Differentiation Of Osteosarcoma Cells Research Articles

Fluorescent monitoring osteogenic differentiation of osteosarcoma cells with an aggregation-induced emission probe

Osteosarcoma is widely believed to be an osteogenic differentiation disorder. In recent years, to further understand this disease, a lot resources were poured into the potential link between differentiation defects and tumorigenesis. Long-term monitoring of the differentiation progress of osteosarcoma cells is of great importance. In order to better promote the research, we have developed a novel fluorescent probe called PTB-EDTA, which exhibits remarkable bio-compatibility and demonstrates high selectivity towards osteosarcoma cells. Not only is the PTB-EDTA is capable of live cell imaging while conventional histology requires to kill the cells, its fluorescence is also enhanced as the osteogenic differentiation proceeding. These properties make PTB-EDTA a promising tool for monitoring osteosarcoma cells.

Osteoblast-derived exosomes promote osteogenic differentiation of osteosarcoma cells via URG4/Wnt signaling pathway

Osteosarcoma is a primary malignant bone tumor. Although surgery and chemotherapy are the main treatment methods, the overall curative effect remains unsatisfactory. Therefore, there is an urgent need to develop new therapeutic options for osteosarcoma. In this study, the effect and molecular mechanism of osteoblast-derived exosomes on the treatment of osteosarcoma were evaluated. Human primary osteoblasts were cultured to observe the effects of osteoblast-derived exosomes on the osteogenic differentiation of osteosarcoma cells both in vitro and in vivo. Alizarin red staining and alkaline phosphatase detection were used to evaluate the degree of osteogenic differentiation, and immunofluorescence and Western blotting were used to detect protein expression. The results showed that osteoblast-derived exosomes effectively inhibited the proliferation of osteosarcoma cells and promoted their mineralization in vitro. The exosomes also significantly inhibited tumor growth and promoted tumor tissue mineralization in vivo. Osteoblast-derived exosomes upregulated the expression of bone sialoprotein, osteonectin, osteopontin, runt-related transcription factor 2, and Wnt inhibitory factor 1, downregulated the expression of cyclin D1, and suppressed the nuclear accumulation of β-catenin and promoted its phosphorylation in vitro and in vivo. However, these effects were significantly reversed by upregulated gene (URG) 4 overexpression. These findings suggest that osteoblast-derived exosomes could activate the osteogenic differentiation process in osteosarcoma cells and promote their differentiation by targeting the URG4/Wnt signaling pathway.

ANXA2 and Rac1 negatively regulates autophagy and osteogenic differentiation in osteosarcoma cells to confer CDDP resistance

BackgroundCisplatin (CDDP) is a mainstay chemotherapeutic agent for OS treatment, but drug resistance has become a hurdle to limit its clinical effect. Autophagy plays an important role in CDDP resistance in OS, and in the present study we explored the role of ANXA2 and Rac1 in dictating CDDP sensitivity in OS cells. MethodsANXA2 and Rac1 expression levels were examined by Western blot and autophagy induction was detected by transmission electron miscroscope (TEM) in the clinical samples and OS cell lines. CDDP resistant cells were established by exposing OS cells to increasing doses of CDDP. The effects of ANXA2 and Rac1 knockdown on CDDP sensitivity were evaluated in the cell and animal models. ResultsReduced autophagy was associated with the increased expression of ANXA2 and Rac1 in CDDP resistant OS tumor samples and cells. Autophagy suppression promoted CDDP resistance and inducing autophagy re-sensitized the resistant cells to CDDP treatment in vitro and in vivo. Further, knocking down ANXA2 or Rac1 re-activated autophagy and attenuated CDDP resistance in OS cells. We further demonstrated that CDDP resistant OS cells displayed a poorer osteogenic differentiation state when compared to the parental cell lines, which was significantly reversed by autophagy re-activation and ANXA2 or Rac1 silencing. ConclusionOur findings revealed a complicated interplay of ANXA2/Rac1, autophagy induction, and osteogenic differentiation in dictating CDDP resistance in OS cells, suggesting ANXA2 and Rac1 as promising targets to modulate autophagy and overcome CDDP resistance in OS cells.

