The FKBP51s Splice Isoform Predicts Unfavorable Prognosis in Patients with Glioblastoma.

The primary treatment for glioblastoma (GBM) is removing the tumor mass as defined by MRI. However, MRI has limited diagnostic and predictive value. Tumor-associated macrophages (TAM) are abundant in GBM tumor microenvironment (TME) and are found in peripheral blood (PB). FKBP51 expression, with its canonical and spliced isoforms, is constitutive in immune cells and aberrant in GBM. Spliced FKBP51s supports M2 polarization. To find an immunologic signature that combined with MRI could advance in diagnosis, we immunophenotyped the macrophages of TME and PB from 37 patients with GBM using FKBP51s and classical M1-M2 markers. We also determined the tumor levels of FKBP51s, PD-L1, and HLA-DR. Tumors expressing FKBP51s showed an increase in various M2 phenotypes and regulatory T cells in PB, indicating immunosuppression. Tumors expressing FKBP51s also activated STAT3 and were associated with reduced survival. Correlative studies with MRI and tumor/macrophages cocultures allowed to interpret TAMs. Tumor volume correlated with M1 infiltration of TME. Cocultures with spheroids produced M1 polarization, suggesting that M1 macrophages may infiltrate alongside cancer stem cells. Cocultures of adherent cells developed the M2 phenotype CD163/FKBP51s expressing pSTAT6, a transcription factor enabling migration and invasion. In patients with recurrences, increased counts of CD163/FKBP51s monocyte/macrophages in PB correlated with callosal infiltration and were accompanied by a concomitant decrease in TME-infiltrating M1 macrophages. PB PD-L1/FKBP51s connoted necrotic tumors. In conclusion, FKBP51s identifies a GBM subtype that significantly impairs the immune system. Moreover, FKBP51s marks PB macrophages associated with MRI features of glioma malignancy that can aid in patient monitoring. SIGNIFICANCE: Our research suggests that by combining imaging with analysis of monocyte/macrophage subsets in patients with GBM, we can enhance our understanding of the disease and assist in its treatment. We discovered a similarity in the macrophage composition between the TME and PB, and through association with imaging, we could interpret macrophages. In addition, we identified a predictive biomarker that drew more attention to immune suppression of patients with GBM.

Scaffold proteins of cancer signaling networks: The paradigm of FK506 binding protein 51 (FKBP51) supporting tumor intrinsic properties and immune escape.

Scaffold proteins are crucial regulators of signaling networks, and their abnormal expression may favor the development of tumors. Among the scaffold proteins, immunophilin covers a unique role as 'protein-philin' (Greek 'philin' = friend) that interacts with proteins to guide their proper assembly. The growing list of human syndromes associated with the immunophilin defect underscores the biological relevance of these proteins that are largely opportunistically exploited by cancer cells to support and enable the tumor's intrinsic properties. Among the members of the immunophilin family, the FKBP5 gene was the only one identified to have a splicing variant. Cancer cells impose unique demands on the splicing machinery, thus acquiring a particular susceptibility to splicing inhibitors. This review article aims to overview the current knowledge of the FKBP5 gene functions in human cancer, illustrating how cancer cells exploit the scaffolding function of canonical FKBP51 to foster signaling networks that support their intrinsic tumor properties and the spliced FKBP51s to gain the capacity to evade the immune system.

FKBP51 plays an essential role in Akt ubiquitination that requires Hsp90 and PHLPP.

FKBP51 plays a relevant role in sustaining cancer cells, particularly melanoma. This cochaperone participates in several signaling pathways. FKBP51 forms a complex with Akt and PHLPP, which is reported to dephosphorylate Akt. Given the recent discovery of a spliced FKBP51 isoform, in this paper, we interrogate the canonical and spliced isoforms in regulation of Akt activation. We show that the TPR domain of FKBP51 mediates Akt ubiquitination at K63, which is an essential step for Akt activation. The spliced FKBP51, lacking such domain, cannot link K63-Ub residues to Akt. Unexpectedly, PHLPP silencing does not foster phosphorylation of Akt, and its overexpression even induces phosphorylation of Akt. PHLPP stabilizes levels of E3-ubiquitin ligase TRAF6 and supports K63-ubiquitination of Akt. The interactome profile of FKBP51 from melanoma cells highlights a relevant role for PHLPP in improving oncogenic hallmarks, particularly, cell proliferation.

Metabolites Profiling of Melanoma Interstitial Fluids Reveals Uridine Diphosphate as Potent Immune Modulator Capable of Limiting Tumor Growth.

