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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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Increase in BIM after combination treatment mediated by Smad3.(A) 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, whole cell lysates were collected and subjected to Western blotting with the indicated antibodies. (B) siRNA against Smad3, and negative control were transfected into A549 cells. After 4-hour incubation, cells were washed and media containing compounds were added to each well. Cells were harvested 48 hours post-transfection for protein extraction preparation and Western blotting analysis. Figure is representative of 3 independent experiments.
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pone-0114131-g006: Increase in BIM after combination treatment mediated by Smad3.(A) 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, whole cell lysates were collected and subjected to Western blotting with the indicated antibodies. (B) siRNA against Smad3, and negative control were transfected into A549 cells. After 4-hour incubation, cells were washed and media containing compounds were added to each well. Cells were harvested 48 hours post-transfection for protein extraction preparation and Western blotting analysis. Figure is representative of 3 independent experiments.

Mentions: Smad3 has been reported to sensitize cells to the apoptotic effects of TGFβ [40], and one of the suggested mechanisms is the induction of the expression of the pro-apoptotic protein BIM (Bcl-2- interacting mediator of cell death)[44]. As shown in Fig. 6A, at 24 hours after combination treatment, we observed an increase in BIM protein expression of the high molecular weight isoform and of the lower molecular and more cytotoxic BIM. It has recently been reported that a BIM deletion polymorphisms (resulting in a BIM isoform without the BH3 domain) are involved in resistance to tyrosine kinase inhibitors in several cancer types [45], [46]. Screening of all 15 cell lines using primers described in the literature [45] we were not able to detect the BIM isoform in any of our cell lines (S5 Figure). However, knockdown of the expression of Smad3 using Smad3 siRNA resulted in a decrease in the amount of BIM after combination treatment, indicating that Smad3 expression is necessary for increased expression of BIM (Fig. 6B). It has been reported that expression of Smad7 prevents apoptosis by inhibiting the expression of BIM [47]. As can be seen in Fig. 6A with the TGFβ-dasatinib treatment that resulted in PARP cleavage, we observed reduced expression of Smad7. This decrease in Smad7 suggests a mechanism to keep the TGFβ pathway active. Our data suggest that the combination treatment of TGFβ and dasatinib induces apoptosis by increasing TGFβ activation of BIM and down-regulation of TGFβ pathway inhibitory signals, such as Smad7.


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)

Increase in BIM after combination treatment mediated by Smad3.(A) 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, whole cell lysates were collected and subjected to Western blotting with the indicated antibodies. (B) siRNA against Smad3, and negative control were transfected into A549 cells. After 4-hour incubation, cells were washed and media containing compounds were added to each well. Cells were harvested 48 hours post-transfection for protein extraction preparation and Western blotting analysis. Figure is representative of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114131-g006: Increase in BIM after combination treatment mediated by Smad3.(A) 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, whole cell lysates were collected and subjected to Western blotting with the indicated antibodies. (B) siRNA against Smad3, and negative control were transfected into A549 cells. After 4-hour incubation, cells were washed and media containing compounds were added to each well. Cells were harvested 48 hours post-transfection for protein extraction preparation and Western blotting analysis. Figure is representative of 3 independent experiments.
Mentions: Smad3 has been reported to sensitize cells to the apoptotic effects of TGFβ [40], and one of the suggested mechanisms is the induction of the expression of the pro-apoptotic protein BIM (Bcl-2- interacting mediator of cell death)[44]. As shown in Fig. 6A, at 24 hours after combination treatment, we observed an increase in BIM protein expression of the high molecular weight isoform and of the lower molecular and more cytotoxic BIM. It has recently been reported that a BIM deletion polymorphisms (resulting in a BIM isoform without the BH3 domain) are involved in resistance to tyrosine kinase inhibitors in several cancer types [45], [46]. Screening of all 15 cell lines using primers described in the literature [45] we were not able to detect the BIM isoform in any of our cell lines (S5 Figure). However, knockdown of the expression of Smad3 using Smad3 siRNA resulted in a decrease in the amount of BIM after combination treatment, indicating that Smad3 expression is necessary for increased expression of BIM (Fig. 6B). It has been reported that expression of Smad7 prevents apoptosis by inhibiting the expression of BIM [47]. As can be seen in Fig. 6A with the TGFβ-dasatinib treatment that resulted in PARP cleavage, we observed reduced expression of Smad7. This decrease in Smad7 suggests a mechanism to keep the TGFβ pathway active. Our data suggest that the combination treatment of TGFβ and dasatinib induces apoptosis by increasing TGFβ activation of BIM and down-regulation of TGFβ pathway inhibitory signals, such as Smad7.

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