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HDAC2 depletion promotes osteosarcoma's stemness both in vitro and in vivo: a study on a putative new target for CSCs
Marcella La Noce1, Francesca Paino2, Luigi Mele1
1Dipartimento di Medicina Sperimentale, Sezione di Biotecnologie, Istologia Medica e Biologia Molecolare, Università degli Studi della Campania "L. Vanvitelli", Napoli, via L. Armanni, 5, 80138, Naples, Italy.
Background:
Cancer stem cells (CSCs) play a key role in cancer initiation, progression and chemoresistance. Epigenetic alterations have been identified as prominent factors that contribute to the CSCs phenotype. Here, we investigated the effects of the HDAC inhibitor valproic acid (VPA) and the demethylating agent, 5'azacytidine (DAC) on the stem phenotype of MG63 and Saos2 osteosarcoma cell lines.
Methods:
Saos2 and MG63 cells were treated with DAC and VPA, alone and in combination. Untreated and treated cells were examined for stemness phenotype by cytometry and real-time PCR. Sarcospheres and colonies formation were also evaluated. Moreover, histone modification and methylation were tested by flow cytomery and western blotting. HDAC2 depleted cells were examined for stemness phenotype and their ability to generate tumors in NOD/SCID IL2R-gamma-0 (NSG) mice. HDAC2 expression on human osteosarcoma tissues was evaluated.
Results:
We found that DAC and VPA induce an increased expression of stem markers including CD133, OCT4, SOX2 and NANOG, and an increased ability in sarcospheres and colonies formation efficiency. Interestingly, we showed that DAC and VPA treatment decreased repressive histone markers, while increased the active ones. These histone modifications were also associated with an increase of acetylation of histones H3, a decrease of DNA global methylation, HDAC2 and DNMT3a. Furthermore, HDAC2 silenced-MG63 and Saos2 cells acquired a stem phenotype, and promoted in vivo tumorigenesis. In human osteosarcoma tissues, HDAC2 was strongly expressed in nucleus.
Conclusions:
Collectively, our results suggest that VPA and DAC induce an expansion of osteosarcoma CSCs, and we report for the first time that HDAC2 is a key factor regulating both CSCs phenotype and in vivo cancer growth. In conclusion, we have identified HDAC2 as a potential therapeutic target in human osteosarcoma treatment.
Insights
Valproic acid (VPA) and 5'azacytidine (DAC) expand cancer stem cells (CSCs) in osteosarcoma by altering epigenetic markers. HDAC2 is identified as a key regulator of CSCs phenotype and tumor growth, suggesting it as a therapeutic target.
Area of Science:
- Oncology
- Epigenetics
- Cancer Stem Cell Biology
Background:
- Cancer stem cells (CSCs) drive tumor initiation, progression, and chemoresistance.
- Epigenetic alterations are crucial in establishing the CSC phenotype.
- Osteosarcoma CSCs represent a significant challenge in treatment.
Purpose of the Study:
- To investigate the effects of valproic acid (VPA) and 5'azacytidine (DAC) on osteosarcoma CSCs.
- To elucidate the role of epigenetic modifications in CSCs.
- To identify potential therapeutic targets for osteosarcoma.
Main Methods:
- Treatment of MG63 and Saos2 osteosarcoma cell lines with VPA and DAC.
- Assessment of stemness markers, sphere and colony formation.
- Analysis of histone modifications, DNA methylation, and HDAC2 expression.
- In vivo tumor formation studies in NSG mice.
- Evaluation of HDAC2 expression in human osteosarcoma tissues.
Main Results:
- VPA and DAC increased stem cell markers (CD133, OCT4, SOX2, NANOG) and sphere/colony formation.
- Epigenetic changes included decreased repressive histone marks, increased active marks, H3 acetylation, and global DNA hypomethylation.
- HDAC2 depletion promoted stem cell phenotype and in vivo tumorigenesis.
- HDAC2 showed strong nuclear expression in human osteosarcoma tissues.
Conclusions:
- VPA and DAC promote osteosarcoma CSC expansion through epigenetic modulation.
- HDAC2 is a critical regulator of CSC phenotype and osteosarcoma tumor growth.
- HDAC2 represents a promising therapeutic target for osteosarcoma.