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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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Oncogenic potential of human cancer-derived TRKB mutants expressed in RIE-1 cells.(A) 1*106 RIE-1 cells expressing TRKB (wild-type or mutant) were subcutaneously injected into both flanks of nude mice. n = 5 for wt and P507L, n = 4 for T695I and D751N. (B) 1*106 RIE-1 cells expressing TRKB (wild-type or mutant) were subcutaneously injected into both flanks of nude mice. n = 4 for both cell lines. (C) 1*104 RIE-1 cells co-expressing BDNF and TRKB (wild-type or mutant) were subcutaneously injected into nude mice. n = 6 for wt and P507L, n = 3 for T695I and D751N. (D) 1*105 RIE-1 cells co-expressing BDNF and TRKB (wild-type or mutant) were subcutaneously injected into nude mice. n = 4 for both cell lines. In all experiments, mice were sacrificed when tumor burden reached 2 cm2 and Kaplan-Meier survival curve is shown. Statistical significance was determined with a Log-Rank test.
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pone-0016871-g005: Oncogenic potential of human cancer-derived TRKB mutants expressed in RIE-1 cells.(A) 1*106 RIE-1 cells expressing TRKB (wild-type or mutant) were subcutaneously injected into both flanks of nude mice. n = 5 for wt and P507L, n = 4 for T695I and D751N. (B) 1*106 RIE-1 cells expressing TRKB (wild-type or mutant) were subcutaneously injected into both flanks of nude mice. n = 4 for both cell lines. (C) 1*104 RIE-1 cells co-expressing BDNF and TRKB (wild-type or mutant) were subcutaneously injected into nude mice. n = 6 for wt and P507L, n = 3 for T695I and D751N. (D) 1*105 RIE-1 cells co-expressing BDNF and TRKB (wild-type or mutant) were subcutaneously injected into nude mice. n = 4 for both cell lines. In all experiments, mice were sacrificed when tumor burden reached 2 cm2 and Kaplan-Meier survival curve is shown. Statistical significance was determined with a Log-Rank test.

Mentions: Next, we tested the oncogenic potential of the TRKB mutants in vivo, in mouse xenograft experiments. We subcutaneously inoculated Balb/c nude mice with wild-type or mutant TRKB-expressing cells. In the absence of BDNF, only wild-type TRKB-, TRKBL138F- and TRKBP507L-expressing RIE-1 cells (Figure 5A,B, Figure S2A,B) and RK3E cells (Figure S2E,F) formed large tumors with short latencies, but none of the TRKBT695I- or TRKBD751N-expressing cells did. Similar to this, also in the presence of co-expressed BDNF, TRKBL138F and TRKBP507L caused tumors similarly to wild-type TRKB in RIE-1 and RK3E cells (Figure 5C,D, Figure S2C,D and Figure S2G,H). In this setting, RIE-1 TRKBT695I+BDNF–expressing cells also formed tumors, but with a statistically significant delay, compared to RIE-1 TRKBwt+BDNF–expressing cells (Figure 5C, S2C). Of note, when 1*105 cells were inoculated, also RIE-1 TRKBD751N+BDNF-expressing cells formed tumors, but with a significant longer latency than those induced by RIE-1 TRKBwt+BDNF–expressing cells (data not shown). In RK3E cells, expression of TRKBD751N did not lead to tumor formation, even with high cell numbers and in the presence of BDNF, whereas RK3E TRKBT695I+BDNF-expressing cells formed tumors with a significant longer latency compared to RK3E TRKBwt+BDNF-expressing cells (Figure S2G). Notwithstanding some differences between the two cell lines, overall, these findings indicate that TRKBT695I and TRKBD751N are impaired in transforming rat epithelial cells also in vivo. Furthermore, neither TRKBL138F nor TRKBP507L displays increased oncogenic activity compared to wild-type TRKB.


Functional characterization of human cancer-derived TRKB mutations.

