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. 2014 Jul 15;120(14):2130-41.
doi: 10.1002/cncr.28668. Epub 2014 Apr 15.

Independent oncogenic and therapeutic significance of phosphatase PRL-3 in FLT3-ITD-negative acute myeloid leukemia

Shuang Qu  1 , Bin Liu, Xiaoling Guo, Hongshun Shi, Meifeng Zhou, Li Li, Shulan Yang, Xiuzhen Tong, Haihe Wang
Affiliations

Independent oncogenic and therapeutic significance of phosphatase PRL-3 in FLT3-ITD-negative acute myeloid leukemia

Shuang Qu et al. Cancer. .

Abstract

Background: Internal tandem duplication of FMS-like tyrosine kinase (FLT3-ITD) is well known to be involved in acute myeloid leukemia (AML) progression, but FLT3-ITD-negative AML cases account for 70% to 80% of AML, and the mechanisms underlying their pathology remain unclear. This study identifies protein tyrosine phophatase PRL-3 as a key mediator of FLT3-ITD-negative AML.

Methods: A total of 112 FLT3-ITD-negative AML patients were sampled between 2010 and 2013, and the occurrence of PRL-3 hyperexpression in FLT3-ITD-negative AML was evaluated by multivariate probit regression analysis. Overexpression or depletion of endogenous PRL-3 expression with the specific small interfering RNAs was performed to investigate the role of PRL-3 in AML progression. Xenograft models were also used to confirm the oncogenic role of PRL-3.

Results: Compared to healthy donors, PRL-3 is upregulated more than 3-fold in 40.2% of FLT3-ITD-negative AML patients. PRL-3 expression level is adversely correlated to the overall survival of the AML patients, and the AML relapses accompany with re-upregulation of PRL-3. Mechanistically, aberrant PRL-3 expression promoted cell cycle progression and enhanced the antiapoptotic machinery of AML cells to drug cytotoxicity through downregulation of p21 and upregulation of Cyclin D1 and CDK2 and activation of STAT5 and AKT. Depletion of endogenous PRL-3 sensitizes AML cells to therapeutic drugs, concomitant with apoptosis by upregulation of cleaved PARP (poly ADP ribose polymerase) and apoptosis-related caspases. Xenograft assays further confirmed PRL-3's oncogenic role in leukemogenesis.

Conclusions: Our results demonstrated that PRL-3 is a novel independent crucial player in both FLT3-ITD-positive and FLT3-ITD-negative AML and could be a potential therapeutic target.

Keywords: FLT3-ITD-negative; PRL-3; acute myeloid leukemia; apoptosis; cell proliferation; drug resistance.

