microRNAs (miRNAs) are small noncoding regulatory RNA molecules with critical roles in cancer. The role of miRNAs in chordomas is mostly unknown. We uncover microRNA-608 (miR-608) and microRNA-34a (miR-34a) as novel tumor suppressive microRNAs that regulate malignancy in chordoma. We find that miR-608 and miR-34a expressions are downregulated in human chordoma cell lines and primary cells at least partially via alteration of their genes’ copy numbers. We identify the commonly deregulated oncogenes EGFR and Bcl-xL as direct targets of miR-608 and the receptor tyrosine kinase MET as direct target of miR-34a. We show that EGFR and MET activations promote chordoma cell proliferation and invasion and that pharmacological inhibition of EGFR and MET inhibits chordoma cell proliferation and survival. We demonstrate that restoration of miR-608 and miR-34a inhibits cell proliferation and invasion and induces apoptosis in chordoma cells. We find that miR-34a inversely correlates with MET expression and miR-608 inversely correlates with EGFR expression in chordoma cells. These findings demonstrate for the first time that miR-608 and miR-34a regulate chordoma malignancy by regulating EGFR, MET and Bcl-xL.</p></div

microRNAs are differentially expressed in chordoma cells.

<p>Small RNAs were extracted from chordoma UCH1 and UCH2 cells and control fibroblasts. miRNA levels were measured using qRT-PCR relative to control U6B snRNA.</p

miR-608 downregulates EGFR and Bcl-xL by directly binding to their mRNA 3′UTR and miR-34a downregulates MET by directly binding MET 3′UTR.

<p>A) Predicted binding sequences of miR-608 in the 3′UTR sequences of EGFR and Bcl-xL mRNA; B) Predicted binding sequences of miR-34a in the 3′UTR sequence of MET mRNA; C), D) UCH1 and C24 cells were transfected with pre-miR-608 (C) or pre-miR-34a (D) or control pre-miR for 48 hrs. Cell lysates were immunoblotted for EGFR or Bcl-xL (C) or MET (D), The results show that the miRNAs significantly inhibited these predicted target proteins in chordoma cells; E), F) UCH1 cells were transfected with pre-miR-608, pre-miR-34a or pre-miR-con and then with either EGFR 3′UTR, Bcl-xL 3′UTR, MET 3′UTR or control reporter plasmids together with β-Galactosidase (β-Gal) plasmid, and 3′UTR reporter activity was measured by a luciferase assay and normalized to β-Gal activity. The results show that miR-608 expression down-regulates EGFR and Bcl-xL luciferase activities (E) and that miR-34a expression repressed MET luciferase activity (F) in UCH1 cells. (* P<0.05)</p

miR-608 and miR-34a are downregulated via gene copy number alteration, and miR-608 and miR-34a expressions inversely correlate with EGFR and MET in chordoma cells.

<p>A) miR-608 and miR-34a levels in chordoma cell lines and primary cells were measured by qRT-PCR and compared with those in fibroblasts, B) lystaes from chordoma cells were immunoblotted for EGFR and normalized to β-actin (left panel), and the level of miR-608 was correlated with EGFR protein level (right panel) (R² = 0.8, P<0.05). C) MET protein levels in chordoma cells were determined by immunoblotting (left panel) and miR-34a levels correlated with MET protein (right panel) (R² = 0.0.61, P<0.05). D) miR-608 gene copy numbers in chordoma cells were determined by q-PCR (left panel) and miR-608 expression levels were correlated with gene copy number (R² = 0.77, P<0.05) (right panel). E) miR-34a gene copy numbers in chordoma cells were determined by q-PCR (left panel) and miR-34a expression levels are correlated with gene copy number (R² = 0.51, P<0.05).</p

EGFR and MET expression and gene amplification in chordoma in published immunohistochemical studies.

