The microbial metabolite desaminotyrosine enhances T-cell priming and cancer immunotherapy with immune checkpoint inhibitors.

BACKGROUND: Inter-individual differences in response to immune checkpoint inhibitors (ICI) remain a major challenge in cancer treatment. The composition of the gut microbiome has been associated with differential ICI outcome, but the underlying molecular mechanisms remain unclear, and therapeutic modulation challenging. METHODS: We established an in vivo model to treat C57Bl/6j mice with the type-I interferon (IFN-I)-modulating, bacterial-derived metabolite desaminotyrosine (DAT) to improve ICI therapy. Broad spectrum antibiotics were used to mimic gut microbial dysbiosis and associated ICI resistance. We utilized genetic mouse models to address the role of host IFN-I in DAT-modulated antitumour immunity. Changes in gut microbiota were assessed using 16S-rRNA sequencing analyses. FINDINGS: We found that oral supplementation of mice with the microbial metabolite DAT delays tumour growth and promotes ICI immunotherapy with anti-CTLA-4 or anti-PD-1. DAT-enhanced antitumour immunity was associated with more activated T cells and natural killer cells in the tumour microenvironment and was dependent on host IFN-I signalling. Consistent with this, DAT potently enhanced expansion of antigen-specific T cells following vaccination with an IFN-I-inducing adjuvant. DAT supplementation in mice compensated for the negative effects of broad-spectrum antibiotic-induced dysbiosis on anti-CTLA-4-mediated antitumour immunity. Oral administration of DAT altered the gut microbial composition in mice with increased abundance of bacterial taxa that are associated with beneficial response to ICI immunotherapy. INTERPRETATION: We introduce the therapeutic use of an IFN-I-modulating bacterial-derived metabolite to overcome resistance to ICI. This approach is a promising strategy particularly for patients with a history of broad-spectrum antibiotic use and associated loss of gut microbial diversity. FUNDING: Melanoma Research Alliance, Deutsche Forschungsgemeinschaft, German Cancer Aid, Wilhelm Sander Foundation, Novartis Foundation.

Targeting nucleic acid sensors in tumor cells to reprogram biogenesis and RNA cargo of extracellular vesicles for T cell-mediated cancer immunotherapy.

Tumor-derived extracellular vesicles (EVs) have been associated with immune evasion and tumor progression. We show that the RNA-sensing receptor RIG-I within tumor cells governs biogenesis and immunomodulatory function of EVs. Cancer-intrinsic RIG-I activation releases EVs, which mediate dendritic cell maturation and T cell antitumor immunity, synergizing with immune checkpoint blockade. Intact RIG-I, autocrine interferon signaling, and the GTPase Rab27a in tumor cells are required for biogenesis of immunostimulatory EVs. Active intrinsic RIG-I signaling governs composition of the tumor EV RNA cargo including small non-coding stimulatory RNAs. High transcriptional activity of EV pathway genes and RIG-I in melanoma samples associate with prolonged patient survival and beneficial response to immunotherapy. EVs generated from human melanoma after RIG-I stimulation induce potent antigen-specific T cell responses. We thus define a molecular pathway that can be targeted in tumors to favorably alter EV immunomodulatory function. We propose "reprogramming" of tumor EVs as a personalized strategy for T cell-mediated cancer immunotherapy.

B-cell lymphoma-extra large is a promising drug target in Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin tumour with neuroendocrine differentiation. Immunotherapies are effective in the treatment of patients with advanced-stage MCC, but for patients whose tumours cannot be controlled by the immune system, alternative approaches are urgently needed. OBJECTIVES: To identify overexpressed oncogenes as potential drug targets for MCC. METHODS: NanoString platform, digital droplet polymerase chain reaction (ddPCR) and fluorescence in situ hybridization (FISH) assays were used to determine copy number variations (CNVs); BCL2L1 and PARP1 mRNA expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), B-cell lymphoma extra-large (Bcl-xL) and poly (ADP-ribose) polymerase 1 (PARP1) protein by immuno-blot. Specific Bcl-xL inhibitors and a PARP1 inhibitor were used alone or in combination to test their antitumour effect. RESULTS: Screening for CNVs in 13 classic Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative MCC cell lines revealed BCL2L1 gains and amplifications, confirmed by ddPCR in 10 cell lines. By ddPCR and FISH, we demonstrated that BCL2L1 gains are present in tumour tissue. BCL2L1 copy number gains were associated with increased Bcl-xL mRNA and protein expression. However, high Bcl-xL expression was not restricted to MCC cells harbouring a BCL2L1 gain/amplification, suggesting additional epigenetic means of regulation. The functional relevance of Bcl-xL in MCC cells was demonstrated by the fact that specific Bcl-xL inhibitors (A1331852 and WEHI-539) led to the induction of apoptosis. Owing to the strong expression and activation of PARP1 in MCC cell lines, we next tested the combination of Bcl-xL inhibitors with the PARP1 inhibitor olaparib, which showed synergistic antitumour effects. CONCLUSIONS: Bcl-xL, which is highly expressed in MCC, appears to be an attractive therapeutic target for the treatment of this tumour, especially as the effect of specific Bcl-xL inhibitors is synergistically enhanced by simultaneous PARP inhibition.

