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Recurrent LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes promote tumor cell motility in human osteosarcoma.

Yang J, Annala M, Ji P, Wang G, Zheng H, Codgell D, Du X, Fang Z, Sun B, Nykter M, Chen K, Zhang W - J Hematol Oncol (2014)

Bottom Line: Nine of 11 samples were found to harbor genetic inactivating alterations in the TP53 pathway.Two recurrent fusion genes associated with the 12q locus, LRP1-SNRNP25 and KCNMB4-CCND3, were identified and validated by RT-PCR, Sanger sequencing and fluorescence in situ hybridization, and were found to be osteosarcoma specific in a validation cohort of 240 other sarcomas.Our study represents the first whole transcriptome analysis of untreated human osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Bone and Soft Tissue Tumor, Tianjin Medical University Cancer Hospital & Institute, Tianjin, 30060, PR China. yangjilong@tjmuch.com.

ABSTRACT

Background: The identification of fusion genes such as SYT-SSX1/SSX2, PAX3-FOXO1, TPM3/TPM4-ALK and EWS-FLI1 in human sarcomas has provided important insight into the diagnosis and targeted therapy of sarcomas. No recurrent fusion has been reported in human osteosarcoma.

Methods: Transcriptome sequencing was used to characterize the gene fusions and mutations in 11 human osteosarcomas.

Results: Nine of 11 samples were found to harbor genetic inactivating alterations in the TP53 pathway. Two recurrent fusion genes associated with the 12q locus, LRP1-SNRNP25 and KCNMB4-CCND3, were identified and validated by RT-PCR, Sanger sequencing and fluorescence in situ hybridization, and were found to be osteosarcoma specific in a validation cohort of 240 other sarcomas. Expression of LRP1-SNRNP25 fusion gene promoted SAOS-2 osteosarcoma cell migration and invasion. Expression of KCNMB4-CCND3 fusion gene promoted SAOS-2 cell migration.

Conclusions: Our study represents the first whole transcriptome analysis of untreated human osteosarcoma. Our discovery of two osteosarcoma specific fusion genes associated with osteosarcoma cellular motility highlights the heterogeneity of osteosarcoma and provides opportunities for new treatment modalities.

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Related in: MedlinePlus

LRP1-SNRNP25andKCNMB4-CCND3fusion genes promoted human osteosarcoma SAOS-2 cells motility relative to empty vector. (A) Transwell migration assay. Both the LRP1-SNRNP25 and KCNMB4-CCND3 fusions significantly promoted cell migration of SAOS-2 cells. (B) Transwell invasion assay. While the LRP1-SNRNP25 promoted invasion of SAOS-2 cells significantly, KCNMB4-CCND3 fusions did not significantly promote cell invasion. (C) Wound healing assay showed that both fusions accelerated the osteosarcoma cell migration.
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Fig4: LRP1-SNRNP25andKCNMB4-CCND3fusion genes promoted human osteosarcoma SAOS-2 cells motility relative to empty vector. (A) Transwell migration assay. Both the LRP1-SNRNP25 and KCNMB4-CCND3 fusions significantly promoted cell migration of SAOS-2 cells. (B) Transwell invasion assay. While the LRP1-SNRNP25 promoted invasion of SAOS-2 cells significantly, KCNMB4-CCND3 fusions did not significantly promote cell invasion. (C) Wound healing assay showed that both fusions accelerated the osteosarcoma cell migration.

Mentions: Since LRP1 and CCND3 play important roles in regulating tumor cell migration, invasion, proliferation and apoptosis in other cancers [19-22], we set out to test whether the chimeric proteins might play any role in osteosarcoma cells. A cell transformation assay with Rat2 fibroblast cells revealed that neither fusion was sufficient to transform Rat2 fibroblast cells (Additional file 1: Figure S12C) or to augment K-Ras V12 driven cell transformation (Additional file 1: Figure S12D). In soft agar colony formation assays, neither fusion promoted anchorage-independent colony formation relative to GFP controls (Additional file 1: Figure S12E). Proliferation of SAOS-2 cells was inhibited rather than increased after fusion transfection (Additional file 1: Figure S12F). However, both the LRP1-SNRNP25 and KCNMB4-CCND3 fusions significantly promoted cell migration of SAOS-2 cells in transwell assays (Figure 4A), and LRP1-SNRNP25 also promoted invasion significantly (Figure 4B). A wound healing assay corroborated these findings, showing that both fusions accelerated cell migration (Figure 4C). Clinically, both LRP1-SNRNP25 positive patients had tumor recurrence 21 months after surgery, and one of the patients developed lung metastases after 6 months. One of the KCNMB4-CCND3 positive patients also had his tumor recur 6 months after surgery.Figure 4


Recurrent LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes promote tumor cell motility in human osteosarcoma.

