Molecular Integrative Clustering of Asian Gastric Cell Lines Revealed Two Distinct Chemosensitivity Clusters

<div><p>Cell lines recapitulate cancer heterogeneity without the presence of interfering tissue found in primary tumor. Their heterogeneous characteristics are reflected in their multiple genetic abnormalities and variable responsiveness to drug treatments. In order to understand the heterogeneity observed in Asian gastric cancers, we have performed array comparative genomic hybridization (aCGH) on 18 Asian gastric cell lines. Hierarchical clustering and single-sample Gene Set Enrichment Analysis were performed on the aCGH data together with public gene expression data of the same cell lines obtained from the Cancer Cell Line Encyclopedia. We found a large amount of genetic aberrations, with some cell lines having 13 fold more aberrations than others. Frequently mutated genes and cellular pathways are identified in these Asian gastric cell lines. The combined analyses of aCGH and expression data demonstrate correlation of gene copy number variations and expression profiles in human gastric cancer cells. The gastric cell lines can be grouped into 2 integrative clusters (ICs). Gastric cells in IC1 are enriched with gene associated with mitochondrial activities and oxidative phosphorylation while cells in IC2 are enriched with genes associated with cell signaling and transcription regulations. The two clusters of cell lines were shown to have distinct responsiveness towards several chemotherapeutics agents such as PI3 K and proteosome inhibitors. Our molecular integrative clustering provides insight into critical genes and pathways that may be responsible for the differences in survival in response to chemotherapy.</p></div

Dot plots of IC₅₀ values for targeted inhibitors that have significant differences in toxicity to the Asian gastric cancer cells between the two integrated clusters.

<p>(A) Targeted inhibitors from the CCLE database. (B) Targeted inhibitors from the Sanger COSMIC database. (C) Selected targeted inhibitors in our lab showing significant differences in sensitivity (except XAV939) towards the two clusters of cell lines. Y-axis is the IC₅₀ values in log10 scale. P-value is computed by Mann Whitney U-test. Horizontal bars are medians for sample distributions.</p

Top 10 cellular pathways having the most genetic aberrations in the Asian gastric cell lines.

<p>The numerics in red boxes are the number of aberrations. Increased intensity of red corresponds to increased number of aberrations.</p

Molecular clustering of the Asian gastric cancer cell lines.

<p>(A) Hierarchical clustering using cell lines with both DNA copy number and mRNA expression data. (B) mRNA expression (mean-centered, normalized) heatmap (upper panel) and copy number (lower panel) of 1,762 putative driver genes from 14 gastric cell lines. (C) ssGSEA pathway enrichment score (mean-centered) heatmap for 380 subtype-specific pathways using 27 gastric cell lines from CCLE. Only selected pathway/genesets are labeled. Color code for mRNA expression: red = high expression, green = low expression. Color code for copy number: green = copy number loss, red = copy number gain, black = normal copy number. Color code for pathway enrichment: red = high enrichment, green = low enrichment.</p

Total and kinome genetics aberrations (consisting of gain, loss and LOH) in the 18 Asian gastric cell lines.

<p>Total and kinome genetics aberrations (consisting of gain, loss and LOH) in the 18 Asian gastric cell lines.</p

TGF-β1 induces EGFR/Ras gene expression signature.