Shedding Light on Osteosarcoma Cell Differentiation: Impact on Biomineralization and Mitochondria Morphology.

Osteosarcoma (OS) is the most common primary malignant bone tumor and its etiology has recently been associated with osteogenic differentiation dysfunctions. OS cells keep a capacity for uncontrolled proliferation showing a phenotype similar to undifferentiated osteoprogenitors with abnormal biomineralization. Within this context, both conventional and X-ray synchrotron-based techniques have been exploited to deeply characterize the genesis and evolution of mineral depositions in a human OS cell line (SaOS-2) exposed to an osteogenic cocktail for 4 and 10 days. A partial restoration of the physiological biomineralization, culminating with the formation of hydroxyapatite, was observed at 10 days after treatment together with a mitochondria-driven mechanism for calcium transportation within the cell. Interestingly, during differentiation, mitochondria showed a change in morphology from elongated to rounded, indicating a metabolic reprogramming of OS cells possibly linked to an increase in glycolysis contribution to energy metabolism. These findings add a dowel to the genesis of OS giving new insights on the development of therapeutic strategies able to restore the physiological mineralization in OS cells.

Membrane-Anchored and Tumor-Targeted IL12 (attIL12)-PBMC Therapy for Osteosarcoma.

Chimeric antigen receptor (CAR) T-cell therapy has shown great promise for treating hematologic malignancies but requires a long duration of T-cell expansion, is associated with severe toxicity, and has limited efficacy for treating solid tumors. We designed experiments to address those challenges. We generated a cell membrane-anchored and tumor-targeted IL12 (attIL12) to arm peripheral blood mononuclear cells (PBMC) instead of T cells to omit the expansion phase for required CAR T cells. This IL12-based attIL12-PBMC therapy showed significant antitumor efficacy in both heterogeneous osteosarcoma patient-derived xenograft tumors and metastatic osteosarcoma tumors with no observable toxic effects. Mechanistically, attIL12-PBMC treatment resulted in tumor-restricted antitumor cytokine release and accumulation of attIL12-PBMCs in tumors. It also induced terminal differentiation of osteosarcoma cells into bone-like cells to impede tumor growth. In summary, attIL12-PBMC therapy is safe and effective against osteosarcoma. Our goal is to move this treatment into a clinical trial. Owing to the convenience of the attIL12-PBMC production process, we believe it will be feasible.

SNHG1 functions as an oncogenic lncRNA and promotes osteosarcoma progression by up-regulating S100A6 via miR-493-5p.

The mechanism behind the aberrant expression of S100A6 in osteosarcoma is seldom reported so far. This study sought to explore the regulatory axis targeting S100A6 involved in osteosarcoma progression. Clinical samples collected from osteosarcoma patients were used to detect the expressions of SNHG1, miR-493-5p, and S100A6 by western bolt analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of S100A6 on proliferation and osteogenic differentiation were investigated by the CCK-8 assay, colony formation assay, Ethynyl deoxyuridine staining, matrix mineralization assay, and alkaline phosphatase assay. The potential of lncRNAs/miRNAs targeting S100A6 was identified by the bioinformatics approach, and the results were verified by the dual luciferase assay and RNA immunoprecipitation assay. Both and rescue experiments were performed to investigate the regulatory relationship between the identified lncRNAs and S100A6. The results showed that S100A6 is highly expressed in osteosarcoma. S100A6 overexpression not only increases the proliferation but also reduces the osteogenic differentiation of osteosarcoma cells, while S1006A silence exerts the opposite effects. Then, SNHG1 is identified to directly interact with miR-493-5p to attenuate miR-493-5p binding to the 3'-untranslated region of S100A6. Notably, S100A6 silence partially rescues the effect of SNHG1 overexpression on proliferation and osteogenic differentiation of osteosarcoma cells. Furthermore, the suppressive role of SNHG1 silence in the growth of osteosarcoma xenograft tumors is countered by S100A6 overexpression. Collectively, this study reveals that S100A6 plays an important role in osteosarcoma progression, and SNHG1 promotes S100A6 expression by competitively sponging miR-493-5p.