Tumor interstitial fluid (TIF) surrounds and perfuses tumors and collects ions, metabolites, proteins, and extracellular vesicles secreted by tumor and stromal cells. Specific metabolites, accumulated within the TIF, could induce metabolic alterations of immune cells and shape the tumor microenvironment. We deployed a metabolomic approach to analyze the composition of melanoma TIF and compared it to the plasma of C57BL6 mice, engrafted or not with B16-melanoma cells. Among the classes of metabolites analyzed, monophosphate and diphosphate nucleotides resulted enriched in TIF compared to plasma samples. The analysis of the effects exerted by guanosine diphosphate (GDP) and uridine diphosphate (UDP) on immune response revealed that GDP and UDP increased the percentage of CD4+CD25+FoxP3- and, on isolated CD4+ T-cells, induced the phosphorylation of ERK, STAT1, and STAT3; increased the activity of NF-κB subunits p65, p50, RelB, and p52; increased the expression of Th1/Th17 markers including IFNγ, IL17, T-bet, and RORγt; and reduced the expression of IL13, a Th2 marker. Finally, we observed that local administrations of UDP in B16-engrafted C57BL6 mice reduced tumor growth and necrotic areas. In addition, UDP-treated tumors showed a higher presence of MHCIIhi tumor-associated macrophage (TAM) and of CD3+CD8+ and CD3+CD4+ tumor-infiltrating T-lymphocytes (TILs), both markers of anti-tumor immune response. Consistent with this, intra-tumoral gene expression analysis revealed in UDP-treated tumors an increase in the expression of genes functionally linked to anti-tumor immune response. Our analysis revealed an important metabolite acting as mediator of immune response, which could potentially represent an additional tool to be used as an adjuvant in cancer immunotherapy.

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.

PD-L1 Expression Fluctuates Concurrently with Cyclin D in Glioblastoma Cells.

Despite Glioblastoma (GBM) frequently expressing programmed cell death ligand-1 (PD-L1), treatment with anti-programmed cell death-1 (PD1) has not yielded brilliant results. Intratumor variability of PD-L1 can impact determination accuracy. A previous study on mouse embryonic fibroblasts (MEFs) reported a role for cyclin-D in control of PD-L1 expression. Because tumor-cell growth within a cancer is highly heterogeneous, we looked at whether PD-L1 and its cochaperone FKBP51s were influenced by cell proliferation, using U251 and SF767 GBM-cell-lines. PD-L1 was measured by Western blot, flow cytometry, confocal-microscopy, quantitative PCR (qPCR), CCND1 by qPCR, FKBP51s by Western blot and confocal-microscopy. Chromatin-Immunoprecipitation assay (xChIp) served to assess the DNA-binding of FKBP51 isoforms. In the course of cell culture, PD-L1 appeared to increase concomitantly to cyclin-D on G1/S transition, to decrease during exponential cell growth progressively. We calculated a correlation between CCND1 and PD-L1 gene expression levels. In the temporal window of PD-L1 and CCND1 peak, FKBP51s localized in ER. When cyclin-D declined, FKBP51s went nuclear. XChIp showed that FKBP51s binds CCND1 gene in a closed-chromatin configuration. Our finding suggests that the dynamism of PD-L1 expression in GBM follows cyclin-D fluctuation and raises the hypothesis that FKBP51s might participate in the events that govern cyclin-D oscillation.

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

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.

Combining Magnetic Resonance Imaging with Systemic Monocyte Evaluation for the Implementation of GBM Management.

Magnetic resonance imaging (MRI) is the gold standard for glioblastoma (GBM) patient evaluation. Additional non-invasive diagnostic modalities are needed. GBM is heavily infiltrated with tumor-associated macrophages (TAMs) that can be found in peripheral blood. FKBP51s supports alternative-macrophage polarization. Herein, we assessed FKBP51s expression in circulating monocytes from 14 GBM patients. The M2 monocyte phenotype was investigated by qPCR and flow cytometry using antibodies against PD-L1, CD163, FKBP51s, and CD14. MRI assessed morphologic features of the tumors that were aligned to flow cytometry data. PD-L1 expression on circulating monocytes correlated with MRI tumor necrosis score. A wider expansion in circulating CD163/monocytes was measured. These monocytes resulted in a dramatic decrease in patients with an MRI diagnosis of complete but not partial surgical removal of the tumor. Importantly, in patients with residual tumor, most of the peripheral monocytes that in the preoperative stage were CD163/FKBP51s- had turned into CD163/FKBP51s+. After Stupp therapy, CD163/FKBP51s+ monocytes were almost absent in a case of pseudoprogression, while two patients with stable or true disease progression showed sustained levels in such circulating monocytes. Our work provides preliminary but meaningful and novel results that deserve to be confirmed in a larger patient cohort, in support of potential usefulness in GBM monitoring of CD163/FKBP51s/CD14 immunophenotype in adjunct to MRI.

Thrombocytopenia Complicating Transcatheter Aortic Valve Implantation: Differences Between Two New-Generation Devices.