<p>(A) A heat map highlighting the results of gene set enrichment analysis of genes significantly altered by TGF-β1 (2.5 ng/ml) in GIF-14 cells (nominal <i>p</i>-values<0.05). Mean gene expression value of leading-edge genes of each gene set is plotted. Lower levels of expression are represented in green and higher expression in red. Gene sets representing differentially enriched pathways are grouped. (B) The Enrichment Plot of a representative EGFR gene set. The relative gene positions of gene set are indicated by the vertical lines (middle) under the graph, which presents the enrichment scores of individual genes (top). Lines clustered to the left represent higher ranked genes in the ranked list. Bottom plot displays the rank matrix of these genes. The position of leading-edge genes suggests a positive correlation between TGF-β1 treatment and EGFR pathway. (C) A heat map depicting the expression levels of genes within the EGFR gene set in response to 24 h of TGF-β1 treatment, as measured by expression microarray analysis. Each column represents the expression data derived from a single replicate (n = 3). Gene expression is normalized for each row; where lower levels of expression are represented in shades of blue and higher expression in red. The arrow highlights an induction of <i>Egfr</i> mRNA in response to TGF-β1. (D) Normalized <i>Egfr</i> mRNA expression level as measured by microarray. (E) Validation of TGF-β1 induction of <i>Egfr</i> expression by quantitative PCR. Expression of <i>Egfr</i> are normalized against that of <i>Gapdh</i> and expressed relative to the untreated control (means ± SEM, n = 4). Student's t-tests are performed and double asterisks represent <i>p</i>-value<0.01. (F) Western blot analysis of total Egfr expression as measured by two separate Egfr-specific antibodies – Ab #1 (Abcam) and Ab #2 (Cell signaling). The expression level of α-tubulin is used as a control for the amounts of protein lysates loaded. (G) Protein band intensities are assessed by densitometric analysis. The band intensities of Egfr in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070427#pone-0070427-g001" target="_blank">Figure 1F</a> were sampled three times and normalized against that of α-tubulin (means ± SEM, n = 3). Student's t-tests are performed in which single and double asterisks denote <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively. (H) Western blot analysis of phosphorylated Egfr and Erk expression in response to TGF-β1 treatment. GIF-14 cells were treated with TGF-β1 (2.5 ng/ml) for 24 h and 48 h before harvesting for Western blotting. Phosphorylation of Egfr at tyrosine residues 1068 and 1092 and Erk1/2 was detected by pEgfr^Y1068/1092- and pErk1/2–specific antibodies. Total Egfr expression levels were measured using anti-Egfr antibody #2 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070427#pone-0070427-g001" target="_blank">Figure 1F</a>. Immunoblots of α-tubulin serves as a control for the amount of proteins loaded.</p

Oligonucleotide primers and Taqman probes used for quantitative RT-PCR.

<p>Oligonucleotide primers and Taqman probes used for quantitative RT-PCR.</p

Proposed model for the contribution of EGFR/Ras/MEK signaling pathway to TGF-β1-induced stemness and tumorigenicity.

<p>The activation of TGF-β pathway triggers <i>Runx3^−/−p53^−/−</i> GIF-14 gastric epithelial cells into EMT, concurrently inducing a stem cell-like and tumorigenic state. The data presented in this report show that TGF-β1 induces an EGFR/Ras gene expression signature marked by increased Egfr expression, which sensitized GIF-14 cells to EGF. The activation of EGFR/Ras pathway promotes stemness and tumorigenicity in GIF-14 cells in a MEK1/2-dependent manner that did not involve increased EMT.</p

TGF-β and EGFR pathways cooperate to induce stemness in GIF-14 cells.

<p>(A) Changes in the expression of stemness- and EMT/mesenchymal-associated marker genes in response to TGF-β1 and EGF. GIF-14 cells were treated with murine EGF (10 ng/ml) or TGF-β1 (2.5 ng/ml) or in combination for 15 h. Quantitative PCR measurements of gene expression levels are normalized against <i>Gapdh</i> levels, and expressed relative to the control sample (means ± SEM, n = 4). Student's t-tests are performed in which single and double asterisks denote <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively and n.s represents not significant (Black bracket: TGF-β1 responsiveness; Red bracket: cooperative induction by TGF-β1 and EGF). (B) Cooperative induction of stemness by EGF and TGF-β1. GIF-14 cells were pretreated with TGF-β1 (2.5 ng/ml) for varying periods at 15 h, 24 h, 48 h and 72 h before the addition of murine EGF (10 ng/ml) for another 15 h. Changes in the mRNA levels of stemness marker <i>Hmga2</i>, regulators of EGF signaling <i>EGFR</i> and <i>Lrig1</i>, EMT markers <i>Snai1</i> and <i>fibronectin1</i> (<i>Fn1</i>) were determined by qRT-PCR. The values are normalized against those of <i>Gapdh</i> and are expressed relative to that of the control (means ± SEM, n = 3). Student's t-tests are performed where indicated. Single and double asterisks represent <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively while n.s denotes not significant (Black bracket: TGF-β1 responsiveness; Blue bracket: EGF responsiveness; Red bracket: cooperative induction by TGF-β1 and EGF). (C) TGF-β1 induction of <i>Hmga2</i> is abrogated by inhibitors of EGFR and MEK1/2. GIF-14 cells were treated with SB431542 (TGF-βRI inhibitor; 10 µM) or AG1478 (EGFR inhibitor; 10 µM) or U0126 (MEK1/2 inhibitor; 10 µM) or in combination with TGF-β1 (2.5 ng/ml) for 48 h. Changes in the mRNA levels of stemness marker <i>Hmga2</i> and EMT marker <i>Snai1</i> were ascertained by qRT-PCR and normalized values are expressed relative to the control values (means ± SEM, n = 4). Student's t-tests are performed where indicated. Single and double asterisks denote <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively. (D) The effects of TGF-β1 and EGF on the phosphorylation states of Egfr and Erk. GIF-14 cells were treated with TGF-β1 (2.5 ng/ml; top panel) or murine EGF (10 ng/ml; bottom panel) for various short periods of time from 15 to 120 min. The expression levels of phosphorylated Egfr at tyrosine residues 1068 and 1092 and Erk1/2 were measured by Western blot analysis using pEgfr^Y1068/1092- and pErk1/2–specific antibodies Total Egfr expression was determined using anti-Egfr antibody #2 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070427#pone-0070427-g001" target="_blank">Figure 1F</a>. The expression level of α-tubulin serves as a control for the amount of proteins loaded.</p

