Follow-Up Study Confirms the Presence of Gastric Cancer DNA Methylation Hallmarks in High-Risk Precursor Lesions

Intestinal metaplasia confers an increased risk of progression to gastric cancer. However, some intestinal metaplasia patients do not develop cancer. The development of robust molecular biomarkers to stratify patients with advanced gastric precursor lesions at risk of cancer progression will contribute to guiding programs for prevention. Starting from a genome-wide methylation study, we have simplified the detection method regarding candidate-methylation tests to improve their applicability in the clinical environment. We identified CpG methylation at the ZNF793 and RPRM promoters as a common event in intestinal metaplasia and intestinal forms of gastric cancer. Furthermore, we also showed that Helicobacter pylori infection influences DNA methylation in early precursor lesions but not in intestinal metaplasia, suggesting that therapeutic strategies to prevent epigenome reprogramming toward a cancer signature need to be adopted early in the precursor cascade. To adopt prevention strategies in gastric cancer, it is imperative to develop robust biomarkers with acceptable costs and feasibility in clinical practice to stratified populations according to risk scores. With this aim, we applied an unbiased genome-wide CpG methylation approach to a discovery cohort composed of gastric cancer (n = 24), and non-malignant precursor lesions (n = 64). Then, candidate-methylation approaches were performed in a validation cohort of precursor lesions obtained from an observational longitudinal study (n = 264), with a 12-year follow-up to identify repression or progression cases. H. pylori stratification and histology were considered to determine their influence on the methylation dynamics. As a result, we ascertained that intestinal metaplasia partially recapitulates patterns of aberrant methylation of intestinal type of gastric cancer, independently of the H. pylori status. Two epigenetically regulated genes in cancer, RPRM and ZNF793, consistently showed increased methylation in intestinal metaplasia with respect to earlier precursor lesions. In summary, our result supports the need to investigate the practical utilities of the quantification of DNA methylation in candidate genes as a marker for disease progression. In addition, the H. pylori-dependent methylation in intestinal metaplasia suggests that pharmacological treatments aimed at H. pylori eradication in the late stages of precursor lesions do not prevent epigenome reprogramming toward a cancer signature

The role of FKBP51 in the activation of Akt oncogenic pathway

FKBP51 is an immunophilin with a relevant role in sustaining cancer cell growth and aggressiveness of different human tumors and, particularly, melanoma. The protein contains two N-terminal FKBP-like domains FK1 and FK2, with FK1 exerting an isomerase activity, and a C-terminal region with a tetratricopeptide repeat (TPR) domain for protein/protein interaction. Based on its scaffold and isomerase activities, it participates in several signaling pathways, including NF-κB and Akt signaling. The role of FKBP51 in the Akt pathway is controversial. In 2009 Pei et al. proposed a role for FKBP51 as a scaffold promoting interaction between Akt and PHLPP phosphatase, thus negatively regulating Akt activation, in a pancreatic cancer context. In 2010, Romano et al. showed an association between FKBP51 upregulation and high pAkt levels in different tumor types. In 2013, Fabian et al. reported an increase in pAkt S473 upon FKBP51 overexpression. Deregulation of the Akt pathway is one of the major mechanisms that sustain cancer survival and progression. Especially in an incurable tumor as melanoma, in depth knowledge of the mechanisms underlying Akt activation is needed. In this thesis work, the interactions of FKBP51 with Akt and PHLPP are investigated along with the mechanism of Akt activation by the immunophilin. The study benefited from the recent identification of the FKBP51 spliced isoform (lacking the TPR domain), that reconciled the diverging results, generating the hypothesis that a unique gene regulated phosphorylation and de-phosphorylation of Akt. Thanks to the use of TPR-mutants, I demonstrated that the TPR domain of the canonical isoform mediates Akt phosphorylation through the activation of Akt-K63-ubiquitination. Conversely, upregulation of the spliced FKBP51s decreased K63-Ub residues binding to Akt. I also show that Hsp90, a known interactor of FKBP51-TPR domain, is a further important element in Akt activation. The pharmacological inhibition of Hsp90, indeed, impaired Akt K63-ubiquitination. Lack of TPR in FKBP51s explains the defective K63-ubiquitination capability. Unexpectedly, PHLPP silencing did not foster phosphorylation of Akt, whereas its overexpression even promoted K63-ubiquitination and phosphorylation of Akt. As TRAF6 is a E3 ubiquitin ligase and a known FKBP51 interactor that undergoes degradation upon phosphorylation, we hypothesized a role for PHLPP in stabilizing TRAF6. The finding that PHLPP silencing decreased TRAF6 level, supported such a hypothesis. Collectively, these results suggest that FKBP51 promotes Akt activation by serving as a scaffold to build the macrocomplex deputed to Akt ubiquitination and phosphorylation. Moreover, the present study introduces an unknown oncogenic role for the phosphatase PHLPP. A preliminary mass spectrometry-based proteome profile of melanoma cells overexpressing PHLPP, highlighted a relevant contribution of PHLPP especially in improvement of melanoma cell proliferation. Our findings have profound implications for designing novel melanoma therapies based on modulation of FKBP51 and FKBP51s that may affect Akt activity in cancer

