YTHDC1 positively regulates PTEN expression and plays a critical role in cisplatin resistance of bladder cancer

Abstract

Activation of PI3K/AKT signalling by PTEN loss significantly enhances chemoresistance in bladder cancer. This study aims to evaluate PTEN regulation and identify targets that could be used to relieve chemoresistance. Expression of YTHDC1, γ-H2AX and PTEN were detected by IHC assay. Cell Counting Kit-8 assay, colony formation assay and tumour xenograft experiment evaluated cisplatin response. Flow cytometry and comet assay estimated cell apoptosis, cell cycle distribution and DNA repair capability. Quantitative real-time polymerase chain reaction, Western blot and RIP assay assessed binding properties between PTEN mRNA and YTHDC1. Silencing YTHDC1 in bladder cancer cells reduced PTEN expression and activated PI3K/AKT signalling by destabilizing PTEN mRNA in an m⁶ A-dependent manner. Low YTHDC1 expression indicated poor cisplatin sensitivity in bladder cancer patients. Reducing YTHDC1 expression promoted drug resistance to cisplatin, while over-expressing YTHDC1 promoted cisplatin sensitivity. Reducing YTHDC1 expression activated DNA damage response, which includes quicker cell cycle recovery, apoptosis evasion and an enhanced DNA repair capability, whereas these effects were attenuated when MK2206, a PI3K/AKT inhibitor was applied. We provide novel evidence that PTEN/PI3K/AKT signalling pathway could be regulated by YTHDC1 in an m⁶ A-dependent manner and highlight a critical role of YTHDC1 in cisplatin resistance of bladder cancer.

FIGURE 1

YTHDC1 positively regulates PTEN expression in bladder cancer. YTHDC1 and PTEN level were detected by an immunohistochemistry assay in tumour sections from 37 bladder cancer patients. The correlation between YTHDC1 and PTEN levels was analysed with Graphpad Prism 8.0 software. (A) Representative images show YTHDC1 and PTEN expression in three identical patients. The scale bar indicates 50 μm. (B) A scatter plot shows the correlation between YTHDC1 and PTEN levels. The expression of YTHDC1 and PTEN levels were analysed by (C and D) quantitative real‐time polymerase chain reaction (PCR) and (E) Western blot. Data are presented as the mean ± SEM, and experiments were performed at least three times. GAPDH was applied as an internal control. (F) Modification of m⁶A on PTEN mRNA was predicted by using SRAMP database (www.cuilab.cn/sramp). Schema illustrates the predicted mode. (G–I) The binding potential between YTHDC1 and PTEN mRNA was evaluated by using a RIP assay. IgG was taken as a negative control. (H) Representative images show pulldown products in T24 bladder cancer cells by performing agarose gel electrophoresis. Data are presented as the mean ± SEM, and experiments were performed at least three times. (J and K) The cells were treated with 5 μg/ml actinomycin D, and the expression of PTEN was detected by quantitative real‐time PCR. Data are presented as the mean ± SEM, and experiments were performed at least three times. (L) The expression of PTEN and p‐AKT(ser473) were evaluated via performing an immunofluorescence assay. T24 bladder cancer cells were captured microscopically and the scale bar indicates 50 μm. Experiments were performed at least three times.

FIGURE 2

Lower YTHDC1 level indicates poor cisplatin response in bladder cancer. YTHDC1 level was detected by immunohistochemistry (IHC) assay in tumour sections from 20 bladder cancer patients that have received cisplatin‐containing chemotherapy. (A) Representative images show YTHDC1 expression in six identical patients. (B and C) Scatter plots show IHC scores for YTHDC1 staining. The scale bar indicates 50 μm. (D and E) Real‐time polymerase chain reaction (PCR) evaluated the expression of YTHDC1 on mRNA level. (F and G) The protein level of YTHDC1 was detected by Western blot. GAPDH was applied as an internal control both for quantitative real‐time PCR and Western blot detection. Data are presented as the mean ± SEM, and experiments were performed at least three times. (H and I) After 48 h of treatment with different doses of cisplatin, cell viabilities were measured by using Cell Counting Kit‐8 assay. Data are presented as the mean ± SEM, and experiments were performed at least three times. (J–M) After treatment with 20 μM cisplatin, the growth of single cells was measured after 2 weeks by a colony formation assay. Representative images are displayed. (K and L) Colonies with over 50 cells were counted. Data are presented as the mean ± SEM, experiments were performed at least three times. Mice that bearing bladder carcinoma xenograft were treated with cisplatin (3 mg/kg) per week. The response to cisplatin treatment was reflected by the change of tumour size. (N) Representative images illustrate dissected tumour samples at the end of experiment. (O) The size of tumour xenografts was measured every 3 days and tumour growth curve was displayed. shYTHDC1‐2 cells were selected and used to construct YTHDC1 silencing xenografts. Data are presented as the mean ± SEM.

