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Functional characterization of human cancer-derived TRKB mutations.

Geiger TR, Song JY, Rosado A, Peeper DS - PLoS ONE (2011)

Bottom Line: Unexpectedly, both colon cancer-derived mutants, TRKB(T695I) and TRKB(D751N), displayed reduced activity compared to that of wild-type TRKB.Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK.In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, the Netherlands.

ABSTRACT
Cancer originates from cells that have acquired mutations in genes critical for controlling cell proliferation, survival and differentiation. Often, tumors continue to depend on these so-called driver mutations, providing the rationale for targeted anticancer therapies. To date, large-scale sequencing analyses have revealed hundreds of mutations in human tumors. However, without their functional validation it remains unclear which mutations correspond to driver, or rather bystander, mutations and, therefore, whether the mutated gene represents a target for therapeutic intervention. In human colorectal tumors, the neurotrophic receptor TRKB has been found mutated on two different sites in its kinase domain (TRKB(T695I) and TRKB(D751N)). Another site, in the extracellular part of TRKB, is mutated in a human lung adenocarcinoma cell line (TRKB(L138F)). Lastly, our own analysis has identified one additional TRKB point mutation proximal to the kinase domain (TRKB(P507L)) in a human melanoma cell line. The functional consequences of all these point mutations, however, have so far remained elusive. Previously, we have shown that TRKB is a potent suppressor of anoikis and that TRKB-expressing cells form highly invasive and metastatic tumors in nude mice. To assess the functional consequences of these four TRKB mutations, we determined their potential to suppress anoikis and to form tumors in nude mice. Unexpectedly, both colon cancer-derived mutants, TRKB(T695I) and TRKB(D751N), displayed reduced activity compared to that of wild-type TRKB. Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK. The two mutants derived from human tumor cell lines (TRKB(L138F) and TRKB(P507L)) were functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo assays. In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

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Related in: MedlinePlus

Identification of human cancer-derived TRKB point mutants and generation of cell systems.(A) Sequencing analysis of gDNA from NCI-H2009 cells harboring the TRKBL138F mutation (left panel) and from MDA-MB-435 cells harboring the TRKBP507L mutation (right panel). Asterisks (*) indicate the respective mutated bases. Black letters denote wild-type residues, red letters denote mutant residues. (B) Schematic overview over all cancer-derived TRKB point mutations analyzed in this study. LRM: Leucine-Rich Motif, Ig: Immunoglobulin-like domain, TM: transmembrane domain. Numbers indicate positions of amino acid residues. (C) Expression levels of mutant or wild-type TRKB in RIE-1 cells, analyzed on immunoblot (IB). Tubulin serves as loading control. Numbers represent quantification of TRKB signal, normalized to alpha-tubulin, and relative to Vector control. (D) Cell surface biotinylation assay showing that at least a significant fraction of all TRKB mutants localizes to the cell membrane. Total cell surface proteins were biotinylated with Sulfo-NHS-LC-Biotin, lysed and TRKB was immunoprecipitated (IP) with TRK antibody (C-14, C-13 for control). After gel electrophoresis, biotinylated TRKB was visualized with streptavidin-HRP and total TRKB with TRK antibody (C-14). All wild-type and mutant TRKB proteins became biotinylated (upper left panel). On the right hand side the specificity of the assay is demonstrated: biotin signal was only detected for full-length TRKB (upper right panel, second lane), but not for cytosolic, truncated TPR-TRKB [25] (third lane, expected at ∼50 kD), and not in the control IP (lane 4) or in the absence of Sulfo-NHS-LC-Biotin (lane 5). IP of total full-length and truncated TRKB is shown in bottom panels. Arrowheads indicate full-length TRKB, arrow indicates truncated TPR-TRKB (just below Ig heavy chains).
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pone-0016871-g001: Identification of human cancer-derived TRKB point mutants and generation of cell systems.(A) Sequencing analysis of gDNA from NCI-H2009 cells harboring the TRKBL138F mutation (left panel) and from MDA-MB-435 cells harboring the TRKBP507L mutation (right panel). Asterisks (*) indicate the respective mutated bases. Black letters denote wild-type residues, red letters denote mutant residues. (B) Schematic overview over all cancer-derived TRKB point mutations analyzed in this study. LRM: Leucine-Rich Motif, Ig: Immunoglobulin-like domain, TM: transmembrane domain. Numbers indicate positions of amino acid residues. (C) Expression levels of mutant or wild-type TRKB in RIE-1 cells, analyzed on immunoblot (IB). Tubulin serves as loading control. Numbers represent quantification of TRKB signal, normalized to alpha-tubulin, and relative to Vector control. (D) Cell surface biotinylation assay showing that at least a significant fraction of all TRKB mutants localizes to the cell membrane. Total cell surface proteins were biotinylated with Sulfo-NHS-LC-Biotin, lysed and TRKB was immunoprecipitated (IP) with TRK antibody (C-14, C-13 for control). After gel electrophoresis, biotinylated TRKB was visualized with streptavidin-HRP and total TRKB with TRK antibody (C-14). All wild-type and mutant TRKB proteins became biotinylated (upper left panel). On the right hand side the specificity of the assay is demonstrated: biotin signal was only detected for full-length TRKB (upper right panel, second lane), but not for cytosolic, truncated TPR-TRKB [25] (third lane, expected at ∼50 kD), and not in the control IP (lane 4) or in the absence of Sulfo-NHS-LC-Biotin (lane 5). IP of total full-length and truncated TRKB is shown in bottom panels. Arrowheads indicate full-length TRKB, arrow indicates truncated TPR-TRKB (just below Ig heavy chains).

