The correlations between clinical stages of gastric cancer and the expressions of proteins.

<p>Although GRP78 and GSTpi were increased in different stages of gastric cancer, no statistical significance could be found. A trend of increase with clinical stage was found in GRP78.</p

The detailed comparison between normal and tumorous tissue from 2D-DIGE.

<p>The selected putative proteins were presented as stereopictures and enlarged scales gel images. These proteins were selected according to the significant difference below 0.05 (<i>p</i><0.05). In addition, the duplicated identification of ATPB, PDIRP5, ApoAI and GSTpi on adjacent spots indicated that they had a different modification.</p

Discovery of Tumor Markers for Gastric Cancer by Proteomics - Figure 3

<p>(A) By Western blotting, 12 paired of gastric cancer and normal tissues was used to validate the putative proteins including GRP78, GSTpi, A1AT, ApoAI and GKN-1. (B) The GSTpi and GRP78 were significantly over-expressed in gastric cancer tissues.Down-expressions of A1AT, ApoAI and GKN-1 were noted. **<i>p</i><0.01. *<i>p</i><0.05.</p

The 2D-DIGE results paired tissue samples.

<p>The differential proteins presented on gels. (left): normal; (right): cancer. Thirty-six proteins were picked by DeCyder statistical software, but only fifteen proteins were identified by PMF or PFF technique. The gel conditions: pI: 4–7; gel: 4–12%.The analytical ranges were from pI 4 to 7 forisoelectric focusing and from 4% to 12% gradient gel for sodium dodecyl sulfate- polyacrylamide gelelectrophoresis.</p

Immunohistochemistric stains were used to identify the location of the putative proteins.

<p>The up-regulated proteins, GRP78 and GSTpi, were specifically located on the gastric adenocarcinoma cells. Contrarily, the down-regulated proteins including ApoAI and A1AT were present on the normal gastric glands. Another down-regulated protein, GKN-1, was shown to appear on the mucosa of gastric tissue.</p

The correlation among the selected proteins.

<p>The GRP78 in these 12 pairs of tissues was expressed correlatively with that of ApoAI (r2: −0.61) and A1AT (r2: −0.49) individually. Meanwhile, ApoAI was expressed correlatively with that of A1AT (r2: 0.62). However, GSTpi and GKN-1 seemed to be independent. **p<0.01. *p<0.05.</p

EGFR induced the formation of HCC827-derived tumorspheres and <i>G9a</i> expression.

<p>To validate that EGFR directly affects the expression of <i>G9a</i> and <i>Oct4</i> involved in tumorsphere formation, we inhibited EGFR by using afatinib and genetic knockdown to demonstrate the EGFR-G9a regulatory relationship. (A) Afatinib treatment with or without 20 ng/mL of EGF blocked <i>G9a</i> expression and H3K9 methylation, accompanied by the reduction in <i>Oct4</i> expression, indicating that EGFR positively regulated <i>G9a</i> in HCC827 cells. (B) EGFR knockdown with lower EGFR autophosphorylation also reduced <i>G9a</i> expression, H3K9 methylation, and <i>Oct4</i> expression. (C) To validate whether the blockade of EGFR phosphorylation can reduce tumorsphere formation, afatinib was used. First, afatinib was treated with parental HCC827 cells at a dose of more than 1 μg/mL, which led to a reduction in the viability of HCC827 cells. (D) However, afatinib (10 ng/mL) clearly inhibited the formation of HCC827-derived tumorspheres by approximately 50% (n = 3), revealing that afatinib preferably inhibited cancer stemness. (E) Moreover, afatinib reduced EGFR autophosphorylation and expression and <i>Oct4</i> expression in the HCC827-derived tumorspheres, implying that EGFR phosphorylation self-regulated EGFR expression in HCC827 cells. Scale bar: 100 μm. *<i>p</i> < 0.05. **<i>p</i> < 0.01,***<i>p</i> < 0.001.</p

YM155 significantly reduced tumorsphere formation and inhibited EGFR autophosphorylation and G9a expression.

