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Metformin and 4SC-202 synergistically promote intrinsic cell apoptosis by accelerating ?Np63 ubiquitination and degradation in oral squamous cell carcinoma.

ABSTRACT: Oral squamous cell carcinoma (OSCC) is the most common and aggressive epithelial tumor in the head and neck region with a rising incidence. Despite the advances in basic science and clinical research, the overall survival rate of OSCC remains low. Thus finding novel effective therapeutic agents for OSCC is necessary. In this study, we investigated the effects and mechanisms of combined metformin and 4SC-202 in OSCC. Our results showed that metformin and 4SC-202 synergistically suppressed the proliferation and promoted the intrinsic apoptosis of OSCC cells in vitro and in vivo. Importantly, the proteasome inhibitor MG132 impeded the ?Np63-decreasing effects after metformin and 4SC-202 treatment, indicating that metformin and 4SC-202 could promote the degradation of ?Np63 protein. Moreover, ubiquitination level of ?Np63 increased after metformin or/and 4SC-202 administration. Furthermore, we revealed that ?Np63 mediated anticancer effects of metformin and 4SC-202, as overexpression or suppression of ?Np63 could attenuate or facilitate the apoptosis rate of OSCC under metformin or/and 4SC-202 treatment. Collectively, metformin and 4SC-202 synergistically promote intrinsic apoptosis through accelerating ubiquitin-mediated degradation of ?Np63 in OSCC, and this co-treatment can serve as a potential therapeutic scheme for OSCC.

SUBMITTER: He Y

PROVIDER: S-EPMC6601594 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Metformin and 4SC-202 synergistically promote intrinsic cell apoptosis by accelerating ΔNp63 ubiquitination and degradation in oral squamous cell carcinoma.

He Yuan Y Tai Shanshan S Deng Miao M Fan Zhaona Z Ping Fan F He Lihong L Zhang Chi C Huang Yulei Y Cheng Bin B Xia Juan J

Cancer medicine 20190425 7

Oral squamous cell carcinoma (OSCC) is the most common and aggressive epithelial tumor in the head and neck region with a rising incidence. Despite the advances in basic science and clinical research, the overall survival rate of OSCC remains low. Thus finding novel effective therapeutic agents for OSCC is necessary. In this study, we investigated the effects and mechanisms of combined metformin and 4SC-202 in OSCC. Our results showed that metformin and 4SC-202 synergistically suppressed the pro ...[more]

PMID: 31025540

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Project description:BackgroundTargeting epigenetic modifiers is effective in cutaneous T cell lymphoma (CTCL). However, there is a need for further improvement of this therapeutic approach. Here, we compared the mode of action of romidepsin (FK228), an established class I histone deacetylase inhibitor, and domatinostat (4SC-202), a novel inhibitor of class I HDACs, which has been reported to also target the lysine-specific histone demethylase 1A (LSD1).MethodsWe performed MTS assays and flow cytometric analyses of propidium iodide or annexin V-stained cells to assess drug impact on cellular proliferation, cell cycle distribution, and survival. Histone acetylation and methylation as well as caspase activation was analyzed by immunoblot. Gene expression analysis was performed using NanosString technology. Knockdown and knockout of LSD1 was achieved with shRNA and CRISPR/Cas9, respectively, while the CRISPR/Cas9 synergistic activation mediator system was used to induce expression of endogenous HDACs and LSD1. Furthermore, time-lapse fluorescence microscopy and an in vitro tubulin polymerization assay were applied.ResultsWhile FK228 as well as 4SC-202 potently induced cell death in six different CTCL cell lines, only in the case of 4SC-202 death was preceded by an accumulation of cells in the G2/M phase of the cell cycle. Surprisingly, apoptosis and accumulation of cells with double DNA content occurred already at 4SC-202 concentrations hardly affecting histone acetylation and methylation, and provoking significantly less changes in gene expression compared to biologically equivalent doses of FK228. Indeed, we provide evidence that the 4SC-202-induced G2/M arrest in CTCL cells is independent of de novo transcription. Furthermore, neither enforced expression of HDAC1 nor knockdown or knockout of LSD1 affected the 4SC-202-induced effects. Since time-lapse microscopy revealed that 4SC-202 could affect mitotic spindle formation, we performed an in vitro tubulin polymerization assay revealing that 4SC-202 can directly inhibit microtubule formation.ConclusionsWe demonstrate that 4SC-202, a drug currently tested in clinical trials, effectively inhibits growth of CTCL cells. The anti-cancer cell activity of 4SC-202 is however not limited to LSD1-inhibition, modulation of histone modifications, and consecutive alteration of gene expression. Indeed, the compound is also a potent microtubule-destabilizing agent.

Project description:The TP63 gene encodes two major protein variants; TAp63 contains a p53-like transcription domain and consequently has tumor suppressor activities whereas ΔNp63 lacks this domain and acts as an oncogene. The two variants show distinct expression patterns in normal tissues and tumors, with lymphocytes and lymphomas/leukemias expressing TAp63, and basal epithelial cells and some carcinomas expressing high levels of ΔNp63, most notably squamous cell carcinomas (SCC). Whilst the transcriptional functions of TAp63 and ΔNp63 isoforms are known, the mechanisms involved in their regulation are poorly understood. Using squamous epithelial cells that contain high levels of ΔNp63 and low/undetectable TAp63, the DNA demethylating agent decitabine (5-aza-2'-deoxycytidine, 5-dAza) caused a dose-dependent increase in TAp63, with a simultaneous reduction in ΔNp63, indicating DNA methylation-dependent regulation at the isoform-specific promoters. The basal cytokeratin KRT5, a direct ΔNp63 transcriptional target, was also reduced, confirming functional alteration of p63 activity after DNA demethylation. We also showed high level methylation of three CpG sites in the TAP63 promoter in these cells, which was reduced by decitabine. DNMT1 depletion using inducible shRNAs partially replicated these effects, including an increase in the ratio of TAP63:ΔNP63 mRNAs, a reduction in ΔNp63 protein and reduced KRT5 mRNA levels. Finally, high DNA methylation levels were found at the TAP63 promoter in clinical SCC samples and matched normal tissues. We conclude that DNA methylation at the TAP63 promoter normally silences transcription in squamous epithelial cells, indicating DNA methylation as a therapeutic approach to induce this tumor suppressor in cancer. That decitabine simultaneously reduced the oncogenic activity of ΔNp63 provides a "double whammy" for SCC and other p63-positive carcinomas. Whilst a variety of mechanisms may be involved in producing the opposite effects of DNA demethylation on TAp63 and ΔNp63, we propose an "either or" mechanism in which TAP63 transcription physically interferes with the ability to initiate transcription from the downstream ΔNP63 promoter on the same DNA strand. This mechanism can explain the observed inverse expression of p63 isoforms in normal cells and cancer.

Dataset Information

Metformin and 4SC-202 synergistically promote intrinsic cell apoptosis by accelerating ?Np63 ubiquitination and degradation in oral squamous cell carcinoma.

Publications

Metformin and 4SC-202 synergistically promote intrinsic cell apoptosis by accelerating ΔNp63 ubiquitination and degradation in oral squamous cell carcinoma.

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