YTH domain family 2 orchestrates epithelial-mesenchymal transition/proliferation dichotomy in pancreatic cancer cells

Abstract

Recent studies show that YTH domain family 2 (YTHDF2) preferentially binds to m⁶A-containing mRNA regulates localization and stability of the bound mRNA. However, the role of YTHDF2 in pancreatic cancers remains to be elucidated. Here, we find that YTHDF2 expression is up-regulated in pancreatic cancer tissues compared with normal tissues at both mRNA and protein levels, and is higher in clinical patients with later stages of pancreatic cancer, indicating that YTHDF2 possesses potential clinical significance for diagnosis and prognosis of pancreatic cancers. Furthermore, we find that YTHDF2 orchestrates two cellular processes: promotes proliferation and inhibits migration and invasion in pancreatic cancer cells, a phenomenon called "migration-proliferation dichotomy", as well as epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. Furthermore, YTHDF2 knockdown significantly increases the total YAP expression, but inhibits TGF-β/Smad signaling, indicating that YTHDF2 regulates EMT probably via YAP signaling. In summary, all these findings suggest that YTHDF2 may be a new predictive biomarker of development of pancreatic cancer, but a serious consideration is needed to treat YTHDF2 as a target for pancreatic cancer.

Keywords: EMT; YAP; YTHDF2; migration-proliferation dichotomy; pancreatic cancer.

Figure 1.

YTHDF2 is up-regulated in pancreatic cancer and associated with patients' poor stage. (A) YTHDF2 protein expression in pancreatic cancer tissues and normal pancreatic tissues was analyzed through the human protein atlas (www.proteinatlas.org). Magnification, × 4; bars, 500 μm. Magnification, × 40; bars, 100 μm. (B) Analysis of YTHDF2 mRNA levels in 52 samples of pancreatic cancer and non-tumor tissues in the Gene Expression Omnibus. N = 16 for non-tumor group, and N = 36 for tumor group. **P < 0.01. (C) Analysis of the TCGA database indicates YTHDF2 is associated with stage in pancreatic cancer. N = 20 for stage I group, N = 140 for stage II group, and N = 4 for stage III group, and N = 3 for stage IV group. *P< 0.05. (D) Analysis of the TCGA database indicates YTHDF2 is associated with PathologicT in pancreatic cancer. N = 27 for PathologicT1 and T2, N = 140 for PathologicT3 and T4. *P < 0.05.

Figure 2.

YTHDF2 Expression in different pancreatic cancer cells. (A) Relative expression levels of YTHDF2 protein and mRNA were assessed in PaTu8988, SW1990 and BxPC3 cells. (B) YTHDF2 protein and mRNA levels were decreased after sh-YTHDF2#1 and sh-YTHDF2#2 was transfected into SW1990 and BxPC3 cells. ***P < 0.001. Data are expressed as mean ± SD. The results are representative of three independent experiments.

Figure 3.

YTHDF2 knockdown inhibits the ability of proliferation via Akt/GSK3β/CyclinD1 pathway in pancreatic cancer cells. (A) Colony-forming assay to examine the effects of knocking down YTHDF2 on the growth of SW1990 and BxPC3 cells. ***P < 0.001. (B) SW1990 and BxPC3 cells were transfected with sh-EGFP or sh-YTHDF2#1/ sh-YTHDF2#2 plasmid and the number of viable cells at the indicated time points was evaluated using the Cell Count Kit-8. The proliferation of cells was suppressed by YTHDF2 depletion. **P < 0.01. (C) Immunoblotting of Akt, p-Akt, GSK3β, p- GSK3β, CyclinD1in SW1990 and BxPC3 cells treated with sh-YTHDF2#1 or sh-EGFP. β-Tubulin was used as a loading control. (D, E) YTHDF2 knockdown in SW1990 and BxPC3 increased the fraction of cells in G1/G0-phasewith a corresponding decrease in S-phase as compared to the control cells. Data are expressed as mean ± SD. The results are representative of three independent experiments. Data are expressed as mean ± SD. The results are representative of three independent experiments.

Figure 4.

YTHDF2 knockdown promotes the migration ability of pancreatic cancer cells. (A) The ability of migration was examined using transwell assay in SW1990 and BxPC3 cells transfected with sh-YTHDF2#1 or sh-EGFP plasmids. Representative images of migrated cells were shown. Magnification, × 20; bars, 50 μm. (B) The graph indicated the average number of migrated cells per field. ***P< 0.001. (C, D) A scrape wound was created in confluent cultures of SW1990 and BxPC3 cells with stable expression of either sh-YTHDF2#1 or sh-EGFP, the distance of cell migration was recorded and the relative migration rate was calculated.***P < 0.001. Data are expressed as mean ± SD. The results are representative of three independent experiments.

Figure 5.

YTHDF2 knockdown promotes the invasion and adhesion ability in pancreatic cancer cells. (A) The invasion ability was examined using BD Matrigel invasion assay in SW1990 and BxPC3 cells transfected with sh-EGFP or sh-YTHDF2#1 plasmids. Magnification, × 20;bars, 50 μm. (B) Invasive cells were counted and analyzed. ***P < 0.001. (C, D)MMP2 and MMP9 were identified using western blotting and real-time PCR in above cells.***P < 0.001. (E) The ability of adhesion was examined using cell adhesion assay in SW1990 and BxPC3 cells transfected with sh-YTHDF2#1 or sh-EGFP plasmids. β-Tubulin was used as a loading control. Data are expressed as mean ± SD. The results are representative of three independent experiments.

Figure 6.

YTHDF2 knockdown promotes EMT through up-regulating of the total YAP expression. (A) Knockdown of YTHDF2 in SW1990 and BxPC3 cells induced EMT, as detected by increases in Vimentin, Snail and a decrease in E-cadherin. (B) Knockdown of YTHDF2 down-regulated the mature TGF-β, Smad2/3, p-Smad2. (C) Knockdown of YTHDF2 down-regulated the LATS1, p-LATS1, MOB1, p-MOB1 and up-regulated the YAP, p-YAP. (D) Knockdown of YAP in stable cell lines-SW1990 and BxPC3 cells up-regulated the E-cadherin and down-regulated the YAP, Vimentin and Snail. β-Tubulin was used as a loading control. Data are expressed as mean ± SD. The results are representative of three independent experiments.

Figure 7.

YTHDF2 is up-regulated in other cancers. (A) Analysis of YTHDF2 mRNA levels in 57 samples of gastric intestinal type adenocarcinoma and non-tumor tissues. N = 31 for non-tumor group, and N = 26 for tumor group. ***P < 0.001. (B) Analysis of YTHDF2 mRNA levels in 552 samples of brain glioblastoma and non-tumor tissues. N = 10 for non-tumor group, and N = 542 for tumor group. ***P < 0.001. (C) Analysis of YTHDF2mRNA levels in 116 samples of lung adenocarcinoma and non-tumor tissues. N = 58 for non-tumor group, and N = 58 for tumor group. ***P < 0.001. (D) Analysis of YTHDF2 mRNA levels in 445 samples of hepatocellular carcinoma and non-tumor tissues. N = 220 for non-tumor group, and N = 225 for tumor group. ***P< 0.001.