KRas robustly induces stemness and tumorigenicity in GIF-14 cells without triggering EMT.

<p>(A) Wild type (KRasWT) or oncogenic (KRasV12) KRas were induced for 48 h in pooled GIF-14/KRasWT and GIF-14/KRasV12 cells by the withdrawal of doxycycline (100 ng/ml). Expression levels EMT/mesenchymal and stemness markers were determined by qRT-PCR, normalized against <i>Gapdh</i> levels and presented relative to those of KRasWT uninduced sample (means ± SEM, n = 3). Student's t-tests are performed where indicated. Single and double asterisks denote <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively. (B) The effects of KRasV12 activation on the expression of mesenchymal and epithelial markers. Changes in the expression levels of mesenchymal marker, Vimentin and epithelial marker, E-cadherin (E-Cad) upon the withdrawal of doxycycline (100 ng/ml) for 24 h and 48 h in GIF-14/KRasV12 cells were measured by Western blot analysis. The expression levels of phosphorylated Erk1/2 were determined to indicate the activation of Ras signaling pathway. Immunoblotting of α-tubulin serves as a control for the amount of proteins loaded. (C) The activation of KRas has no discernable effect on cell morphology of GIF-14/KRasV12 cells 48 h after doxycycline (100 ng/ml) was withdrawn, compared with those treated with TGF-β1 (2.5 ng/ml). Phase contrast images were captured. Scale bars = 50 µm. (D) The activation of KRas does not significantly alter the migratory properties of GIF-14 cells. GIF-14/KRasV12 cells were concurrently induced for KRas expression and co-treated with TGF-β1 (2.5 ng/ml) or carrier control for 24 h before the creation of scratch wounds. Phase contrast images were captured at 0 h and 12 h and representative pictures 12 h post-wounding are shown. Scale bars = 50 µm. (E) Graphical representation of wound healing assay data as analysed by the Tscratch software. Results presented are compiled from four replicates (means ± SEM, n = 4). Student's t-tests are performed and double asterisks denote <i>p</i> value<0.01 while n.s represents not significant. (F) KRasV12 promotes sphere formation in GIF-14 cells. Sphere-forming potentials of GIF-14/KRasV12 cells were determined upon the activation of KRasV12 in the presence of SB431542 (10 µM) or U0126 (10 µM). Representative images of the spheres are shown. Scale bars = 50 µm. (G) Graphical presentation of sphere assay results. The number of spheres ≥250 µm in size were scored after 6 days (means ± SEM, n = 3). Student's t-tests are performed. Single and double asterisks denote <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively, (H) Anchorage-independent growth of GIF-14/KRasV12 cells was ascertained by soft agar assay following the induction of KRasV12. Representative images of the colonies at the seeding density of 200,000 cells/well are shown. Scale bars = 100 µm. (I) Graphical presentation of colony counts of soft agar assays in which each replicate was seeded at 200,000 cells/well and cultured for 2 weeks (mean ± SEM, n = 3). Student's t-tests are performed and double asterisks denote <i>p</i>-value<0.01.</p