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Clonality and evolutionary history of rhabdomyosarcoma.

Chen L, Shern JF, Wei JS, Yohe ME, Song YK, Hurd L, Liao H, Catchpoole D, Skapek SX, Barr FG, Hawkins DS, Khan J - PLoS Genet. (2015)

Bottom Line: Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype.We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1.Our findings provide information critical to the understanding of tumorigenesis of RMS.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, Oncogenomics Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.

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PKN1 E216K mutation prevented muscle differentiation in C2C12 mouse myoblasts.(a) C2C12 cells were transduced with viral constructs containing either empty vector (pBabe), wild type PKN1 (HA-PKN1 wt) or PKN1 E216 mutant (HA-PKN1 E216K). Immunofluorescence performed for myosin heavy chain showed a striking arrest of terminal muscle differentiation. The size bar represents 400um. (b) Quantitation of the observed phenotype showed a significant decrease myosin heavy chain positive cells when PKN1 E216K is expressed. (c) Immunoblot demonstrating expression of HA tagged wild type and mutant PKN1. (d) Gene set enrichment analysis (GSEA) of gene expression profiles demonstrated that PKN1 E216K mutant activated YAP1 target genes as well as cancer invasion-associated genes; while repressed muscle differentiation program in the myoblasts.
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pgen.1005075.g005: PKN1 E216K mutation prevented muscle differentiation in C2C12 mouse myoblasts.(a) C2C12 cells were transduced with viral constructs containing either empty vector (pBabe), wild type PKN1 (HA-PKN1 wt) or PKN1 E216 mutant (HA-PKN1 E216K). Immunofluorescence performed for myosin heavy chain showed a striking arrest of terminal muscle differentiation. The size bar represents 400um. (b) Quantitation of the observed phenotype showed a significant decrease myosin heavy chain positive cells when PKN1 E216K is expressed. (c) Immunoblot demonstrating expression of HA tagged wild type and mutant PKN1. (d) Gene set enrichment analysis (GSEA) of gene expression profiles demonstrated that PKN1 E216K mutant activated YAP1 target genes as well as cancer invasion-associated genes; while repressed muscle differentiation program in the myoblasts.

Mentions: Recurrent and early mutation of PKN1 in two of the evaluated tumors led us to hypothesize that these changes were involved in myogenic differentiation. To test our hypothesis and assess the potential of our method in identifying driver mutations, we conducted a cell line study described in this section. PKN1 is a member of the AGC-subfamily of serine/threonine kinases. The protein product of the PKN1 gene is composed of a C-terminal kinase domain with significant homology to that of the protein kinase C isoforms, but a unique auto-inhibitory N-terminus made up of 3 homologous stretches of anti-parallel coiled-coil folds (ACC1–3) known to bind to Rho-family GTPases in a nucleotide-dependent manner followed by a C2-like region, known to bind phospholipids and fatty acids. Rho-GTPase binding to the ACC regions causes a conformational change that allows PKN1 to be phosphorylated and activated by PDK1. Active PKN1 plays a role in diverse cellular processes such as regulation of the actin cytoskeleton, cell adhesion, vesicular transport and glucose metabolism [30]. PKN1 represses WNT/CTNNB1 signaling [31] and stimulates the ATF2 and MEF2A transcription factors via a signaling pathway that involves MAP2K3/MAP2K6 and MAPK12 [32]. The observed mutations in RMS202 (E216K) and RMS2035 (A298T) occurred prior to 30% and 42% of molecular lifetime of the tumors, respectively. Both mutations occur in the region of the third ACC domain and could potentially interfere with regulation of the kinase activity of PKN1. To test the functional consequences of the PKN1 E216K mutation, wild type and mutant PKN1 viral constructs were made and transduced into the mouse skeletal muscle precursor cell line C2C12. Using a differentiation assay described by [33], defects in terminal differentiation, reflected in expression of myosin heavy chain (MHC) were observed with the mutated version of PKN1, (Fig. 5A-C), suggesting that the mutant PKN1 prevented the C2C12 cell differentiation. C2C12 cells expressing wild type PKN1 could be induced to express MHC although cell fusion, as determined by the number of nuclei per MHC positive cell, was significantly inhibited. Since PKN1 is known to regulate the activity of several transcription factors known to play a role in myogenic differentiation, we performed expression analysis of the constructed cell lines to determined differentially expressed genes among the constructs. Interestingly, gene set enrichment analysis showed that YAP1 target genes were induced in myoblasts and skeletal muscle genes were repressed in myotubes (Fig. 5D) when the PKN1 mutation was present.


