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Cross-species array comparative genomic hybridization identifies novel oncogenic events in zebrafish and human embryonal rhabdomyosarcoma.

Chen EY, Dobrinski KP, Brown KH, Clagg R, Edelman E, Ignatius MS, Chen JY, Brockmann J, Nielsen GP, Ramaswamy S, Keller C, Lee C, Langenau DM - PLoS Genet. (2013)

Bottom Line: Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS--identifying critical genes associated with tumor maintenance.PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth.By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America.

ABSTRACT
Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS--identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer.

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Genes contained within low copy CNAs are expressed in primary human rhabdomyosaroma but not normal fetal muscle.Immunohistochemistry of human primary RMS and fetal muscle tissue samples. Hematoxylin and Eosin stained sections (A–C). Expression of HOXC6, CCND2, PLXNA1 and VEGFA in embryonal rhabdomyosaroma (D, G, J, M), alveolar rhabdomyosaroma (E, H, K, N) and fetal muscle (F, I, L, O). Magnified views of staining are shown in insets. In fetal muscle, PLXNA1 and VEGFA are only expressed in the vasculature (examples indicated by arrowheads in L and O and corresponding insets). The cumulative frequency of positive staining within tumor subtype is shown in the top right corner of each panel (pediatric and adult samples combined). Scale bar (panel A) = 50 µm.
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pgen-1003727-g006: Genes contained within low copy CNAs are expressed in primary human rhabdomyosaroma but not normal fetal muscle.Immunohistochemistry of human primary RMS and fetal muscle tissue samples. Hematoxylin and Eosin stained sections (A–C). Expression of HOXC6, CCND2, PLXNA1 and VEGFA in embryonal rhabdomyosaroma (D, G, J, M), alveolar rhabdomyosaroma (E, H, K, N) and fetal muscle (F, I, L, O). Magnified views of staining are shown in insets. In fetal muscle, PLXNA1 and VEGFA are only expressed in the vasculature (examples indicated by arrowheads in L and O and corresponding insets). The cumulative frequency of positive staining within tumor subtype is shown in the top right corner of each panel (pediatric and adult samples combined). Scale bar (panel A) = 50 µm.

Mentions: Having established roles for CCND2, HOXC6, PLXNA1 and VEGFA in ERMS growth, we next wanted to assess the extent to which these proteins are expressed in human primary RMS. Immunohistochemistry was performed using antibodies to CCND2, HOXC6, PLXNA1 and VEGFA in primary human tumors and fetal muscle (Supplemental Table S2). In all, 8 pediatric and 11 adult ERMS and 3 pediatric and 4 adult alveolar RMS (ARMS) were analyzed. Remarkably, CCND2, HOXC6, PLXNA1 and VEGFA protein expression were detected in a majority of RMS samples while antibody staining for each was largely negative in fetal muscle (Fig. 6). Specifically, HOXC6 protein expression was detected in 14 of 19 ERMS with strong, diffuse staining being found in 6 of the 14 cases (1 adult and 5 pediatric). In contrast, only 2 of 7 ARMS showed weak, positive staining for HOXC6, consistent with lower-level gene transcript levels being detected in pediatric ARMS compared to ERMS (Supplemental Fig. S7). CCND2, PLXNA and VEGFA were expressed at comparable frequency in both subtypes of RMS. For example, CCND2 was detected in 15 of 19 ERMS and 5 of 7 ARMS, while PLXNA1 expression was found in 17 of 19 ERMS and 6 of 7 ARMS. VEGFA antibody staining was detected in the tumor cells and the vasculature in 10 of 19 ERMS (strong staining in 1 adult and 1 pediatric case), while 6 of 7 ARMS exhibited weak staining in all cases analyzed. Additional immunohistochemical analysis of a tissue microarray from Children's Oncology Group revealed positive VEGF expression in 31 of 38 ERMS and 3 of 6 ARMS (Table S3). Of the 38 cases of ERMS, 29 cases showed strong and diffuse staining. Our analysis suggests that despite these oncogenes being infrequently amplified in human disease, their protein expression levels are elevated in a majority of human ERMS. These data imply important roles for these genes in regulating tumor growth in a large fraction of human ERMS and suggesting additional, as of yet undiscovered mechanisms that regulate expression of these genes.


