Hypoxia-inducible factor-1 promotes cancer progression through activating AKT/Cyclin D1 signaling pathway in osteosarcoma.

Osteosarcoma is an aggressive malignant neoplasm, which commonly afflicts patients of 20-30 years of age, and its morbidity has increased markedly in recent years. Certain genes and signal pathways have been identified to exert key roles in osteosarcoma progression. Here, we set out to characterize in more detail of the role of HIF-1/AKT/Cyclin D1 pathway in the progression of osteosarcoma. Immunohistochemistry, western blot and qPCR were used to test the protein or mRNA levels of HIF-1 in osteosarcoma tissues or adjacent nontumor tissues. MTT, clone formation, wound healing, Transwell, in vivo tumorigenesis, flow cytometry and western blot analysis were used to determine cell proliferation, clone formation ability, migration, invasion, tumorigenesis, and cell apoptosis in MG63 and U2OS cells, respectively. Immunoprecipitation and immunofluorescence assays were performed to investigate the protein-protein interaction between HIF-1α and proteins related to signal pathways. HIF-1 was overexpressed in osteosarcoma tissues and cell lines, which promoted cell proliferation, clone formation, migration, invasion and inhibited cell apoptosis. Results also demonstrated that HIF-1 combined with AKT, and there might be a positive loop between the two proteins of HIF-1 and AKT, then the protein-protein interaction up-regulated the expression of Cyclin D1 in protein level, but not mRNA level, made Cyclin D1 protein more stable, triggered cell proliferation, clone formation, tumorigenesis, but inhibited cell apoptosis. The present study showed that HIF-1 modulated Cyclin D1 expression might through shaping a positive loop with AKT proteins. Additionally, HIF-1α promoted the tumor cells growth, migration and invasion in osteosarcoma through the activation of the AKT/Cyclin D1 signal cascade. We proposed that HIF-1 could be served as a marker for distinguishing osteosarcoma and an effective therapeutic target for osteosarcoma.

Zhang B ，Li YL ，Zhao JL ，Zhen O ，Yu C ，Yang BH ，Yu XR ... -  《-》

SIX1 reduces the expression of PTEN via activating PI3K/AKT signal to promote cell proliferation and tumorigenesis in osteosarcoma.

Osteosarcoma is the most common form of primary malignant bone cancer which is most prevalent in children and adolescents. Dysregulated expressions of SIX1 and PTEN/PI3K/AKT have been demonstrated in bone malignancies including osteosarcoma. However, the mechanism of SIX1/PTEN/PI3K/AKT on osteosarcoma progression remains unknown. Therefore, this study aims to investigate the molecular mechanism of SIX1 and PTEN/PI3K/AKT on osteosarcoma progression. In this study, we first examined the expression of SIX1 and PTEN in human osteosarcoma tissues or blood samples and cell lines by immunohistochemistry, western blot analysis and qPCR. MTT, clone formation assay, wound healing assay, Transwell assay, in vivo tumorigenesis, flow cytometry and western blot were used to determine the function of SIX1/PTEN on cell proliferation, clone formation ability, migration, invasion, tumorigenesis, and cell apoptosis in SAOS2 and U2OS cells, respectively. Results showed that SIX1 was overexpressed in osteosarcoma tissues, blood samples and cell lines, whereas PTEN expression was reduced. SIX1 promoted cell growth, migration, invasion, and suppressed cell apoptosis. Up-regulation of SIX1 associated with reduced expression of PTEN and activation of PI3K/AKT signaling pathway. Down-regulated the expression of PTEN using gene transfer in U2OS and SAOS2 cells increased cell proliferation and inhibited cell apoptosis through activating PI3K/AKT signaling cascade. In addition, the tumorigenesis of U2OS and SAOS2 cells was suppressed when the cells were stably overexpressed SIX1 and PTEN simultaneously, compared with that in cells stably overexpressed SIX1 only. SIX1 promoted the progression of osteosarcoma via regulating PTEN/PI3K/AKT signaling cascade, which might provide a new potent therapeutic target for osteosarcoma.

Yu C ，Zhang B ，Li YL ，Yu XR ... -  《-》

Microvesicles derived from human bone marrow mesenchymal stem cells promote U2OS cell growth under hypoxia: the role of PI3K/AKT and HIF-1α.

