Limits...
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.

Show MeSH

Related in: MedlinePlus

Knockdown of endogenous TRKBL138F and TRKBP507L in tumor cell lines.(A) qRT-PCR demonstrating knockdown of TRKB mRNA levels with two non-overlapping short hairpins (sh). Average values of three independent measurements are shown, error bars represent standard deviations. (B) Knockdown of TRKB neither affects morphology of adherently growing NCI-H2009 cells, photographed at 50x magnification (top panel), nor anoikis resistance of NCI-H2009 cells in suspension (bottom panel). ULC plates were scanned at 1x magnification 6 days after seeding. (C) qRT-PCR demonstrating knockdown of TRKB mRNA levels with two non-overlapping short hairpins. Average values of three independent measurements are shown, error bars represent standard deviations. (D) Knockdown of TRKB neither affects morphology of adherently growing MDA-MB-435 cells, photographed at 50x magnification, nor anoikis resistance of MDA-MB-435 cells in suspension (bottom panel). ULC plates were scanned at 1x magnification 6 days after seeding. (E) Knockdown of TRKB in NCI-H2009 and MDA-MB-435 cells does not affect E-cadherin protein levels, as assessed by immunoblot (IB) analysis. Long and short exposures of the same blot are shown. β-actin serves as loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3040757&req=5

pone-0016871-g007: Knockdown of endogenous TRKBL138F and TRKBP507L in tumor cell lines.(A) qRT-PCR demonstrating knockdown of TRKB mRNA levels with two non-overlapping short hairpins (sh). Average values of three independent measurements are shown, error bars represent standard deviations. (B) Knockdown of TRKB neither affects morphology of adherently growing NCI-H2009 cells, photographed at 50x magnification (top panel), nor anoikis resistance of NCI-H2009 cells in suspension (bottom panel). ULC plates were scanned at 1x magnification 6 days after seeding. (C) qRT-PCR demonstrating knockdown of TRKB mRNA levels with two non-overlapping short hairpins. Average values of three independent measurements are shown, error bars represent standard deviations. (D) Knockdown of TRKB neither affects morphology of adherently growing MDA-MB-435 cells, photographed at 50x magnification, nor anoikis resistance of MDA-MB-435 cells in suspension (bottom panel). ULC plates were scanned at 1x magnification 6 days after seeding. (E) Knockdown of TRKB in NCI-H2009 and MDA-MB-435 cells does not affect E-cadherin protein levels, as assessed by immunoblot (IB) analysis. Long and short exposures of the same blot are shown. β-actin serves as loading control.

Mentions: The fact that TRKBL138F and TRKBP507L are endogenously expressed in tumor cell lines gave us the possibility to investigate whether they are required for their oncogenic phenotype. We depleted TRKBL138F and TRKBP507L from NCI-H2009 and MDA-MB-435 cells, respectively, by RNA interference with two independent, non-overlapping short hairpin (sh) RNA constructs. We achieved ∼75% downregulation of TRKB in NCI-H2009 cells (Figure 7A). However, this affected neither cell morphology (Figure 7B top panel) nor anoikis resistance (Figure 7B bottom panel). Likewise, ∼80% downregulation of TRKB in MDA-MB-435 cells (Figure 7C) had no effect on cell morphology (Figure 7D upper panel) and anoikis resistance (Figure 7D bottom panel). Consistent with these observations, E-cadherin levels were not changed upon TRKB knockdown in either cell line (Figure 7E). Furthermore, no effect on cell proliferation was observed (data not shown). Notwithstanding that TRKB was not fully depleted in either cell line, these results suggest that (mutant) TRKB in NCI-H2009 and MDA-MB-435 cells is not a major driver of their transformed phenotype.


Functional characterization of human cancer-derived TRKB mutations.

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

Knockdown of endogenous TRKBL138F and TRKBP507L in tumor cell lines.(A) qRT-PCR demonstrating knockdown of TRKB mRNA levels with two non-overlapping short hairpins (sh). Average values of three independent measurements are shown, error bars represent standard deviations. (B) Knockdown of TRKB neither affects morphology of adherently growing NCI-H2009 cells, photographed at 50x magnification (top panel), nor anoikis resistance of NCI-H2009 cells in suspension (bottom panel). ULC plates were scanned at 1x magnification 6 days after seeding. (C) qRT-PCR demonstrating knockdown of TRKB mRNA levels with two non-overlapping short hairpins. Average values of three independent measurements are shown, error bars represent standard deviations. (D) Knockdown of TRKB neither affects morphology of adherently growing MDA-MB-435 cells, photographed at 50x magnification, nor anoikis resistance of MDA-MB-435 cells in suspension (bottom panel). ULC plates were scanned at 1x magnification 6 days after seeding. (E) Knockdown of TRKB in NCI-H2009 and MDA-MB-435 cells does not affect E-cadherin protein levels, as assessed by immunoblot (IB) analysis. Long and short exposures of the same blot are shown. β-actin serves as loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016871-g007: Knockdown of endogenous TRKBL138F and TRKBP507L in tumor cell lines.(A) qRT-PCR demonstrating knockdown of TRKB mRNA levels with two non-overlapping short hairpins (sh). Average values of three independent measurements are shown, error bars represent standard deviations. (B) Knockdown of TRKB neither affects morphology of adherently growing NCI-H2009 cells, photographed at 50x magnification (top panel), nor anoikis resistance of NCI-H2009 cells in suspension (bottom panel). ULC plates were scanned at 1x magnification 6 days after seeding. (C) qRT-PCR demonstrating knockdown of TRKB mRNA levels with two non-overlapping short hairpins. Average values of three independent measurements are shown, error bars represent standard deviations. (D) Knockdown of TRKB neither affects morphology of adherently growing MDA-MB-435 cells, photographed at 50x magnification, nor anoikis resistance of MDA-MB-435 cells in suspension (bottom panel). ULC plates were scanned at 1x magnification 6 days after seeding. (E) Knockdown of TRKB in NCI-H2009 and MDA-MB-435 cells does not affect E-cadherin protein levels, as assessed by immunoblot (IB) analysis. Long and short exposures of the same blot are shown. β-actin serves as loading control.
Mentions: The fact that TRKBL138F and TRKBP507L are endogenously expressed in tumor cell lines gave us the possibility to investigate whether they are required for their oncogenic phenotype. We depleted TRKBL138F and TRKBP507L from NCI-H2009 and MDA-MB-435 cells, respectively, by RNA interference with two independent, non-overlapping short hairpin (sh) RNA constructs. We achieved ∼75% downregulation of TRKB in NCI-H2009 cells (Figure 7A). However, this affected neither cell morphology (Figure 7B top panel) nor anoikis resistance (Figure 7B bottom panel). Likewise, ∼80% downregulation of TRKB in MDA-MB-435 cells (Figure 7C) had no effect on cell morphology (Figure 7D upper panel) and anoikis resistance (Figure 7D bottom panel). Consistent with these observations, E-cadherin levels were not changed upon TRKB knockdown in either cell line (Figure 7E). Furthermore, no effect on cell proliferation was observed (data not shown). Notwithstanding that TRKB was not fully depleted in either cell line, these results suggest that (mutant) TRKB in NCI-H2009 and MDA-MB-435 cells is not a major driver of their transformed phenotype.

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