HDAC2 Expression Research Articles

Zingerone Facilitates Apoptosis in Triple Negative Breast Cancer Cells by Inducing Autophagy

ABSTRACTTriple negative breast cancer (TNBC) is characterized by high heterogenicity and aggressiveness and autophagy plays a complicated role in cancer development. Zingerone is reported to possess multiple pharmacological activities, including antitumors. This study explored the biological role and the relevant mechanisms of zingerone in TNBC. Following zingerone treatment, the viability of normal breast cancer cells MCF‐10A and TNBC cells (MDA‐MB‐231 and MDA‐MB‐468) was detected with CCK‐8 assay. The proliferation, migration and invasion of TNBC cells were detected with colony formation, wound healing, and transwell assays. Western blot was used to detect the expressions of migration‐, apoptosis‐ and autophagy‐related proteins. Flow cytometry was used to detect the cell apoptotic level and immunofluorescence assay measured the autophagy. The experimental data revealed that zingerone with varying concentrations suppressed cell viability, proliferation, migration and invasion while promoting the apoptosis in TNBC, which might be mediated by autophagy activation. Besides, zingerone decreased HDAC1 expression in TNBC cells and regulated autophagy via HDAC1. Collectively, zingerone impeded the malignant progression of TNBC via inducing HDAC1‐mediated autophagy.

Adipose stem cell exosomes, stimulated by pro-inflammatory factors, enhance immune evasion in triple-negative breast cancer by modulating the HDAC6/STAT3/PD-L1 pathway through the transporter UCHL1

BackgroundTriple-negative breast cancer (TNBC) is characterized by high invasiveness and metastasis potential. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is strongly associated with breast cancer progression, although the underlying mechanisms are largely unknown.MethodsThe gene expression profiles of TNBC samples were downloaded from the TCGA database, and ubiquitination enzymes related to immune regulation were screened. UCHL1 expression in the TNBC tissues and in adipose-derived mesenchymal stem cells (ADSCs) stimulated in vitro with pro-inflammatory cytokines were analyzed. Exosomes were isolated from these stimulated ADSCs and transfected with scrambled (si-NC) or UCHL1-specific (si-UCHL1) siRNA constructs. TNBC cells were treated with the ADSCs-derived exosomes (ADSCs-Exos) and then co-cultured with macrophages or T cells. Finally, the tumorigenic potential of the ADSCs-Exos was evaluated by injecting the exosomes into mice bearing TNBC xenografts.ResultsUCHL1 was highly expressed in TNBC tissues and the stimulated ADSCs. The exosomes derived from stimulated ADSCs increased the viability and migration capacity of TNBC cells in vitro, and significantly increased Ki-67 expression through UCHL1. Furthermore, ADSCs-Exos induced M2 polarization of THP-1 monocytes by upregulating CD206 and Arg-1, and downregulating TNF-α and iNOS, and also decreased the proportion of CD3+CD8+ T cells. Mechanistically, UCHL1 regulated the STAT3 and PD-L1 signaling pathways through HDAC6. Exosomes derived from the control and cytokine-stimulated ADSCs also promoted tumor growth in vivo, and increased the expression of UCHL1, CD206, HDAC6, STAT3, and PD-L1. However, UCHL1 knockdown reversed the pro-tumorigenic effects of the ADSCs-derived exosomes in vivo and in vitro.ConclusionPro-inflammatory factors (IFN-γ + TNF-α) stimulating ADSCs-Exos enhance immune evasion in triple-negative breast cancer by regulating the HDAC6/STAT3/PD-L1 pathway via UCHL1 transporter. Thus, UCHL1 inhibition may enhance the response of TNBC to immunotherapy.Graphical

N6-Methyladenosine Regulates Cilia Elongation in Cancer Cells by Modulating HDAC6 Expression.

Primary cilia are microtubule-based organelles that function as cellular antennae to address multiple metabolic and extracellular cues. The past decade has seen significant advances in understanding the pro-tumorigenic role of N6-methyladenosine (m6A) modification in tumorigenesis. Nevertheless, whether m6A modification modulates the cilia dynamics during cancer progression remains unclear. Here, the results show that m6A methyltransferase METTL3regulates cilia length in cancer cells via HDAC6-dependent deacetylation of axonemal α-tubulin, thereby controlling cancer development. Mechanically, METTL3 positively regulates the translation of HDAC6 in an m6A-dependent manner, while m6A methylation of A3678 in the coding sequence (CDS) of HDAC6 ameliorates its translation efficiency via facilitating the binding with YTHDF3. The upregulation of HDAC6 induced by METTL3 over-expression is capable of inhibitingcilia elongation and acetylation of α-tubulin, thereby shortening cilia length and accelerating the progression of cervical cancer both in vitro and in vivo. Collectively, depletion of METTL3-mediated m6A modification leads to abnormally elongated cilia via suppressing HDAC6-dependent deacetylation of axonemal α-tubulin, ultimately attenuating cell growth and cervical cancer development.