Cell stemness, epithelial-to-mesenchymal transition, and immunoevasion: Intertwined aspects in cancer metastasis.

Recent advances in tumor immunology, fostered by dramatic outcomes with cancer immunotherapy, have opened new scenarios in cancer metastasis. The cancer stemness/mesenchymal phenotype and an excess of immune suppressive signals are emerging as Intertwined aspects of human tumors. This review examines recent studies that explored the mechanistic links between cancer cell stemness and immunoevasion, and the evidence points to these key events in cancer metastasis as two sides of the same coin. This review also covers the mechanisms involved in tumor expression of programmed cell death ligand 1 (PD-L1), a major factor exploited by human neoplasias to suppress immune control. We highlight the convergence of mesenchymal traits and PD-L1 expression and examine the functions of this immune inhibitory molecule, which confers cancer cell resistance and aggressiveness

FKBP51 Affects TNF-Related Apoptosis Inducing Ligand Response in Melanoma

Melanoma is one of the most immunogenic tumors and has the highest potential to elicit specific adaptive antitumor immune responses. Immune cells induce apoptosis of cancer cells either by soluble factors or by triggering cell-death pathways. Melanoma cells exploit multiple mechanisms to escape immune system tumoricidal control. FKBP51 is a relevant pro-oncogenic factor of melanoma cells supporting NF-κB-mediated resistance and cancer stemness/invasion epigenetic programs. Herein, we show that FKBP51-silencing increases TNF-related apoptosis-inducing ligand (TRAIL)-R2 (DR5) expression and sensitizes melanoma cells to TRAIL-induced apoptosis. Consistent with the general increase in histone deacetylases, as by the proteomic profile, the immune precipitation assay showed decreased acetyl-Yin Yang 1 (YY1) after FKBP51 depletion, suggesting an impaired repressor activity of this transcription factor. ChIP assay supported this hypothesis. Compared with non-silenced cells, a reduced acetyl-YY1 was found on the DR5 promoter, resulting in increased DR5 transcript levels. Using Crispr/Cas9 knockout (KO) melanoma cells, we confirmed the negative regulation of DR5 by FKBP51. We also show that KO cells displayed reduced levels of acetyl-EP300 responsible for YY1 acetylation, along with reduced acetyl-YY1. Reconstituting FKBP51 levels contrasted the effects of KO on DR5, acetyl-YY1, and acetyl-EP300 levels. In conclusion, our finding shows that FKBP51 reduces DR5 expression at the transcriptional level by promoting YY1 repressor activity. Our study supports the conclusion that targeting FKBP51 increases the expression level of DR5 and sensitivity to TRAIL-induced cell death, which can improve the tumoricidal action of immune cells

Study of PDL-1 regulation and expression in glioblastoma and its role in cancer resistance

