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Transforming growth factor β signaling overcomes dasatinib resistance in lung cancer.

Gordian E, Li J, Pevzner Y, Mediavilla-Varela M, Luddy K, Ohaegbulam K, Daniel KG, Haura EB, Muñoz-Antonia T - PLoS ONE (2014)

Bottom Line: As a result, TKIs often developed for a specific purpose have been found to act on other targets.We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFβ and dasatinib, apoptosis was induced.Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFβ-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology Program, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612, United States of America.

ABSTRACT
Lung cancer is the second most common cancer and the leading cause of cancer-related deaths. Despite recent advances in the development of targeted therapies, patients with advanced disease remain incurable, mostly because metastatic non-small cell lung carcinomas (NSCLC) eventually become resistant to tyrosine kinase inhibitors (TKIs). Kinase inhibitors have the potential for target promiscuity because the kinase super family is the largest family of druggable genes that binds to a common substrate (ATP). As a result, TKIs often developed for a specific purpose have been found to act on other targets. Drug affinity chromatography has been used to show that dasatinib interacts with the TGFβ type I receptor (TβR-I), a serine-threonine kinase. To determine the potential biological relevance of this association, we studied the combined effects of dasatinib and TGFβ on lung cancer cell lines. We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFβ and dasatinib, apoptosis was induced. Combined TGFβ-1 + dasatinib treatment had no effect on the activity of Smad2 or other non-canonical TGFβ intracellular mediators. Interestingly, combined TGFβ and dasatinib treatment resulted in a transient increase in p-Smad3 (seen after 3 hours). In addition, when NSCLC cells were treated with this combination, the pro-apoptotic protein BIM was up-regulated. Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFβ-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM. Dasatinib is only effective in killing EGFR mutant cells, which is shown in only 10% of NSCLCs. Therefore, the observation that wild-type EGFR lung cancers can be manipulated to render them sensitive to killing by dasatinib could have important implications for devising innovative and potentially more efficacious treatment strategies for this disease.

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Related in: MedlinePlus

Dasatinib does not affect the intracellular localization of phosphorylated Smads phosphorylation of non-canonical intermediaries after TGFβ treatment.A549 cells were treated with DMSO, 5 ng/mL TGFβ-1, 100 nM dasatinib, or a combination of 5 ng/mL TGFβ-1 and 100 nM dasatinib for 48 hours. After treatment, cell lysates were collected and the nuclear and cytoplasmic fractions were separated as described before [35]. After fractionation, lysates were subjected to Western blotting with the indicated antibodies.
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pone-0114131-g005: Dasatinib does not affect the intracellular localization of phosphorylated Smads phosphorylation of non-canonical intermediaries after TGFβ treatment.A549 cells were treated with DMSO, 5 ng/mL TGFβ-1, 100 nM dasatinib, or a combination of 5 ng/mL TGFβ-1 and 100 nM dasatinib for 48 hours. After treatment, cell lysates were collected and the nuclear and cytoplasmic fractions were separated as described before [35]. After fractionation, lysates were subjected to Western blotting with the indicated antibodies.

Mentions: Localization and activity of Smads proteins are dependent of the phosphorylation status in the COOH and linker regions [41]. To determine whether the increase in apoptosis observed with the combination treatment is due to alterations in the location of the Smads, we examined the effect of the combined TGFβ-dasatinib treatment on localization of the active Smads. After 48 hours of TGFβ-1 or combination treatment of TGFβ-dasatinib, cell extracts were fractionated and the phosphorylated Smads were examined in each fraction. As shown in Fig. 5A, the Smad2 phosphorylated in the carboxy termini (p-Smad2 COOH) increases predominantly in the nucleus. The Smad2 phosphorylated in the linker region (p-Smad2 Linker) also increases after treatment with TGFβ-1, but a greater amount remains in the cytoplasm. Interestingly, the amount of phosphorylated Smad3 after TGFβ-1 treatment also accumulates in the nucleus, independent of dasatinib treatment (Fig. 5B).


