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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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Inhibition of cell growth of A549 cells treated with TGFβ-1 and dasatinib.(A) A549 cells were treated with DMSO, different concentrations of TGFβ-1 (0–10 ng/mL; white bars), different concentrations of dasatinib (0–400 nM; black bars), or a combination of TGFβ-1 and dasatinib (dashed bars) for 48 hours. Treatment of cells with both agents resulted in increase inhibition (**), as compared to when cells were treated with either agent alone (*). (B) 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 48 hours, propidium iodide stained cells were analyzed to determine cell cycle distribution and analyzed as described in Materials and Methods.
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pone-0114131-g002: Inhibition of cell growth of A549 cells treated with TGFβ-1 and dasatinib.(A) A549 cells were treated with DMSO, different concentrations of TGFβ-1 (0–10 ng/mL; white bars), different concentrations of dasatinib (0–400 nM; black bars), or a combination of TGFβ-1 and dasatinib (dashed bars) for 48 hours. Treatment of cells with both agents resulted in increase inhibition (**), as compared to when cells were treated with either agent alone (*). (B) 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 48 hours, propidium iodide stained cells were analyzed to determine cell cycle distribution and analyzed as described in Materials and Methods.

Mentions: To determine the potential biological relevance of the association between dasatinib and TβR-I, A549 lung cancer cells grown in complete media containing TGFβ-1 were incubated in the presence or absence of different concentrations of dasatinib as described before[9]. Single treatment with TGFβ-1 or dasatinib inhibited cell proliferation (45% and 34%, respectively; Fig. 2A, asterisks). Interestingly, this inhibition of proliferation increased when cells were treated with a combination of TGFβ-1 and dasatinib (75% inhibition; Fig. 2A, double asterisks). The increase in inhibition of proliferation was dependent on the dasatinib dose. Similar results were obtained when cell viability was used to determine number of cells (S1 Figure). To further understand the observed inhibition in cell proliferation, we examined the effect of this combined treatment on cell cycle progression. After 48 hours of treatment with TGFβ-1 and dasatinib, the increase in cells in G1 was not significantly different from when the cells were treated with TGFβ-1 alone (Fig. 2B, solid line). Similarly, with the double treatment, there was an increase in the number of cells in G2/M after TGFβ-1 treatment, but the increase in cells was not different from when cells were treated with dasatinib alone (Fig. 2B, dashed line). Therefore, the reduction in cell number after the TGFβ-dasatinib combination treatment cannot be explained by changes in cell cycle.


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)

Inhibition of cell growth of A549 cells treated with TGFβ-1 and dasatinib.(A) A549 cells were treated with DMSO, different concentrations of TGFβ-1 (0–10 ng/mL; white bars), different concentrations of dasatinib (0–400 nM; black bars), or a combination of TGFβ-1 and dasatinib (dashed bars) for 48 hours. Treatment of cells with both agents resulted in increase inhibition (**), as compared to when cells were treated with either agent alone (*). (B) 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 48 hours, propidium iodide stained cells were analyzed to determine cell cycle distribution and analyzed as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114131-g002: Inhibition of cell growth of A549 cells treated with TGFβ-1 and dasatinib.(A) A549 cells were treated with DMSO, different concentrations of TGFβ-1 (0–10 ng/mL; white bars), different concentrations of dasatinib (0–400 nM; black bars), or a combination of TGFβ-1 and dasatinib (dashed bars) for 48 hours. Treatment of cells with both agents resulted in increase inhibition (**), as compared to when cells were treated with either agent alone (*). (B) 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 48 hours, propidium iodide stained cells were analyzed to determine cell cycle distribution and analyzed as described in Materials and Methods.
Mentions: To determine the potential biological relevance of the association between dasatinib and TβR-I, A549 lung cancer cells grown in complete media containing TGFβ-1 were incubated in the presence or absence of different concentrations of dasatinib as described before[9]. Single treatment with TGFβ-1 or dasatinib inhibited cell proliferation (45% and 34%, respectively; Fig. 2A, asterisks). Interestingly, this inhibition of proliferation increased when cells were treated with a combination of TGFβ-1 and dasatinib (75% inhibition; Fig. 2A, double asterisks). The increase in inhibition of proliferation was dependent on the dasatinib dose. Similar results were obtained when cell viability was used to determine number of cells (S1 Figure). To further understand the observed inhibition in cell proliferation, we examined the effect of this combined treatment on cell cycle progression. After 48 hours of treatment with TGFβ-1 and dasatinib, the increase in cells in G1 was not significantly different from when the cells were treated with TGFβ-1 alone (Fig. 2B, solid line). Similarly, with the double treatment, there was an increase in the number of cells in G2/M after TGFβ-1 treatment, but the increase in cells was not different from when cells were treated with dasatinib alone (Fig. 2B, dashed line). Therefore, the reduction in cell number after the TGFβ-dasatinib combination treatment cannot be explained by changes in cell cycle.

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