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

Qu S, Liu B, Guo X, Shi H, Zhou M, Li L, Yang S, Tong X, Wang H - Cancer (2014)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China; Department of Hematology, Fujian Provincial Hospital, Fujian Medical University, Fuzhou, China.

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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.
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fig04: 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.

Mentions: To further investigate the effect of PRL-3 in drug therapy of the AML, PRL-3 overexpressing U937 and ML-1 cells were treated with Ara-C for 72 hours. PRL-3 overexpression counteracted the drug sensitivity of AML cells to Ara-C, but the PRL-3 mutant (C104S) did not (Fig. 4A,B). In addition, knockdown of the endogenous PRL-3 obviously sensitized cells to the cytotoxicity of the drug (Fig. 4C). PRL-3 effects on drug resistance and cell apoptosis was further explored. Aberrant upregulation of wild-type PRL3 but not its mutant form sufficiently neutralized Ara-C and daunorubicin (DNR)-induced apoptosis (P < .05, Fig. 4D,E). As expected, PRL-3 depletion significantly sensitized cells to death by this drug (P < .05, Fig. 4F). Given that PRL-3 is implicated in antiapoptosis,31 the expression of apoptosis-related proteins were checked. Upon Ara-C treatment, the apoptotic proteins PARP, caspase-9, and caspase-3 were clearly activated in ML-1 cells where the expression of endogenous PRL-3 was silenced by siRNA (Fig. 4G). Because PRL-3 can activate STAT5 to confer antiapoptosis effect in FLT3-ITD positive AML cell,27 we asked if PRL-3 functions in FLT3-ITD–negative cells in a similar manner. Our immunoblotting results showed that PRL-3 can effectively enhance STAT5 phosphorylation (Fig. 4H). Moreover, activation of PI3K/AKT pathway is well known to be involved in survival, the phosphorylation status of AKT and its downstream effectors were checked in these FLT3-ITD–negative AML cells. In line with our observation in other solid tumor cells,22 PRL-3 overexpression could activate AKT to some extent, rather than the further downstream effectors, including 4E-BP1 and p70S6K in both ML-1 and U937 cells (Fig. 4H). Taken together, all above results demonstrate that PRL-3 exerts its antiapoptotic effects on drug resistance in FLT3-ITD–negative AML via regulating phosphorylation of STAT5 and AKT.


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

Qu S, Liu B, Guo X, Shi H, Zhou M, Li L, Yang S, Tong X, Wang H - Cancer (2014)

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.
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fig04: 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.
Mentions: To further investigate the effect of PRL-3 in drug therapy of the AML, PRL-3 overexpressing U937 and ML-1 cells were treated with Ara-C for 72 hours. PRL-3 overexpression counteracted the drug sensitivity of AML cells to Ara-C, but the PRL-3 mutant (C104S) did not (Fig. 4A,B). In addition, knockdown of the endogenous PRL-3 obviously sensitized cells to the cytotoxicity of the drug (Fig. 4C). PRL-3 effects on drug resistance and cell apoptosis was further explored. Aberrant upregulation of wild-type PRL3 but not its mutant form sufficiently neutralized Ara-C and daunorubicin (DNR)-induced apoptosis (P < .05, Fig. 4D,E). As expected, PRL-3 depletion significantly sensitized cells to death by this drug (P < .05, Fig. 4F). Given that PRL-3 is implicated in antiapoptosis,31 the expression of apoptosis-related proteins were checked. Upon Ara-C treatment, the apoptotic proteins PARP, caspase-9, and caspase-3 were clearly activated in ML-1 cells where the expression of endogenous PRL-3 was silenced by siRNA (Fig. 4G). Because PRL-3 can activate STAT5 to confer antiapoptosis effect in FLT3-ITD positive AML cell,27 we asked if PRL-3 functions in FLT3-ITD–negative cells in a similar manner. Our immunoblotting results showed that PRL-3 can effectively enhance STAT5 phosphorylation (Fig. 4H). Moreover, activation of PI3K/AKT pathway is well known to be involved in survival, the phosphorylation status of AKT and its downstream effectors were checked in these FLT3-ITD–negative AML cells. In line with our observation in other solid tumor cells,22 PRL-3 overexpression could activate AKT to some extent, rather than the further downstream effectors, including 4E-BP1 and p70S6K in both ML-1 and U937 cells (Fig. 4H). Taken together, all above results demonstrate that PRL-3 exerts its antiapoptotic effects on drug resistance in FLT3-ITD–negative AML via regulating phosphorylation of STAT5 and AKT.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China; Department of Hematology, Fujian Provincial Hospital, Fujian Medical University, Fuzhou, China.

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Related in: MedlinePlus