Transforming growth factor β signaling overcomes dasatinib resistance in lung cancer.

Gordian E, Li J, Pevzner Y, Mediavilla-Varela M, Luddy K, Ohaegbulam K, Daniel KG, Haura EB, Muñoz-Antonia T - PLoS ONE (2014)

Dasatinib does not affect the intracellular localization of phosphorylated Smads phosphorylation of non-canonical intermediaries after TGFβ treatment.A549 cells were treated with DMSO, 5 ng/mL TGFβ-1, 100 nM dasatinib, or a combination of 5 ng/mL TGFβ-1 and 100 nM dasatinib for 48 hours. After treatment, cell lysates were collected and the nuclear and cytoplasmic fractions were separated as described before [35]. After fractionation, lysates were subjected to Western blotting with the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263601&req=5

pone-0114131-g005: Dasatinib does not affect the intracellular localization of phosphorylated Smads phosphorylation of non-canonical intermediaries after TGFβ treatment.A549 cells were treated with DMSO, 5 ng/mL TGFβ-1, 100 nM dasatinib, or a combination of 5 ng/mL TGFβ-1 and 100 nM dasatinib for 48 hours. After treatment, cell lysates were collected and the nuclear and cytoplasmic fractions were separated as described before [35]. After fractionation, lysates were subjected to Western blotting with the indicated antibodies.
Mentions: Localization and activity of Smads proteins are dependent of the phosphorylation status in the COOH and linker regions [41]. To determine whether the increase in apoptosis observed with the combination treatment is due to alterations in the location of the Smads, we examined the effect of the combined TGFβ-dasatinib treatment on localization of the active Smads. After 48 hours of TGFβ-1 or combination treatment of TGFβ-dasatinib, cell extracts were fractionated and the phosphorylated Smads were examined in each fraction. As shown in Fig. 5A, the Smad2 phosphorylated in the carboxy termini (p-Smad2 COOH) increases predominantly in the nucleus. The Smad2 phosphorylated in the linker region (p-Smad2 Linker) also increases after treatment with TGFβ-1, but a greater amount remains in the cytoplasm. Interestingly, the amount of phosphorylated Smad3 after TGFβ-1 treatment also accumulates in the nucleus, independent of dasatinib treatment (Fig. 5B).

Bottom Line: As a result, TKIs often developed for a specific purpose have been found to act on other targets.We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFβ and dasatinib, apoptosis was induced.Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFβ-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology Program, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612, United States of America.

ABSTRACT
Lung cancer is the second most common cancer and the leading cause of cancer-related deaths. Despite recent advances in the development of targeted therapies, patients with advanced disease remain incurable, mostly because metastatic non-small cell lung carcinomas (NSCLC) eventually become resistant to tyrosine kinase inhibitors (TKIs). Kinase inhibitors have the potential for target promiscuity because the kinase super family is the largest family of druggable genes that binds to a common substrate (ATP). As a result, TKIs often developed for a specific purpose have been found to act on other targets. Drug affinity chromatography has been used to show that dasatinib interacts with the TGFβ type I receptor (TβR-I), a serine-threonine kinase. To determine the potential biological relevance of this association, we studied the combined effects of dasatinib and TGFβ on lung cancer cell lines. We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFβ and dasatinib, apoptosis was induced. Combined TGFβ-1 + dasatinib treatment had no effect on the activity of Smad2 or other non-canonical TGFβ intracellular mediators. Interestingly, combined TGFβ and dasatinib treatment resulted in a transient increase in p-Smad3 (seen after 3 hours). In addition, when NSCLC cells were treated with this combination, the pro-apoptotic protein BIM was up-regulated. Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFβ-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM. Dasatinib is only effective in killing EGFR mutant cells, which is shown in only 10% of NSCLCs. Therefore, the observation that wild-type EGFR lung cancers can be manipulated to render them sensitive to killing by dasatinib could have important implications for devising innovative and potentially more efficacious treatment strategies for this disease.

Show MeSH
Related in: MedlinePlus