Mentions: Two non-synonymous point mutations within the kinase domain of the TRKB gene have been discovered in colorectal tumors: TRKBT695I and TRKBD751N [17] (the numbering of all amino acid sequences refers to the full length human TRKB protein, accession number NP_006171.2). Another TRKB point mutation, TRKBL138F, was identified in the lung adenocarcinoma cell line NCI-H2009 [19]. This mutation lies within the leucine-rich domain of the extracellular part of the receptor, which has been shown to be required for binding of the TRKB ligand brain-derived neurotrophic factor (BDNF) and activation of the TRKB receptor [25], [27], [28]. We confirmed the presence of this mutation by sequencing genomic DNA (gDNA) isolated from NCI-H2009 cells (Figure 1A, left panel). The presence of a double peak (thymidine and cytosine) indicates that the mutation is heterozygous, consistent with the original report [19]. It results in the substitution of leucine by phenylalanine. To search for more cancer-associated TRKB point mutations, we sequenced the exons comprising the TRKB kinase domain from genomic DNA of 28 human tumor cell lines, from several tissue origins (data not shown). This analysis revealed one additional TRKB point mutation, in the MDA-MB-435 melanoma cell line. (The MDA-MB-435 cell line seems to have been classified wrongly in the past [29]. Whereas it was originally isolated from a patient with breast cancer [30], [31], recent analysis indicated that the currently available cell line is of melanoma origin [32], [33]). The TRKB mutation identified in MDA-MB-435 cells is C1520T, resulting in a substitution of proline 507 with leucine (TRKBP507L). In this case, the sequencing of gDNA revealed a single peak only (Figure 1A right panel), suggesting that the mutation is homozygous. The P507 residue is located proximal to the kinase domain in the intracellular part of the receptor. Figure 1B shows a schematic overview of the positions of all human cancer-derived TRKB point mutations analyzed in this study.


Functional characterization of human cancer-derived TRKB mutations.

Geiger TR, Song JY, Rosado A, Peeper DS - PLoS ONE (2011)

Identification of human cancer-derived TRKB point mutants and generation of cell systems.(A) Sequencing analysis of gDNA from NCI-H2009 cells harboring the TRKBL138F mutation (left panel) and from MDA-MB-435 cells harboring the TRKBP507L mutation (right panel). Asterisks (*) indicate the respective mutated bases. Black letters denote wild-type residues, red letters denote mutant residues. (B) Schematic overview over all cancer-derived TRKB point mutations analyzed in this study. LRM: Leucine-Rich Motif, Ig: Immunoglobulin-like domain, TM: transmembrane domain. Numbers indicate positions of amino acid residues. (C) Expression levels of mutant or wild-type TRKB in RIE-1 cells, analyzed on immunoblot (IB). Tubulin serves as loading control. Numbers represent quantification of TRKB signal, normalized to alpha-tubulin, and relative to Vector control. (D) Cell surface biotinylation assay showing that at least a significant fraction of all TRKB mutants localizes to the cell membrane. Total cell surface proteins were biotinylated with Sulfo-NHS-LC-Biotin, lysed and TRKB was immunoprecipitated (IP) with TRK antibody (C-14, C-13 for control). After gel electrophoresis, biotinylated TRKB was visualized with streptavidin-HRP and total TRKB with TRK antibody (C-14). All wild-type and mutant TRKB proteins became biotinylated (upper left panel). On the right hand side the specificity of the assay is demonstrated: biotin signal was only detected for full-length TRKB (upper right panel, second lane), but not for cytosolic, truncated TPR-TRKB [25] (third lane, expected at ∼50 kD), and not in the control IP (lane 4) or in the absence of Sulfo-NHS-LC-Biotin (lane 5). IP of total full-length and truncated TRKB is shown in bottom panels. Arrowheads indicate full-length TRKB, arrow indicates truncated TPR-TRKB (just below Ig heavy chains).
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Related In: Results  -  Collection

