Promoter Region Binding Research Articles

Abstract 3307: Multiomics analysis to uncover the mechanism of radiosensitization of AR-positive triple negative breast cancers with AR inhibition

Abstract Purpose: Expression of the androgen receptor (AR) has been identified as a driver of tumor growth in triple negative breast cancers (TNBC), and previous work has nominated AR inhibition as a strategy for radiosensitization in AR+ TNBC. Despite its role in radioresistance in AR+ TNBC, the mechanistic role of AR and specifically its role in mediating DNA damage repair in response to radiation therapy (RT) remains unknown. Methods: Nuclear fractionation experiments were performed to assess cellular localization of AR protein in AR+ TNBC cell lines (ACC-422, MDA-MB-453). Cells were cultured in media containing hormones (FBS) with treatment of enzalutamide (ENZA), apalutamide (APA), or darolutamide (DARO). Cells were alternatively cultured in media containing charcoal stripped serum (CSS) without hormones with R1881 stimulation. RNA-sequencing was performed to compare AR+ TNBC cells treated with CSS or R1881 stimulation alone or in combination with ionizing radiation. Reverse phase protein arrays were performed in cells treated with ENZA, RT, or combination treatment. Results: While stimulation with R1881 was sufficient to induce nuclear translocation of AR in MDA-MB-453 cells, AR inhibition with ENZA, APA, or DARO blocked AR nuclear translocation under CSS or FBS growth conditions. When cells were treated with R1881+RT, AR nuclear translocation was induced at similar or greater levels compared to R1881 alone in MDA-MB-453 and ACC-422 cells. Combination treatment of RT with ENZA in the presence of hormones reduced AR nuclear localization (39% reduction in MDA-MB-453 cells and 32% reduction in ACC-422 cells) compared to RT alone. These results suggest that decreased promoter region binding, and gene expression upregulation may be a mechanism of radiosensitization with AR inhibition. In addition, transcriptomic analyses demonstrated at least 979 genes differentially expressed in multiple models. Pathway analyses in these models showed common affected pathways included ECM-receptor interaction, PPAR-gamma activation, PI3K-Akt signaling pathway, and the MAPK/ERK signaling pathway. Proteomic analysis in the same cell lines identified apoptosis, DNA damage, and cell cycle pathway changes after RT when AR-signaling was blocked. Common affected pathways in combined analyses identified PI3K-Akt and MAPK/ERK signaling pathway changes that may be responsible for this radiosensitizing phenotype. Conclusions: Our data suggest that AR inhibition in AR+ TNBC is sufficient to inhibit AR nuclear translocation suggesting that AR may play a nuclear role in response to RT to promote DNA repair and radioresistance. We identify potential pathways, including ECM-receptor interaction, PI3K-Akt signaling pathway, and the MAPK/ERK signaling pathway that may be regulated by AR in response to RT and therefore may be responsible for radioresistance. Citation Format: Anna R. Michmerhuizen, Andrea M. Pesch, Benjamin C. Chandler, Lynn M. Lerner, Connor Ward, Leah Moubadder, Stephanie The, Breanna McBean, Caleb Cheng, Lori J. Pierce, Corey W. Speers. Multiomics analysis to uncover the mechanism of radiosensitization of AR-positive triple negative breast cancers with AR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3307.

Inhibition of HDAC4 by GSK3\u03b2 leads to downregulation of KLF5 and ASK1 and prevents the progression of intravertebral disc degeneration

BackgroundIntervertebral disc degeneration (IDD) is a major cause of lower back pain. This study aimed at exploring the effects of histone deacetylase 4 (HDAC4) and its upstream and downstream signaling molecules on IDD development.MethodsA murine IDD model was established by inducing a needle puncture injury to the vertebrate, whereupon we isolated and transfected of nucleus pulposus (NP) cells. Disc height index (DHI) of the mice was determined by X-ray tomography, while the pain experienced by the IDD mice was evaluated by mechanical and thermal sensitivity tests. Next, the interaction between GSK3β and HDAC4 as well as that between HDAC4 and KLF5 acetylation was assessed by co-immunoprecipitation, while the promoter region binding was assessed identified by chromatin immunoprecipitation. By staining methods with TUNEL, Safranin O fast green, and hematoxylin and eosin, the NP cell apoptosis, degradation of extracellular matrix, and morphology of intervertebral disc tissues were measured. Furthermore, mRNA and protein expressions of GSK3β, HDAC4, KLF5, and ASK1, as well as the extent of HDAC4 phosphorylation, were determined by RT-qPCR and Western blotting.ResultsGSK3β was identified to be downregulated in the intervertebral disc tissues obtained from IDD mice, while HDAC4, KLF5, and ASK1 were upregulated. HDAC4 silencing alleviated IDD symptoms. It was also found that GSK3β promoted the phosphorylation of HDAC4 to increase its degradation, while HDAC4 promoted ASK1 expression through upregulating the expression of KLF5. In IDD mice, GSK3β overexpression resulted in increased DHI, inhibition of NP cell apoptosis, alleviation of disc degeneration, and promoted mechanical and thermal pain thresholds. However, HDAC4 overexpression reversed these effects by promoting ASK1 expression.ConclusionBased on the key findings of the current study, we conclude that GSK3β can promote degradation of HDAC4, which lead to an overall downregulation of the downstream KLF5/ASK1 axis, thereby alleviating the development of IDD.