<p>YM155 is reported to suppress cancer stemness; therefore, we used this compound to investigate the cellular mechanism of tumorsphere formation. To compare the cytotoxic capacity of YM155 against tumorsphere formation and parental cell lines, YM155 was applied to HCC827 and A549 cells in the stemness cultured or integrated medium. (A) YM155 significantly reduced HCC827 cells when used at a dose of 100 ng/mL (**<i>p</i> < 0.01) and (B) A549 cells when used at a dose of 1 ng/mL (***<i>p</i> < 0.001). (C) Microscopy revealed that 10 ng/mL of YM155 considerably inhibited the formation of HCC827 and (D) A549CSC tumorspheres and resulted in a lower cell viability. (E) Because EGFR overexpresses in HCC827 and A549 cells, and because YM155 has been reported to suppress EGFR, we investigated the autophosphorylation status of EGFR in HCC827- and A549-derived tumorspheres. EGFR phosphorylation was blocked by YM155 in HCC827-derived tumorspheres, whereas afatinib, a tyrosine kinase inhibitor, was used as a control. (F) Moreover, YM155 inhibited EGFR autophosphorylation in A549-derived tumorspheres and reduced <i>Oct4</i> expression. (G) The methylation on H3K9 was high in HCC827-derived tumorspheres; therefore, we investigated the mRNA levels of the SET domain-containing proteins, particularly those functioning in H3K9 methylation, in HCC827 CSCs compared with parental HCC827 cells. The results revealed that the mRNA of <i>SUV39H2</i>, <i>G9a</i>, <i>GLP</i>, and <i>SETDB2</i> increased in HCC827 CSCs. (H) In addition, YM155 significantly reduced the mRNA levels of <i>G9a</i> and <i>GLP</i> but increased those of <i>SUV39H2</i> and <i>SETDB2</i>. (I) To confirm the significant roles of tumorsphere-expressed <i>SUV39H2</i>, <i>G9a</i>, <i>GLP</i>, and <i>SETDB2</i> in lung adenocarcinoma, the Kaplan–Meier method was used for investigating the relationship between the mRNA levels of <i>SUV39H2</i>, <i>G9a</i>, <i>GLP</i>, and <i>SETDB2</i> and the survival rate in 2,437 patients with lung cancer; the mean follow-up period was 49 months. The results revealed that an elevated mRNA level of <i>G9a</i> was associated with a lower overall survival rate in the patients with lung adenocarcinoma, but the remaining three genes yielded controversial results. Higher gene expression levels are indicated in red. (J) We investigated whether EGFR could downregulate <i>G9a</i> expression in HCC827 cells. We found that YM155 reduced EGF-induced <i>G9a</i> expression, H3K9 methylation, and <i>Oct4</i> expression in HCC827 cells. HCC827 cells were treated with 20 ng/mL of EGF with or without 10 ng/mL of YM155. (K) HCC827, an EGFR-mutant strain, can induce autophosphorylation; therefore, we investigated and validated EGFR-regulated <i>G9a</i> expression in EGFR wild-type A549 cells. A549 cells treated with 20 ng/mL of EGF expressed immediate EGFR autophosphorylation in 0.5 h and subsequent <i>G9a</i>, mH3K9, and <i>Oct4</i> expression. The results indicated that EGF induced the expression of <i>G9a</i> and <i>Oct4</i> in lung cancer. Scale bar: 100 μm. *<i>p</i> < 0.05.</p

G9a–GLP inhibitor, UNC0642, and G9a knockdown inhibited G9a-mediated H3K9 methylation and reduced stemness in lung cancer cells.

<p>To determine that <i>G9a</i> is a regulator of stemness in lung cancer, the G9a–GLP inhibitor and genetic knockdown of G9a were applied to inhibit the formation of tumorspheres and expression of stemness markers, respectively. (A) We found that UNC0642 significantly reduced HCC827 cells when used at a dose of less than 1 μg/mL and (B) A549 cells when used at a dose of less than 0.1 μg/mL. (C) UNC0642 also significantly reduced the formation of HCC827 CSCs when used at a dose of 10 μg/mL and (D) A549 CSCs. The reduction in cell viability was similar to that observed for parental cells. (E) Therefore, to validate that the reduction in tumorsphere formation was derived from the inhibition of the cancer stemness property, <i>Oct4</i> was detected in UNC0642-treated HCC827 cells with or without 20 ng/mL of EGF. We found that UNC0642 reduced EGF-mediated mH3K9 and <i>Oct4</i> expression in HCC827 cells. (F) To validate the significant role of G9a in regulating the stemness property, we knocked down G9a by using shRNA technique and investigated the methylation of H3K9 and expression of <i>Oct4</i> in EGFR wild-type A549 cells. We found a marked reduction in H3K9 methylation and a slight reduction in <i>Oct4</i> expression in the <i>G9a</i> knockdown A549 cells. (G) The inhibition of <i>G9a</i> expression led to a reduction in the mRNA levels of (H) <i>CD133</i>, (J) <i>Oct4</i>, and (K) <i>Nanog</i>, indicating that <i>G9a</i> played a significant role in regulating the stemness property of lung cancer cells. Scale bar: 100 μm. *<i>p</i> < 0.05. **<i>p</i> < 0.01. ***<i>p</i> < 0.001.</p

Additional file 4: of STAT3 exacerbates survival of cancer stem-like tumorspheres in EGFR-positive colorectal cancers: RNAseq analysis and therapeutic screening

Figure S3. Higher levels of PDGFA are associated with overall survival in patients with CRC in the database PROGgeneV2. (TIF 226 kb