Fig 1.
Elevated phosphorylation of EGFR and G9a-mediated methylation on H3K9 in HCC827-derived CSCs.
(A) HCC827++, A549+, and H520− were selected for analyzing EGFR phosphorylation. (++) high expression; (+) moderate expression; (−) no expression. The results indicated higher EGFR expression in HCC827 cells than in A549 and H520 cells, whereas autophosphorylation of EGFR in HCC827 cells. (B and C) HCC827- or A549-derived tumorspheres were detected and imaged through inverted microscopy when the cells were cultured in a low attached dish with FBS-free B27 supplement medium. For triggering the formation of temporary CSCs, four additive growth factors (4 GFs) were added, namely EGF, bFGF, insulin, and heparin. (D) HCC827- and A549-derived tumorspheres were investigated to detect the expression of stemness markers, ALDH1, CD133, Oct4, and Nanog, for identifying the stemness property. These markers were increased in HCC827 CSCs (n = 3) compared with parental HCC827 cells (n = 3), but (E) only CD133, Oct4, and Nanog were increased in A549 CSCs. (F) In addition, HCC827 cells cultured in serum-free B27 supplement medium without the aforementioned factors formed tumorspheres with (G) elevated EGFR phosphorylation. (H) EGFR phosphorylation was higher in HCC827 CSCs than in parental HCC827 cells, accompanied by higher G9a expression and H3K9 methylation, whereas Oct4 was used as a stemness marker. Scale bar: 100 μm. *p < 0.05 and ***p < 0.001.
Fig 2.
YM155 significantly reduced tumorsphere formation and inhibited EGFR autophosphorylation and G9a expression.
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 (**p < 0.01) and (B) A549 cells when used at a dose of 1 ng/mL (***p < 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 Oct4 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 SUV39H2, G9a, GLP, and SETDB2 increased in HCC827 CSCs. (H) In addition, YM155 significantly reduced the mRNA levels of G9a and GLP but increased those of SUV39H2 and SETDB2. (I) To confirm the significant roles of tumorsphere-expressed SUV39H2, G9a, GLP, and SETDB2 in lung adenocarcinoma, the Kaplan–Meier method was used for investigating the relationship between the mRNA levels of SUV39H2, G9a, GLP, and SETDB2 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 G9a 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 G9a expression in HCC827 cells. We found that YM155 reduced EGF-induced G9a expression, H3K9 methylation, and Oct4 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 G9a 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 G9a, mH3K9, and Oct4 expression. The results indicated that EGF induced the expression of G9a and Oct4 in lung cancer. Scale bar: 100 μm. *p < 0.05.
Fig 3.
EGFR induced the formation of HCC827-derived tumorspheres and G9a expression.
To validate that EGFR directly affects the expression of G9a and Oct4 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 G9a expression and H3K9 methylation, accompanied by the reduction in Oct4 expression, indicating that EGFR positively regulated G9a in HCC827 cells. (B) EGFR knockdown with lower EGFR autophosphorylation also reduced G9a expression, H3K9 methylation, and Oct4 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 Oct4 expression in the HCC827-derived tumorspheres, implying that EGFR phosphorylation self-regulated EGFR expression in HCC827 cells. Scale bar: 100 μm. *p < 0.05. **p < 0.01,***p < 0.001.
Fig 4.
G9a–GLP inhibitor, UNC0642, and G9a knockdown inhibited G9a-mediated H3K9 methylation and reduced stemness in lung cancer cells.
To determine that G9a 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, Oct4 was detected in UNC0642-treated HCC827 cells with or without 20 ng/mL of EGF. We found that UNC0642 reduced EGF-mediated mH3K9 and Oct4 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 Oct4 in EGFR wild-type A549 cells. We found a marked reduction in H3K9 methylation and a slight reduction in Oct4 expression in the G9a knockdown A549 cells. (G) The inhibition of G9a expression led to a reduction in the mRNA levels of (H) CD133, (J) Oct4, and (K) Nanog, indicating that G9a played a significant role in regulating the stemness property of lung cancer cells. Scale bar: 100 μm. *p < 0.05. **p < 0.01. ***p < 0.001.