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pone-0016871-g001: Identification of human cancer-derived TRKB point mutants and generation of cell systems.(A) Sequencing analysis of gDNA from NCI-H2009 cells harboring the TRKBL138F mutation (left panel) and from MDA-MB-435 cells harboring the TRKBP507L mutation (right panel). Asterisks (*) indicate the respective mutated bases. Black letters denote wild-type residues, red letters denote mutant residues. (B) Schematic overview over all cancer-derived TRKB point mutations analyzed in this study. LRM: Leucine-Rich Motif, Ig: Immunoglobulin-like domain, TM: transmembrane domain. Numbers indicate positions of amino acid residues. (C) Expression levels of mutant or wild-type TRKB in RIE-1 cells, analyzed on immunoblot (IB). Tubulin serves as loading control. Numbers represent quantification of TRKB signal, normalized to alpha-tubulin, and relative to Vector control. (D) Cell surface biotinylation assay showing that at least a significant fraction of all TRKB mutants localizes to the cell membrane. Total cell surface proteins were biotinylated with Sulfo-NHS-LC-Biotin, lysed and TRKB was immunoprecipitated (IP) with TRK antibody (C-14, C-13 for control). After gel electrophoresis, biotinylated TRKB was visualized with streptavidin-HRP and total TRKB with TRK antibody (C-14). All wild-type and mutant TRKB proteins became biotinylated (upper left panel). On the right hand side the specificity of the assay is demonstrated: biotin signal was only detected for full-length TRKB (upper right panel, second lane), but not for cytosolic, truncated TPR-TRKB [25] (third lane, expected at ∼50 kD), and not in the control IP (lane 4) or in the absence of Sulfo-NHS-LC-Biotin (lane 5). IP of total full-length and truncated TRKB is shown in bottom panels. Arrowheads indicate full-length TRKB, arrow indicates truncated TPR-TRKB (just below Ig heavy chains).
Mentions: Two non-synonymous point mutations within the kinase domain of the TRKB gene have been discovered in colorectal tumors: TRKBT695I and TRKBD751N [17] (the numbering of all amino acid sequences refers to the full length human TRKB protein, accession number NP_006171.2). Another TRKB point mutation, TRKBL138F, was identified in the lung adenocarcinoma cell line NCI-H2009 [19]. This mutation lies within the leucine-rich domain of the extracellular part of the receptor, which has been shown to be required for binding of the TRKB ligand brain-derived neurotrophic factor (BDNF) and activation of the TRKB receptor [25], [27], [28]. We confirmed the presence of this mutation by sequencing genomic DNA (gDNA) isolated from NCI-H2009 cells (Figure 1A, left panel). The presence of a double peak (thymidine and cytosine) indicates that the mutation is heterozygous, consistent with the original report [19]. It results in the substitution of leucine by phenylalanine. To search for more cancer-associated TRKB point mutations, we sequenced the exons comprising the TRKB kinase domain from genomic DNA of 28 human tumor cell lines, from several tissue origins (data not shown). This analysis revealed one additional TRKB point mutation, in the MDA-MB-435 melanoma cell line. (The MDA-MB-435 cell line seems to have been classified wrongly in the past [29]. Whereas it was originally isolated from a patient with breast cancer [30], [31], recent analysis indicated that the currently available cell line is of melanoma origin [32], [33]). The TRKB mutation identified in MDA-MB-435 cells is C1520T, resulting in a substitution of proline 507 with leucine (TRKBP507L). In this case, the sequencing of gDNA revealed a single peak only (Figure 1A right panel), suggesting that the mutation is homozygous. The P507 residue is located proximal to the kinase domain in the intracellular part of the receptor. Figure 1B shows a schematic overview of the positions of all human cancer-derived TRKB point mutations analyzed in this study.

Bottom Line: Unexpectedly, both colon cancer-derived mutants, TRKB(T695I) and TRKB(D751N), displayed reduced activity compared to that of wild-type TRKB.Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK.In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, the Netherlands.

ABSTRACT
Cancer originates from cells that have acquired mutations in genes critical for controlling cell proliferation, survival and differentiation. Often, tumors continue to depend on these so-called driver mutations, providing the rationale for targeted anticancer therapies. To date, large-scale sequencing analyses have revealed hundreds of mutations in human tumors. However, without their functional validation it remains unclear which mutations correspond to driver, or rather bystander, mutations and, therefore, whether the mutated gene represents a target for therapeutic intervention. In human colorectal tumors, the neurotrophic receptor TRKB has been found mutated on two different sites in its kinase domain (TRKB(T695I) and TRKB(D751N)). Another site, in the extracellular part of TRKB, is mutated in a human lung adenocarcinoma cell line (TRKB(L138F)). Lastly, our own analysis has identified one additional TRKB point mutation proximal to the kinase domain (TRKB(P507L)) in a human melanoma cell line. The functional consequences of all these point mutations, however, have so far remained elusive. Previously, we have shown that TRKB is a potent suppressor of anoikis and that TRKB-expressing cells form highly invasive and metastatic tumors in nude mice. To assess the functional consequences of these four TRKB mutations, we determined their potential to suppress anoikis and to form tumors in nude mice. Unexpectedly, both colon cancer-derived mutants, TRKB(T695I) and TRKB(D751N), displayed reduced activity compared to that of wild-type TRKB. Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK. The two mutants derived from human tumor cell lines (TRKB(L138F) and TRKB(P507L)) were functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo assays. In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

Show MeSH
Related in: MedlinePlus