Thrombocytopenia after TAVI is common and clinically detrimental. Retrospectively, we observed Portico recipients had a more profound platelet drop than Evolut recipients. We thus investigated periprocedural platelet damage and/orpro-inflammatory state in 64 TAVI recipients at baseline and after implantation. Platelet damage was assessed by annexin V staining and monocyte-phagocytic phenotype was assessed according to CD14/CD36 expression. Serum cytokines were measured in 20 patients. The formaldehyde-based storage solution altered platelets. When, before being loaded onto the delivery system, Portico underwent one additional flushing to those recommended, the receiving patients showed thrombocytopenia, platelet damage, and CD36-monocyte count were mitigated. A general increase in IL-6 was recorded in overall TAVI recipients, but a high serum level of IL-8, a potent thrombocytopenia inducer, was measured in Portico recipients only, including those with extra-rinsed valve. Our study suggests a platelet-injury effect by storage-solution and generates the hypothesis of a role for the biomaterial in stimulating innate-immunity. Larger prospective studies are needed. Graphical Abstract.

Alternative macrophage polarisation associated with resistance to anti-PD1 blockade is possibly supported by the splicing of FKBP51 immunophilin in melanoma patients.

BACKGROUND: FKBP51 immunophilin is abundantly expressed by immune cells. Co-inhibitory immune receptor signalling generates the splicing isoform FKBP51s. Tregs stained by FKBP51s are increased in melanoma patients and their counts are associated with anti-CTLA-4 response. An expansion of FKBP51s+PD-L1+ monocytes was measured in a group of non-responding patients to anti-CTLA-4. The aim of this work was to confirm the predictive value of response of FKBP51s+Tregs in a cohort of patients undergoing anti-PD1 treatment and shed light on a monocyte subset co-expressing PD-L1/FKBP51s. METHODS: Co-cultures of organoids and autologous lymphocytes were used to confirm that tumour T-cell interaction can induce FKBP51s. PBMC immunophenotype and flow cytometry served to assess and monitor FKBP51s+Treg and FKBP51s+PD-L1+ monocytes in 22 advanced melanoma patients treated with anti-PD1. Silencing and overexpression of FKBP51s in human macrophages served to address the protein role in the tolerant macrophages' behaviour. RESULTS: FKBP51s+Tregs count was increased in responders and had a prognostic value. Non-responders showed an early increase in FKBP51s+ PD-L1+ monocytes during anti-PD1 treatment. Manipulation of FKBP51s modulated the macrophage-phenotype, with forced protein expression promoting aspects associated with tolerance. CONCLUSIONS: FKBP51s may guide in the selection and monitoring of melanoma patient candidates to immune-checkpoint-targeted therapy. Manipulation of FKBP51s may overcome resistance.

Manipulation of the Immune System for Cancer Defeat: A Focus on the T Cell Inhibitory Checkpoint Molecules.

The immune system actively counteracts the tumorigenesis process; a breakout of the immune system function, or its ability to recognize transformed cells, can favor cancer development. Cancer becomes able to escape from immune system control by using multiple mechanisms, which are only in part known at a cellular and molecular level. Among these mechanisms, in the last decade, the role played by the so-called "inhibitory immune checkpoints" is emerging as pivotal in preventing the tumor attack by the immune system. Physiologically, the inhibitory immune checkpoints work to maintain the self-tolerance and attenuate the tissue injury caused by pathogenic infections. Cancer cell exploits such immune-inhibitory molecules to contrast the immune intervention and induce tumor tolerance. Molecular agents that target these checkpoints represent the new frontier for cancer treatment. Despite the heterogeneity and multiplicity of molecular alterations among the tumors, the immune checkpoint targeted therapy has been shown to be helpful in selected and even histologically different types of cancer, and are currently being adopted against an increasing variety of tumors. The most frequently used is the moAb-based immunotherapy that targets the Programmed Cell Death 1 protein (PD-1), the PD-1 Ligand (PD-L1) or the cytotoxic T lymphocyte antigen-4 (CTLA4). However, new therapeutic approaches are currently in development, along with the discovery of new immune checkpoints exploited by the cancer cell. This article aims to review the inhibitory checkpoints, which are known up to now, along with the mechanisms of cancer immunoediting. An outline of the immune checkpoint targeting approaches, also including combined immunotherapies and the existing trials, is also provided. Notwithstanding the great efforts devoted by researchers in the field of biomarkers of response, to date, no validated FDA-approved immunological biomarkers exist for cancer patients. We highlight relevant studies on predictive biomarkers and attempt to discuss the challenges in this field, due to the complex and largely unknown dynamic mechanisms that drive the tumor immune tolerance.

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.

The splicing FK506-binding protein-51 isoform plays a role in glioblastoma resistance through programmed cell death ligand-1 expression regulation.