Abstract 4129974: Increased Expression of Histone Deacetylases-2 and -6 in Left Ventricular Myocardium of Dogs with Chronic Heart Failure

Background: Histone deacetylases (HDACs) -2 and -6 are localized in the nucleus and cytoplasm of cells and have been implicated in the development of left ventricular (LV) hypertrophy, cardiac interstitial fibrosis, inflammation and worsening of LV systolic and diastolic function thus contributing to progressive worsening of heart failure (HF) with reduced ejection fraction (HFrEF) as well as HF with preserved ejection fraction (HFpEF). There is considerable interest in the application of small molecules that inhibit the activity of HDAC proteins in HF in the hope of fostering clinical benefit. Purpose: In the present study we examined the expression of HDAC-2 and -6 in LV tissue from normal (NL) dogs and dogs with chronic HF (LVEF <35%) produced by multiple sequential intracoronary microembolizations. Methods: Studies were performed in LV tissue from 7 NL dogs and 7 HF dogs. HDAC-2 and -6 protein levels were determined by Western blotting in the lysate of homogenate and 9000g supernatant (cytosolic) fractions prepared from powder of approximately 200 mg frozen LV tissue. After determining the protein by Lowry method, approximately 30 μg homogenate and 15 μg supernatant for HDAC-2 and 8 μg protein for both homogenate and supernatant for HDAC-6 were separated on 4%-20% SDS-PAGE and transferred on PVDF membrane. Band intensities were quantified in densitometric units (du). All data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results: HDAC-2 proteins levels normalized to GAPDH were higher in the cytosol (0.59 ± 0.10 vs. 0.17 ± 0.04, p<0.05 vs. NL) and homogenate (0.75 ± 0.12 vs. 0.55 ± 0.10, not statistically significant) of LV tissue of dogs with HF compared to NL dogs. HDAC-6 protein levels were higher in the homogenate (2.54 ± 0.36 vs. 0.57 ± 0.19, p<0.05 vs. NL) and the cytosol (0.96 ± 0.15 vs. 0.78 ± 0.13, not statistically significant) of LV tissue of dogs with HF compared to NL dogs. Conclusions: These findings indicate upregulation of protein levels of HDAC-2 and -6 isoforms in LV myocardium of dogs with chronic HF. These abnormalities are likely to contribute to worsening HF. Selective inhibition of HDAC-2 and -6 represents a potential therapeutic approach for the treatment of both HFrEF and HFpEF.

HDAC6 as a Prognostic Factor and Druggable Target in HER2-Positive Breast Cancer.

Adjuvant trastuzumab is the standard of care for HER2+ breast cancer (BC) patients. However, >50% of patients become resistant. This study aimed at the identification of the molecular factors associated with disease relapse and their further investigation as therapeutically exploitable targets. Analyses were conducted on formalin-fixed paraffin-embedded tissues of the primary tumors of relapsed (cases) and not relapsed (controls) HER2+ BC patients treated with adjuvant trastuzumab. The nCounter Human Breast Cancer Panel 360 was used. Logistic regression and partitioning around medoids were employed to identify the genes associated with disease recurrence. Cytotoxicity experiments using trastuzumab-resistant cell lines and a network pharmacology approach were carried out to investigate drug efficacy. A total of 52 patients (26 relapsed and 26 not relapsed) were analyzed. We found that a higher expression of HDAC6 was significantly associated with an increased risk of recurrence, with an adjusted OR of 3.20 (95% CI 1.38-9.91, p = 0.016). Then, we investigated the cytotoxic activity of the selective HDAC6 inhibitor Nexturastat A (NextA) on HER2+ cell lines, which were both sensitive and trastuzumab-resistant. A sub-cytotoxic concentration of NextA, combined with trastuzumab, showed a synergistic effect on BC cell lines. Finally, using a network pharmacology approach, we identified HSP90AA1 as the putative molecular candidate responsible for the synergism observed in vitro. Our findings encourage the exploration of the role of HDAC6 as a prognostic factor and the combinatorial use of HDAC6 selective inhibitors combined with trastuzumab in HER2+ BC, in particular for those patients experiencing drug resistance.

Histone deacetylase 9 promotes osteogenic trans-differentiation of vascular smooth muscle cells via ferroptosis in chronic kidney disease vascular calcification.