Cell Differentiation Trajectory-Associated Molecular Classification of Osteosarcoma.

This study aims to investigate the differentiation trajectory of osteosarcoma cells and to construct molecular subtypes with their respective characteristics and generate a multi-gene signature for predicting prognosis. Integrated single-cell RNA-sequencing (scRNA-seq) data, bulk RNA-seq data and microarray data from osteosarcoma samples were used for analysis. Via scRNA-seq data, time-related as well as differentiation-related genes were recognized as osteosarcoma tumor stem cell-related genes (OSCGs). In Gene Expression Omnibus (GEO) cohort, osteosarcoma patients were classified into two subtypes based on prognostic OSCGs and it was found that molecular typing successfully predicted overall survival, tumor microenvironment and immune infiltration status. Further, available drugs for influencing osteosarcoma via prognostic OSCGs were revealed. A 3-OSCG-based prognostic risk score signature was generated and by combining other clinic-pathological independent prognostic factor, stage at diagnosis, a nomogram was established to predict individual survival probability. In external independent TARGET cohort, the molecular types, the 3-gene signature as well as nomogram were validated. In conclusion, osteosarcoma cell differentiation occupies a crucial position in many facets, such as tumor prognosis and microenvironment, suggesting promising therapeutic targets for this disease.

The EMT transcription factor ZEB1 blocks osteoblastic differentiation in bone development and osteosarcoma.

Osteosarcoma is an often-fatal mesenchyme-derived malignancy in children and young adults. Overexpression of EMT-transcription factors (EMT-TFs) has been associated with poor clinical outcome. Here, we demonstrated that the EMT-TF ZEB1 is able to block osteoblastic differentiation in normal bone development as well as in osteosarcoma cells. Consequently, overexpression of ZEB1 in osteosarcoma characterizes poorly differentiated, highly metastatic subgroups and its depletion induces differentiation of osteosarcoma cells. Overexpression of ZEB1 in osteosarcoma is frequently associated with silencing of the imprinted DLK-DIO3 locus, which encodes for microRNAs targeting ZEB1. Epigenetic reactivation of this locus in osteosarcoma cells reduces ZEB1 expression, induces differentiation, and sensitizes to standard treatment, thus indicating therapeutic options for ZEB1-driven osteosarcomas. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

TRIM21-regulated Annexin A2 plasma membrane trafficking facilitates osteosarcoma cell differentiation through the TFEB-mediated autophagy

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, which is characterized by dysfunctional autophagy and poor differentiation. Our recent studies have suggested that the tripartite motif containing-21 (TRIM21) plays a crucial role in regulating OS cell senescence and proliferation via interactions with several proteins. Yet, its implication in autophagy and differentiation in OS is largely unknown. In the present study, we first showed that TRIM21 could promote OS cell autophagy, as determined by the accumulation of LC3-II, and the degradation of cargo receptor p62. Further, we were able to identify that Annexin A2 (ANXA2), as a novel interacting partner of TRIM21, was critical for TIRM21-induced OS cell autophagy. Although TRIM21 had a negligible effect on the mRNA and protein expressions of ANXA2, we did find that TRIM21 facilitated the translocation of ANXA2 toward plasma membrane (PM) in OS cells through a manner relying on TRIM21-mediated cell autophagy. This functional link has been confirmed by observing a nice co-expression of TRIM21 and ANXA2 (at the PM) in the OS tissues. Mechanistically, we demonstrated that TRIM21, via facilitating the ANXA2 trafficking at the PM, enabled to release the transcription factor EB (TFEB, a master regulator of autophagy) from the ANXA2-TFEB complex, which in turn entered into the nucleus for the regulation of OS cell autophagy. In accord with previous findings that autophagy plays a critical role in the control of differentiation, we also demonstrated that autophagy inhibited OS cell differentiation, and that the TRIM21/ANXA2/TFEB axis is implicated in OS cell differentiation through the coordination with autophagy. Taken together, our results suggest that the TRIM21/ANXA2/TFEB axis is involved in OS cell autophagy and subsequent differentiation, indicating that targeting this signaling axis might lead to a new clue for OS treatment.