Gliomas aberrantly express programmed cell death ligand-1 (PD-L1), which has a pivotal role in immunoevasion. The splicing isoform of FKBP5, termed FKBP51s, is a PD-L1 foldase, assisting the immune checkpoint molecule in maturation and expression on the plasma membrane. The concept that PD-L1 supports tumor-intrinsic properties is increasingly emerging. The aim of the present work was to confirm the pro-tumoral effect of PD-L1 on human glioma cell survival, stemness capacity and resistance, and to address the issue of whether, by targeting its foldase either chemically or by silencing, the aggressive tumor features could be attenuated. PD-L1-depleted glioma cells have a reduced threshold for apoptosis, while PD-L1 forced expression increases resistance. Similar results were obtained with FKBP51s modulation. The ability of PD-L1 to counteract cell death was hampered by FKBP51s silencing. PD-L1 expression was particularly high in glioma cells with a cancer-stem-cell profile. Moreover, PD-L1 sustained the spheroid formation capability of glioma cells. Targeting of FKBP51s by small-interfering RNA (siRNA) or the specific inhibitor SAFit2, reduced the number of formed spheroids, along with PD-L1 expression. Finally, in an orthotopic mouse model of glioblastoma, daily treatment with SAFit2 significantly reduced tumor PD-L1 expression, and tumor growth. In treated mice, caspase-3 activation and reduced vimentin expression were observed in excised tumors. In conclusion, targeting of FKBP51s hampers PD-L1 and its pro-tumoral properties, thereby affecting the self-renewal and growth capacities of glioblastoma cells in vitro and in vivo.

TRAF2 and FKBP51 as possible markers for identification of suitable melanoma tumors for tumor necrosis factor-α inhibition.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, whose role in melanoma is controversial. Although high-dose TNF-α is approved for the treatment of patients with in transit-metastatic melanoma confined to the limb, diverse preclinical models of melanoma have shown that TNF-α can induce cell invasion. Biomarkers that can differentiate between the dual role of TNF-α are needed. TRAF2 is critical to TNF receptor-induced activation of nuclear factor-κB (NF-κB), allowing shifting from death to survival-signaling cascades. The large immunophilin FKBP51 acts as a scaffold and catalyst in the IκB kinase complex assembly and activation. Here, using microscopy and an electrophoretic mobility-shift assay, we provide further evidence in support of the essential role of FKBP51 in sustaining the TNF-α NF-κB signaling in melanoma. Through the cross-linking reaction with the chemical linker disuccinimidyl glutarate, we show that a direct interaction occurs between FKBP51 and TRAF2 in melanoma cells. Immunohistochemistry of tumor samples from 24 patients with cutaneous melanomas showed a correlation between the expressions of the two proteins. Given the association of FKBP51 and TRAF2 with TNF-α-induced NF-κB signaling and their correlation in tumor samples, we propose that the two proteins can be exploited as useful markers for the identification of those melanoma tumors that can benefit from TNF-α inhibition. Future studies will address this hypothesis.

A regulatory role for the co-chaperone FKBP51s in PD-L1 expression in glioma.

BACKGROUND: FKBP51 is a co-chaperone with isomerase activity, abundantly expressed in glioma. We previously identified a spliced isoform (FKBP51s) and highlighted a role for this protein in the upregulation of Programmed Death Ligand 1 (PD-L1) expression in melanoma. Because gliomas can express PD-L1 causing a defective host anti-tumoral immunity, we investigated whether FKBP51s was expressed in glioma and played a role in PD-L1 regulation in this tumour. METHODS: We used D54 and U251 glioblastoma cell lines that constitutively expressed PD-L1. FKBP51s was measured by immunoblot, flow cytometry and microscopy. In patient tumours, IHC and qPCR were used to measure protein and mRNA levels respectively. FKBP51s depletion was achieved by siRNAs, and its enzymatic function was inhibited using selective inhibitors (SAFit). We investigated protein maturation using N-glycosidase and cell fractionation approaches. RESULTS: FKBP51s was expressed at high levels in glioma cells. Glycosylated-PD-L1 was increased and reduced by FKBP51s overexpression or silencing, respectively. Naïve PD-L1 was found in the endoplasmic reticulum (ER) of glioma cells complexed with FKBP51s, whereas the glycosylated form was measured in the Golgi apparatus. SAFit reduced PD-L1 levels (constitutively expressed and ionizing radiation-induced). SAFit reduced cell death of PBMC co-cultured with glioma. CONCLUSIONS: Here we addressed the mechanism of post-translational regulation of PD-L1 protein in glioma. FKBP51s upregulated PD-L1 expression on the plasma membrane by catalysing the protein folding required for subsequent glycosylation. Inhibition of FKBP51s isomerase activity by SAFit decreased PD-L1 levels. These findings suggest that FKBP51s is a potential target of immunomodulatory strategies for glioblastoma treatment.