Vascular calcification, a common complication of chronic kidney disease (CKD), remains an unmet therapeutic challenge. The trans-differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells is crucial in the pathogenesis of vascular calcification in CKD. Despite ferroptosis promotes vascular calcification in CKD, the upstream or downstream regulatory mechanisms involved remains unclear. In this study, we aimed to investigate the regulatory mechanism involved in ferroptosis in CKD vascular calcification. Transcriptome sequencing revealed a potential relationship between HDAC9 and ferroptosis in CKD vascular calcification. Subsequently, we observed increased expression of HDAC9 in calcified arteries of patients undergoing hemodialysis and in a rat model of CKD. We further demonstrated that knockout of HDAC9 attenuates osteogenic trans-differentiation and ferroptosis in VSMCs stimulated by high calcium and phosphorus. In addition, RSL3 exacerbated ferroptosis and osteogenic trans-differentiation of VSMCs exposed to high levels of calcium and phosphorus, and these effects were suppressed to some extent by HDAC9 knockout. In summary, our findings suggest that HDAC9 promotes VSMCs osteogenic trans-differentiation involving ferroptosis, providing new insights for the therapy of CKD vascular calcification.

Inhibition of cytokine-like protein 1 transcription hinders wound-healing process in diabetic rats.

This study explored the function and mechanism of cytokine-like protein 1 (CYTL1) in regulating the wound-healing process of rats with diabetes mellitus (DM). A wound was made in diabetic rats, in which CYTL1 overexpression or HDAC1 expression-interfering adenovirus was injected. The wound area on day 0, 7, 14 and 21 was observed and photographed to calculate the wound-healing rate. The wound tissues were collected for H&E, Masson staining and CD31 immunohistochemistry. The HDAC1 and CYTL1 mRNA and protein expressions in wound tissues were detected by RT-qPCR and western blot. The regulation of HDAC1 on CYTL1 was predicted by hTFtarget and AnimalTFDB database. The H3K27Ac level in the CYTL1 promoter was detected by chromatin immunoprecipitation (ChIP). Diabetic rats with CYTL1 overexpression or interfered HDAC1 expression had accelerated the wound-healing rate, in which massive fibroblasts, attenuated inflammatory infiltration and increased collagen and microvessel density were observed. Further experiments found that HDAC1 can inhibit CYTL1 transcription and expression by inhibiting H3K27Ac expression in CYTL1 promoter. Collected evidence showed HDAC1 can inhibit CYTL1 transcription by down-regulating the H3K27Ac level in CYTL1 promoter to slow down the wound-healing process in diabetic ulcer rats.

Updated Survival Follow-Up for Phase Ib Trial of the Histone Deacetylase Inhibitor Abexinostat With Pazopanib in Patients With Solid Tumor Malignancies.

Histone deacetylase (HDAC) inhibition downregulates hypoxia-inducible factor-1α and modulates multiple metabolomic pathways relevant in cancer. Here we report a potential novel biomarker to predict exceptional responders (>3 years) in patients receiving HDAC and vascular endothelial growth factor (VEGF) inhibition. Patients with solid tumor malignancies were enrolled in this phase Ib trial of abexinostat (4/7 ×21 days) and pazopanib (28/28 days), with a dose expansion in renal cell carcinoma (RCC). Plasma was analyzed for metabolomics and peripheral blood mononuclear cells (PBMCs) for VEGF and HDAC2 expression levels. Fifty-one patients were enrolled: n = 36 patients in dose escalation and n = 15 in dose expansion. After the initial report in 2017, six patients had remained on study: four with RCC and one each with medullary thyroid and thymic neuroendocrine carcinoma. One patient with RCC remains on treatment for >11 years after progression on five systemic therapies. Overall, the median duration of therapy measured 5.6 (1-133) months. The median duration of therapy in exceptional responders measured 44.1 (39.8-133+) months. The median overall survival in patients with high PBMC HDAC2 expression versus low HDAC2 was 32.3 versus 9.2 months (P = .004) for all patients and 43.3 versus 25.1 months for patients with RCC (P = .09). Exceptional responders had lower kynurenine levels both pre- and post-treatment (P = .002, P < .001, respectively). HDAC2 and kynurenine expression levels were inversely correlated (P = .02). Abexinostat added to pazopanib shows extended tolerability and long-term responses and survival. PBMC HDAC2 levels, the abexinostat target, are relevant predictors of response. In addition, metabolomic assessment points to kynurenine as a predictor for exceptional response to combined VEGF plus HDAC inhibition.

Kaempferol alleviates myocardial ischemia injury by reducing oxidative stress via the HDAC3-mediated Nrf2 signaling pathway