Induction of osteogenesis in bone tumour cells by purine-conjugated zinc-hydroxyapatite

This study aimed to improve the biocompatibility and osteogenic property of hydroxyapatite (HAP). So HAP nanoparticles were doped with zinc (Zn), and their surface was modified with a purine nucleotide, guanosine 5′-triphosphate (GTP). GTP-loaded nanoparticles (GTP@ZnHAP) were characterised by field emission scanning electron microscopy, Fourier transform infrared, thermogravimetric analysis, zeta potential and ultraviolet–visible spectroscopy. Biological experiments revealed that GTP@ZnHAP nanoparticles were internalised by the cells, inhibiting tumour cell (osteoblast-like cells, Saos-2) expansion with an efficiency more than that observed for ZnHAP nanoparticles and GTP alone. Furthermore, Saos-2 cells were committed to differentiate into the normal osteoblast cells under the influence of GTP@ZnHAP nanoparticles demonstrated by the quantitative assessment of bone-related protein expression (Runx2 and osteocalcin) and cell morphological changes. Moreover, high-performance liquid chromatography analyses disclosed a significant enhancement of intracellular GTP content in GTP@ZnHAP-treated cells, proposing perturbation of intracellular nucleotide equilibrium during the process of osteogenesis induced by GTP@ZnHAP nanoparticles. Overall, GTP@ZnHAP exhibits a better synergistic effect on the modulation of cell growth and induction of osteogenic differentiation in osteosarcoma cells than ZnHAP nanoparticles and GTP alone do. Therefore, GTP@ZnHAP may be regarded as a promising biomaterial for the treatment of bone-related diseases.

Mifamurtide and TAM-like macrophages: effect on proliferation, migration and differentiation of osteosarcoma cells.

Tumor-associated macrophages and their alternative activation states together with cytokines and growth factors trapped in tumor microenvironment contribute to the progression of OS. In contrast to other tumor types, M2 polarized macrophages, reduce metastasis and improve survival in osteosarcoma patients. Mifamurtide is an immunomodulatory drug given together with standard adjuvant chemotherapy in high-grade osteosarcoma to improve outcome. Macrophages obtained from peripheral blood mononucleated cells of healthy donors and MG63 cells were cultured alone and together, and treated with Mifamurtide. We analyzed the effects of Mifamurtide on macrophage polarization and on MG63 proliferation, migration and differentiation, evaluating the expression of M1/M2 and osteoblast markers and molecules involved in metastasis and proliferation pathways.Our data suggest that Mifamurtide, switching macrophage polarization towards a TAM-like intermediate M1/M2 phenotype, may modulate the delicate balance between pro-inflammatory and immunomodulatory macrophage functions. Moreover, Mifamurtide may inhibit the cellular proliferation and induce the tumor cell differentiation, probably through the down regulation of pSTAT3, pAKT and IL-17R.

Advances in differentiation therapy for osteosarcoma

Differentiation therapy involves the use of agents that can induce differentiation in cancer cells, with the irreversible loss of tumour phenotype. The application of differentiation therapy in osteosarcoma has made progress because of a better understanding of the potential links between differentiation defects and tumorigenesis. Here, we review recent studies on differentiation therapy for osteosarcoma, describing a variety of differentiation inducers. By highlighting these examples of drug-induced osteosarcoma cell differentiation, we can acquire unique insights into not only osteosarcoma treatment, but also novel approaches to transform differentiating drugs into more effective therapies for other solid tumours.

