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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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Induction of apoptosis after treatment with TGFβ-1 and dasatinib.(A) A549 cells were treated with 100 nM of dasatinib, 1000 nM of AZD0530, or erlotinib with or without 5 ng/mL TGFβ-1 for 48 hours. (B) A549 cells were pre-treated with 10 µg/mL cycloheximide (CHX) for 1 hour, followed by TGFβ-1 plus or minus dasatinib. After incubation, cells were harvested, lysed, and PARP cleavage detected by Western Blot analysis. (C) A549 cells were seeded in 96-well plates at 5×103 per well. Cells were treated, and Cell Player 96-Well Kinetic Caspase 3/7 Reagent was added simultaneously. Treatments were done in triplicate. Values are shown as the average number of caspase 3/7 positive cells from 3 independent experiments.
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pone-0114131-g003: Induction of apoptosis after treatment with TGFβ-1 and dasatinib.(A) A549 cells were treated with 100 nM of dasatinib, 1000 nM of AZD0530, or erlotinib with or without 5 ng/mL TGFβ-1 for 48 hours. (B) A549 cells were pre-treated with 10 µg/mL cycloheximide (CHX) for 1 hour, followed by TGFβ-1 plus or minus dasatinib. After incubation, cells were harvested, lysed, and PARP cleavage detected by Western Blot analysis. (C) A549 cells were seeded in 96-well plates at 5×103 per well. Cells were treated, and Cell Player 96-Well Kinetic Caspase 3/7 Reagent was added simultaneously. Treatments were done in triplicate. Values are shown as the average number of caspase 3/7 positive cells from 3 independent experiments.

Mentions: TGFβ has been implicated in pro-apoptotic responses, as well as anti-apoptotic responses [38]; therefore, we examined whether the inhibition of proliferation seen after combined TGFβ + dasatinib in A549 cells was the result of apoptotic cell death. Apoptosis was measured by poly(ADP-ribose) polymerase (PARP) cleavage after treatment with the TGFβ-dasatinib combination. Dasatinib or TGFβ-1 alone did not induce apoptosis as measured by PARP cleavage (Fig. 3A). This is in agreement with what has been reported before, where treatment of A549 cells with dasatinib did not result in apoptosis [4]. In contrast, the combination of TGFβ + dasatinib resulted in apoptosis, specific to co-treatment with dasatinib, as incubation with an EGFR inhibitor (erlotinib) or another SRC inhibitor (AZD0530) did not result in PARP cleavage (Fig. 3A). To assess whether de novo protein synthesis is required for TGFβ + dasatinib-induced apoptosis, A549 cells were pre-treated with 10 µg/mL cycloheximide for 1 hour, followed by TGFβ-1 treatment with or without dasatinib. As shown in Fig. 3B, addition of cycloheximide did not inhibit the increase in apoptosis, suggesting that de novo protein synthesis is not required. We examined the effect of the combination treatment in 15 NSCLC cell lines (13 EGFR WT and 2 EGFR MU) and found increased PARP cleavage in 5 of them when treated with the combination TGFβ + dasatinib treatment (S2 Figure).


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)

Induction of apoptosis after treatment with TGFβ-1 and dasatinib.(A) A549 cells were treated with 100 nM of dasatinib, 1000 nM of AZD0530, or erlotinib with or without 5 ng/mL TGFβ-1 for 48 hours. (B) A549 cells were pre-treated with 10 µg/mL cycloheximide (CHX) for 1 hour, followed by TGFβ-1 plus or minus dasatinib. After incubation, cells were harvested, lysed, and PARP cleavage detected by Western Blot analysis. (C) A549 cells were seeded in 96-well plates at 5×103 per well. Cells were treated, and Cell Player 96-Well Kinetic Caspase 3/7 Reagent was added simultaneously. Treatments were done in triplicate. Values are shown as the average number of caspase 3/7 positive cells from 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114131-g003: Induction of apoptosis after treatment with TGFβ-1 and dasatinib.(A) A549 cells were treated with 100 nM of dasatinib, 1000 nM of AZD0530, or erlotinib with or without 5 ng/mL TGFβ-1 for 48 hours. (B) A549 cells were pre-treated with 10 µg/mL cycloheximide (CHX) for 1 hour, followed by TGFβ-1 plus or minus dasatinib. After incubation, cells were harvested, lysed, and PARP cleavage detected by Western Blot analysis. (C) A549 cells were seeded in 96-well plates at 5×103 per well. Cells were treated, and Cell Player 96-Well Kinetic Caspase 3/7 Reagent was added simultaneously. Treatments were done in triplicate. Values are shown as the average number of caspase 3/7 positive cells from 3 independent experiments.
Mentions: TGFβ has been implicated in pro-apoptotic responses, as well as anti-apoptotic responses [38]; therefore, we examined whether the inhibition of proliferation seen after combined TGFβ + dasatinib in A549 cells was the result of apoptotic cell death. Apoptosis was measured by poly(ADP-ribose) polymerase (PARP) cleavage after treatment with the TGFβ-dasatinib combination. Dasatinib or TGFβ-1 alone did not induce apoptosis as measured by PARP cleavage (Fig. 3A). This is in agreement with what has been reported before, where treatment of A549 cells with dasatinib did not result in apoptosis [4]. In contrast, the combination of TGFβ + dasatinib resulted in apoptosis, specific to co-treatment with dasatinib, as incubation with an EGFR inhibitor (erlotinib) or another SRC inhibitor (AZD0530) did not result in PARP cleavage (Fig. 3A). To assess whether de novo protein synthesis is required for TGFβ + dasatinib-induced apoptosis, A549 cells were pre-treated with 10 µg/mL cycloheximide for 1 hour, followed by TGFβ-1 treatment with or without dasatinib. As shown in Fig. 3B, addition of cycloheximide did not inhibit the increase in apoptosis, suggesting that de novo protein synthesis is not required. We examined the effect of the combination treatment in 15 NSCLC cell lines (13 EGFR WT and 2 EGFR MU) and found increased PARP cleavage in 5 of them when treated with the combination TGFβ + dasatinib treatment (S2 Figure).

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