A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

Gene Expression of the D-Series Resolvin Pathway Predicts Activation of Anti-Tumor Immunity and Clinical Outcomes in Head and Neck Cancer.

In human medicine, the progression from early neoplasia development to either complete resolution of tumorigenesis and associated inflammation or chronicity and fatal outcomes remain difficult to predict. Resolution of inflammation is an active process that stimulates the termination of the inflammatory response and promotes return to homeostasis, while failure in resolution contributes to the development of a number of diseases. To understand how resolution pathways contribute to tumorigenesis, we defined and employed a cumulative score based on the expression level of genes involved in synthesis, signaling, and metabolism of the D-series resolvin (RvD). This score was used for comparative analyses of clinical, cellular, and molecular features of tumors, based on RNA-sequencing (RNA-seq) datasets collected within The Cancer Genome Atlas (TCGA) program. Our results indicate that higher RvD scores are associated with better clinical outcome of patients with head and neck squamous cell carcinoma (HNSC), and with molecular and cellular signatures indicative of enhanced anti-tumor immunity and better response to immune-checkpoint inhibitors (ICI), also in human papilloma virus (HPV) negative HNSC subtypes. Thus, higher activity of the RvD pathway identifies patients with improved resolution and a more efficient immune reaction against cancer.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems.

Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.

Synergistic antitumour activity of HDAC inhibitor SAHA and EGFR inhibitor gefitinib in head and neck cancer: a key role for ΔNp63α.

BACKGROUND: Epidermal growth factor receptor (EGFR) overexpression is associated with the development of head and neck cancer (HNC) and represents one of the main therapeutic targets for this disease. The use of EGFR inhibitors has limited efficacy due to their primary and acquired resistance, partially because of increased epithelial to mesenchymal transition (EMT). The HDAC inhibitor SAHA has been shown to revert EMT in different tumours, including HNC. In this study, we investigated the cooperative role of SAHA and the EGFR tyrosine kinase inhibitor gefitinib in both HPV-positive and HPV-negative HNC cell lines. METHODS: A panel of 12 HPV-positive and HPV-negative HNC cell lines were screened for cell viability upon treatment with SAHA, gefitinib and the combination of the two. Epithelial/mesenchymal marker expression, as well as activation of signalling pathway, were assessed upon SAHA treatment. ΔNp63α silencing with shRNA lentiviral particles was used to determine its role in cell proliferation, migration and TGFß pathway activation. RESULTS: We found that both SAHA and gefitinib have antitumour activity in both HPV-positive and HPV-negative HNC cell lines and that their combination has a synergistic effect in inhibiting cell growth. SAHA treatment reverts EMT and inhibits the expression of the transcription factor ΔNp63α. Suppression of ΔNp63α reduces EGFR protein levels and decreases cell proliferation and TGFß-dependent migration in both HPV-positive and HPV-negative HNC cell lines. CONCLUSIONS: Our results, by giving a clear molecular mechanism at the basis of the antitumour activity of SAHA in HNC cell lines, provide a rationale for the clinical evaluation of SAHA in combination with gefitinib in both HPV-positive and HPV-negative HNC patients. Further knowledge is key to devising additional lines of combinatorial treatment strategies for this disease.

Autophagy regulates UBC9 levels during viral-mediated tumorigenesis.

UBC9, the sole E2-conjugating enzyme required for SUMOylation, is a key regulator of essential cellular functions and, as such, is frequently altered in cancers. Along these lines, we recently reported that its expression gradually increases during early stages of human papillomavirus (HPV)-mediated cervical lesions transformation. However, a better understanding of how UBC9 is exploited by transforming viral oncoproteins is still needed. In the present study, we show that in human samples HPV drives UBC9 up-regulation also in very early steps of head and neck tumorigenesis, pointing to the important role for UBC9 in the HPV-mediated carcinogenic program. Moreover, using HPV-infected pre-cancerous tissues and primary human keratinocytes as the natural host of the virus, we investigate the pathological meaning and the cellular mechanisms responsible for UBC9 de-regulation in an oncoviral context. Our results show that UBC9 overexpression is promoted by transforming viral proteins to increase host cells' resistance to apoptosis. In addition, ultrastuctural, pharmacological and genetic approaches crucially unveil that UBC9 is physiologically targeted by autophagy in human cells. However, the presence of HPV E6/E7 oncoproteins negatively impacts the autophagic process through selective inhibition of autophagosome-lysosome fusion, finally leading to p53 dependent UBC9 accumulation during viral-induced cellular transformation. Therefore, our study elucidates how UBC9 is manipulated by HPV oncoproteins, details the physiological mechanism by which UBC9 is degraded in cells, and identifies how HPV E6/E7 impact on autophagy. These findings point to UBC9 and autophagy as novel hallmarks of HPV oncogenesis, and open innovative avenues towards the treatment of HPV-related malignancies.

