Overexpression of RPS27a contributes to enhanced chemoresistance of CML cells to imatinib by the transactivated STAT3

Abstract

STAT3 plays a pivotal role in the hematopoietic system, which constitutively activated by BCR-ABL via JAK and Erk/MAP-kinase pathways. Phospho-STAT3 was overexpressed in imatinib-resistant CML patients as relative to imatinib responsive ones. By activation of the STAT3 pathway, BCR-ABL can promote cell cycling, and inhibit differentiation and apoptosis. Ribosomal protein S27a (RPS27a) performs extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. RPS27a can promote proliferation, regulate cell cycle progression and inhibit apoptosis of leukemia cells. However, the relationship between STAT3 and RPS27a has not been reported. In this study, we detected a significantly increased expression of STAT3 and RPS27a in bone marrow samples from CML-AP/BP patients compared with those from CML-CP. In addition, we also demonstrated that it was a positive correlation between the level of STAT3 and that of RPS27a. Imatinib-resistant K562/G01 cells expressed significantly higher levels of STAT3 and RPS27a compared with those of K562 cells. RPS27a could be transactivated by p-STAT3 through the specific p-STAT3-binding site located nt -633 to -625 and -486 to -478 of the RPS27a gene promoter in a dose-dependent manner. The transactivated RPS27a could decrease the percentage of apoptotic CML cells induced by imatinib. And the effect of STAT3 overexpression could be counteracted by the p-STAT3 inhibitor WP1066 or RPS27a knockdown. These results suggest that drugs targeting STAT3/p-STAT3/RPS27a combining with TKI might represent a novel therapy strategy in patients with TKI-resistant CML.

Keywords: CML; RPS27a; STAT3; apoptosis; imatinib.

Figure 1. Relationship of STAT3 and RPS27a and their expression in CML patients

(A) The DNA sequence of 1.6-kb full-length human RPS27a gene was cloned into luciferase reporter constructs. HEK-293 cells were transfected with FL-RPS27 a along with either vector control or express ion plasmi ds for P-gp, AXL, Hsp-70, STAT3, STAT5, CIP2A, BMI-1 and ABCG2. Cells were harvested after 48 h and total cell lysates were used for the luciferase assay. (B and C) Differential expression of STAT3 and RPS27a mRNA in patients with CML-CP or CML-AP/BP was illustrated in scatter plots. (D and E) The correlation of STAT3 and RPS27a was analyzed by Spearman correlation test. Columns and bars represent mean from 3 parallel experiments and SD, respectively. *P < 0.05; **P < 0.01.

Figure 2. Expression of STAT3 and RPS27a in different phases of two CML patients and K562, K562/G01 cells

(A and B) Relative mRNA expression of STAT3 and RPS27a was assessed in different phases of two CML patients by qRT-PCR. (Cand D) Lysates of bone marrow cells from two CML patients in different phases were analyzed for STAT3, p-STAT3 and RPS27a protein by Western blot. (E) The qRT-PCR was performed to determine the mRNA levels of STAT3 and RPS27a in K562 and K562/G01cells. (F) The Western blot was performed to determine the protein levels of STAT3, p-STAT3 and RPS27a in K562 and K562/G01cells. Columns and bars represent mean from 3 parallel experiments and SD, respectively. *P < 0.05; **P < 0.01.

Figure 3. Upregulation of RPS27a by p-STAT3 in HEK293T cells

(A) Relative RPS27a mRNA expression was assessed in HEK293T cells transfected with different dose of pcDNA3.1-STAT3 plasmid by qRT-PCR. (B) Lysates of HEK293T cells transfected with different dose of pcDNA3.1-STAT3 plasmid were analyzed for STAT3, p-STAT3 and RPS27a protein by Western blot. (C) Relative RPS27a mRNA expression was assessed in HEK293T cells transfected with different dose of pcDNA3.1-STAT3 plasmid and incubated with protein phosphatase inhibitor (PPI) by qRT-PCR. (D) Lysates of HEK293T cells transfected with different dose of pcDNA3.1-STAT3 plasmid and incubated with PPI were analyzed for STAT3, p-STAT3 and RPS27a protein by Western blot. (E) Lysates of HEK293T cells transfected with the same dose of pcDNA3.1-STAT3 plasmid and incubated with different dose of P-STAT3 inhibitor WP1066 were analyzed for STAT3, p-STAT3 and RPS27a protein by Western blot. Columns and bars represent mean from 3 parallel experiments and SD, respectively. *P < 0.05; **P < 0.01.

