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DEC2 expression is positively correlated with HIF-1 activation and the invasiveness of human osteosarcomas.

Hu T, He N, Yang Y, Yin C, Sang N, Yang Q - J. Exp. Clin. Cancer Res. (2015)

Bottom Line: Compared with the low invasive parental U2OS, U2OS-M showed higher levels of DEC2 expression which were confirmed at both mRNA and protein levels.Importantly, we found that the increased DEC2 expression resulted in a more rapid accumulation of HIF-1α in U2OS-M cells in response to hypoxia.Taken together, whereas DEC2 was found to promote HIF-1α degradation in other types of tumors, our data indicate that DEC2 facilitates HIF-1α stabilization and promotes HIF-1 activation in osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, No.600, Yishan Road, Shanghai, 200233, China. zzuhutu@163.com.

ABSTRACT

Background: Osteosarcoma is the most common malignancy of bone. HIF-1 (hypoxia-inducible factor 1) activation is critical for the metabolic reprogramming and progression of solid tumors, and DEC2 (differentiated embryonic chondrocyte gene 2) has been recently reported to suppress HIF-1 in human breast and endometrial cancers. However, the roles of HIF-1 and DEC2 in human osteosarcomas remain unclear.

Methods: We evaluated the correlation of DEC2 and HIF-1 expression to the prognosis, and studied the roles of DEC2 and HIF-1 activation in the invasiveness of osteosarcoma. Multiple approaches including immunohistochemical staining of clinical osteosarcoma tissues, siRNA-based knockdown and other molecular biology techniques were used. Particularly, by using a repetitive trans-well culture-based in vitro evolution system, we selected a more invasive subpopulation (U2OS-M) of osteosarcoma cells from U2OS and used it as a model to study the roles of DEC2 and HIF-1 in the invasiveness of osteosarcoma.

Results: We found that the expression of DEC2 was positively correlated with HIF-1α levels, and HIF-1α expression positively correlated with poor prognosis in osteosarcomas. DEC2 knockdown in osteosarcoma cell lines (U2OS, MNNG and 143B) attenuated HIF-1α accumulation and impaired the up-regulation of HIF-1 target genes in response to hypoxia. Compared with the low invasive parental U2OS, U2OS-M showed higher levels of DEC2 expression which were confirmed at both mRNA and protein levels. Importantly, we found that the increased DEC2 expression resulted in a more rapid accumulation of HIF-1α in U2OS-M cells in response to hypoxia. Finally, we found that HIF-1 activation is sufficient to upregulate DEC2 expression in osteosarcoma cells.

Conclusion: Taken together, whereas DEC2 was found to promote HIF-1α degradation in other types of tumors, our data indicate that DEC2 facilitates HIF-1α stabilization and promotes HIF-1 activation in osteosarcoma. This implies that DEC2 may contribute to the progression and metastasis of human osteosarcoma by sensitizing tumor cells to hypoxia. On the other hand, HIF-1 activation may contribute to the expression of DEC2 in osteosarcoma. This is the first demonstration of a novel DEC2-HIF-1 vicious cycle in osteosarcoma and a tumor-type specific role for DEC2.

No MeSH data available.


Related in: MedlinePlus

DEC2 is required for HIF-1α transactivation activity in U2OS. Cells were transfected with either control (NC) or DEC2 siRNAs and exposed to either 21% or 1% oxygen for 8 h. A. Confirmation of DEC2 knockdown by qRT-PCR. B-F. Analyses of the expression levels of HIF-1α downstream target genes ANGPTL4 (B), VEGFA (C), HK2 (D), PGK1 (E) in U2OS cells and PGK1 (F) in MNNG cells by qRT-PCR.
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Fig3: DEC2 is required for HIF-1α transactivation activity in U2OS. Cells were transfected with either control (NC) or DEC2 siRNAs and exposed to either 21% or 1% oxygen for 8 h. A. Confirmation of DEC2 knockdown by qRT-PCR. B-F. Analyses of the expression levels of HIF-1α downstream target genes ANGPTL4 (B), VEGFA (C), HK2 (D), PGK1 (E) in U2OS cells and PGK1 (F) in MNNG cells by qRT-PCR.

Mentions: Under hypoxia or when driven by oncogenic signaling, HIF-1α levels are stabilized to activate a set of target genes, such as angiopoietin-like 4 (ANGPTL4), vascular endothelial growth factor A (VEGFA) and hexokinase 2 (HK2), which promote adaptations at cellular, tissue and organismal levels [6,31,32]. In this study, we also found that hypoxia promoted U2OS cell invasiveness and migration (Additional file 3: Figure S2). We asked if DEC2 knockdown impairs hypoxia-stimulated HIF-1α accumulation and HIF-1-dependent gene expression. We knocked down DEC2 expression in U2OS cells which have undetectable basal levels of HIF-1α under normoxic condition, and cultured them under either normoxic or hypoxic condition for 48 h. Using qRT-PCR, we confirmed that the siRNA efficiently knocked down DEC2 (Figure 3A, P > 0.05), and that DEC2 knockdown did not affect HIF-1α mRNA levels (Figure 1). The mRNA levels of ANGPTL4, VEGFA, HK2 and PGK1, four classical HIF-1 downstream target genes, were stimulated by hypoxia in U2OS cells; however, DEC2 knockdown partially suppressed the hypoxia-stimulated expression of these HIF-1 target genes (Figures 3B-E). Similar results were obtained in MNNG and 143B cells (Figure 3F and Additional file 4: Figure S3). These results indicate a distinctive role of DEC2 in osteosarcoma, which facilitates HIF-1 activation and is apparently different from those observed in other types of tumors [22,23].Figure 3


DEC2 expression is positively correlated with HIF-1 activation and the invasiveness of human osteosarcomas.