Among the high-grade malignant gliomas (III-IV), grade IV glioma, or glioblastoma (GBM), is the most common and destructive form of brain cancer. Despite rigorous molecular, preclinical, and clinical research these tumors remain a challenge to treat and 70% of patients diagnosed with GBM will succumb to the disease within 2 years. New immune-based cancer treatments are currently in development, among which, anti-PD-1 is in a phase II clinical trial for recurrent glioblastoma. Tumor cells exploit PDL-1/PD1 pathway to induce T cell exhaustion and evade host antitumour immunity. PDL-1 expression on glioblastoma has been shown to correlate with a bad prognosis. There is strong demand for knowledge about the mechanisms regulating PDL-1 expression in glioblastoma. Our research group, has previously identified a relevant role for FKBP51, and its spliced isoform FKBP51s, in PDL-1 expression and melanoma resistance to the therapy. FKBP51 is an immunophilin capable of immunosuppression, originally cloned in lymphocytes. Because FKBP51 was identified as highly expressed in glioblastoma, correlating with tumor aggressiveness, we investigated whether this protein and/or its spliced isoform FKBP51s could be involved in regulation of PDL-1 expression in glioblastoma. In addition, we looked at the role of these protein-isoforms in glioblastoma resistance to chemotherapy. To this aim, we used two different glioblastoma cell lines termed D54 and U251. Our results suggest that, differently from melanoma, in which PDL-1 is inducible and associated with FKBP51 splicing, glioblastoma cell lines express high basal levels of PDL-1. Interestingly, both the canonical and the spliced FKBP51 isoforms were highly expressed in the glioblastoma cell lines. Expression of PDL-1 was regulated by FKBP51s knockin and knockout and the levels changed in accordance with FKBP51s levels. Moreover, in glioblastoma cells silenced for FKBP51s, the decrease in PDL-1 levels was associated with a significant increase in apoptosis, either spontaneous apoptosis and the one stimulated by etoposide or temozolomide. Two recently developed compounds, selective inhibitor of FKBP51, termed SAFit1 and SAFit2 were able to reduce expression of PDL-1 on glioblastoma cell lines. Particularly, SAFit 2 was found to be more effective, in accordance with the notion that it is more brain-permeable than SAFit1. SaFit 2 was also shown to increase sensitivity to etoposide of glioblastoma cell, although to a lesser extent when compared to FKBP51siRNAs. In conclusion, our preliminary results suggest that FKBP51s plays a relevant role in glioblastoma resistance and PDL-1 expression. Future studies are needed to shed light on the mechanisms underlying FKBP51s-induced PDL-1 expression and signaling pathway downstream to FKBP51s/PDL-1 that sustain glioblastoma resistance

SARS-CoV-2 pandemic tracing in Italy highlights lineages with mutational burden in growing subsets

Tracing the appearance and evolution of virus variants is essential in the management of the COVID-19 pandemic. Here, we focus on SARS-CoV-2 spread in Italian patients by using viral sequences deposited in public databases and a tracing procedure which is used to monitor the evolution of the pandemic and detect the spreading, within the infected population of emergent sub-clades with a potential positive selection. Analyses of a collection of monthly samples focused on Italy highlighted the appearance and evolution of all the main viral sub-trees emerging at the end of the first year of the pandemic. It also identified additional expanding subpopulations which spread during the second year (i.e., 2021). Three-dimensional (3D) modelling of the main amino acid changes in mutated viral proteins, including ORF1ab (nsp3, nsp4, 2’-o-ribose methyltransferase, nsp6, helicase, nsp12 [RdRp]), N, ORF3a, ORF8, and spike proteins, shows the potential of the analysed structural variations to result in epistatic modulation and positive/negative selection pressure. These analyzes will be of importance to the early identification of emerging clades, which can develop into new “variants of concern” (i.e., VOC). These analyses and settings will also help SARS-CoV-2 coronet genomic centers in other countries to trace emerging worldwide varian