Yang J, Annala M, Ji P, Wang G, Zheng H, Codgell D, Du X, Fang Z, Sun B, Nykter M, Chen K, Zhang W - J Hematol Oncol (2014)

LRP1-SNRNP25andKCNMB4-CCND3fusion genes promoted human osteosarcoma SAOS-2 cells motility relative to empty vector. (A) Transwell migration assay. Both the LRP1-SNRNP25 and KCNMB4-CCND3 fusions significantly promoted cell migration of SAOS-2 cells. (B) Transwell invasion assay. While the LRP1-SNRNP25 promoted invasion of SAOS-2 cells significantly, KCNMB4-CCND3 fusions did not significantly promote cell invasion. (C) Wound healing assay showed that both fusions accelerated the osteosarcoma cell migration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4197299&req=5

Fig4: LRP1-SNRNP25andKCNMB4-CCND3fusion genes promoted human osteosarcoma SAOS-2 cells motility relative to empty vector. (A) Transwell migration assay. Both the LRP1-SNRNP25 and KCNMB4-CCND3 fusions significantly promoted cell migration of SAOS-2 cells. (B) Transwell invasion assay. While the LRP1-SNRNP25 promoted invasion of SAOS-2 cells significantly, KCNMB4-CCND3 fusions did not significantly promote cell invasion. (C) Wound healing assay showed that both fusions accelerated the osteosarcoma cell migration.
Mentions: Since LRP1 and CCND3 play important roles in regulating tumor cell migration, invasion, proliferation and apoptosis in other cancers [19-22], we set out to test whether the chimeric proteins might play any role in osteosarcoma cells. A cell transformation assay with Rat2 fibroblast cells revealed that neither fusion was sufficient to transform Rat2 fibroblast cells (Additional file 1: Figure S12C) or to augment K-Ras V12 driven cell transformation (Additional file 1: Figure S12D). In soft agar colony formation assays, neither fusion promoted anchorage-independent colony formation relative to GFP controls (Additional file 1: Figure S12E). Proliferation of SAOS-2 cells was inhibited rather than increased after fusion transfection (Additional file 1: Figure S12F). However, both the LRP1-SNRNP25 and KCNMB4-CCND3 fusions significantly promoted cell migration of SAOS-2 cells in transwell assays (Figure 4A), and LRP1-SNRNP25 also promoted invasion significantly (Figure 4B). A wound healing assay corroborated these findings, showing that both fusions accelerated cell migration (Figure 4C). Clinically, both LRP1-SNRNP25 positive patients had tumor recurrence 21 months after surgery, and one of the patients developed lung metastases after 6 months. One of the KCNMB4-CCND3 positive patients also had his tumor recur 6 months after surgery.Figure 4

Bottom Line: Nine of 11 samples were found to harbor genetic inactivating alterations in the TP53 pathway.Two recurrent fusion genes associated with the 12q locus, LRP1-SNRNP25 and KCNMB4-CCND3, were identified and validated by RT-PCR, Sanger sequencing and fluorescence in situ hybridization, and were found to be osteosarcoma specific in a validation cohort of 240 other sarcomas.Our study represents the first whole transcriptome analysis of untreated human osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Bone and Soft Tissue Tumor, Tianjin Medical University Cancer Hospital & Institute, Tianjin, 30060, PR China. yangjilong@tjmuch.com.

ABSTRACT

Background: The identification of fusion genes such as SYT-SSX1/SSX2, PAX3-FOXO1, TPM3/TPM4-ALK and EWS-FLI1 in human sarcomas has provided important insight into the diagnosis and targeted therapy of sarcomas. No recurrent fusion has been reported in human osteosarcoma.

Methods: Transcriptome sequencing was used to characterize the gene fusions and mutations in 11 human osteosarcomas.

Results: Nine of 11 samples were found to harbor genetic inactivating alterations in the TP53 pathway. Two recurrent fusion genes associated with the 12q locus, LRP1-SNRNP25 and KCNMB4-CCND3, were identified and validated by RT-PCR, Sanger sequencing and fluorescence in situ hybridization, and were found to be osteosarcoma specific in a validation cohort of 240 other sarcomas. Expression of LRP1-SNRNP25 fusion gene promoted SAOS-2 osteosarcoma cell migration and invasion. Expression of KCNMB4-CCND3 fusion gene promoted SAOS-2 cell migration.

Conclusions: Our study represents the first whole transcriptome analysis of untreated human osteosarcoma. Our discovery of two osteosarcoma specific fusion genes associated with osteosarcoma cellular motility highlights the heterogeneity of osteosarcoma and provides opportunities for new treatment modalities.

Show MeSH
Related in: MedlinePlus