Corrigendum] All-trans retinoic acid restored the osteogenic ability of BMP9 in osteosarcoma through the p38 MAPK pathway.

Subsequently to the publication of the above paper, the authors have realized that the images presented in Fig.1A were selected erroneously (essentially, the images for group 'AdBMP9+++' were chosen to represent the group 'AdGFP'). A corrected version of Fig.1, including the correct data for the experiments depicted in Fig.1A, is shown opposite. Note that this change does not affect the results or the conclusions reported in this paper, and all the authors agree to this correction. The authors apologize to the Editor and to the readership of the Journal for any inconvenience caused. [the original article was published in International Journal of Oncology 50: 1363‑1371, 2017; DOI: 10.3892/ijo.2017.3910].

Abstract 1505: GATA3 expression is associated with poor differentiation of osteosarcoma cells

Abstract Objective: GATA3, a transcriptional factor promoting differentiation of Th2 cells, was expressed in breast cancer cells or bladder cancer cells. We reported that Gata3 expression in soft tissue sarcoma is an independent factor for poor prognosis. However, the role of GATA3 in osteosarcoma is unknown. We investigated whether GATA3 expression is associated with cell differentiation or clinical prognosis of osteosarcoma. Methods: We examined whether decreased GATA3 expression in MG63 osteosarcoma cells by induction of siRNA GATA3 using electroporation affects cell differentiation. We also evaluated GATA3 expression in biopsy samples of 31 osteosarcoma cases using immunohistochemical analysis, and statistically compared clinicopathological characteristics of GATA3-positive and GATA3-negative cases. In particular, the correlation between osteoid formation and GATA3 expression was examined. Result: GATA3 expression was observed in MG63 cells by Western blotting and real-time PCR. Decreased GATA3 expression caused by induction of siRNA promoted the expression of the osteoblast differentiation markers; osteopontin, osteocalcin and RUNX2; in MG63 cells, while ALP and DMP-1, pre-osteocyte markers, had not increased. The expression of p21, one of the differentiation markers of osteosarcoma, is also elevated by decreased GATA3 expression. GATA3 significantly expressed in osteosarcoma cases with less osteoid formation (P<0.001), although no significant correlation between GATA3 expression and clinical prognosis was observed. Conclusion: These data indicated that GATA3 expression might be associated with poor differentiation of osteosarcoma cells. GATA3 may be a target to treat for osteosarcoma through promotion of cell differentiation. Citation Format: Koji Hiraoka. GATA3 expression is associated with poor differentiation of osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1505.

The potential of magnetic hyperthermia for triggering the differentiation of cancer cells.

Magnetic hyperthermia is a potential technique for cancer therapy that exploits heat generated by magnetic nanoparticles to kill cancerous cells. Many studies have shown that magnetic hyperthermia is effective at killing cancer cells both in vitro and in vivo, however little attention has been paid to the cellular functioning of the surviving cells. We report here new evidence demonstrating the onset of thermally triggered differentiation in osteosarcoma cancer cells that survive magnetic hyperthermia treatment. This raises the possibility that in addition to causing cell death, magnetic hyperthermia could induce surviving cancer cells to form more mature cell types and thereby inhibit their capacity to self-renew. Such processes could prove to be as important as cell death when considering magnetic hyperthermia for treating cancer.

