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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 BDNF-activated TRKB point mutants in vitro.(A) RIE-1 cells expressing mutant or wild-type TRKB were transduced with BDNF and analyzed on immunoblot (IB). Arrowhead indicates position of BDNF. Tubulin serves as loading control. (B) Morphologic transformation of RIE-1 cells co-expressing mutant or wild-type TRKB and BDNF, photographed at 50x magnification. (C) The epithelial marker E-cadherin is downregulated in TRKB-induced, morphologically transformed, cells, as assessed by immunoblot (IB) analysis. β-actin serves as loading control. (D) Anoikis suppression by mutant or wild-type TRKB+BDNF in cells described in (A). Cells were scanned at 1x magnification 9 days after seeding onto ULC plates.
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pone-0016871-g003: Transforming potential of BDNF-activated TRKB point mutants in vitro.(A) RIE-1 cells expressing mutant or wild-type TRKB were transduced with BDNF and analyzed on immunoblot (IB). Arrowhead indicates position of BDNF. Tubulin serves as loading control. (B) Morphologic transformation of RIE-1 cells co-expressing mutant or wild-type TRKB and BDNF, photographed at 50x magnification. (C) The epithelial marker E-cadherin is downregulated in TRKB-induced, morphologically transformed, cells, as assessed by immunoblot (IB) analysis. β-actin serves as loading control. (D) Anoikis suppression by mutant or wild-type TRKB+BDNF in cells described in (A). Cells were scanned at 1x magnification 9 days after seeding onto ULC plates.

Mentions: Next, we activated the wild-type and mutant receptors by stably co-expressing human BDNF, both in RIE-1 (Figure 3A) and RK3E cells (Figure S1D). Consistent with our previous observations [24]–[26], for cells expressing wild-type TRKB+BDNF this resulted in EMT-like morphologic transformation (Figure 3B, Figure S1E), downregulation of E-cadherin (Figure 3C and Figure S1F) and anoikis suppression (Figure 3D, Figure S1G). The same was seen for cells expressing TRKBL138F+BDNF and TRKBP507L+BDNF. By contrast, TRKBT695I– and TRKBD751N–expressing cells were impaired in their response to BDNF (Figure 3B,C,D, Figure S1E,F,G). These results show that, unexpectedly, TRKBT695I and TRKBD751N are impaired in their ability to transform rat epithelial cells in vitro. Furthermore, TRKBL138F and TRKBP507L are indistinguishable from wild-type TRKB in this setting.


Functional characterization of human cancer-derived TRKB mutations.

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

Transforming potential of BDNF-activated TRKB point mutants in vitro.(A) RIE-1 cells expressing mutant or wild-type TRKB were transduced with BDNF and analyzed on immunoblot (IB). Arrowhead indicates position of BDNF. Tubulin serves as loading control. (B) Morphologic transformation of RIE-1 cells co-expressing mutant or wild-type TRKB and BDNF, photographed at 50x magnification. (C) The epithelial marker E-cadherin is downregulated in TRKB-induced, morphologically transformed, cells, as assessed by immunoblot (IB) analysis. β-actin serves as loading control. (D) Anoikis suppression by mutant or wild-type TRKB+BDNF in cells described in (A). Cells were scanned at 1x magnification 9 days after seeding onto ULC plates.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016871-g003: Transforming potential of BDNF-activated TRKB point mutants in vitro.(A) RIE-1 cells expressing mutant or wild-type TRKB were transduced with BDNF and analyzed on immunoblot (IB). Arrowhead indicates position of BDNF. Tubulin serves as loading control. (B) Morphologic transformation of RIE-1 cells co-expressing mutant or wild-type TRKB and BDNF, photographed at 50x magnification. (C) The epithelial marker E-cadherin is downregulated in TRKB-induced, morphologically transformed, cells, as assessed by immunoblot (IB) analysis. β-actin serves as loading control. (D) Anoikis suppression by mutant or wild-type TRKB+BDNF in cells described in (A). Cells were scanned at 1x magnification 9 days after seeding onto ULC plates.
Mentions: Next, we activated the wild-type and mutant receptors by stably co-expressing human BDNF, both in RIE-1 (Figure 3A) and RK3E cells (Figure S1D). Consistent with our previous observations [24]–[26], for cells expressing wild-type TRKB+BDNF this resulted in EMT-like morphologic transformation (Figure 3B, Figure S1E), downregulation of E-cadherin (Figure 3C and Figure S1F) and anoikis suppression (Figure 3D, Figure S1G). The same was seen for cells expressing TRKBL138F+BDNF and TRKBP507L+BDNF. By contrast, TRKBT695I– and TRKBD751N–expressing cells were impaired in their response to BDNF (Figure 3B,C,D, Figure S1E,F,G). These results show that, unexpectedly, TRKBT695I and TRKBD751N are impaired in their ability to transform rat epithelial cells in vitro. Furthermore, TRKBL138F and TRKBP507L are indistinguishable from wild-type TRKB in this setting.

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