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

Oncogenic potential of human cancer-derived TRKB mutants expressed in RIE-1 cells.(A) 1*106 RIE-1 cells expressing TRKB (wild-type or mutant) were subcutaneously injected into both flanks of nude mice. n = 5 for wt and P507L, n = 4 for T695I and D751N. (B) 1*106 RIE-1 cells expressing TRKB (wild-type or mutant) were subcutaneously injected into both flanks of nude mice. n = 4 for both cell lines. (C) 1*104 RIE-1 cells co-expressing BDNF and TRKB (wild-type or mutant) were subcutaneously injected into nude mice. n = 6 for wt and P507L, n = 3 for T695I and D751N. (D) 1*105 RIE-1 cells co-expressing BDNF and TRKB (wild-type or mutant) were subcutaneously injected into nude mice. n = 4 for both cell lines. In all experiments, mice were sacrificed when tumor burden reached 2 cm2 and Kaplan-Meier survival curve is shown. Statistical significance was determined with a Log-Rank test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040757&req=5

pone-0016871-g005: Oncogenic potential of human cancer-derived TRKB mutants expressed in RIE-1 cells.(A) 1*106 RIE-1 cells expressing TRKB (wild-type or mutant) were subcutaneously injected into both flanks of nude mice. n = 5 for wt and P507L, n = 4 for T695I and D751N. (B) 1*106 RIE-1 cells expressing TRKB (wild-type or mutant) were subcutaneously injected into both flanks of nude mice. n = 4 for both cell lines. (C) 1*104 RIE-1 cells co-expressing BDNF and TRKB (wild-type or mutant) were subcutaneously injected into nude mice. n = 6 for wt and P507L, n = 3 for T695I and D751N. (D) 1*105 RIE-1 cells co-expressing BDNF and TRKB (wild-type or mutant) were subcutaneously injected into nude mice. n = 4 for both cell lines. In all experiments, mice were sacrificed when tumor burden reached 2 cm2 and Kaplan-Meier survival curve is shown. Statistical significance was determined with a Log-Rank test.
Mentions: Next, we tested the oncogenic potential of the TRKB mutants in vivo, in mouse xenograft experiments. We subcutaneously inoculated Balb/c nude mice with wild-type or mutant TRKB-expressing cells. In the absence of BDNF, only wild-type TRKB-, TRKBL138F- and TRKBP507L-expressing RIE-1 cells (Figure 5A,B, Figure S2A,B) and RK3E cells (Figure S2E,F) formed large tumors with short latencies, but none of the TRKBT695I- or TRKBD751N-expressing cells did. Similar to this, also in the presence of co-expressed BDNF, TRKBL138F and TRKBP507L caused tumors similarly to wild-type TRKB in RIE-1 and RK3E cells (Figure 5C,D, Figure S2C,D and Figure S2G,H). In this setting, RIE-1 TRKBT695I+BDNF–expressing cells also formed tumors, but with a statistically significant delay, compared to RIE-1 TRKBwt+BDNF–expressing cells (Figure 5C, S2C). Of note, when 1*105 cells were inoculated, also RIE-1 TRKBD751N+BDNF-expressing cells formed tumors, but with a significant longer latency than those induced by RIE-1 TRKBwt+BDNF–expressing cells (data not shown). In RK3E cells, expression of TRKBD751N did not lead to tumor formation, even with high cell numbers and in the presence of BDNF, whereas RK3E TRKBT695I+BDNF-expressing cells formed tumors with a significant longer latency compared to RK3E TRKBwt+BDNF-expressing cells (Figure S2G). Notwithstanding some differences between the two cell lines, overall, these findings indicate that TRKBT695I and TRKBD751N are impaired in transforming rat epithelial cells also in vivo. Furthermore, neither TRKBL138F nor TRKBP507L displays increased oncogenic activity compared to wild-type TRKB.

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