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A critical role of CDKN3 in Bcr-Abl-mediated tumorigenesis.

Chen Q, Chen K, Guo G, Li F, Chen C, Wang S, Nalepa G, Huang S, Chen JL - PLoS ONE (2014)

Bottom Line: Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence.Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression.Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China.

ABSTRACT
CDKN3 (cyclin-dependent kinase inhibitor 3), a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs) and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML) remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

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Overexpression of CDKN3 dramatically inhibits K562 xenografted tumor growth in nude mice.(A) Nude mice were subcutaneously injected with K562 cells stably expressing CDKN3-WT or EV. The tumor volumes were measured at indicated time points. Plotted are results from three independent experiments. Error bars, SEM; n = 9; *P<0.05. (B) Tumors were excised from mice. Shown are representative images from four independent experiments with similar results. (C) Relative volume of tumors excised from nude mice injected with K562 cells expressing CDKN3-WT or EV (control). The average volume of control tumors is set to 100%. Error bars, SEM; n = 9; *P<0.05. (D) Over a 21-day period after inoculation, tumors formed by control or CDKN3-WT overexpressing K562 cells were measured by bioluminescent imaging. Shown are representative images from at least three independent experiments with similar results. (E) CDKN3 expression in representative tumors expressing CDKN3-WT or EV was examined by Western blotting. (F) Nude mice were subcutaneously injected with K562 cells stably expressing CDKN3-C140S or EV. The tumor volumes were measured at indicated time points. Shown are volumes of tumors excised from nude mice injected with K562 cells expressing CDKN3-C140S or EV. Plotted are results from three independent experiments. Error bars, SEM; n = 9. (G) Tumors from nude mice injected with K562 cells expressing CDKN3-C140S or EV were excised from mice. Shown are representative images from three independent experiments with similar results.
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pone-0111611-g002: Overexpression of CDKN3 dramatically inhibits K562 xenografted tumor growth in nude mice.(A) Nude mice were subcutaneously injected with K562 cells stably expressing CDKN3-WT or EV. The tumor volumes were measured at indicated time points. Plotted are results from three independent experiments. Error bars, SEM; n = 9; *P<0.05. (B) Tumors were excised from mice. Shown are representative images from four independent experiments with similar results. (C) Relative volume of tumors excised from nude mice injected with K562 cells expressing CDKN3-WT or EV (control). The average volume of control tumors is set to 100%. Error bars, SEM; n = 9; *P<0.05. (D) Over a 21-day period after inoculation, tumors formed by control or CDKN3-WT overexpressing K562 cells were measured by bioluminescent imaging. Shown are representative images from at least three independent experiments with similar results. (E) CDKN3 expression in representative tumors expressing CDKN3-WT or EV was examined by Western blotting. (F) Nude mice were subcutaneously injected with K562 cells stably expressing CDKN3-C140S or EV. The tumor volumes were measured at indicated time points. Shown are volumes of tumors excised from nude mice injected with K562 cells expressing CDKN3-C140S or EV. Plotted are results from three independent experiments. Error bars, SEM; n = 9. (G) Tumors from nude mice injected with K562 cells expressing CDKN3-C140S or EV were excised from mice. Shown are representative images from three independent experiments with similar results.

Mentions: To understand whether CDKN3 regulates Bcr-Abl-mediated tumorigenesis in vivo, nude mice were subcutaneously injected with K562 cells stably expressing CDKN3-WT or empty vector as control. Tumor volumes were measured each week after inoculation. Remarkably, we observed that the tumors formed by K562 cells overexpressing the CDKN3 phosphatase grew clearly slower than those formed by control cells (Figure 2A and 2B). Statistical analysis revealed that the tumor growth was significantly inhibited by exogenous expression of CDKN3-WT in K562 cells (Figures 2C). This finding was validated via bioluminescent imaging analysis (Figure 2D) and confirmed in three independent experiments. Additionally, Western blotting analysis demonstrated the overexpression of CDKN3 in the slow growth tumors (Figure 2E). In contrast, no significant difference was observed in the growth of tumors formed by K562 cells overexpressing phosphatase-dead CDKN3-C140S and control cells (Figures 2F and 2G). Together, these findings revealed that ectopic expression of wild type CDKN3, but not the phosphatase-deficient CDKN3-C140S mutant, significantly impeded the growth of the Bcr-Abl-driven K562 cells in the nude mouse xenograft model.


