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Tyrosine kinase ETK/BMX is up-regulated in bladder cancer and predicts poor prognosis in patients with cystectomy.

Guo S, Sun F, Guo Z, Li W, Alfano A, Chen H, Magyar CE, Huang J, Chai TC, Qiu S, Qiu Y - PLoS ONE (2011)

Bottom Line: Deregulation of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors.Immunohistochemistry analysis on tissue microarrays containing 619 human bladder tissue samples shows that ETK is significantly upregulated during bladder cancer development and progression and ETK expression level predicts the survival rate of patients with cystectomy.Taken together, our results suggest that ETK may potentially serve as a new drug target for bladder cancer treatment as well as a biomarker which could be used to identify patients with higher mortality risk, who may be benefited from therapeutics targeting ETK activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Therapeutics and The Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Deregulation of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors. In this report, we demonstrated that ETK expression is progressively increased during bladder cancer progression. We found that down-regulation of ETK in bladder cancer cells attenuated STAT3 and AKT activity whereas exogenous overexpression of ETK had opposite effects, suggesting that deregulation of ETK may attribute to the elevated activity of STAT3 and AKT frequently detected in bladder cancer. The survival, migration and invasion of bladder cancer cells were significantly compromised when ETK expression was knocked down by a specific shRNA. In addition, we showed that ETK localizes to mitochondria in bladder cancer cells through interacting with Bcl-XL and regulating ROS production and drug sensitivity. Therefore, ETK may play an important role in regulating survival, migration and invasion by modulating multiple signaling pathways in bladder cancer cells. Immunohistochemistry analysis on tissue microarrays containing 619 human bladder tissue samples shows that ETK is significantly upregulated during bladder cancer development and progression and ETK expression level predicts the survival rate of patients with cystectomy. Taken together, our results suggest that ETK may potentially serve as a new drug target for bladder cancer treatment as well as a biomarker which could be used to identify patients with higher mortality risk, who may be benefited from therapeutics targeting ETK activity.

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Regulation of survival, migration and invasion by ETK in bladder cancer cells.A, Down-regulation of ETK expression induced apoptosis in bladder cancer cells. UM-UC-3 and T24 cells were infected with lentivirus encoding the specific shRNA for ETK for 96 h and apoptosis was evaluated by TUNEL assays. Apoptotic cells were quantified by counting TUNEL-positive cells from five random independent fields. B, ETK regulates ROS activity and cytotoxicity in bladder cancer cells in response to Doxorubucin (DOX) treatment. Cells were infected with lentivirus encoding the shRNA specific for ETK, Etk T7-tagged ETK or a vector control. At 48 h postinfection, cells were treated with DOX (UM-UC-3, 0.5 µg/ml; T24 cells, 2.5 µg/ml) for 20 h and then incubated with 10 nM 5(6)-CDCFDA for 30 min. ROS-positive cells were counted under the fluorescence microscopy. The results were expressed as percentage of the control. C, ETK regulates cytotoxicity in bladder cancer cells in response to DOX treatment. Cells were infected as described in B and treated with DOX (T24, 5 µg/ml; UM-UC-3, 0.5 µg/ml) for 48 h. Cell viability was assessed by WST-1 assays (Top panel). The level of ETK expression was monitored by immunoblotting with anti-ETK (Bottom panel). *, p<0.05 compared with the control; **, p<0.01 compared with the control. D, Knockdown of ETK inhibited migration and invasion in vitro. Cell migration and invasion assay were described as Methods. The results were expressed as percentage of the control. *, p<0.05 compared with the control; **, p<0.01 compared with the control.
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pone-0017778-g003: Regulation of survival, migration and invasion by ETK in bladder cancer cells.A, Down-regulation of ETK expression induced apoptosis in bladder cancer cells. UM-UC-3 and T24 cells were infected with lentivirus encoding the specific shRNA for ETK for 96 h and apoptosis was evaluated by TUNEL assays. Apoptotic cells were quantified by counting TUNEL-positive cells from five random independent fields. B, ETK regulates ROS activity and cytotoxicity in bladder cancer cells in response to Doxorubucin (DOX) treatment. Cells were infected with lentivirus encoding the shRNA specific for ETK, Etk T7-tagged ETK or a vector control. At 48 h postinfection, cells were treated with DOX (UM-UC-3, 0.5 µg/ml; T24 cells, 2.5 µg/ml) for 20 h and then incubated with 10 nM 5(6)-CDCFDA for 30 min. ROS-positive cells were counted under the fluorescence microscopy. The results were expressed as percentage of the control. C, ETK regulates cytotoxicity in bladder cancer cells in response to DOX treatment. Cells were infected as described in B and treated with DOX (T24, 5 µg/ml; UM-UC-3, 0.5 µg/ml) for 48 h. Cell viability was assessed by WST-1 assays (Top panel). The level of ETK expression was monitored by immunoblotting with anti-ETK (Bottom panel). *, p<0.05 compared with the control; **, p<0.01 compared with the control. D, Knockdown of ETK inhibited migration and invasion in vitro. Cell migration and invasion assay were described as Methods. The results were expressed as percentage of the control. *, p<0.05 compared with the control; **, p<0.01 compared with the control.