IntroductionKaempferol (KAE) is a flavonoid found in various plants. Recent studies showed that high dietary intake of KAE was associated with a lower risk of myocardial infarction; however, the cardioprotective mechanism of KAE remains unknown. ObjectivesTo determine the effect of KAE on cardiac injury in isoproterenol (ISO)-induced rats and cobalt chloride (CoCl2)-treated cardiomyocytes, and the underlying mechanisms. MethodsMale rats were pretreated with different doses of KAE for 14 days, and then injected with ISO to induce myocardial ischemia injury. We also established a model of myocardial cell injury using rat H9c2 cardiomyocytes stimulated with CoCl2. ResultsWe found that KAE pretreatment significantly alleviated myocardial injury and improved cardiac function in ISO-injected rats. In addition, KAE reduced oxidative stress in rats with myocardial ischemia by decreasing malondialdehyde concentration and increasing superoxide dismutase activity, and protection of the myocardial mitochondrial structure. KAE also attenuated CoCl2-induced injuryof H9c2 cardiomyocytes via suppression ofoxidative stress. With regard to the mechanism, we found that KAE down-regulated HDAC3 expression and up-regulated Nrf2 expression in ISO-induced rats and CoCl2-stimulated cardiomyocytes. Incubation of cardiomyocytes with HDAC3-selective inhibitor RGFP966 augmented the protective effect of KAE and reduced oxidative stress. By contrast, HDAC3 overexpression by adenovirus attenuated the effect of KAE on oxidative stress compared with KAE treatment group. HDAC3 also regulated Nrf2 expression in the cardiomyocytes with RGFP966 or an adenovirus overexpressing HDAC3; but Nrf2 inhibition reduced the effect of KAE on ROS generation in CoCl2-induced cardiomyocytes. Immunoprecipitation assay showed that HDAC3 interacted with Nrf2 in cardiomyocytes. Further studies found that KAE increased the acetylation level of Nrf2, while HDAC3 overexpression decreased the acetylation of Nrf2 compared with KAE treatment group. ConclusionOur data show that KAE ameliorates cardiac injury by reducing oxidative stress via the HDAC3-mediated Nrf2 signaling pathway in cardiomyocytes.

Chidamide triggers pyroptosis in T-cell lymphoblastic lymphoma/leukemia via the FOXO1/GSDME axis.

T-cell lymphoblastic lymphoma/leukemia (T-LBL/ALL) is an aggressive form of hematological malignancy associated with poor prognosis in adult patients. Histone deacetylases (HDACs) are aberrantly expressed in T-LBL/ALL and are considered potential therapeutic targets. Here, we investigated the antitumor effect of a novel HDAC inhibitor, chidamide, on T-LBL/ALL. HDAC1, HDAC2 and HDAC3 levels in T-LBL/ALL cell lines and patient samples were compared with those in normal controls. Flow cytometry, transmission electron microscopy and lactate dehydrogenase release assays were conducted in Jurkat and MOLT-4 cells to assess apoptosis and pyroptosis. A specific forkhead box O1 (FOXO1) inhibitor was used to rescue pyroptosis and upregulated gasdermin E (GSDME) expression caused by chidamide treatment. The role of the FOXO1 transcription factor was evaluated by dual-luciferase reporter and chromatin immunoprecipitation assays. The efficacy of chidamide in vivo was evaluated in a xenograft mouse. The expression of HDAC1, HDAC2 and HDAC3 was significantly upregulated in T-LBL/ALL. Cell viability was obviously inhibited after chidamide treatment. Pyroptosis, characterized by cell swelling, pore formation on the plasma membrane and lactate dehydrogenase leakage, was identified as a new mechanism of chidamide treatment. Chidamide triggered pyroptosis through caspase 3 activation and GSDME transcriptional upregulation. Chromatin immunoprecipitation assays confirmed that chidamide led to the increased transcription of GSDME through a more relaxed chromatin structure at the promoter and the upregulation of FOXO1 expression. Moreover, we identified the therapeutic effect of chidamide in vivo. Our study suggested that chidamide exerts an antitumor effect on T-LBL/ALL and promotes a more inflammatory form of cell death via the FOXO1/GSDME axis, which provides a novel choice of targeted therapy for patients with T-LBL/ALL.

HDAC7 promotes ovarian cancer malignancy via AKT/mTOR signalling pathway.

Ovarian cancer is of the most lethal malignancy and causes serious threat to women health worldwide. A deep understanding of molecular mechanisms underlying ovarian cancer progression is critical for the development of promising therapeutic strategies. In this study, we aimed to employ immunohistochemistry to determine the protein level of HDAC7 in patient tissues, our data showed HDAC7 levels are upregulated in tumour tissues. In addition, we also performed Kaplan-Meier survival analysis to investigate the association between HDAC7 expression and clinical prognosis, and found that HDAC7 expression was associated with poor prognosis in ovarian cancer patients. Inhibition of HDAC7 cells resulted in lower cell proliferation, invasion and colony formation. Furthermore, we also found that HDAC7 inhibition suppressed PI3K/AKT/mTOR pathway. In contrast, exogenous HDAC7 expression activated the PI3K/AKT/mTOR pathway in HDAC7 knockout cells and rescued the cell proliferation, invasion and colony formation. However, inhibition of p-AKT induced lower cell proliferation, metastasis and colony formation abilities. In murine model, HDAC7 KO significantly decreased the tumour burden. These data indicate that HDAC7 is involved in regulation of PI3K/AKT/mTOR pathway and targeting of HDAC7 could be potential therapeutic strategy in the treatment of ovarian cancer.

