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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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BDNF responsiveness of human colon cancer-derived TRKBT695I and TRKBD751N expressed in colon cancer cells.(A) COLO 205 cells and (B) Caco-2 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho-tyrosine (pY) content by immunoprecipitation (IP) and subsequent immunoblot (IB) analysis. (C) Cell lysates of COLO 205 cells from (A) and, (D) lysates of Caco-2 cells from (B) were analyzed for phospho (p) and total TRK and (p)ERK. The MEK inhibitor U0126 was applied to confirm the identity of the pERK signals. β-actin serves as loading control.
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pone-0016871-g006: BDNF responsiveness of human colon cancer-derived TRKBT695I and TRKBD751N expressed in colon cancer cells.(A) COLO 205 cells and (B) Caco-2 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho-tyrosine (pY) content by immunoprecipitation (IP) and subsequent immunoblot (IB) analysis. (C) Cell lysates of COLO 205 cells from (A) and, (D) lysates of Caco-2 cells from (B) were analyzed for phospho (p) and total TRK and (p)ERK. The MEK inhibitor U0126 was applied to confirm the identity of the pERK signals. β-actin serves as loading control.

Mentions: One possible explanation for the impaired activity of TRKBT695I and TRKBD751N in the assays described above is that we performed them in an aphysiological cellular context. Indeed, the wiring of the intracellular signaling networks and the expression pattern of TRKB regulatory factors may not be identical across cell types. Ideally, one should assess the function of the TRKB mutants in their original context, that is, in the tumor cells in which they were originally identified. However, there are no cell lines available that express TRKBT695I or TRKBD751N endogenously. Moreover, to our knowledge there is no primary human colon epithelial cell line available. Aiming to use a physiologically relevant cell system, we transduced COLO 205 and Caco-2 human colon carcinoma cells with TRKBT695I or TRKBD751N and obtained polyclonal populations stably expressing either receptor. We stimulated these cells with recombinant BDNF and subsequently measured TRKB autophosphorylation and activation of downstream effectors. Similar to what was observed in RIE-1 cells, in both COLO 205 and Caco-2 cells, TRKBT695I and TRKBD751N were less abundantly phosphorylated than wild-type TRKB upon stimulation with BDNF (Figure 6). ERK was phosphorylated upon TRKB stimulation only in Caco-2 cells, but not in COLO 205 cells (Figure 6C,D). This is probably because COLO 205 cells harbor a BRAFV600E mutation (www.sanger.ac.uk/genetics/CGP/cosmic), which is constitutively active and stimulates MAPK signaling [16]. Upon stimulation of Caco-2 cells with BDNF, neither TRKBT695I nor TRKBD751N induced ERK phosphorylation (Figure 6D). Together, these results show that the TRKBT695I and TRKBD751N mutants are less active not only in rat epithelial cells but also in two human colon carcinoma cell lines.


Functional characterization of human cancer-derived TRKB mutations.

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

BDNF responsiveness of human colon cancer-derived TRKBT695I and TRKBD751N expressed in colon cancer cells.(A) COLO 205 cells and (B) Caco-2 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho-tyrosine (pY) content by immunoprecipitation (IP) and subsequent immunoblot (IB) analysis. (C) Cell lysates of COLO 205 cells from (A) and, (D) lysates of Caco-2 cells from (B) were analyzed for phospho (p) and total TRK and (p)ERK. The MEK inhibitor U0126 was applied to confirm the identity of the pERK signals. β-actin serves as loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040757&req=5

pone-0016871-g006: BDNF responsiveness of human colon cancer-derived TRKBT695I and TRKBD751N expressed in colon cancer cells.(A) COLO 205 cells and (B) Caco-2 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho-tyrosine (pY) content by immunoprecipitation (IP) and subsequent immunoblot (IB) analysis. (C) Cell lysates of COLO 205 cells from (A) and, (D) lysates of Caco-2 cells from (B) were analyzed for phospho (p) and total TRK and (p)ERK. The MEK inhibitor U0126 was applied to confirm the identity of the pERK signals. β-actin serves as loading control.
Mentions: One possible explanation for the impaired activity of TRKBT695I and TRKBD751N in the assays described above is that we performed them in an aphysiological cellular context. Indeed, the wiring of the intracellular signaling networks and the expression pattern of TRKB regulatory factors may not be identical across cell types. Ideally, one should assess the function of the TRKB mutants in their original context, that is, in the tumor cells in which they were originally identified. However, there are no cell lines available that express TRKBT695I or TRKBD751N endogenously. Moreover, to our knowledge there is no primary human colon epithelial cell line available. Aiming to use a physiologically relevant cell system, we transduced COLO 205 and Caco-2 human colon carcinoma cells with TRKBT695I or TRKBD751N and obtained polyclonal populations stably expressing either receptor. We stimulated these cells with recombinant BDNF and subsequently measured TRKB autophosphorylation and activation of downstream effectors. Similar to what was observed in RIE-1 cells, in both COLO 205 and Caco-2 cells, TRKBT695I and TRKBD751N were less abundantly phosphorylated than wild-type TRKB upon stimulation with BDNF (Figure 6). ERK was phosphorylated upon TRKB stimulation only in Caco-2 cells, but not in COLO 205 cells (Figure 6C,D). This is probably because COLO 205 cells harbor a BRAFV600E mutation (www.sanger.ac.uk/genetics/CGP/cosmic), which is constitutively active and stimulates MAPK signaling [16]. Upon stimulation of Caco-2 cells with BDNF, neither TRKBT695I nor TRKBD751N induced ERK phosphorylation (Figure 6D). Together, these results show that the TRKBT695I and TRKBD751N mutants are less active not only in rat epithelial cells but also in two human colon carcinoma cell lines.

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