GSK3β inhibits HDAC4 to Alleviate Intravertebral Disc Degeneration Through KLF5 and ASK1 Downregulation

Intervertebral disc degeneration (IDD) is a major cause of lower back pain, yet the molecular mechanisms are not fully understood. Therefore, the effects of HDAC4 and its upstream and downstream signaling molecules on mediating IDD are determined in this study. IDD mouse model was established by injury of the vertebrate. Nucleus pulposus (NP) cells were collected for culture and transfection. Disc height index (DHI) was determined by X-ray. Pain in IDD mice was evaluated by mechanical and thermal sensitivity testes. Interaction between GSK3β and HDAC4 was assessed by co-immunoprecipitation. The effect of HDAC4 on KLF5 acetylation was determined by immunoprecipitation. Promoter region binding was assessed by chromatin immunoprecipitation. NP cells apoptosis was determined by TUNEL assay. Degradation of extracellular matrix was studied by safranin O fast green staining. Morphology of intervertebral disc tissue was determined by HE staining. mRNA and protein expression of GSK3β, HDAC4, phosphorylated HDAC4, KLF5, and ASK1 were determined by qRT-PCR and Western blotting, respectively. HDAC4 was upregulated while GSK3β was downregulated in IDD mice. HDAC4 silencing alleviated IDD. GSK3β promoted HDAC4 phosphorylation and degradation. HDAC4 promoted ASK1 expressed through, leading to enhanced IDD. GSK3β overexpression increased DHI, inhibited NP cell apoptosis, alleviated disc degeneration, and increased mechanical and thermal pain thresholds in IDD mice. The addition of HDAC4 overexpression reversed these effects of GSK3β. In conclusion, HDAC4 was degraded by GSK3β, leading to reduced IDD through KLF5 and ASK1 downregulation. Funding Statement: None. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The above experiments were approved by the Animal Ethics Committee in Guangxi University of Chinese Medicine, Guangxi International Zhuang Medicine Hospital.

I4, a synthetic anti-diabetes agent, attenuates atherosclerosis through its lipid-lowering, anti-inflammatory and anti-apoptosis properties

Here, we investigated whether I4, which was initially developed as a hypoglycemic agent, possesses anti-atherosclerotic activity and attempted to elucidate the probable mechanism of action underlying this activity. ApoE−/− mice were fed a Western diet and simultaneously administered I4, glimepiride, or pioglitazone once daily for 12 weeks, and the atherosclerotic vascular lesions, lipid content, and expression levels of LOX-1, ICAM-1, VCAM-1 and Bax/Bcl-2 in mouse aortas were assessed. RAW264.7 macrophage-derived foam cells were obtained via ox-LDL stimulation to investigate the lipid-lowering, anti-atherosclerotic inflammation and anti-apoptotic effect of I4. The data indicated that I4 significantly decreased the lipid accumulation in the circulation and tissue, especially for TG and FFA levels (p < 0.05 vs model group), alleviating the arterial and liver lesions induced by lipotoxicity. Its lipid-reducing effects may due to LOX-1and CD36 expression suppression. I4, at doses of 20 mg/kg and 10 mg/kg, significantly decreased serum IL-6, IL-1β, and TNF-α production and suppressed the expression of p-ERK, p-p38, VCAM-1 and ICAM-1 protein. I4 attenuated atherosclerotic inflammation by blocking NF-κB nuclear translocation, suppressing MAPK/NF-κB signaling pathway and diminishing NF-κB-VCAM-1 promoter region binding. Additionally, I4 suppressed p-p53 and cleaved-caspase-3 expression to inhibit foam cell apoptosis induced by ox-LDL uptake. Overall, I4 exerts potent inhibitory effects on atherosclerosis onset and development.