Lin S ，Zhu B ，Huang G ，Zeng Q ，Wang C ... -  《Human Cell》

Highly-expressed P2X7 receptor promotes growth and metastasis of human HOS/MNNG osteosarcoma cells via PI3K/Akt/GSK3β/β-catenin and mTOR/HIF1α/VEGF signaling.

The P2X7 receptor, an ATP-gated ion channel, is critical for cancer cell growth, invasiveness, and angiogenesis. Previous studies indicate that P2X7 regulates osteoblast proliferation and osteodeposition and that high P2X7 expression has a pro-growth effect in osteosarcoma. However, how it functions in osteosarcoma cell growth and metastasis is not clear. Thus, we elucidated molecular mechanisms of P2X7-dependent positive regulation of osteosarcoma cell proliferation, invasion, migration, epithelial to mesenchymal transition (EMT), and angiogenesis using in vitro and in vivo models. We confirm that P2X7 is highly-expressed in human osteosarcoma tumor tissues and HOS/MNNG, MG63, U2OS, SW1353 and SAOS-2 cell lines. P2X7 receptor stimulation enhanced HOS/MNNG and SAOS-2 cell proliferation, migration and invasion; but knockdown of P2X7 expression or receptor inhibition had opposite effects. P2X7 positively regulated glycogen content, epithelial to mesenchymal transition and stemness of HOS/MNNG cells. P2X7 activation promoted PI3K/Akt/GSK3β/β-catenin and mTOR/HIF1α/VEGF signaling, thereby mediating pro-tumor effects of osteosarcoma cells. Consistent with data from in vitro experiments, systemic administration of P2X7 agonist induced tumor growth, metastasis and tumor-associated bone destruction in osteosarcoma-bearing nude mice, whereas a P2X7 antagonist reversed these effects. Thus, the P2X7 receptor participates in regulation of osteosarcoma growth and metastasis and we offer evidence that P2X7 may be a promising therapeutic target for treating osteosarcoma.

Zhang Y ，Cheng H ，Li W ，Wu H ，Yang Y ... -  《-》

Knockdown of TBRG4 suppresses proliferation, invasion and promotes apoptosis of osteosarcoma cells by downregulating TGF-β1 expression and PI3K/AKT signaling pathway.

Transforming growth factor beta regulator 4 (TBRG4) is a novel regulator in tumorigenic progression of several tumors. However, so far, the expression and functions of TBRG4 in osteosarcoma are unknown. The aim of this study was to investigate the potential biological functions of TBRG4 in osteosarcoma. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of TBRG4 in osteosarcoma tissues and cell lines. The levels of TBRG4 protein in osteosarcoma tissues were assessed by immunohistochemistry. Lentivirus-mediated short hairpin (sh) RNA was employed to knock down TBRG4 in osteosarcoma cells, and the expressions of TBRG4 mRNA and protein were determined by qRT-PCR and Western blot assay, respectively. Subsequently, the proliferation, clonogenic ability, apoptosis and invasion of osteosarcoma cells were measured using high content screening analysis and CCK8 assay, tumor sphere formation assay, flow cytometry and Transwell invasion assays, respectively. Furthermore, the osteosarcoma cells growth and metastasis in vivo were detected, and the effect of TBRG4 on the transforming growth factor β1 (TGF-β1) and PI3K/AKT signaling pathway was explored by qRT-PCR and Western blot assay, respectively. The results showed the levels of TBRG4 were overexpressed in osteosarcoma tissues and cell lines, confirming that the high TBRG4 expression was related to advanced tumor stages, large tumor size, and lymph node metastasis. Functional assays showed knockdown of TBRG4 could inhibit proliferation, invasion and induce apoptosis of osteosarcoma cells in vitro, and could also suppress osteosarcoma growth and metastasis in vivo. By examining the expression levels of TGF-β1, p-PI3K, PI3K, p-AKT and AKT, it showed that the suppression of TBRG4 would reduce TGF-β1 expression and inactivate the PI3K/AKT signaling pathway. These results showed for the first time that TBRG4 knockdown could suppress osteosarcoma progression, suggesting TBRG4 might be a promising therapeutic target for osteosarcoma treatment.

Huang F ，Zhou P ，Wang Z ，Zhang XL ，Liao FX ，Hu Y ，Chang J ... -  《-》