<p>EGFR and MET expression and gene amplification in chordoma in published immunohistochemical studies.</p

Additional file 1: of Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

Figure S1. T cell stimulated with αCD3/αCD28 microbeads proliferate in the presence of dexamethasone.Healthy donor T cells were cultured for four days with the indicated ratio of αCD3/αCD28 microbeads:total T cells in the presence of vehicle or dexamethasone. A, Representative flow cytometry plots of CellTrace violet dilution. Plots were derived from gated CD4 (top row) or CD8 (bottom row) T cells. B-D, Proliferation analyses of CD4 T cells (top) and CD8 T cells (bottom) performed on the samples shown in (A). Precursor Frequency (B), Expansion Index (C), and Proliferation Index (D) are shown. Samples were plated in duplicate and analyzed with an unpaired students T test. Data are representative of three independent experiments. (PDF 3563 kb

Additional file 5: of Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

Figure S5 PD-1 blockade does not rescue dexamethasone-mediated proliferation defects. A, Flow cytometry analysis of PD-1 surface expression on CD4 (left) or CD8 (right) T cells stimulated with αCD3/αCD28 microbeads. Unstimulated (dashed line), stimulated in presence of vehicle (solid line), and stimulated in presence of dexamethasone (filled red line) are shown. B, Geometric median fluorescence intensity (gMFI) of PD-1 staining on CD4 or CD8 T cells. Cells cultured with vehicle (black bars) and dexamethasone (red bars) are shown. Data are an average of duplicate samples. C, Expression of PD-1 by qPCR of T cells stimulated in the presence of vehicle or dexamethasone. Data are representative of four independent experiments. D-E. Healthy donor T cells were stimulated for four days in the presence of vehicle or dexamethasone and nivolumab or ipilimumab F(ab’)2 antibody as indicated. Precursor frequency of CD4 and CD8 T cells was quantified by FlowJo. The ratio of dexamethasone to vehicle for CD4 (C) and CD8 (D) T cells is shown. All samples were plated in duplicate and the ratios were analyzed with a one-way ANOVA. Data are representative of n = 4 healthy donors. (PDF 2522 kb

Additional file 4: of Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

Figure S4. Increased co-stimulation ameliorates the inhibitory effects of dexamethasone. Negatively-selected healthy donor T cells were cultured with 5 μg/mL αCD3 and increasing concentrations of CD80 in the presence of vehicle or dexamethasone. A-B. CD8 T cells cultured with vehicle (A) or dexamethasone (B). Flow cytometry plots showing proliferation of cells cultured with the indicated concentration of CD80 (left) and total numbers of naïve (TN), central memory (TCM), effector memory (TEM), and terminal effector (TTE) T cells following four days of culture (right) are shown. Differentiation subsets were assessed by CD45RO and CCR7 staining. Each condition was plated in duplicate, and data are representative of three independent experiments. Data were analyzed with an unpaired, two-tailed T Test. (PDF 2573 kb

Additional file 3: of Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

Figure S3. T cell differentiation subsets formed during in vitro stimulation with ιCD3/CD80 stimulation. Negatively-selected healthy donor T cells were cultured with 5 Οg/mL ιCD3 and the indicated concentration of CD80. T cell differentiation subsets were quantified following four days of culture. A, Flow plot of gating strategy to identify the indicated T cell differentiation subsets. B, Flow plots of CD4 (top) and CD8 (bottom) T cells cultured under the indicated conditions. (PDF 3995 kb

Additional file 7: of Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

Figure S7. Quantification of Treg and checkpoint molecules in tumor-bearing mice. GL261 ffluc-mCherry tumor-bearing mice were randomized into the indicated cohorts based on bioluminescence values from tumor. Vehicle or dexamethasone treatment was initiated on day 7, and isotype or CTLA-4 blocking antibody were administered on days 13, 16, and 19 following tumor implantation. Mice were euthanized on day 23 and tissues were harvested for flow cytometry analysis. A, Treg cell number from tumor-bearing brain hemisphere (left; n = 8) or the cervical tumor-draining lymph nodes (right; n = 10). B, The percentage of CD4 (top two plots) or CD8 (bottom two plots) T cells expressing the indicated checkpoint molecules. Co-expression of molecules was quantified using a Boolean gating strategy. Data were analyzed using a unpaired students T test. (PDF 1891 kb