Cross-species array comparative genomic hybridization identifies novel oncogenic events in zebrafish and human embryonal rhabdomyosarcoma.

Chen EY, Dobrinski KP, Brown KH, Clagg R, Edelman E, Ignatius MS, Chen JY, Brockmann J, Nielsen GP, Ramaswamy S, Keller C, Lee C, Langenau DM - PLoS Genet. (2013)

Genes contained within low copy CNAs are expressed in primary human rhabdomyosaroma but not normal fetal muscle.Immunohistochemistry of human primary RMS and fetal muscle tissue samples. Hematoxylin and Eosin stained sections (A–C). Expression of HOXC6, CCND2, PLXNA1 and VEGFA in embryonal rhabdomyosaroma (D, G, J, M), alveolar rhabdomyosaroma (E, H, K, N) and fetal muscle (F, I, L, O). Magnified views of staining are shown in insets. In fetal muscle, PLXNA1 and VEGFA are only expressed in the vasculature (examples indicated by arrowheads in L and O and corresponding insets). The cumulative frequency of positive staining within tumor subtype is shown in the top right corner of each panel (pediatric and adult samples combined). Scale bar (panel A) = 50 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757044&req=5

pgen-1003727-g006: Genes contained within low copy CNAs are expressed in primary human rhabdomyosaroma but not normal fetal muscle.Immunohistochemistry of human primary RMS and fetal muscle tissue samples. Hematoxylin and Eosin stained sections (A–C). Expression of HOXC6, CCND2, PLXNA1 and VEGFA in embryonal rhabdomyosaroma (D, G, J, M), alveolar rhabdomyosaroma (E, H, K, N) and fetal muscle (F, I, L, O). Magnified views of staining are shown in insets. In fetal muscle, PLXNA1 and VEGFA are only expressed in the vasculature (examples indicated by arrowheads in L and O and corresponding insets). The cumulative frequency of positive staining within tumor subtype is shown in the top right corner of each panel (pediatric and adult samples combined). Scale bar (panel A) = 50 µm.
Mentions: Having established roles for CCND2, HOXC6, PLXNA1 and VEGFA in ERMS growth, we next wanted to assess the extent to which these proteins are expressed in human primary RMS. Immunohistochemistry was performed using antibodies to CCND2, HOXC6, PLXNA1 and VEGFA in primary human tumors and fetal muscle (Supplemental Table S2). In all, 8 pediatric and 11 adult ERMS and 3 pediatric and 4 adult alveolar RMS (ARMS) were analyzed. Remarkably, CCND2, HOXC6, PLXNA1 and VEGFA protein expression were detected in a majority of RMS samples while antibody staining for each was largely negative in fetal muscle (Fig. 6). Specifically, HOXC6 protein expression was detected in 14 of 19 ERMS with strong, diffuse staining being found in 6 of the 14 cases (1 adult and 5 pediatric). In contrast, only 2 of 7 ARMS showed weak, positive staining for HOXC6, consistent with lower-level gene transcript levels being detected in pediatric ARMS compared to ERMS (Supplemental Fig. S7). CCND2, PLXNA and VEGFA were expressed at comparable frequency in both subtypes of RMS. For example, CCND2 was detected in 15 of 19 ERMS and 5 of 7 ARMS, while PLXNA1 expression was found in 17 of 19 ERMS and 6 of 7 ARMS. VEGFA antibody staining was detected in the tumor cells and the vasculature in 10 of 19 ERMS (strong staining in 1 adult and 1 pediatric case), while 6 of 7 ARMS exhibited weak staining in all cases analyzed. Additional immunohistochemical analysis of a tissue microarray from Children's Oncology Group revealed positive VEGF expression in 31 of 38 ERMS and 3 of 6 ARMS (Table S3). Of the 38 cases of ERMS, 29 cases showed strong and diffuse staining. Our analysis suggests that despite these oncogenes being infrequently amplified in human disease, their protein expression levels are elevated in a majority of human ERMS. These data imply important roles for these genes in regulating tumor growth in a large fraction of human ERMS and suggesting additional, as of yet undiscovered mechanisms that regulate expression of these genes.

Bottom Line: Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS--identifying critical genes associated with tumor maintenance.PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth.By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America.

ABSTRACT
Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS--identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer.

Show MeSH
Related in: MedlinePlus