Hu T, He N, Yang Y, Yin C, Sang N, Yang Q - J. Exp. Clin. Cancer Res. (2015)

DEC2 is required for HIF-1α transactivation activity in U2OS. Cells were transfected with either control (NC) or DEC2 siRNAs and exposed to either 21% or 1% oxygen for 8 h. A. Confirmation of DEC2 knockdown by qRT-PCR. B-F. Analyses of the expression levels of HIF-1α downstream target genes ANGPTL4 (B), VEGFA (C), HK2 (D), PGK1 (E) in U2OS cells and PGK1 (F) in MNNG cells by qRT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4379712&req=5

Fig3: DEC2 is required for HIF-1α transactivation activity in U2OS. Cells were transfected with either control (NC) or DEC2 siRNAs and exposed to either 21% or 1% oxygen for 8 h. A. Confirmation of DEC2 knockdown by qRT-PCR. B-F. Analyses of the expression levels of HIF-1α downstream target genes ANGPTL4 (B), VEGFA (C), HK2 (D), PGK1 (E) in U2OS cells and PGK1 (F) in MNNG cells by qRT-PCR.
Mentions: Under hypoxia or when driven by oncogenic signaling, HIF-1α levels are stabilized to activate a set of target genes, such as angiopoietin-like 4 (ANGPTL4), vascular endothelial growth factor A (VEGFA) and hexokinase 2 (HK2), which promote adaptations at cellular, tissue and organismal levels [6,31,32]. In this study, we also found that hypoxia promoted U2OS cell invasiveness and migration (Additional file 3: Figure S2). We asked if DEC2 knockdown impairs hypoxia-stimulated HIF-1α accumulation and HIF-1-dependent gene expression. We knocked down DEC2 expression in U2OS cells which have undetectable basal levels of HIF-1α under normoxic condition, and cultured them under either normoxic or hypoxic condition for 48 h. Using qRT-PCR, we confirmed that the siRNA efficiently knocked down DEC2 (Figure 3A, P > 0.05), and that DEC2 knockdown did not affect HIF-1α mRNA levels (Figure 1). The mRNA levels of ANGPTL4, VEGFA, HK2 and PGK1, four classical HIF-1 downstream target genes, were stimulated by hypoxia in U2OS cells; however, DEC2 knockdown partially suppressed the hypoxia-stimulated expression of these HIF-1 target genes (Figures 3B-E). Similar results were obtained in MNNG and 143B cells (Figure 3F and Additional file 4: Figure S3). These results indicate a distinctive role of DEC2 in osteosarcoma, which facilitates HIF-1 activation and is apparently different from those observed in other types of tumors [22,23].Figure 3

Bottom Line: Compared with the low invasive parental U2OS, U2OS-M showed higher levels of DEC2 expression which were confirmed at both mRNA and protein levels.Importantly, we found that the increased DEC2 expression resulted in a more rapid accumulation of HIF-1α in U2OS-M cells in response to hypoxia.Taken together, whereas DEC2 was found to promote HIF-1α degradation in other types of tumors, our data indicate that DEC2 facilitates HIF-1α stabilization and promotes HIF-1 activation in osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, No.600, Yishan Road, Shanghai, 200233, China. zzuhutu@163.com.

ABSTRACT

Background: Osteosarcoma is the most common malignancy of bone. HIF-1 (hypoxia-inducible factor 1) activation is critical for the metabolic reprogramming and progression of solid tumors, and DEC2 (differentiated embryonic chondrocyte gene 2) has been recently reported to suppress HIF-1 in human breast and endometrial cancers. However, the roles of HIF-1 and DEC2 in human osteosarcomas remain unclear.

Methods: We evaluated the correlation of DEC2 and HIF-1 expression to the prognosis, and studied the roles of DEC2 and HIF-1 activation in the invasiveness of osteosarcoma. Multiple approaches including immunohistochemical staining of clinical osteosarcoma tissues, siRNA-based knockdown and other molecular biology techniques were used. Particularly, by using a repetitive trans-well culture-based in vitro evolution system, we selected a more invasive subpopulation (U2OS-M) of osteosarcoma cells from U2OS and used it as a model to study the roles of DEC2 and HIF-1 in the invasiveness of osteosarcoma.

Results: We found that the expression of DEC2 was positively correlated with HIF-1α levels, and HIF-1α expression positively correlated with poor prognosis in osteosarcomas. DEC2 knockdown in osteosarcoma cell lines (U2OS, MNNG and 143B) attenuated HIF-1α accumulation and impaired the up-regulation of HIF-1 target genes in response to hypoxia. Compared with the low invasive parental U2OS, U2OS-M showed higher levels of DEC2 expression which were confirmed at both mRNA and protein levels. Importantly, we found that the increased DEC2 expression resulted in a more rapid accumulation of HIF-1α in U2OS-M cells in response to hypoxia. Finally, we found that HIF-1 activation is sufficient to upregulate DEC2 expression in osteosarcoma cells.

Conclusion: Taken together, whereas DEC2 was found to promote HIF-1α degradation in other types of tumors, our data indicate that DEC2 facilitates HIF-1α stabilization and promotes HIF-1 activation in osteosarcoma. This implies that DEC2 may contribute to the progression and metastasis of human osteosarcoma by sensitizing tumor cells to hypoxia. On the other hand, HIF-1 activation may contribute to the expression of DEC2 in osteosarcoma. This is the first demonstration of a novel DEC2-HIF-1 vicious cycle in osteosarcoma and a tumor-type specific role for DEC2.

No MeSH data available.


Related in: MedlinePlus