Mentions: When we examined cellular localization of ETK in bladder cancer cells, we observed a puncate distribution of ETK in the cytoplasm. Interestingly, ETK staining was partially overlapped with Mitotracker labeling in these cells (Fig. 2A). We also detected a significant amount of ETK protein in the mitochondrial fraction, along with other known mitochondrial proteins Bcl-XL and VDAC (Fig. 2B). As ETK lacks a mitochondria targeting signal, we wondered whether it could localize to mitochondria through a protein-protein interaction. As shown in Fig. 2C, endogenous Bcl-XL was co-precipitated with ETK in both bladder cancer cell lines examined. Similar results were also obtained in 293T cells overexpressing both T7-tagged ETK and GFP-tagged Bcl-XL. These data suggested that ETK may form a complex with Bcl-XL and localize to mitochondria. Due to Bcl-XL's anti-apoptotic function, we examined the role of ETK in regulating apoptosis in these cells. As shown in Fig. 3A, knock-down of ETK expression by a specific shRNA significantly increased the number of apoptotic cells in UM-UC-3 and T24 cells. In addition, inhibition of ETK activity enhanced ROS production and cytotoxicity in bladder cancer cells in response to treatment of Doxorubucin, while overexpression of ETK had protective effects (Fig. 3B & 3C). Tumor growth and metastasis is regulated in part by the ability of tumor cells to degrade surrounding matrix tissue and migrate. To test whether ETK upregulation may cause such a phenotype, we further examined the effects of ETK knock-down on bladder cancer cell migration and invasion using the Boyden chamber assays. Fig. 3D shows that migration of ETK-knockdown UM-UC-3 and T24 cells was reduced to 40% and 60% respectively compared with the control shRNA treated cells. Similar results were also observed when we examined their ability to invade through matrigel. These data suggested that inhibition of ETK expression in bladder cancer cells compromised both migration and invasion.


Tyrosine kinase ETK/BMX is up-regulated in bladder cancer and predicts poor prognosis in patients with cystectomy.

Guo S, Sun F, Guo Z, Li W, Alfano A, Chen H, Magyar CE, Huang J, Chai TC, Qiu S, Qiu Y - PLoS ONE (2011)