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Figures

Figure 1
Figure 1
PRL-3 highly expresses in FLT3-ITD–negative AML patients and cells. (A-C) Comparison of PRL-3 mRNA expression in bone marrow of the 112 FLT3-ITD–negative AML patients to that of the 16 normal healthy donors (A), or the relapsed/refractory AML patients to the initially diagnosed patients (B), or the patients with complete remission to that of the initially diagnosed patients (C) by quantitative RT-PCR analysis. Statistical analysis of PRL-3 mRNA expression level was performed with SPSS, version 16.0, software. The data are indicated as mean ± standard error of the mean. (D) Detection of PRL-3 protein expression in bone marrow of the indicated FLT3-ITD–negative AML patients and healthy donors by western blotting. The standardized relative PRL-3 expression to beta-actin (ST) is indicated below each lane. (E) PRL-3 expression in the indicated human AML cell lines by western blotting. (F) PRL-3 protein expression in ML-1 cells viewed by confocal microscope (magnification, 40× times 10×).
Figure 2
Figure 2
The effect of PRL-3 on FLT3-ITD–negative AML cell proliferation. (A,B) Detection of the ectopic GFP-PRL-3 expression in U937 cells (A) and ML-1 cells (B) stably transduced with either pLVX-puro-EGFP-PRL-3 wt or EGFP-PRL-3 (C104S) expression constructs by western blotting. (C) Depletion of endogenous PRL-3 expression in ML-1 cells transfected by either 2 PRL-3–specific shRNAs (shPRL-3-1 and shPRL-3-2), respectively, or by control (Ctrl) shRNA by western blotting. (D,E) The effects of PRL-3 overexpression on the proliferation of U937 (D) and ML-1 cells (E) assayed by CCK8 cell proliferation methods. (F) The effects of endogenous PRL-3 depletion on ML-1 cell proliferation by CCK8 cell proliferation methods. (G-I) Colony formation of ML-1 (G) and U937 (H) cells transfected with either PRL-3 or its mutant, or ML-1 cells (I) with endogenous PRL-3 silencing by specific shRNA1 and shRNA2. Data were shown as mean ± standrad deviation of triplicate experiments. *P < .05; **P < .01.
Figure 3
Figure 3
The effect of PRL-3 on cell cycle and apoptosis. (A,B) The effect of PRL-3 overexpression on cell cycle progression of U937 (A) and ML-1 (B) cells with the typical histograms. The indicated cells were fixed and analyzed by flow cytometry after propidium iodide staining. The statistical analyses are indicated at the right. Data were shown as mean ± SD of triplicate experiments. *P < .05, n=3. (C) The effect of endogenous PRL-3 silenced by either 2 PRL-3–specific shRNA-1,-2, or by control shRNA on ML-1 cell cycle progression. The analysis is similar to that in (A) and (B). (D) The effect of PRL-3 overexpression on the expression of cell cycle–related CDK2, CyclinD1 and p21 by western blotting in ML-1 and U937 whole-cell lysates.
Figure 4
Figure 4
The effect of PRL-3 on drug sensitivity and drug-induced apoptosis. (A-C) Cytotoxicity analysis of U937 (A) and ML-1 (B) cells with ectopic expression of PRL-3 or its mutant, or with silenced endogenous PRL-3 by the specific shRNAs as shown (C). The cells were treated with Ara-C or doxorubicin (DNR) in the indicated concentrations for 72 hours, and assayed by CCK8 methods. (D-F) Annexin V/7-AAD staining and flow cytometry analysis of U937 (D) and ML-1 (E,F) in the indicated conditions. Cells were treated with 0.4 μM Ara-C or 0.04 μM DNR for 24 hours and analyzed with flow cytomery. The representive histograms and statistical analysis are indicated. Data were shown as mean ± SD of triplicate experiments. *P < .05; **P < .01, n=3. (G) Western blots of the apoptosis-related proteins as indicated in ML-1 whole-cell lysates. The indicated cells were treated with 0.4 μM Ara-C for 24 hours and lyzed for immunoblotting. (H) Western blots of the survival-related proteins as indicated in ML-1 and U937 whole-cell lysates.
Figure 5
Figure 5
The function of PRL-3 in tumorigenesis and leukemogenesis. (A,B) Tumor formation induced by ML-1 cells stably transfected with scrambled ShRNAs (Ctrl shRNA) or with PRL-3–specific RNAs (ShPRL-3) on the 20th day post-subcutaneous injection (psi) (A), or with empty vector (Vector) or PRL-3 (PRL-3) on the 14th day psi (B) in nude mice. Upper panel shows the dissected paired tumors from each mouse; Lower panel shows the weight (g) of the indicated tumors. *P < .05; **P < .01; n = 5 (paired Student t test). (C) Tumor formation induced by U937 cells transfected [as in (B)] in nude mice on 14th day psi. **P < .01, n = 5 (paired Student t test). (D,E) Livers (D) and spleens (E) weight (g) of the nude mice injected with the indicated ML-1 cells through tail-vein transplantation. Mice were sacrificed on the 15th day post inoculation, and the organs were dissected and measured. *P < .05; **P < .01, n = 5 (unpaired Student t test). (F) IF staining of GFP on the frozen sections of the spleens as shown in (E) to discriminate the injected GFP-labeled AML cells from mice cells. Nuclei were stained with DAPI. Bar represents 25 μm. (G,H) Livers (G) and Spleens (H) weight (g) of the nude mice injected with U937 cells stably transfected with the indicated constructs as in (D) through (E). Mice were sacrificed and dissected on the 15th day post-inoculation as in (D) through (E). *P < .05;**P < .01, n = 4-5 (unpaired Student t test).
Figure 6
Figure 6
The Kaplan-Meier survival analysis of de novo AML patients with high PRL-3 expression and low PRL-3 expression. P = .001, n = 80.
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