BC-box protein domain-related mechanism for VHL protein degradation.

The tumor suppressor VHL (von Hippel-Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasome-dependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins.

HPV-mediated regulation of SMAD4 modulates the DNA damage response in head and neck cancer.

BACKGROUND: Head and Neck cancer (HNC) is a fatal malignancy with poor prognosis. Human Papillomavirus (HPV) infection is becoming the prominent cause of HNC in the western world, and studying the molecular mechanisms underlying its action in cancers is key towards targeted therapy. To replicate, HPV regulates the host DNA damage repair (DDR) pathway. SMAD4 is also involved in the regulation of the DDR machinery and likely plays important role in maintaining cell viability upon genotoxic stress. In this study, we investigated the role of HPV in the upregulation of SMAD4 to control the DDR response and facilitate its lifecycle. METHODS: SMAD4, Rad51 and CHK1 expression was assessed in HPV-positive and HPV-negative HNC using TCGA data, a panel of 14 HNC cell lines and 8 fresh tumour tissue samples from HNC patients. HPV16 expression was modulated by E6/E7 siRNA knock-down or transduction in HPV-positive HNC cell lines and Human Primary keratinocytes respectively. SMAD4 half-life was assessed by cycloheximide treatment in HNC cell lines, together with ßTRCP1-dependent SMAD4 ubiquitination. SMAD4 siRNA knock-down was used to determine its role in HPV-mediated regulation of DDR machinery and to assess cisplatin sensitivity in HPV-positive HNC cell lines. RESULTS: We found that HPV increases SMAD4 expression is both HPV-positive HNC tumours and cell lines, impairing its degradation which is mediated by the E3 ubiquitin ligase ßTRCP1. SMAD4 expression highly correlates with the expression of two main players of the DDR pathway, CHK1 and Rad51, which expression is also upregulated by the presence of HPV. In particular, we demonstrate that HPV stabilizes SMAD4 to increase CHK1 and Rad51 expression. In addition, SMAD4-deficient HPV-positive cells have increased sensitivity to cisplatin treatment. CONCLUSIONS: Our results give a clear molecular mechanism at the basis of HPV regulation of the DDR pathway. In particular, we show how HPV stabilizes SMAD4 to promote DDR protein expression, which may be used to facilitate viral replication and HNC onset. Moreover, we found that SMAD4 silencing in HPV-positive HNC cell lines increases sensitivity to cisplatin treatment, suggesting that HPV-positive HNC with low SMAD4 expression may be preferentially susceptible to similar treatments.

The UBC9/SUMO pathway affects E-cadherin cleavage in HPV-positive head and neck cancer.

Functional loss of E-cadherin is frequent during tumor progression and occurs through a variety of mechanisms, including proteolytic cleavage. E-cadherin downregulation leads to the conversion of a more malignant phenotype promoting Epithelial to Mesenchymal Transition (EMT). The UBC9/SUMO pathway has been also shown to be involved in the regulation of EMT in different cancers. Here we found an increased expression of UBC9 in the progression of Head and Neck Cancer (HNC) and uncovered a role for UBC9/SUMO in hampering the HPV-mediated E-cadherin cleavage in HNC.

Vitamin D and SARS-CoV2 infection, severity and mortality: A systematic review and meta-analysis.