Recombinant adenoviruses expressing HPV16/18 E7 upregulate the HDAC6 and DNMT3B genes in C33A cells.

High-risk human papillomavirus (HPV) is a carcinogenic virus associated with nearly all cases of cervical cancer, as well as an increasing number of anal and oral cancers. The two carcinogenic proteins of HPV, E6 and E7, can immortalize keratinocytes and are essential for HPV-related cellular transformation. Currently, the global regulatory effects of these oncogenic proteins on the host proteome are not fully understood, and further exploration of the functions and carcinogenic mechanisms of E6 and E7 proteins is needed. We used a previously established platform in our laboratory for constructing recombinant adenoviral plasmids expressing the HPV16 E7 gene to further construct recombinant virus particles expressing HPV16/18 E6, E7, and both E6 and E7 genes. These recombinant viruses were used to infect C33A cells to achieve sustained expression of the HPV16/18 E6/E7 genes. Subsequently, total RNA was extracted and RNA-Seq technology was employed for transcriptome sequencing to identify differentially expressed genes associated with HPV infection in cervical cancer. RNA-Seq analysis revealed that overexpression of the HPV16/18 E6/E7 genes upregulated GP6, CD36, HDAC6, ESPL1, and DNMT3B among the differentially expressed genes (DEGs) associated with cervical cancer. Spearman correlation analysis revealed a statistically significant correlation between the HDAC6 and DNMT3B genes and key pathways, including DNA replication, tumor proliferation signature, G2M checkpoint, p53 pathways, and PI3K/AKT/mTOR signaling pathways. Further, qRT-PCR and Western blot analyses indicated that both HPV16/18 E7 can upregulate the expression of HDAC6 and DNMT3B, genes associated with HPV infection-related cervical cancer. The successful expression of HPV16/18 E6/E7 in cells indicates that the recombinant viruses retain the replication and infection capabilities of Ad4. Furthermore, the recombinant viruses expressing HPV16/18 E7 can upregulate the HDAC6 and DNMT3B genes involved in cervical cancer pathways, thereby influencing the cell cycle. Additionally, HDAC6 and DNMT3B are emerging as important therapeutic targets for cancer. This study lays the foundation for further exploration of the oncogenic mechanisms of HPV E6/E7 and may provide new directions for the treatment of HPV-related cancers.

Histone deacetylase expression following cisplatin-induced acute kidney injury in male and female mice.

The chemotherapeutic agent cisplatin accumulates in the kidneys, leading to acute kidney injury (AKI). Preclinical and clinical studies have demonstrated sex-dependent outcomes of cisplatin-AKI. Deranged histone deacetylase (HDAC) activity is hypothesized to promote the pathogenesis of male murine cisplatin-AKI; however, it is unknown whether there are sex differences in the kidney HDACs. We hypothesized that there would be sex-specific Hdac expression, localization, or enzymatic activity, which may explain sexual dimorphic responses to cisplatin-AKI. In normal human kidney RNA samples, HDAC10 was significantly greater in the kidneys of women compared with men, whereas HDAC1, HDAC6, HDAC10, and HDAC11 were differentially expressed between the kidney cortex and medulla, regardless of sex. In a murine model of cisplatin-AKI (3 days after a 15 mg/kg injection), we found few sex- or cisplatin-related differences in Hdac kidney transcripts among the mice. Although Hdac9 was significantly greater in female mice compared with male mice, HDAC9 protein localization did not differ. Hdac7 transcripts were greater in the inner medulla of cisplatin-AKI mice, regardless of sex, and this agreed with a greater HDAC7 abundance. HDAC activity within the cortex, outer medulla, and inner medulla was significantly lower in cisplatin-AKI mice but did not differ between the sexes. In agreement with these findings, a class I HDAC inhibitor did not improve kidney injury or function. In conclusion, even though cisplatin-AKI was evident and there were transcript level differences among the different kidney regions in this model, there were few sex- or cisplatin-dependent effects on kidney HDAC localization or activity.NEW & NOTEWORTHY Kidney histone deacetylases (HDACs) are abundant in male and female mice, and the inner medulla has the greatest HDAC activity. A low dose of cisplatin caused acute kidney injury (AKI) in these mice, but there were few changes in kidney HDACs at the RNA/protein/activity level. A class I HDAC inhibitor failed to improve AKI outcomes. Defining the HDAC isoform, cellular source, and interventional timing is necessary to determine whether HDAC inhibition is a therapeutic strategy to prevent cisplatin-AKI in both sexes.

Astragaloside I from Astragalus Attenuates Diabetic Kidney Disease by Regulating HDAC3/Klotho/TGF-β1 Loop.

