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Comparative oncogenomic analysis of copy number alterations in human and zebrafish tumors enables cancer driver discovery.

Zhang G, Hoersch S, Amsterdam A, Whittaker CA, Beert E, Catchen JM, Farrington S, Postlethwait JH, Legius E, Hopkins N, Lees JA - PLoS Genet. (2013)

Bottom Line: Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs.We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes.For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs.

View Article: PubMed Central - PubMed

Affiliation: David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts, United States of America.

ABSTRACT
The identification of cancer drivers is a major goal of current cancer research. Finding driver genes within large chromosomal events is especially challenging because such alterations encompass many genes. Previously, we demonstrated that zebrafish malignant peripheral nerve sheath tumors (MPNSTs) are highly aneuploid, much like human tumors. In this study, we examined 147 zebrafish MPNSTs by massively parallel sequencing and identified both large and focal copy number alterations (CNAs). Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs. We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes. For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs. We conclude that zebrafish-human comparative analysis represents a powerful, and broadly applicable, tool to enrich for evolutionarily conserved cancer drivers.

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Functional testing of candidate driver gene nf2 and putative passenger genes.(A,B) nf2b loss cooperates with rp or tp53 mutation to promote MPNST development. Kaplan-Meier curves showing tumor-free survival of cohorts of single and double heterozygotes derived from (A) rpL36a+/−×nf2b+/− crosses and (B) tp53M214K/+×nf2b+/− crosses. (C, D) Neither tln1 nor mcm3 mutations affect tumor onset in zebrafish MPNST models, suggesting that these genes are passengers in CNAs. Kaplan-Meier curves showing tumor-free survival of cohorts of single and double heterozygotes derived from (C) rpL36a+/−×tln1+/− and (D) tp53M214K/M214K×mcm3+/−. Fish from all crosses were genotyped by PCR for each relevant mutation at 6–8 weeks of age and housed segregated by genotype. In all panels, the numbers of fish of each genotype are shown in parenthesis, and the p values between the rp or tp53 single heterozygotes and the double mutants are shown.
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pgen-1003734-g004: Functional testing of candidate driver gene nf2 and putative passenger genes.(A,B) nf2b loss cooperates with rp or tp53 mutation to promote MPNST development. Kaplan-Meier curves showing tumor-free survival of cohorts of single and double heterozygotes derived from (A) rpL36a+/−×nf2b+/− crosses and (B) tp53M214K/+×nf2b+/− crosses. (C, D) Neither tln1 nor mcm3 mutations affect tumor onset in zebrafish MPNST models, suggesting that these genes are passengers in CNAs. Kaplan-Meier curves showing tumor-free survival of cohorts of single and double heterozygotes derived from (C) rpL36a+/−×tln1+/− and (D) tp53M214K/M214K×mcm3+/−. Fish from all crosses were genotyped by PCR for each relevant mutation at 6–8 weeks of age and housed segregated by genotype. In all panels, the numbers of fish of each genotype are shown in parenthesis, and the p values between the rp or tp53 single heterozygotes and the double mutants are shown.

Mentions: The candidate driver was NF2, a gene whose loss-function mutation is known to cause human schwannomas. Importantly, our analysis detected NF2 within large CNAs in both human and zebrafish tumors (Figure S5). In human, NF2 is on chromosome 22, which is under-represented as a whole. Zebrafish have two paralogs of NF2, nf2a, which is on an underrepresented zebrafish chromosome (5), and nf2b, which is on a neutral chromosome (21). If the nf2a and nf2b paralogs have similar roles, we reasoned that loss of either might cooperate with the initiating rp or tp53 mutations to promote MPNST development. No nf2a mutant currently exists, but we previously identified an nf2b zebrafish mutant that has a weak tumor phenotype [27]. To test if this could synergize with the rp or tp53M214K mutations, we intercrossed these lines. In both cases, double heterozygotes developed MPNSTs faster than any of the sibling single heterozygotes, affirming nf2 as a valid MPNST driver (Figure 4A, B). These findings fit with prior reports that loss of murine Nf2 and Trp53 cooperate to yield MPNST [68]. In parallel, we also tested two genes that were identified as putative passengers, based on the fact that they were gained in fish tumors but either lost (tln1; zebrafish chromosome 10, human chromosome 9) or not recurrently altered (mcm3; zebrafish chromosome 20, human chromosome 6) in human MPNSTs (Figure 4C, D). Consistent with our designation as likely passengers, the heterozygous mutation of these genes had no significant impact on the development of MPNSTs resulting from rp or tp53M214K mutation. We believe that this general methodology can employed to systematically screen the identified candidate drivers.