Effect of Matrigel on the establishment of human osteosarcoma animal model

Objective To investigate the effects of cell suspension including Matrigel, normal culture medium and phosphate buffer saline (PBS) on the xenograft model establishment of human osteosarcoma. The function of Matrigel on regulating human osteosarcoma cell differentiation and proliferation was analyzed. Methods Twenty-four BALB/c-nu/nu nude mice were randomly divided into three groups with 8 animals in every group: Matrigel and RPMI 1640 suspension (group M), RPMI 1640 culture medium (group R), PBS (group P). Human osteosarcoma cell-SaOS-2 was suspended in the three groups respectively. 1×106/ml equivalent cell counts were injected into the back of each anesthetized nude mouse (400 μl per mouse). Xenograft tumors were measured at regular intervals and the tumor volume was calculated. After 5 weeks of inoculation, the tumor parts were dissected. Paraffin-embedded sections from xenograft tumor tissues were fixed in 4 % paraformaldehyde and pathological study was made after paraffin embedding and cutting under microscope by HE stains. Results Eight nude mice formed tumor lumps at 0 day in group M and were gradually increased over time. Xenograft tumors of group R and group P disappeared in 2-4 days and some appeared again after 1 week with an increase of tumor size. After 5 weeks, the tumor volume in the group M was significantly larger than that in the group P and group R [(3 185 ± 488), (598 ± 189), (512 ± 109) mm3 respectively, F= 85.7, P 0.05). Histopathological analysis showed that cells in the group M could keep original degrees of pathological differentiation in osteosarcoma cells. Besides, cells suspension of culture medium or PBS in the group P and group R were poorly differentiated. Conclusions Matrigel can promote high tumor growth rate and good uniformity of human osteosarcoma cells in experimental animals. The histological state is similar to original structure, which conforms to the occurrence and development of human osteosarcoma. Key words: Osteosarcoma; Models, animal; Transplantation; Matrigel

CD49b inhibits osteogenic differentiation and plays an important role in osteosarcoma progression.

Osteosarcoma is a cancer whose cell of origin lies in the differentiation pathway between the mesenchymal stem cell (MSC) and the osteoblast (OB). In this study, we sought to determine if surface markers associated with osteoblastic differentiation are involved in osteosarcoma progression. cDNA expression arrays were performed on MSCs and osteoblasts to identify differentially expressed genes. The specificity of candidate genes for osteoblast differentiation was assessed through time course experiments in differentiation media with confirmation utilizing CD49b transfected MSCs. In addition, CD49b was transfected into osteosarcoma cell lines to determine its impact on cell proliferation, motility, and invasion. Finally, the expression of CD49b was assessed in osteosarcoma patient samples and correlated with survival outcomes. cDNA expression arrays identified a list of genes differentially expressed between MSCs and osteoblasts with a subset of those genes encoding cell surface proteins. Three genes were selected for further analysis, based on qPCR validation, but only CD49b was selective for osteoblastic differentiation. Forced expression of CD49b in MSCs led to delayed osteoblastic differentiation. Down-regulation of CD49b expression in osteosarcoma cell lines resulted in inhibition of their migration and invasion capacity. CD49b expression in osteosarcoma patients was associated with presence of metastases and inferior 5 year overall survival (31.4% vs. 57.4%, p=0.03). Surface proteins involved in osteosarcoma cell differentiation, such as CD49b, have the potential to serve as prognostic biomarkers, and may lead to the identification of new therapeutic targets.

All-trans retinoic acid restored the osteogenic ability of BMP9 in osteosarcoma through the p38 MAPK pathway Corrigendum in /10.3892/ijo.2019.4817.