A critical role of CDKN3 in Bcr-Abl-mediated tumorigenesis.

Chen Q, Chen K, Guo G, Li F, Chen C, Wang S, Nalepa G, Huang S, Chen JL - PLoS ONE (2014)

Overexpression of CDKN3 dramatically inhibits K562 xenografted tumor growth in nude mice.(A) Nude mice were subcutaneously injected with K562 cells stably expressing CDKN3-WT or EV. The tumor volumes were measured at indicated time points. Plotted are results from three independent experiments. Error bars, SEM; n = 9; *P<0.05. (B) Tumors were excised from mice. Shown are representative images from four independent experiments with similar results. (C) Relative volume of tumors excised from nude mice injected with K562 cells expressing CDKN3-WT or EV (control). The average volume of control tumors is set to 100%. Error bars, SEM; n = 9; *P<0.05. (D) Over a 21-day period after inoculation, tumors formed by control or CDKN3-WT overexpressing K562 cells were measured by bioluminescent imaging. Shown are representative images from at least three independent experiments with similar results. (E) CDKN3 expression in representative tumors expressing CDKN3-WT or EV was examined by Western blotting. (F) Nude mice were subcutaneously injected with K562 cells stably expressing CDKN3-C140S or EV. The tumor volumes were measured at indicated time points. Shown are volumes of tumors excised from nude mice injected with K562 cells expressing CDKN3-C140S or EV. Plotted are results from three independent experiments. Error bars, SEM; n = 9. (G) Tumors from nude mice injected with K562 cells expressing CDKN3-C140S or EV were excised from mice. Shown are representative images from three independent experiments with similar results.
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pone-0111611-g002: Overexpression of CDKN3 dramatically inhibits K562 xenografted tumor growth in nude mice.(A) Nude mice were subcutaneously injected with K562 cells stably expressing CDKN3-WT or EV. The tumor volumes were measured at indicated time points. Plotted are results from three independent experiments. Error bars, SEM; n = 9; *P<0.05. (B) Tumors were excised from mice. Shown are representative images from four independent experiments with similar results. (C) Relative volume of tumors excised from nude mice injected with K562 cells expressing CDKN3-WT or EV (control). The average volume of control tumors is set to 100%. Error bars, SEM; n = 9; *P<0.05. (D) Over a 21-day period after inoculation, tumors formed by control or CDKN3-WT overexpressing K562 cells were measured by bioluminescent imaging. Shown are representative images from at least three independent experiments with similar results. (E) CDKN3 expression in representative tumors expressing CDKN3-WT or EV was examined by Western blotting. (F) Nude mice were subcutaneously injected with K562 cells stably expressing CDKN3-C140S or EV. The tumor volumes were measured at indicated time points. Shown are volumes of tumors excised from nude mice injected with K562 cells expressing CDKN3-C140S or EV. Plotted are results from three independent experiments. Error bars, SEM; n = 9. (G) Tumors from nude mice injected with K562 cells expressing CDKN3-C140S or EV were excised from mice. Shown are representative images from three independent experiments with similar results.
Mentions: To understand whether CDKN3 regulates Bcr-Abl-mediated tumorigenesis in vivo, nude mice were subcutaneously injected with K562 cells stably expressing CDKN3-WT or empty vector as control. Tumor volumes were measured each week after inoculation. Remarkably, we observed that the tumors formed by K562 cells overexpressing the CDKN3 phosphatase grew clearly slower than those formed by control cells (Figure 2A and 2B). Statistical analysis revealed that the tumor growth was significantly inhibited by exogenous expression of CDKN3-WT in K562 cells (Figures 2C). This finding was validated via bioluminescent imaging analysis (Figure 2D) and confirmed in three independent experiments. Additionally, Western blotting analysis demonstrated the overexpression of CDKN3 in the slow growth tumors (Figure 2E). In contrast, no significant difference was observed in the growth of tumors formed by K562 cells overexpressing phosphatase-dead CDKN3-C140S and control cells (Figures 2F and 2G). Together, these findings revealed that ectopic expression of wild type CDKN3, but not the phosphatase-deficient CDKN3-C140S mutant, significantly impeded the growth of the Bcr-Abl-driven K562 cells in the nude mouse xenograft model.

Bottom Line: Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence.Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression.Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China.

ABSTRACT
CDKN3 (cyclin-dependent kinase inhibitor 3), a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs) and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML) remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

Show MeSH
Related in: MedlinePlus