Comparative oncogenomic analysis of copy number alterations in human and zebrafish tumors enables cancer driver discovery.

Zhang G, Hoersch S, Amsterdam A, Whittaker CA, Beert E, Catchen JM, Farrington S, Postlethwait JH, Legius E, Hopkins N, Lees JA - PLoS Genet. (2013)

Functional testing of candidate driver gene nf2 and putative passenger genes.(A,B) nf2b loss cooperates with rp or tp53 mutation to promote MPNST development. Kaplan-Meier curves showing tumor-free survival of cohorts of single and double heterozygotes derived from (A) rpL36a+/−×nf2b+/− crosses and (B) tp53M214K/+×nf2b+/− crosses. (C, D) Neither tln1 nor mcm3 mutations affect tumor onset in zebrafish MPNST models, suggesting that these genes are passengers in CNAs. Kaplan-Meier curves showing tumor-free survival of cohorts of single and double heterozygotes derived from (C) rpL36a+/−×tln1+/− and (D) tp53M214K/M214K×mcm3+/−. Fish from all crosses were genotyped by PCR for each relevant mutation at 6–8 weeks of age and housed segregated by genotype. In all panels, the numbers of fish of each genotype are shown in parenthesis, and the p values between the rp or tp53 single heterozygotes and the double mutants are shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3757083&req=5

pgen-1003734-g004: Functional testing of candidate driver gene nf2 and putative passenger genes.(A,B) nf2b loss cooperates with rp or tp53 mutation to promote MPNST development. Kaplan-Meier curves showing tumor-free survival of cohorts of single and double heterozygotes derived from (A) rpL36a+/−×nf2b+/− crosses and (B) tp53M214K/+×nf2b+/− crosses. (C, D) Neither tln1 nor mcm3 mutations affect tumor onset in zebrafish MPNST models, suggesting that these genes are passengers in CNAs. Kaplan-Meier curves showing tumor-free survival of cohorts of single and double heterozygotes derived from (C) rpL36a+/−×tln1+/− and (D) tp53M214K/M214K×mcm3+/−. Fish from all crosses were genotyped by PCR for each relevant mutation at 6–8 weeks of age and housed segregated by genotype. In all panels, the numbers of fish of each genotype are shown in parenthesis, and the p values between the rp or tp53 single heterozygotes and the double mutants are shown.
Mentions: The candidate driver was NF2, a gene whose loss-function mutation is known to cause human schwannomas. Importantly, our analysis detected NF2 within large CNAs in both human and zebrafish tumors (Figure S5). In human, NF2 is on chromosome 22, which is under-represented as a whole. Zebrafish have two paralogs of NF2, nf2a, which is on an underrepresented zebrafish chromosome (5), and nf2b, which is on a neutral chromosome (21). If the nf2a and nf2b paralogs have similar roles, we reasoned that loss of either might cooperate with the initiating rp or tp53 mutations to promote MPNST development. No nf2a mutant currently exists, but we previously identified an nf2b zebrafish mutant that has a weak tumor phenotype [27]. To test if this could synergize with the rp or tp53M214K mutations, we intercrossed these lines. In both cases, double heterozygotes developed MPNSTs faster than any of the sibling single heterozygotes, affirming nf2 as a valid MPNST driver (Figure 4A, B). These findings fit with prior reports that loss of murine Nf2 and Trp53 cooperate to yield MPNST [68]. In parallel, we also tested two genes that were identified as putative passengers, based on the fact that they were gained in fish tumors but either lost (tln1; zebrafish chromosome 10, human chromosome 9) or not recurrently altered (mcm3; zebrafish chromosome 20, human chromosome 6) in human MPNSTs (Figure 4C, D). Consistent with our designation as likely passengers, the heterozygous mutation of these genes had no significant impact on the development of MPNSTs resulting from rp or tp53M214K mutation. We believe that this general methodology can employed to systematically screen the identified candidate drivers.

Bottom Line: Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs.We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes.For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs.

View Article: PubMed Central - PubMed

Affiliation: David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts, United States of America.

ABSTRACT
The identification of cancer drivers is a major goal of current cancer research. Finding driver genes within large chromosomal events is especially challenging because such alterations encompass many genes. Previously, we demonstrated that zebrafish malignant peripheral nerve sheath tumors (MPNSTs) are highly aneuploid, much like human tumors. In this study, we examined 147 zebrafish MPNSTs by massively parallel sequencing and identified both large and focal copy number alterations (CNAs). Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs. We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes. For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs. We conclude that zebrafish-human comparative analysis represents a powerful, and broadly applicable, tool to enrich for evolutionarily conserved cancer drivers.

Show MeSH
Related in: MedlinePlus