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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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Transforming potential of human cancer-derived TRKB point mutants in vitro.(A) Morphology of RIE-1 cells expressing mutant or wild-type TRKB, photographed at 50x magnification. (B) Anoikis assay, in which cells described in (A) were seeded onto ULC plates and scanned at 1x magnification 9 days later (7 days for TPR-TRKB).
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pone-0016871-g002: Transforming potential of human cancer-derived TRKB point mutants in vitro.(A) Morphology of RIE-1 cells expressing mutant or wild-type TRKB, photographed at 50x magnification. (B) Anoikis assay, in which cells described in (A) were seeded onto ULC plates and scanned at 1x magnification 9 days later (7 days for TPR-TRKB).

Mentions: Like most kinases, TRKB needs a minimal level of activation to transform epithelial cells. We hypothesized that if the cancer-derived TRKB mutations confer a gain of function, they may transform epithelial cells even in the absence (or with reduced levels) of BDNF. However, when we tested the various TRKB mutants in the absence of co-expressed ligand, none of them induced a spindle-shaped cell morphology (Figure 2A for RIE-1 cells, Figure S1B for RK3E cells) or suppressed anoikis (Figure 2B, Figure S1C) in the absence of BDNF. This was in contrast to what was observed for the constitutively active and ligand-independent TPR-TRKB mutant, which did transform RIE-1 and RK3E cells and suppressed anoikis in the absence of exogenous BDNF, consistent with our previous report [25].


Functional characterization of human cancer-derived TRKB mutations.

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

Transforming potential of human cancer-derived TRKB point mutants in vitro.(A) Morphology of RIE-1 cells expressing mutant or wild-type TRKB, photographed at 50x magnification. (B) Anoikis assay, in which cells described in (A) were seeded onto ULC plates and scanned at 1x magnification 9 days later (7 days for TPR-TRKB).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016871-g002: Transforming potential of human cancer-derived TRKB point mutants in vitro.(A) Morphology of RIE-1 cells expressing mutant or wild-type TRKB, photographed at 50x magnification. (B) Anoikis assay, in which cells described in (A) were seeded onto ULC plates and scanned at 1x magnification 9 days later (7 days for TPR-TRKB).
Mentions: Like most kinases, TRKB needs a minimal level of activation to transform epithelial cells. We hypothesized that if the cancer-derived TRKB mutations confer a gain of function, they may transform epithelial cells even in the absence (or with reduced levels) of BDNF. However, when we tested the various TRKB mutants in the absence of co-expressed ligand, none of them induced a spindle-shaped cell morphology (Figure 2A for RIE-1 cells, Figure S1B for RK3E cells) or suppressed anoikis (Figure 2B, Figure S1C) in the absence of BDNF. This was in contrast to what was observed for the constitutively active and ligand-independent TPR-TRKB mutant, which did transform RIE-1 and RK3E cells and suppressed anoikis in the absence of exogenous BDNF, consistent with our previous report [25].

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