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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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Combination TGFβ-1 and dasatinib treatment effect on phosphorylation of canonical and non-canonical TGFβ pathway intermediaries.A549 NSCLC 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 different amounts of time (1 hour for detection of pSmad2 and pSmad3, and 48 hours for detection of pSrc). After incubation, whole cell lysates were collected and subjected to Western blotting with the indicated antibodies.
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pone-0114131-g004: Combination TGFβ-1 and dasatinib treatment effect on phosphorylation of canonical and non-canonical TGFβ pathway intermediaries.A549 NSCLC 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 different amounts of time (1 hour for detection of pSmad2 and pSmad3, and 48 hours for detection of pSrc). After incubation, whole cell lysates were collected and subjected to Western blotting with the indicated antibodies.

Mentions: TGFβ has been shown to stimulate several intracellular pathways, including the Smad pathway (canonical pathway) and other intracellular mediators, including p38 mitogen-activated protein kinase (MAPK), AKT, and ERK (non-canonical pathways)[39]. Therefore, we next examined whether TGFβ-dasatinib treatment activated these pathways by using antibodies that detect the phosphorylated (activated) form of the mediators of both the canonical and non-canonical pathways. As shown in Fig. 4, expression of activated Smad2 or Smad3 was not observed in whole cell extracts of either untreated or dasatinib-treated cells but was observed after TGFβ-1 treatment or after treatment with TGFβ + dasatinib. Interestingly, levels of phosphorylated Smad3 after treatment with TGFβ-dasatinib are higher than levels seen with TGFβ-1 treatment alone. The increase in p-Smad3 seen after 1 hour of TGFβ-dasatinib combination treatment in Smad3 activation is transient as it cannot be seen 48 hours after treatment (data not shown). This is in contrast to Smad2 phosphorylation, which can be seen as early as 5 minutes and remains phosphorylated until 48 hours. This could be due to the considerably shorter half-life of Smad3 (4.7 hours) compared to Smad2 (12.5 hours)[40]. Pre-treatment with the TβR-I inhibitor LY364947, blocks Smad2 phosphorylation in the presence or absence of dasatinib (Fig. 4B). As expected, SRC phosphorylation is inhibited in the presence of dasatinib, and incubation with TGFβ-1 does not affect this inhibition.


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)

Combination TGFβ-1 and dasatinib treatment effect on phosphorylation of canonical and non-canonical TGFβ pathway intermediaries.A549 NSCLC 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 different amounts of time (1 hour for detection of pSmad2 and pSmad3, and 48 hours for detection of pSrc). After incubation, whole cell lysates were collected and 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-g004: Combination TGFβ-1 and dasatinib treatment effect on phosphorylation of canonical and non-canonical TGFβ pathway intermediaries.A549 NSCLC 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 different amounts of time (1 hour for detection of pSmad2 and pSmad3, and 48 hours for detection of pSrc). After incubation, whole cell lysates were collected and subjected to Western blotting with the indicated antibodies.
Mentions: TGFβ has been shown to stimulate several intracellular pathways, including the Smad pathway (canonical pathway) and other intracellular mediators, including p38 mitogen-activated protein kinase (MAPK), AKT, and ERK (non-canonical pathways)[39]. Therefore, we next examined whether TGFβ-dasatinib treatment activated these pathways by using antibodies that detect the phosphorylated (activated) form of the mediators of both the canonical and non-canonical pathways. As shown in Fig. 4, expression of activated Smad2 or Smad3 was not observed in whole cell extracts of either untreated or dasatinib-treated cells but was observed after TGFβ-1 treatment or after treatment with TGFβ + dasatinib. Interestingly, levels of phosphorylated Smad3 after treatment with TGFβ-dasatinib are higher than levels seen with TGFβ-1 treatment alone. The increase in p-Smad3 seen after 1 hour of TGFβ-dasatinib combination treatment in Smad3 activation is transient as it cannot be seen 48 hours after treatment (data not shown). This is in contrast to Smad2 phosphorylation, which can be seen as early as 5 minutes and remains phosphorylated until 48 hours. This could be due to the considerably shorter half-life of Smad3 (4.7 hours) compared to Smad2 (12.5 hours)[40]. Pre-treatment with the TβR-I inhibitor LY364947, blocks Smad2 phosphorylation in the presence or absence of dasatinib (Fig. 4B). As expected, SRC phosphorylation is inhibited in the presence of dasatinib, and incubation with TGFβ-1 does not affect this inhibition.

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