Diabetic kidney disease (DKD) has become the primary cause of end-stage renal disease (ESRD), causing an urgent need for preventive strategies for DKD. Astragaloside I (ASI), a bioactive saponin extracted from Astragalus membranaceus (Fisch.) Bunge has been demonstrated to possess a variety of biological activities. This study investigates the therapeutic potential of ASI in DKD and the underlying molecular mechanism using db/db mice in vivo and high glucose (HG)-induced SV40-MES-13 cells in vitro. The results indicated that ASI significantly ameliorated renal dysfunction and mitigated the pathological alterations in the renal tissues of db/db mice. Moreover, ASI was found to reduce the levels of renal fibrosis makers and suppress the activation of TGF-[Formula: see text]1/Smad2/3 pathway in both db/db mice and HG-induced SV40-MES-13 cells. Furthermore, ASI downregulated HDAC3 expression, upregulated Klotho expression, and enhanced Klotho release. ASI is directly bound to HDAC3, and the beneficial effects of ASI on Klotho/TGF-[Formula: see text]1/Smad2/3-mediciated renal fibrosis in DKD were reversed by the HDAC3 agonist ITSA-1. In conclusion, ASI attenuates renal fibrosis in DKD, and may act through concurrently inhibiting HDAC3 and TGF-[Formula: see text]1, thereby regulating HDAC3-mediciated Klotho/TGF-[Formula: see text]1/Smad2/3 pathway.

Abstract B062: Low dose dual epigenetic therapy utilizing hypomethylating agents and HDAC inhibitors prolong survival in an orthotopic PDAC model and alter the tumor microenvironment

Abstract Background: We have previously studied the effect of hypomethylating agent Decitabine (5- aza-dC; DAC) on pancreatic cancer using the KPC model (KrasG12D/+;Trp53R172H/+;Pdx1- Cre) and observed increased CD4+/CD8+ TILs, greater PD1 expression and slowed tumor growth with increased tumor necrosis. Subsequently we showed a significant survival benefit with DAC+αPD1 compared to either single agent. However, we identified a specific myeloid cell population initially identified by Chi3l3 expression that may be responsible for treatment resistance or escape. Based on prior observations of synergy specifically affecting tumor infiltrating myeloid and lymphoid cells, we focused on identifying a dual epigenetic strategy (a combination of DAC plus histone deacetylase inhibitors (HDACi)) to enhance immunological synergy and test their treatment efficacy with αPD1. Methods: We used a combination primary KPC-derived cell lines, bone marrow (BMDM) and peritoneum derived (PM) myeloid cells and T cells in co-culture systems to assess cytotoxic synergy (Bliss/Lowe consensus) and immune effects (cytokine release, myeloid cell polarization and T cell activation) to evaluate combinations of DAC+HDACi. The KPC orthotopic model relying on US monitoring of tumor size for standardized initiation of therapy was used for survival analysis and profiling in treatment combinations of DAC+HDACi+αPD1. Results: We showed that hypomethylation therapy exerts a primarily paracrine effect on myeloid cells and this polarization results in a decrease in DAC–induced T cell activation. We therefore selected HDAC inhibitors that in combination with DAC alter this paracrine effect. We selected specific HDAC inhibitors based on induction of class of HDAC expression following hypomethylation therapy, highest Bliss synergy scores at low doses and reversal of cytokine release, M2 polarization and T cell inactivation. Three HDAC inhibitors were chosen for in vivo testing, Domatinostat, Pracinostat and Entinostat. Using the treatment paradigm of ultra-low dose DAC+ HDACi (mainly to minimize toxicity) combined with αPD1 in the KPC orthotopic model we show that the triple combination (DAC + Entinostat + αPD1) is well-tolerated and results in a significant survival benefit compared to single and dual agent combinations. In addition, we also demonstrate a reduction in the Chi3l3 expressing myeloid cell population. Conclusions: Our results show that the combination of hypomethylating agent plus HDAC inhibitor has several beneficial effects on the tumor immune response in PDAC. HDAC inhibitors specifically reduce the suppressive myeloid cell population we previously identified after DAC therapy. The combination of dual low dose epigenetic therapy using a hypomethylating agent plus HDAC inhibitor followed by αPD1 results in significant survival benefit in the orthotopic KPC model. Citation Format: Stephen Muzyka, Arturo Orlacchio, Amanda Ackermann, Yasmine Chinda, Yoona Shim, Igor Dolgalev, Tamas Gonda. Low dose dual epigenetic therapy utilizing hypomethylating agents and HDAC inhibitors prolong survival in an orthotopic PDAC model and alter the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B062.

