Limits...
Acquired genetic alterations in tumor cells dictate the development of high-risk neuroblastoma and clinical outcomes.

Khan FH, Pandian V, Ramraj S, Natarajan M, Aravindan S, Herman TS, Aravindan N - BMC Cancer (2015)

Bottom Line: Under serum-free conditions, MSDACs demonstrated profound tumorosphere formation ex vivo.Tissue microarray analysis coupled with automated IHC revealed significant association of RALYL to the tumor grade in a cohort of 25 neuroblastoma patients.Clinical outcome association analysis showed a strong correlation between the expression of CFHR3, CSMD3, MDFIC, FOXP2, RALYL, POLDIP3, SLC25A17, SERHL, MGAT3, TTLL1, or LOC400927 and overall and relapse-free survival in patients with neuroblastoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of Oklahoma Health Sciences Science Center, 940 Stanton L. Young Blvd., BMSB 737, Oklahoma City, OK, 73104, USA. Faizan-Khan@ouhsc.edu.

ABSTRACT

Background: Determining the driving factors and molecular flow-through that define the switch from favorable to aggressive high-risk disease is critical to the betterment of neuroblastoma cure.

Methods: In this study, we examined the cytogenetic and tumorigenic physiognomies of distinct population of metastatic site- derived aggressive cells (MSDACs) from high-risk tumors, and showed the influence of acquired genetic rearrangements on poor patient outcomes.

Results: Karyotyping in SH-SY5Y and MSDACs revealed trisomy of 1q, with additional non-random chromosomal rearrangements on 1q32, 8p23, 9q34, 15q24, 22q13 (additions), and 7q32 (deletion). Array CGH analysis of individual clones of MSDACs revealed genetic alterations in chromosomes 1, 7, 8, and 22, corresponding to a gain in the copy numbers of LOC100288142, CD1C, CFHR3, FOXP2, MDFIC, RALYL, CSMD3, SAMD12-AS1, and MAL2, and a loss in ADAM5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT, and TTLL1. QPCR analysis and immunoblotting showed a definite association between DNA-copy number changes and matching transcriptional/translational expression in clones of MSDACs. Further, MSDACs exert a stem-like phenotype. Under serum-free conditions, MSDACs demonstrated profound tumorosphere formation ex vivo. Moreover, MSDACs exhibited high tumorigenic capacity in vivo and prompted aggressive metastatic disease. Tissue microarray analysis coupled with automated IHC revealed significant association of RALYL to the tumor grade in a cohort of 25 neuroblastoma patients. Clinical outcome association analysis showed a strong correlation between the expression of CFHR3, CSMD3, MDFIC, FOXP2, RALYL, POLDIP3, SLC25A17, SERHL, MGAT3, TTLL1, or LOC400927 and overall and relapse-free survival in patients with neuroblastoma.

Conclusion: Together, these data highlight the ongoing acquired genetic rearrangements in undifferentiated tumor-forming neural crest cells, and suggest that these alterations could switch favorable neuroblastoma to high-risk aggressive disease, promoting poor clinical outcomes.

No MeSH data available.


Related in: MedlinePlus

Transcriptional and translational validation array CGH outcomes. a Histograms of QPCR analysis showing transcriptional amplification of CD1C, FOXP2, RALYL, NBPF20, and MAL2, and suppression of APOBEC3B, SLC25A17, EP300, L3MBTL2, SERHL, A4GALT, POLDIP3, and ADAM5 in clones of MSDACs compared with SH-SY5Y cells. b Representative immunoblots showing the expression level of RALYL and FOXP2 (both showed gain in Array CGH analysis) in two different clones of metastatic site derived aggressive cells (MSDAC) in comparison with the parental SH-SY5Y cells and in the metastatic tumors derived from three different animals bearing high-risk aggressive neuroblastoma (NB-MT-AD) in comparison with the non-metastatic primary xenograft (NB-NM-PX). Side Panel: Histograms of Quantity one densitometry analysis showing robust increase in RALYL and FOXP2 expression in MSDACs as well as in metastatic tumors in vivo
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4496850&req=5