FIGURE 3

Lower expression of YTHDC1 alleviates DNA damage and apoptosis in bladder cancer. The YTHDC1 and γ‐H2AX levels were detected by an immunohistochemistry assay in tumour sections from 36 bladder cancer patients. The correlation between YTHDC1 and γ‐H2AX levels were analysed by using Graphpad Prism 8.0 software. (A) Representative images show YTHDC1 and γ‐H2AX expression levels in three identical patients. The scale bar indicates 50 μm. (B) Scatter plots show the correlation between YTHDC1 and γ‐H2AX level. Bladder cancer cells were treated with 20 μM cisplatin and 10 μM etoposide for 4 h, and 48 h later, the cells were fixed. The expression of γ‐H2AX was detected by using an immunofluorescence assay. (C) Representative images show the level of γ‐H2AX in different T24 bladder cancer cells. The scale bar indicates 40 μm. Experiments were performed at least three times. Annexin V‐AF647/PI staining was performed to evaluate the effect of cisplatin on cell apoptosis. Cells were treated with 40 μM cisplatin for 4 h and tested 48 h later. Cell positivity for Annexin V‐AF647/PI was detected by flow cytometry (D and E). The total apoptotic rate includes the sum of early and late apoptotic cells, which are shown in (F) and (G). Data are presented as the mean ± SEM, and experiments were performed at least three times.

FIGURE 4

Lower expression of YTHDC1 affects cisplatin‐induced DNA damage response in bladder cancer. Cell aliquots were collected at 24‐h intervals after treatment with 20 μM cisplatin. The cell cycle distribution was analysed by flow cytometry. (A) Representative images show G1, S and G2 populations in T24 bladder cancer cells. The grouped histogram displays the dynamic mobility of the cell cycle distribution after cisplatin treatment in (B) T24 bladder cancer cells and (C) U3 bladder cancer cells. Data are presented as the mean ± SEM, and experiments were performed at least three times. For the comet assay, alkaline single‐cell electrophoresis was conducted 48 h after cisplatin or etoposide treatment, and the results were observed and photographed via microscopy. (D) Representative images show cell DNA fragments after cisplatin or etoposide treatment in T24 bladder cancer cells. Tail DNA content (E and F) and tail length (G and H) were calculated by using a CometScore 2.0 software. Data are presented as the mean ± SEM, and experiments were performed at least three times.

FIGURE 5

Activation of PI3K/AKT signalling is responsible for YTHDC1 regulated DNA damage response (DDR) after cisplatin treatment in bladder cancer. Bladder cancer cells were treated with 20 μM cisplatin for 4 h. (A) After 48 h, cells were collected and protein was extracted. The expression of p‐AKT(ser473) was examined by using Western blot assay. GAPDH was applied as an internal control, and experiments were performed at least three times. (B) To test the effect of MK2206 on the YTHDC1 associated DDR, cells were first treated with 20 μM cisplatin for 4 h, and then, the culture medium was changed to complete medium containing 10 μM MK2206. The expression of γ‐H2AX was examined by Western blot assay. GAPDH was applied as an internal control, and the experiments were performed at least three times. (C) Cell aliquots were collected 24 h after treatment with 20 μM cisplatin and 10 μM MK2206. The cell cycle distribution was analysed by flow cytometry. (C) Representative images show cell cycle distributions in T24 bladder cancer cells. The grouped histogram displays the dynamic mobility of the cell cycle distribution after treatment in (D) T24 bladder cancer cells and (E) U3 bladder cancer cells. Data are presented as the mean ± SEM, and experiments were performed at least three times. (F) Annexin V‐AF647/PI staining was performed to evaluate the synergistic effects of MK2206 on cisplatin‐induced cell apoptosis. Cells were treated with 40 μM cisplatin for 4 h and then 10 μM MK2206 for 48 h. Cell positivity for Annexin V‐AF647/PI was detected by flow cytometry. (F) Representative images show apoptosis in T24 bladder cancer cells. (G and H) The total apoptotic rate includes the sum of early and late apoptotic cells. Data are presented as the mean ± SEM, and experiments were performed at least three times. (I) and (J) present graphic abstract of this research. As illustrated, low YTHDC1 expression in cancer cells indicates poor cisplatin therapy outcome in bladder cancer patients, and low YTHDC1 expression results in destabilization of PTEN mRNA, which activates the AKT‐associated DDR and attenuates cisplatin‐induced DNA damage.