Figure 4. Effects of p-STAT3 on the transcriptional activity of RPS27a promoter

(A) The constructions of various truncated RPS27a promoter-luciferase reporter plasmids and the transcriptional activity analysis. Different RPS27a promoter DNA fragments were fused to a luciferase reporter vector pGL3-promoter or pGL3-basic. The putative p-STAT3-binding sites were presented by black spots. Mu934B has six bases (underlined) different from those in S934B. P: pGL3-promoter, B: pGL3-basic. Each of the constructs was co-transfected with pCMV5-STAT3 into HEK293 cells. At 48 h after transfection, a six-fold increase in luciferase activity of S934B was observed in cells transfected with pCMV5-STAT3 at a dose of 80 ng. No changes were observed in Mu934B groups. (B and C) The luciferase transcriptional activity of S934B was activated by p-STAT3 in a dose-dependant manner. (D) Chromatin immunoprecipitation analysis of p-STAT3 binding to the RPS27a promoter in k562/G01 cells. The blot is representative of three experiments. The promoter fragment of 184 bp containing the putative p-STAT3-binding site at nt −633 to −625 and nt −486 to −478 of RPS27a was successfully amplified, both from the input DNA and the chromatin immunoprecipitated by anti-p-STAT3 antibody, whereas no amplified product was obtained in the anti-STAT3 antibody group, the immunoglobulin G isotype control group and H2O blank group. Columns and bars represent mean from 3 parallel experiments and SD, respectively. *P < 0.05; **P < 0.01.

Figure 5. Validation of STAT3 overexpression and its effect on apoptosis in K562 cells

(A) Relative STAT3 and RPS27a mRNA expression was assessed in K562 cells transfected with pcDNA3.1-STAT3 plasmids by qRT-PCR. (B) Lysates of K562 cells transfected with indicated plasmids were analyzed for STAT3, p-STAT3 and RPS27a protein by Western blot. (C) IC50s of K562-con and K562-STAT3 cells to imatinib at 48 h and 72 h was detected by MTT assays. (D) K562-con and K562-STAT3 cells were treated with or without 0.5 μM imatinib and 1.0 μM imatinib for 48 h and subjected to cell apoptosis analysis by flow cytometry analysis of Annexin-V labeling. Columns and bars represent mean from 3 parallel experiments and SD, respectively. *P < 0.05; **P < 0.01.

Figure 6. Validation of p-STAT3/RPS27a pathway blocking and its effect on apoptosis in K562-STAT3 cells

(A) Lysates of K562-STAT3 cells incubated with or without WP1066 were analyzed for STAT3, p-STAT3 and RPS27a protein by Western blot. (B) K562-STAT3 cells were treated with or without 0.5 μM imatinib and 1.0 μM imatinib in the presence of 5 μM WP1066 for 48 h and subjected to cell apoptosis analysis by flow cytometry analysis of Annexin-V labeling. (C) Relative STAT3 and RPS27a mRNA expression was assessed in K562-STAT3 cells transfected with RPS27a shRNA plasmids by qRT-PCR. (D) Lysates of K562-STAT3-RPS27a-scr and K562-STAT3-RPS27a-sh cells were analyzed for STAT3, p-STAT3 and RPS27a protein by Western blot. (E) IC50s of K562-STAT3-RPS27a-scr and K562-STAT3-RPS27a-sh cells to imatinib at 48 h and 72 h was detected by MTT assays. (F) K562-STAT3-RPS27a-scr and K562-STAT3-RPS27a-sh cells were treated with or without 0.5 μM imatinib and 1.0 μM imatinib for 48 h and subjected to cell apoptosis analysis by flow cytometry analysis of Annexin-V labeling. Columns and bars represent mean from 3 parallel experiments and SD, respectively. *P < 0.05; **P < 0.01.