Fig5: Transcriptional and translational validation array CGH outcomes. a Histograms of QPCR analysis showing transcriptional amplification of CD1C, FOXP2, RALYL, NBPF20, and MAL2, and suppression of APOBEC3B, SLC25A17, EP300, L3MBTL2, SERHL, A4GALT, POLDIP3, and ADAM5 in clones of MSDACs compared with SH-SY5Y cells. b Representative immunoblots showing the expression level of RALYL and FOXP2 (both showed gain in Array CGH analysis) in two different clones of metastatic site derived aggressive cells (MSDAC) in comparison with the parental SH-SY5Y cells and in the metastatic tumors derived from three different animals bearing high-risk aggressive neuroblastoma (NB-MT-AD) in comparison with the non-metastatic primary xenograft (NB-NM-PX). Side Panel: Histograms of Quantity one densitometry analysis showing robust increase in RALYL and FOXP2 expression in MSDACs as well as in metastatic tumors in vivo

Mentions: To determine any acquired genetic rearrangements and to underscore their impact on disease progression, we utilized high-throughput whole genome array CGH analysis (Fig. 3a) coupled with quantitative transcriptional expression (QPCR). High resolution array CGH analysis showed unique yet extensive copy-number variations (CNVs), including insertions, deletions, and more complex changes that involve gain (duplication) or loss (deletion) at the same locus in MSDAC clones (Fig. 3a, Fig. 4). However, in order to characterize the association of acquired genetic rearrangements with disease progression, we considered only the common genetic variations across the investigated clones of MSDACs. Forty-five common CNVs were observed with gain in 30 (Chr.1,7; Chr.2, 3; Chr.4, 1; Chr.6, 1; Chr.7, 6; Chr.8, 8; Chr.11,2; Chr.17,2) regions and loss in 15 (Chr.4,1; Chr.8,1; Chr.14,1; Chr.22,12) regions (Fig. 3b, Fig. 4). Interestingly, these CNVs correspond to the gain in the coding regions of CD1C, CFHR3, FOXP2, MDFIC, ADAM5, RALYL, CSMD3, SAMD12-AS1, MAL2, OR52N5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT, and TTLL1 genes. (Fig. 3b, Fig. 4). Unlike the healthy genome, in which changes in gene expression are carefully controlled through transcription factors, the cancer genome adapts through the duplication of CD1C, CFHR3, FOXP2, MDFIC, RALYL, CSMD3, SAMD12-AS1, MAL2, and OR52N5, and loss in the coding regions of ADAM5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT, and TTLL1 genes. QPCR analysis revealed a CNV gain with a corresponding increase in transcriptional expression of CD1C, FOXP2, RALYL, and MAL2 in MSDACs, but not in SH-SY5Y cells (Fig. 5a). Likewise, we observed a transcriptional repression of ADAM5, A4GALT, ABPOBEC3B, EP300, L3MBTL2, SERHL, SLC25A17, and POLDIP3, consistent with the CNV loss in MSDACs (Fig. 5a). Moreover, immunoblotting analysis revealed a profound increase in RALYL and FOXP2 translation in aggressive MSDAC clones as opposed to the parental SH-SY5Y cells (Fig. 5b). Like-wise we observed a robust increase in RALYL and FOXP2 expression in metastatic tumors compared to the non-metastatic primary xenograft (Fig. 5b). Quantity one densitometry analysis revealed consistent increase in RALYL and FOXP2 expression both in ex vivo and in vivo settings (Fig. 5b side panel). Together, the definite genetic changes (CNV loss/gain) in the coding regions of specific genes and their subsequent transcriptional/translational modulations across MSDACs highlight the acquired genetic rearrangements in neuroblastoma progression.Fig. 4


Acquired genetic alterations in tumor cells dictate the development of high-risk neuroblastoma and clinical outcomes.