Osteosarcoma (OS) is the most common malignant bone tumour and is considered to be a disease caused by a dysfunction in differentiation. Bone morphogenetic protein9(BMP9) is the most potent osteogenic factor in mesenchymal stem cells, but it cannot induce osteogenic differentiation in OS cells; this might be one of the determinants in the pathogenesis of OS. All-trans retinoic acid(ATRA) can induce osteogenic differentiation of OS cells and potentiate BMP9-induced osteogenesis in preadipocytes. However, the concomitant effect of ATRA and BMP9 in OS cells is unclear; therefore, in the present study, we focused on this topic. The results showed that BMP9 significantly promoted the proliferation of human OS 143Bcells and did not induce osteogenic differentiation of cells invitro (p<0.01). ATRA inhibited proliferation and induced osteogenesis in 143Bcells; these effects could be enhanced by BMP9 overexpression (p<0.05). ATRA could significantly increase the level of phosphorylated p38 MAPK (p-p38) in 143Bcells, while BMP9 did not have any significant effect. Notably, BMP9 overexpression enhanced the ability of ATRA to increase the levels of p-p38. Both the osteogenic differentiation and the anti-proliferative activity of BMP9 in the presence of ATRA decreased upon treatment with a specific inhibitor of p38 MAPK (SB203580) (p<0.01). This study indicates that the osteogenic differentiation ability of BMP9 in 143Bcells can be restored by ATRA, and the combination of BMP9 and ATRA generated a stronger anti-proliferative effect on 143Bcells than ATRA alone. This result may be due to the activation of the p38 MAPK pathway.

The Emerging Roles of Forkhead Box (FOX) Proteins in Osteosarcoma.

Osteosarcoma is the most common bone cancer primarily occurring in children and young adults. Over the past few years, the deregulation of a superfamily transcription factors, known as forkhead box (FOX) proteins, has been demonstrated to contribute to the pathogenesis of osteosarcoma. Molecular mechanism studies have demonstrated that FOX family proteins participate in a variety of signaling pathways and that their expression can be regulated by multiple factors. The dysfunction of FOX genes can alter osteosarcoma cell differentiation, metastasis and progression. In this review, we summarized the evidence that FOX genes play direct or indirect roles in the development and progression of osteosarcoma, and evaluated the emerging role of FOX proteins as targets for therapeutic intervention.

Abstract 1142: E2F1 impairs all-trans retinoic acid-induced osteogenic differentiation of osteosarcoma by promoting ubiquitination-mediated degradation of RARa

Abstract Objective: All-trans retinoic acid (ATRA) is a widely-used differentiation drug that can effectively induce osteogenic differentiation of osteosarcoma cells, but the underlying mechanism remains elusive, which limits the clinical application for ATRA in osteosarcoma patients. In this study, we identified E2F1 as a novel regulator involved in ATRA induced osteogenic differentiation of osteosarcoma cells. Methods: Osteosarcoma cell lines were used as our research models. (1) Protein levels were detected by Western blotting; (2) The interactions between E2F1 and RARalpha were determined by immunofluorescence, immunoprecipitation and GST pull-down assay; (3) The mRNA levels were determined by RT-PCR; (4) Related proteins and genes were investigated via transfection; (5) RARE activity were detected by luciferase reporter assay. (6) Alkaline phosphatase activity was assessed by colorimetric assay using BCIP/NBT Alkaline Phosphatase Color Development kit. Results: We observed that osteosarcoma cells are coupled with individual differences in the expression levels of E2F1 in patients, and E2F1 impairs ATRA-induced differentiation of osteosarcoma cells. Moreover, remarkable anti-proliferative and differentiation-inducing effects of ATRA treatment are only observed in E2F1 low to negative expressed primary osteosarcoma cultures. These results strongly suggested that E2F1 may serve as a potent indicator for the effectiveness of ATRA treatment in osteosarcoma. Interestingly, E2F1 is found to down-regulate retinoic acid receptor (RAR), a key factor determines the effectiveness of ATRA. E2F1 specifically binds to RAR and promotes its ubiquitination-mediated degradation; as a consequence, RAR-mediated differentiation is inhibited in osteosarcoma. Conclusion: E2F1 may serve as a potent biomarker as well as a therapeutic target for ATRA-based differentiation therapeutics, and raise the hope of using differentiation-based approaches for osteosarcoma patients. Citation Format: Qian Zhou, Meidan Ying, Bo Yang, Qiaojun He. E2F1 impairs all-trans retinoic acid-induced osteogenic differentiation of osteosarcoma by promoting ubiquitination-mediated degradation of RARa. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1142.