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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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Interactions of dasatinib and bosutinib with TβR-I.Ligand interaction diagram of (A) bosutinib and (B) dasatinib docked into TβR-1. Ligand is represented in black. Hydrogen bonds are labeled by numbers next to the bond. Distances and angles of each hydrogen bond as labeled in the diagram are given in tables within each figure. (C) Site 1 IFD results. Average (black) and best (white) IFD scores of the four compounds docked into site 1 based on ∼50 reported poses for each compound. IFDScore is multiplied by -1 for clarity of presentation.
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pone-0114131-g001: Interactions of dasatinib and bosutinib with TβR-I.Ligand interaction diagram of (A) bosutinib and (B) dasatinib docked into TβR-1. Ligand is represented in black. Hydrogen bonds are labeled by numbers next to the bond. Distances and angles of each hydrogen bond as labeled in the diagram are given in tables within each figure. (C) Site 1 IFD results. Average (black) and best (white) IFD scores of the four compounds docked into site 1 based on ∼50 reported poses for each compound. IFDScore is multiplied by -1 for clarity of presentation.

Mentions: Experiments using drug affinity chromatography to identify molecules that interact directly with dasatinib have identified over 40 kinases, including tyrosine kinases, receptor tyrosine kinases, serine/threonine kinases, and MAP kinases. One of the serine/threonine kinases identified using this approach is TβR-I [17]. TβR-I has a cysteine-rich N-terminal domain involved in ligand binding, a single transmembrane helix, a regulatory cytoplasmic juxtamembrane region, and a C-terminal serine threonine kinase domain [36]. To examine the binding of dasatinib to TβR-I, we performed docking studies using the previously reported crystal structure of the TβR-I cytoplasmic domain [18]. As described in Materials and Methods, the site designated as Site 1 was chosen as the more likely binding site. In our studies, we compared the binding of dasatinib, bosutinib (structurally related to dasatinib), LY-364947 (TβR-I kinase commercially available inhibitor [37]), and dorsomorphin (originally used in the TβR-I crystallization studies). Each ligand was docked into Site 1 defined by the ligand's best scoring pose from the initial standard precision docking into Site 1. For each ligand, Induced Fit Docking (IFD) was scored, with multiple (∼50) protein-ligand conformations reported from which the average IFD score was calculated (Fig. 1C). Our docking results of the four ligands of interest showed that dasatinib scored the highest. IFD protocol reported that TβR-I -dasatinib formed the best scoring complex (IFD score of approximately -14,742 kcal/mol) as well as scored better on average (IFD score of approximately -14,694 kcal/mol) over all reported complexes. The best TβR-I-dorsomorphin complex had an IFD score of about −14,519 kcal/mol with the average of −14,469 kcal/mol. Bosutinib and LY-364947 performed the poorest, with best protein-ligand complexes scoring at approximately -14,353 kcal/mol and −14,154 kcal/mol, respectively, and averaging −14,313 kcal/mol and −14,119 kcal/mol. Analysis of the top pose produced by the flexible docking model reveals that bosutinib's interaction with the binding site involves four hydrogen bonds with residues Pro-439, Thr-378, Asn-341 and Tyr-219 at distances of 2.4, 2.18, 2.37 and 2.25 Å and with respective angles of 120.7, 144.8, 154.1 and 151.6 degrees (Fig. 1A). Conversely, dasatinib forms three hydrogen bonds with Arg-218, Asn-341 and His-284 at distances of 1.97, 2.00 and 2.32 Å and angles of 145.9, 150.3 and 139.7 degrees (Fig. 1B).


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)

Interactions of dasatinib and bosutinib with TβR-I.Ligand interaction diagram of (A) bosutinib and (B) dasatinib docked into TβR-1. Ligand is represented in black. Hydrogen bonds are labeled by numbers next to the bond. Distances and angles of each hydrogen bond as labeled in the diagram are given in tables within each figure. (C) Site 1 IFD results. Average (black) and best (white) IFD scores of the four compounds docked into site 1 based on ∼50 reported poses for each compound. IFDScore is multiplied by -1 for clarity of presentation.
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Related In: Results  -  Collection

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

pone-0114131-g001: Interactions of dasatinib and bosutinib with TβR-I.Ligand interaction diagram of (A) bosutinib and (B) dasatinib docked into TβR-1. Ligand is represented in black. Hydrogen bonds are labeled by numbers next to the bond. Distances and angles of each hydrogen bond as labeled in the diagram are given in tables within each figure. (C) Site 1 IFD results. Average (black) and best (white) IFD scores of the four compounds docked into site 1 based on ∼50 reported poses for each compound. IFDScore is multiplied by -1 for clarity of presentation.
Mentions: Experiments using drug affinity chromatography to identify molecules that interact directly with dasatinib have identified over 40 kinases, including tyrosine kinases, receptor tyrosine kinases, serine/threonine kinases, and MAP kinases. One of the serine/threonine kinases identified using this approach is TβR-I [17]. TβR-I has a cysteine-rich N-terminal domain involved in ligand binding, a single transmembrane helix, a regulatory cytoplasmic juxtamembrane region, and a C-terminal serine threonine kinase domain [36]. To examine the binding of dasatinib to TβR-I, we performed docking studies using the previously reported crystal structure of the TβR-I cytoplasmic domain [18]. As described in Materials and Methods, the site designated as Site 1 was chosen as the more likely binding site. In our studies, we compared the binding of dasatinib, bosutinib (structurally related to dasatinib), LY-364947 (TβR-I kinase commercially available inhibitor [37]), and dorsomorphin (originally used in the TβR-I crystallization studies). Each ligand was docked into Site 1 defined by the ligand's best scoring pose from the initial standard precision docking into Site 1. For each ligand, Induced Fit Docking (IFD) was scored, with multiple (∼50) protein-ligand conformations reported from which the average IFD score was calculated (Fig. 1C). Our docking results of the four ligands of interest showed that dasatinib scored the highest. IFD protocol reported that TβR-I -dasatinib formed the best scoring complex (IFD score of approximately -14,742 kcal/mol) as well as scored better on average (IFD score of approximately -14,694 kcal/mol) over all reported complexes. The best TβR-I-dorsomorphin complex had an IFD score of about −14,519 kcal/mol with the average of −14,469 kcal/mol. Bosutinib and LY-364947 performed the poorest, with best protein-ligand complexes scoring at approximately -14,353 kcal/mol and −14,154 kcal/mol, respectively, and averaging −14,313 kcal/mol and −14,119 kcal/mol. Analysis of the top pose produced by the flexible docking model reveals that bosutinib's interaction with the binding site involves four hydrogen bonds with residues Pro-439, Thr-378, Asn-341 and Tyr-219 at distances of 2.4, 2.18, 2.37 and 2.25 Å and with respective angles of 120.7, 144.8, 154.1 and 151.6 degrees (Fig. 1A). Conversely, dasatinib forms three hydrogen bonds with Arg-218, Asn-341 and His-284 at distances of 1.97, 2.00 and 2.32 Å and angles of 145.9, 150.3 and 139.7 degrees (Fig. 1B).

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