Impact of HDAC6-mediated progesterone receptor expression on the response of breast cancer cells to hormonal therapy

Modulation of estrogen receptor (ER) and progesterone receptor (PR) expression, as well as their emerging functional crosstalk, remains a potential approach for enhancing the response to hormonal therapy in breast cancer. Aberrant epigenetic alterations induced by histone deacetylases (HDACs) were massively implicated in dysregulating the function of hormone receptors in breast cancer. Although much is known about the regulation of ER signaling by HDAC, the precise role of HDAC in modulating the expression of PR and its impact on the outcomes of hormonal therapy is poorly defined. Here, we demonstrate the involvement of HDAC6 in regulating PR expression in breast cancer cells. The correlation between HDAC6 and hormone receptors was investigated in patients’ tissues by immunohistochemistry (n = 80) and publicly available data (n = 3260) from breast cancer patients. We explored the effect of modulating the expression of HDAC6 as well as its catalytic inhibition on the level of hormone receptors by a variety of molecular analyses, including Western blot, immunofluorescence, Real-time PCR, RNA-seq analysis and chromatin immunoprecipitation. Based on our in-silico and immunohistochemistry analyses, HDAC6 levels were negatively correlated with PR status in breast cancer tissues. The downregulation of HDAC6 enhanced the expression of PR-B in hormone receptor-positive and triple-negative breast cancer (TNBC) cells. The selective targeting of HDAC6 by tubacin resulted in the enrichment of the H3K9 acetylation mark at the PGR-B gene promoter region and enhanced the expression of PR-B. Additionally, transcriptomic analysis of tubacin-treated cells revealed enhanced activity of acetyltransferase and growth factor signaling pathways, along with the enrichment of transcription factors involved in the transcriptional activity of ER, underscoring the crucial role of HDAC6 in regulating hormone receptors. Notably, the addition of HDAC6 inhibitor potentiated the effects of anti-ER and anti-PR drugs mainly in TNBC cells. Together, these data highlight the role of HDAC6 in regulating PR expression and provide a promising therapeutic approach for boosting breast cancer sensitivity to hormonal therapy.

Identification of HDAC10 as a candidate oncogene in clear cell renal carcinoma that facilitates tumor proliferation and metastasis

BackgroundClear cell renal cell carcinoma (ccRCC) remains one of the most lethal urological malignancies even though a great number of improvements in diagnosis and management have achieved over the past few decades. Accumulated evidence revealed that histone deacetylases (HDACs) play vital role in cell proliferation, differentiation and apoptosis. Nevertheless, the biological functions of histone deacetylation modification related genes in ccRCC remains poorly understood.MethodBulk transcriptomic data and clinical information of ccRCC patients were obtained from the TCGA database and collected from the Chinese PLA General Hospital. A total of 36 histone deacetylation genes were selected and studied in our research. Univariate cox regression analysis, least absolute shrinkage and selection operator (LASSO) regression, random forest (RF) analysis, and protein-protein interaction (PPI) network analysis were applied to identify key genes affecting the prognosis of ccRCC. The ‘oncoPredict’ algorithm was utilized for drug-sensitive analysis. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to explore the potential biological function. The ssGSEA algorithm was used for tumor immune microenvironment analysis. The expression levels of HDAC10 were validated by RT-PCR and immunohistochemistry (IHC). 5-ethynyl-2′-deoxyuridine (EdU assay), CCK-8 assay, cell transwell migration and invasion assay and colony formation assay were performed to detect the proliferation and invasion ability of ccRCC cells. A nomogram incorporating HDAC10 and clinicopathological characteristics was established to predict the prognosis of ccRCC patients.ResultTwo machine learning algorithms and PPI analysis identified four histone deacetylation genes that have a significant association with the prognosis of ccRCC, with HDAC10 being the key gene among them. HDAC10 is highly expressed in ccRCC and its high expression is associated with poor prognosis for ccRCC patients. Pathway enrichment and the experiments of EdU staining, CCK-8 assay, cell transwell migration and invasion assay and colony formation assay demonstrated that HDAC10 mediated the proliferation and metastasis of ccRCC cells and involved in reshaping the tumor microenvironment (TME) of ccRCC. A clinically reliable prognostic predictive model was established by incorporating HDAC10 and other clinicopathological characteristics ( https://nomogramhdac10.shinyapps.io/HDAC10_Nomogram/ ).ConclusionOur study found the increased expression of HDAC10 was closely associated with poor prognosis of ccRCC patients. HDAC10 showed a pro-tumorigenic effect on ccRCC and promote the proliferation and metastasis of ccRCC, which may provide new light on targeted therapy for ccRCC.