Regulation of survival, migration and invasion by ETK in bladder cancer cells.A, Down-regulation of ETK expression induced apoptosis in bladder cancer cells. UM-UC-3 and T24 cells were infected with lentivirus encoding the specific shRNA for ETK for 96 h and apoptosis was evaluated by TUNEL assays. Apoptotic cells were quantified by counting TUNEL-positive cells from five random independent fields. B, ETK regulates ROS activity and cytotoxicity in bladder cancer cells in response to Doxorubucin (DOX) treatment. Cells were infected with lentivirus encoding the shRNA specific for ETK, Etk T7-tagged ETK or a vector control. At 48 h postinfection, cells were treated with DOX (UM-UC-3, 0.5 µg/ml; T24 cells, 2.5 µg/ml) for 20 h and then incubated with 10 nM 5(6)-CDCFDA for 30 min. ROS-positive cells were counted under the fluorescence microscopy. The results were expressed as percentage of the control. C, ETK regulates cytotoxicity in bladder cancer cells in response to DOX treatment. Cells were infected as described in B and treated with DOX (T24, 5 µg/ml; UM-UC-3, 0.5 µg/ml) for 48 h. Cell viability was assessed by WST-1 assays (Top panel). The level of ETK expression was monitored by immunoblotting with anti-ETK (Bottom panel). *, p<0.05 compared with the control; **, p<0.01 compared with the control. D, Knockdown of ETK inhibited migration and invasion in vitro. Cell migration and invasion assay were described as Methods. The results were expressed as percentage of the control. *, p<0.05 compared with the control; **, p<0.01 compared with the control.
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pone-0017778-g003: Regulation of survival, migration and invasion by ETK in bladder cancer cells.A, Down-regulation of ETK expression induced apoptosis in bladder cancer cells. UM-UC-3 and T24 cells were infected with lentivirus encoding the specific shRNA for ETK for 96 h and apoptosis was evaluated by TUNEL assays. Apoptotic cells were quantified by counting TUNEL-positive cells from five random independent fields. B, ETK regulates ROS activity and cytotoxicity in bladder cancer cells in response to Doxorubucin (DOX) treatment. Cells were infected with lentivirus encoding the shRNA specific for ETK, Etk T7-tagged ETK or a vector control. At 48 h postinfection, cells were treated with DOX (UM-UC-3, 0.5 µg/ml; T24 cells, 2.5 µg/ml) for 20 h and then incubated with 10 nM 5(6)-CDCFDA for 30 min. ROS-positive cells were counted under the fluorescence microscopy. The results were expressed as percentage of the control. C, ETK regulates cytotoxicity in bladder cancer cells in response to DOX treatment. Cells were infected as described in B and treated with DOX (T24, 5 µg/ml; UM-UC-3, 0.5 µg/ml) for 48 h. Cell viability was assessed by WST-1 assays (Top panel). The level of ETK expression was monitored by immunoblotting with anti-ETK (Bottom panel). *, p<0.05 compared with the control; **, p<0.01 compared with the control. D, Knockdown of ETK inhibited migration and invasion in vitro. Cell migration and invasion assay were described as Methods. The results were expressed as percentage of the control. *, p<0.05 compared with the control; **, p<0.01 compared with the control.
Mentions: When we examined cellular localization of ETK in bladder cancer cells, we observed a puncate distribution of ETK in the cytoplasm. Interestingly, ETK staining was partially overlapped with Mitotracker labeling in these cells (Fig. 2A). We also detected a significant amount of ETK protein in the mitochondrial fraction, along with other known mitochondrial proteins Bcl-XL and VDAC (Fig. 2B). As ETK lacks a mitochondria targeting signal, we wondered whether it could localize to mitochondria through a protein-protein interaction. As shown in Fig. 2C, endogenous Bcl-XL was co-precipitated with ETK in both bladder cancer cell lines examined. Similar results were also obtained in 293T cells overexpressing both T7-tagged ETK and GFP-tagged Bcl-XL. These data suggested that ETK may form a complex with Bcl-XL and localize to mitochondria. Due to Bcl-XL's anti-apoptotic function, we examined the role of ETK in regulating apoptosis in these cells. As shown in Fig. 3A, knock-down of ETK expression by a specific shRNA significantly increased the number of apoptotic cells in UM-UC-3 and T24 cells. In addition, inhibition of ETK activity enhanced ROS production and cytotoxicity in bladder cancer cells in response to treatment of Doxorubucin, while overexpression of ETK had protective effects (Fig. 3B & 3C). Tumor growth and metastasis is regulated in part by the ability of tumor cells to degrade surrounding matrix tissue and migrate. To test whether ETK upregulation may cause such a phenotype, we further examined the effects of ETK knock-down on bladder cancer cell migration and invasion using the Boyden chamber assays. Fig. 3D shows that migration of ETK-knockdown UM-UC-3 and T24 cells was reduced to 40% and 60% respectively compared with the control shRNA treated cells. Similar results were also observed when we examined their ability to invade through matrigel. These data suggested that inhibition of ETK expression in bladder cancer cells compromised both migration and invasion.

Bottom Line: Deregulation of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors.Immunohistochemistry analysis on tissue microarrays containing 619 human bladder tissue samples shows that ETK is significantly upregulated during bladder cancer development and progression and ETK expression level predicts the survival rate of patients with cystectomy.Taken together, our results suggest that ETK may potentially serve as a new drug target for bladder cancer treatment as well as a biomarker which could be used to identify patients with higher mortality risk, who may be benefited from therapeutics targeting ETK activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Therapeutics and The Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Deregulation of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors. In this report, we demonstrated that ETK expression is progressively increased during bladder cancer progression. We found that down-regulation of ETK in bladder cancer cells attenuated STAT3 and AKT activity whereas exogenous overexpression of ETK had opposite effects, suggesting that deregulation of ETK may attribute to the elevated activity of STAT3 and AKT frequently detected in bladder cancer. The survival, migration and invasion of bladder cancer cells were significantly compromised when ETK expression was knocked down by a specific shRNA. In addition, we showed that ETK localizes to mitochondria in bladder cancer cells through interacting with Bcl-XL and regulating ROS production and drug sensitivity. Therefore, ETK may play an important role in regulating survival, migration and invasion by modulating multiple signaling pathways in bladder cancer cells. Immunohistochemistry analysis on tissue microarrays containing 619 human bladder tissue samples shows that ETK is significantly upregulated during bladder cancer development and progression and ETK expression level predicts the survival rate of patients with cystectomy. Taken together, our results suggest that ETK may potentially serve as a new drug target for bladder cancer treatment as well as a biomarker which could be used to identify patients with higher mortality risk, who may be benefited from therapeutics targeting ETK activity.

Show MeSH
Related in: MedlinePlus