Clonality and evolutionary history of rhabdomyosarcoma.

Chen L, Shern JF, Wei JS, Yohe ME, Song YK, Hurd L, Liao H, Catchpoole D, Skapek SX, Barr FG, Hawkins DS, Khan J - PLoS Genet. (2015)

PKN1 E216K mutation prevented muscle differentiation in C2C12 mouse myoblasts.(a) C2C12 cells were transduced with viral constructs containing either empty vector (pBabe), wild type PKN1 (HA-PKN1 wt) or PKN1 E216 mutant (HA-PKN1 E216K). Immunofluorescence performed for myosin heavy chain showed a striking arrest of terminal muscle differentiation. The size bar represents 400um. (b) Quantitation of the observed phenotype showed a significant decrease myosin heavy chain positive cells when PKN1 E216K is expressed. (c) Immunoblot demonstrating expression of HA tagged wild type and mutant PKN1. (d) Gene set enrichment analysis (GSEA) of gene expression profiles demonstrated that PKN1 E216K mutant activated YAP1 target genes as well as cancer invasion-associated genes; while repressed muscle differentiation program in the myoblasts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358975&req=5

pgen.1005075.g005: PKN1 E216K mutation prevented muscle differentiation in C2C12 mouse myoblasts.(a) C2C12 cells were transduced with viral constructs containing either empty vector (pBabe), wild type PKN1 (HA-PKN1 wt) or PKN1 E216 mutant (HA-PKN1 E216K). Immunofluorescence performed for myosin heavy chain showed a striking arrest of terminal muscle differentiation. The size bar represents 400um. (b) Quantitation of the observed phenotype showed a significant decrease myosin heavy chain positive cells when PKN1 E216K is expressed. (c) Immunoblot demonstrating expression of HA tagged wild type and mutant PKN1. (d) Gene set enrichment analysis (GSEA) of gene expression profiles demonstrated that PKN1 E216K mutant activated YAP1 target genes as well as cancer invasion-associated genes; while repressed muscle differentiation program in the myoblasts.
Mentions: Recurrent and early mutation of PKN1 in two of the evaluated tumors led us to hypothesize that these changes were involved in myogenic differentiation. To test our hypothesis and assess the potential of our method in identifying driver mutations, we conducted a cell line study described in this section. PKN1 is a member of the AGC-subfamily of serine/threonine kinases. The protein product of the PKN1 gene is composed of a C-terminal kinase domain with significant homology to that of the protein kinase C isoforms, but a unique auto-inhibitory N-terminus made up of 3 homologous stretches of anti-parallel coiled-coil folds (ACC1–3) known to bind to Rho-family GTPases in a nucleotide-dependent manner followed by a C2-like region, known to bind phospholipids and fatty acids. Rho-GTPase binding to the ACC regions causes a conformational change that allows PKN1 to be phosphorylated and activated by PDK1. Active PKN1 plays a role in diverse cellular processes such as regulation of the actin cytoskeleton, cell adhesion, vesicular transport and glucose metabolism [30]. PKN1 represses WNT/CTNNB1 signaling [31] and stimulates the ATF2 and MEF2A transcription factors via a signaling pathway that involves MAP2K3/MAP2K6 and MAPK12 [32]. The observed mutations in RMS202 (E216K) and RMS2035 (A298T) occurred prior to 30% and 42% of molecular lifetime of the tumors, respectively. Both mutations occur in the region of the third ACC domain and could potentially interfere with regulation of the kinase activity of PKN1. To test the functional consequences of the PKN1 E216K mutation, wild type and mutant PKN1 viral constructs were made and transduced into the mouse skeletal muscle precursor cell line C2C12. Using a differentiation assay described by [33], defects in terminal differentiation, reflected in expression of myosin heavy chain (MHC) were observed with the mutated version of PKN1, (Fig. 5A-C), suggesting that the mutant PKN1 prevented the C2C12 cell differentiation. C2C12 cells expressing wild type PKN1 could be induced to express MHC although cell fusion, as determined by the number of nuclei per MHC positive cell, was significantly inhibited. Since PKN1 is known to regulate the activity of several transcription factors known to play a role in myogenic differentiation, we performed expression analysis of the constructed cell lines to determined differentially expressed genes among the constructs. Interestingly, gene set enrichment analysis showed that YAP1 target genes were induced in myoblasts and skeletal muscle genes were repressed in myotubes (Fig. 5D) when the PKN1 mutation was present.

Bottom Line: Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype.We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1.Our findings provide information critical to the understanding of tumorigenesis of RMS.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, Oncogenomics Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.

Show MeSH
Related in: MedlinePlus