MiRNA-105–5p regulates the histone deacetylase HDAC2 through FOXG1 to affect the malignant biological behavior of triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC) is a specific subtype of breast cancer (BC). Some potential molecular targets have been identified, and miR-105–5p was found to be abnormally expressed in TNBC tissues. ObjectiveThe objective of this study was to probe the effect of miR-105–5p on TNBC via FOXG1/HDAC2-mediated acetylation. MethodsAn animal model of TNBC was established by injecting BC cells into the axillary area of nude mice. The levels of miR-105–5p, FOXG1, HDAC2, Bcl-2, Bax, and Ki67 were detected via RT‒qPCR, Western blotting and immunohistochemistry. Flow cytometry, CCK-8, Transwell and colony formation assays were used to measure apoptosis, proliferation and migration, respectively. Total histone acetylation levels were measured by ELISA. The binding of FOXG1 to HDAC2 was detected by co-immunoprecipitation. The binding relationship between miR-105–5p and FOXG1 was verified using a dual-luciferase reporter gene assay. ResultsIn this study, miR-105–5p and HDAC2 were highly expressed in the MDA-MB-231 and BT-549 BC cell lines, whereas FOXG1 was expressed at low levels. The inhibition of miR-105–5p inhibited the proliferation and migration of MDA-MB-231 and BT-549 cells and promoted their apoptosis. Bioinformatics analysis revealed that miR-105–5p and FOXG1 had a negative targeting regulatory relationship. FOXG1 overexpression had a similar effect on cancer cells as the inhibition of miR-105–5p. Moreover, experiments revealed that FOXG1 and HDAC2 could bind to each other and that HDAC2 overexpression or treatment with the histone acetyltransferase inhibitor Garcinol weakened the effect of FOXG1 overexpression. In addition, FOXG1 knockdown inhibited the effect of the miR-105–5p inhibitor, while Garcinol treatment further enhanced the effect of FOXG1 knockdown, inhibited histone acetylation, promoted the proliferation and migration of cancer cells, and inhibited apoptosis. Moreover, the in vivo results confirmed the in vitro results. ConclusionmiR-105–5p promotes HDAC2 expression by reducing FOXG1, inhibits histone acetylation, and aggravates the malignant biological behavior of TNBC cells.

Expression patterns of HDAC6 in correlation to ARID1A status in different subtypes of endometriosis: A retrospective tissue microarray analysis

Endometriosis is a disease affecting approximately 10% of reproductive age women. Loss of the tumor suppressor gene AT-rich interactive domain-containing protein 1A (ARID1A) occurs in some endometriosis cases. Histone deacetylase 6 (HDAC-6) is an enzyme with implication in several diseases including different cancer types and immunological disorders, where it is involved in protein trafficking and degradation, cell shape, and migration. In ARID1A-deficient ovarian cancer increased HDAC-6 expression lead to apoptosis-inhibiting post-translational modification of p53. It is not known if HDAC-6 expression is also altered in ARID1A-deficient endometriosis. The aim of this study was to assess HDAC-6 expression in endometriotic lesions in correlation to ARID1A-status.Two tissue-microarrays with 168 endometriotic lesions, including ovarian (64/168, 38 %), peritoneal (66/168, 39 %) and deep-infiltrating (38/168, 23 %) subtypes, and 73 endometrium of women without endometriosis were assessed. Mean ARID1A immunoreactivity score (IRS) in endometriosis group was 10.83 (±2.36) and 10.78 (±1.94) in the epithelium and stroma, respectively, while the respective mean HDAC6 IRS were 9.16 (±2.76) and 5.94 (±2.88). The comparison of the HDAC6 expression between endometriosis subtypes showed higher expression in deep-infiltrating endometriosis, in both, epithelium (p = 0.032) and stroma (p = 0.007). In ARID1A negative cases, epithelial expression of HDAC6 was higher in endometriosis compared to women without endometriosis (p = 0.031), and this was also specifically observed in the subset of ovarian endometriosis (p = 0.037). There were no significant differences in the stromal expression of HDAC6.In conclusion, our results demonstrate a complex expression pattern of HDAC6 depending on ARID1A status in different endometriosis subtypes. Further studies on HDAC6 and ARID1A are important to elucidate mechanisms involved in malignant transformation of endometriosis.

Differential Expression of DNA Methyltransferase (DNMT1 and DNMT3), Histone Deacetylase (HDAC1 and HDAC2), and Upstream Target Regulators MiR-145 and Mir-152 among Oral Cancers

Epigenetic modulation of DNA and histones facilitated by and histone deacetylases (HDAC) is associated with the development and progression of many cancers, although less is known about DNA methyltransferase (DNMT) in oral cancers and the regulation of these targets. Using commercially available cell lines, oral squamous cell carcinomas (SCC4, SCC9, SCC15, SCC25, and CAL27), and normal gingival fibroblasts (HGF-1), growth assays and mRNA expression were evaluated using ANOVA. These results revealed homeostasis enzyme DNMT1 expression was significantly higher among slow-growing HGF-1 cells than among fast-growing oral cancers, p &lt; 0.05. In contrast, DNMT3A and DNMT3B expression was significantly higher among oral cancers compared with HGF-1 cells, p &lt; 0.05. However, differential expression of HDAC1 and HDAC2 was observed among SCC4, SCC25, and CAL27 cells. Further analysis of miR-152 (regulation and control of DNMT expression) and miR-21, miR-221, and miR-145 (regulation of HDAC expression) revealed all oral cancers produced miR-21, but none produced miR-221. However, differential expression of miR-145 (SCC15) and miR-152 (SCC25) suggested alternative epigenetic pathways and mechanisms of DNMT and HDAC regulation may be responsible for some of the observations revealed in this study.