To assess the evidence on SARS-CoV2 infection and Covid-19 in relation to deficiency and supplementation of vitamin D, we conducted a systematic review up to April 2021. We summarised data from 38 eligible studies, which presented risk estimates for at least one endpoint, including two RCT and 27 cohort-studies: 205565 patients with information on 25OHD status and 2022 taking vitamin D supplementation with a total of 1197 admitted to the ICU or who needed invasive mechanical ventilation or intubation and hospital stay, and more than 910 Covid-19 deaths. Primary outcomes were severity and mortality and the main aim was to evaluate the association with vitamin D supplementation. Random effects models showed that supplementation was associated with a significant lower risk of both Covid-19 severe disease (SRR 0.38, 95% CI 0.20-0.72, 6 studies) and mortality (SRR 0.35, 95% CI 0.17-0.70, 8 studies). There were no statistically significant dose differences between studies: summary estimates with regular doses remain statistically significant, suggesting that higher doses are not necessary. For patients on vitamin D supplementation, a greater reduction in mortality risk emerged in older individuals and at higher latitudes. Regarding the quality of studies, assessed using the New Castle-Ottawa quality scale, the analysis revealed in most cases no statistically significant differences between low, medium or high quality studies. We found significant associations of vitamin D supplementation with Covid-19, encompassing risks of disease worsening and mortality, especially in seasons characterized by 25OHD deficiency and with not severe patients. Dedicated randomized clinical studies are encouraged to confirm these results.

NF-Y Subunits Overexpression in HNSCC.

NF-Y is the CCAAT-binding trimer formed by the histone fold domain (HFD), NF-YB/NF-YC and NF-YA. The CCAAT box is generally prevalent in promoters of "cancer" genes. We reported the overexpression of NF-YA in BRCA, LUAD and LUSC, and of all subunits in HCC. Altered splicing of NF-YA was found in breast and lung cancer. We analyzed RNA-seq datasets of TCGA and cell lines of head and neck squamous cell carcinomas (HNSCC). We partitioned all TCGA data into four subtypes, deconvoluted single-cell RNA-seq of tumors and derived survival curves. The CCAAT box was enriched in the promoters of overexpressed genes. The "short" NF-YAs was overexpressed in all subtypes and the "long" NF-YAl in Mesenchymal. The HFD subunits are overexpressed, except Basal (NF-YB) and Atypical (NF-YC); NF-YAl is increased in p53 mutated tumors. In HPV-positive tumors, high levels of NF-YAs, p16 and ΔNp63 correlate with better prognosis. Deconvolution of single cell RNA-seq (scRNA-seq) found a correlation of NF-YAl with Cancer Associated Fibroblasts (CAFs) and p-EMT cells, a population endowed with metastatic potential. We conclude that overexpression of HFD subunits and NF-YAs is protective in HPV-positive tumors; expression of NF-YAl is largely confined to mutp53 tumors and malignant p-EMT cells.

Vitamin D Supplementation and Cancer Mortality: Narrative Review of Observational Studies and Clinical Trials.

Several studies have investigated the beneficial effects of vitamin D on survival of cancer patients. Overall evidence has been accumulating with contrasting results. This paper aims at narratively reviewing the existing articles examining the link between vitamin D supplementation and cancer mortality. We performed two distinct searches to identify observational (ObS) studies and randomized clinical trials (RCTs) of vitamin D supplementation (VDS) in cancer patients and cohorts of general population, which included cancer mortality as an outcome. Published reports were gathered until March 2021. We identified 25 papers published between 2003 and 2020, including n. 8 RCTs on cancer patients, n. 8 population RCTs and n. 9 ObS studies. There was some evidence that the use of VDS in cancer patients could improve cancer survival, but no significant effect was found in population RCTs. Some ObS studies reported evidence that VDS was associated with a longer survival among cancer patients, and only one study found an opposite effect. The findings do not allow conclusive answers. VDS may have the potential as treatment to improve survival in cancer patients, but further investigations are warranted. We strongly support investment in well-designed and sufficiently powered RCTs to fully evaluate this association.

Conversion of the BASE prion strain into the BSE strain: the origin of BSE?

Atypical neuropathological and molecular phenotypes of bovine spongiform encephalopathy (BSE) have recently been identified in different countries. One of these phenotypes, named bovine "amyloidotic" spongiform encephalopathy (BASE), differs from classical BSE for the occurrence of a distinct type of the disease-associated prion protein (PrP), termed PrP(Sc), and the presence of PrP amyloid plaques. Here, we show that the agents responsible for BSE and BASE possess different biological properties upon transmission to transgenic mice expressing bovine PrP and inbred lines of nontransgenic mice. Strikingly, serial passages of the BASE strain to nontransgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species, including humans.

Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development.

Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology.

PI3K/mTOR mediate mitogen-dependent HDAC1 phosphorylation in breast cancer: a novel regulation of estrogen receptor expression.

Histone deacetylase 1 (HDAC1) is an important epigenetic controller involved in transcriptional regulation through modification of chromatin structure. Genetic and epigenetic changes and deregulation of signal transduction pathways have been implicated in the development of breast cancer. Downregulation of estrogen receptor α (ERα) expression is one of the mechanisms behind the acquisition of endocrine resistance. Sustained and increased hormone and growth factor receptor signaling in breast cancer cells contribute to resistance to endocrine therapy. Both HDACs and the PI3K/mTOR signaling pathway are becoming promising targets in breast cancer, reversing also acquired hormone resistance. Here we show how mitogens, activating the PI3K/mTOR pathway, trigger the phosphorylation of HDAC1 in breast cancer cells, which is completely dependent on the activity of the p70 S6 kinase (S6K1). Our findings show that S6K1, overexpressed in many breast cancers, controls HDAC1-dependent transcriptional regulation of ERα levels upon mitogenic stimuli, controlling HDAC1 recruitment to the ERα promoter. Furthermore, cell treatment with both mTOR and HDACs inhibitors shows an additive effect in inhibiting breast cancer proliferation. This confirms the novel cross-talk between the HDAC1 and PI3K pathways with clinical implications towards the treatment of this malignant disease.

Redox-Mediated Suberoylanilide Hydroxamic Acid Sensitivity in Breast Cancer.

AIMS: Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the potential to be effective also in breast cancer. We investigated the responsiveness to SAHA in human breast primary tumors and cancer cell lines. RESULTS: We observed a differential response to drug treatment in both human breast primary tumors and cancer cell lines. Gene expression analysis of the breast cancer cell lines revealed that genes involved in cell adhesion and redox pathways, especially glutathione metabolism, were differentially expressed in the cell lines resistant to SAHA compared with the sensitive ones, indicating their possible association with drug resistance mechanisms. Notably, such an association was also observed in breast primary tumors. Indeed, addition of buthionine sulfoximine (BSO), a compound capable of depleting cellular glutathione, significantly enhanced the cytotoxicity of SAHA in both breast cancer cell lines and primary breast tumors. INNOVATION: We identify and validate transcriptional differences in genes involved in redox pathways, which include potential predictive markers of sensitivity to SAHA. CONCLUSION: In breast cancer, it could be relevant to evaluate the expression of antioxidant genes that may favor tumor resistance as a factor to consider for potential clinical application and treatment with epigenetic drugs (HDACis).

Molecular and biologic analysis of histone deacetylase inhibitors with diverse specificities.

Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited.

The efficacy of tetracyclines in peripheral and intracerebral prion infection.

We have previously shown that tetracyclines interact with and reverse the protease resistance of pathological prion protein extracted from scrapie-infected animals and patients with all forms of Creutzfeldt-Jakob disease, lowering the prion titre and prolonging survival of cerebrally infected animals. To investigate the effectiveness of these drugs as anti-prion agents Syrian hamsters were inoculated intramuscularly or subcutaneously with 263K scrapie strain at a 10(-4) dilution. Tetracyclines were injected intramuscularly or intraperitoneally at the dose of 10 mg/kg. A single intramuscular dose of doxycycline one hour after infection in the same site of inoculation prolonged median survival by 64%. Intraperitoneal doses of tetracyclines every two days for 40 or 44 days increased survival time by 25% (doxycycline), 32% (tetracycline); and 81% (minocycline) after intramuscular infection, and 35% (doxycycline) after subcutaneous infection. To extend the therapeutic potential of tetracyclines, we investigated the efficacy of direct infusion of tetracyclines in advanced infection. Since intracerebroventricular infusion of tetracycline solutions can cause overt acute toxicity in animals, we entrapped the drugs in liposomes. Animals were inoculated intracerebrally with a 10(-4) dilution of the 263K scrapie strain. A single intracerebroventricular infusion of 25 microg/20 microl of doxycycline or minocycline entrapped in liposomes was administered 60 days after inoculation, when 50% of animals showed initial symptoms of the disease. Median survival increased of 8.1% with doxycycline and 10% with minocycline. These data suggest that tetracyclines might have therapeutic potential for humans.