Khan FH, Pandian V, Ramraj S, Natarajan M, Aravindan S, Herman TS, Aravindan N - BMC Cancer (2015)

Transcriptional and translational validation array CGH outcomes. a Histograms of QPCR analysis showing transcriptional amplification of CD1C, FOXP2, RALYL, NBPF20, and MAL2, and suppression of APOBEC3B, SLC25A17, EP300, L3MBTL2, SERHL, A4GALT, POLDIP3, and ADAM5 in clones of MSDACs compared with SH-SY5Y cells. b Representative immunoblots showing the expression level of RALYL and FOXP2 (both showed gain in Array CGH analysis) in two different clones of metastatic site derived aggressive cells (MSDAC) in comparison with the parental SH-SY5Y cells and in the metastatic tumors derived from three different animals bearing high-risk aggressive neuroblastoma (NB-MT-AD) in comparison with the non-metastatic primary xenograft (NB-NM-PX). Side Panel: Histograms of Quantity one densitometry analysis showing robust increase in RALYL and FOXP2 expression in MSDACs as well as in metastatic tumors in vivo
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4496850&req=5

Fig5: Transcriptional and translational validation array CGH outcomes. a Histograms of QPCR analysis showing transcriptional amplification of CD1C, FOXP2, RALYL, NBPF20, and MAL2, and suppression of APOBEC3B, SLC25A17, EP300, L3MBTL2, SERHL, A4GALT, POLDIP3, and ADAM5 in clones of MSDACs compared with SH-SY5Y cells. b Representative immunoblots showing the expression level of RALYL and FOXP2 (both showed gain in Array CGH analysis) in two different clones of metastatic site derived aggressive cells (MSDAC) in comparison with the parental SH-SY5Y cells and in the metastatic tumors derived from three different animals bearing high-risk aggressive neuroblastoma (NB-MT-AD) in comparison with the non-metastatic primary xenograft (NB-NM-PX). Side Panel: Histograms of Quantity one densitometry analysis showing robust increase in RALYL and FOXP2 expression in MSDACs as well as in metastatic tumors in vivo
Mentions: To determine any acquired genetic rearrangements and to underscore their impact on disease progression, we utilized high-throughput whole genome array CGH analysis (Fig. 3a) coupled with quantitative transcriptional expression (QPCR). High resolution array CGH analysis showed unique yet extensive copy-number variations (CNVs), including insertions, deletions, and more complex changes that involve gain (duplication) or loss (deletion) at the same locus in MSDAC clones (Fig. 3a, Fig. 4). However, in order to characterize the association of acquired genetic rearrangements with disease progression, we considered only the common genetic variations across the investigated clones of MSDACs. Forty-five common CNVs were observed with gain in 30 (Chr.1,7; Chr.2, 3; Chr.4, 1; Chr.6, 1; Chr.7, 6; Chr.8, 8; Chr.11,2; Chr.17,2) regions and loss in 15 (Chr.4,1; Chr.8,1; Chr.14,1; Chr.22,12) regions (Fig. 3b, Fig. 4). Interestingly, these CNVs correspond to the gain in the coding regions of CD1C, CFHR3, FOXP2, MDFIC, ADAM5, RALYL, CSMD3, SAMD12-AS1, MAL2, OR52N5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT, and TTLL1 genes. (Fig. 3b, Fig. 4). Unlike the healthy genome, in which changes in gene expression are carefully controlled through transcription factors, the cancer genome adapts through the duplication of CD1C, CFHR3, FOXP2, MDFIC, RALYL, CSMD3, SAMD12-AS1, MAL2, and OR52N5, and loss in the coding regions of ADAM5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT, and TTLL1 genes. QPCR analysis revealed a CNV gain with a corresponding increase in transcriptional expression of CD1C, FOXP2, RALYL, and MAL2 in MSDACs, but not in SH-SY5Y cells (Fig. 5a). Likewise, we observed a transcriptional repression of ADAM5, A4GALT, ABPOBEC3B, EP300, L3MBTL2, SERHL, SLC25A17, and POLDIP3, consistent with the CNV loss in MSDACs (Fig. 5a). Moreover, immunoblotting analysis revealed a profound increase in RALYL and FOXP2 translation in aggressive MSDAC clones as opposed to the parental SH-SY5Y cells (Fig. 5b). Like-wise we observed a robust increase in RALYL and FOXP2 expression in metastatic tumors compared to the non-metastatic primary xenograft (Fig. 5b). Quantity one densitometry analysis revealed consistent increase in RALYL and FOXP2 expression both in ex vivo and in vivo settings (Fig. 5b side panel). Together, the definite genetic changes (CNV loss/gain) in the coding regions of specific genes and their subsequent transcriptional/translational modulations across MSDACs highlight the acquired genetic rearrangements in neuroblastoma progression.Fig. 4

Bottom Line: Under serum-free conditions, MSDACs demonstrated profound tumorosphere formation ex vivo.Tissue microarray analysis coupled with automated IHC revealed significant association of RALYL to the tumor grade in a cohort of 25 neuroblastoma patients.Clinical outcome association analysis showed a strong correlation between the expression of CFHR3, CSMD3, MDFIC, FOXP2, RALYL, POLDIP3, SLC25A17, SERHL, MGAT3, TTLL1, or LOC400927 and overall and relapse-free survival in patients with neuroblastoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of Oklahoma Health Sciences Science Center, 940 Stanton L. Young Blvd., BMSB 737, Oklahoma City, OK, 73104, USA. Faizan-Khan@ouhsc.edu.

ABSTRACT

Background: Determining the driving factors and molecular flow-through that define the switch from favorable to aggressive high-risk disease is critical to the betterment of neuroblastoma cure.

Methods: In this study, we examined the cytogenetic and tumorigenic physiognomies of distinct population of metastatic site- derived aggressive cells (MSDACs) from high-risk tumors, and showed the influence of acquired genetic rearrangements on poor patient outcomes.

Results: Karyotyping in SH-SY5Y and MSDACs revealed trisomy of 1q, with additional non-random chromosomal rearrangements on 1q32, 8p23, 9q34, 15q24, 22q13 (additions), and 7q32 (deletion). Array CGH analysis of individual clones of MSDACs revealed genetic alterations in chromosomes 1, 7, 8, and 22, corresponding to a gain in the copy numbers of LOC100288142, CD1C, CFHR3, FOXP2, MDFIC, RALYL, CSMD3, SAMD12-AS1, and MAL2, and a loss in ADAM5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT, and TTLL1. QPCR analysis and immunoblotting showed a definite association between DNA-copy number changes and matching transcriptional/translational expression in clones of MSDACs. Further, MSDACs exert a stem-like phenotype. Under serum-free conditions, MSDACs demonstrated profound tumorosphere formation ex vivo. Moreover, MSDACs exhibited high tumorigenic capacity in vivo and prompted aggressive metastatic disease. Tissue microarray analysis coupled with automated IHC revealed significant association of RALYL to the tumor grade in a cohort of 25 neuroblastoma patients. Clinical outcome association analysis showed a strong correlation between the expression of CFHR3, CSMD3, MDFIC, FOXP2, RALYL, POLDIP3, SLC25A17, SERHL, MGAT3, TTLL1, or LOC400927 and overall and relapse-free survival in patients with neuroblastoma.

Conclusion: Together, these data highlight the ongoing acquired genetic rearrangements in undifferentiated tumor-forming neural crest cells, and suggest that these alterations could switch favorable neuroblastoma to high-risk aggressive disease, promoting poor clinical outcomes.

No MeSH data available.


Related in: MedlinePlus