Merkel cell carcinoma-derived exosome-shuttle miR-375 induces fibroblast polarization by inhibition of RBPJ and p53.

Merkel cell carcinoma (MCC) is a highly invasive and metastatic skin cancer. While high expression of miR-375 is a characteristic of MCC, it seems not to contribute to the malignant phenotype of MCC cells. miR-375 enrichment in MCC-derived extracellular vesicles suggests its intercellular signaling function. Here, we demonstrate that horizontally transferred miR-375 causes fibroblast polarization toward cancer-associated fibroblasts (CAFs). The polarization is evidenced by phenotypic changes and induction of α-SMA, CXCL2, and IL-1ß. Fibroblast polarization is inhibited by specific antagomirs and mimicked by experimental miR-375 expression. Mechanistically, miR-375 downregulates RBPJ and p53, two key players regulating fibroblast polarization. In clinical MCC samples, in situ hybridization located miR-375 in CAFs, which correlated with high α-SMA protein and low RBPJ and TP53 expression; single-cell RNAseq revealed a disparate fibroblast polarization negatively correlating with p53 pathway-related gene expression. Thus, the functional role of miR-375 in MCC is to generate a pro-tumorigenic microenvironment by inducing fibroblast polarization.

UV-type specific alteration of miRNA expression and its association with tumor progression and metastasis in SCC cell lines.

PURPOSE: UV exposure is the main risk factor for development of cutaneous squamous cell carcinoma (cSCC). While early detection greatly improves cSCC prognosis, locally advanced or metastatic cSCC has a severely impaired prognosis. Notably, the mechanisms of progression to metastatic cSCC are not well understood. We hypothesized that UV exposure of already transformed epithelial cSCC cells further induces changes which might be involved in the progression to metastatic cSCCs and that UV-inducible microRNAs (miRNAs) might play an important role. METHODS: Thus, we analyzed the impact of UV radiation of different quality (UVA, UVB, UVA + UVB) on the miRNA expression pattern in established cell lines generated from primary and metastatic cSCCs (Met-1, Met-4) using the NanoString nCounter platform. RESULTS: This analysis revealed that the expression pattern of miRNAs depends on both the cell line used per se and on the quality of UV radiation. Comparison of UV-induced miRNAs in cSCC cell lines established from a primary tumor (Met-1) and the respective (un-irradiated) metastasis (Met-4) suggest that miR-7-5p, miR-29a-3p and miR-183-5p are involved in a UV-driven pathway of progression to metastasis. This notion is supported by the fact that these three miRNAs build up a network of 81 potential target genes involved e.g. in UVA/UVB-induced MAPK signaling and regulation of the epithelial-mesenchymal transition. As an example, PTEN, a target of UV-upregulated miRNAs (miR-29a-3p, miR-183-5p), could be shown to be down-regulated in response to UV radiation. We further identified CNOT8, the transcription complex subunit 8 of the CCR4-NOT complex, a deadenylase removing the poly(A) tail from miRNA-destabilized mRNAs, in the center of this network, targeted by all three miRNAs. CONCLUSION: In summary, our results demonstrate that UV radiation induces an miRNA expression pattern in primary SCC cell line partly resembling those of metastatic cell line, thus suggesting that UV radiation impacts SCC progression beyond initiation.

Highly Expressed miR-375 is not an Intracellular Oncogene in Merkel Cell Polyomavirus-Associated Merkel Cell Carcinoma.

miR-375 is a highly abundant miRNA in Merkel cell carcinoma (MCC). In other cancers, it acts as either a tumor suppressor or oncogene. While free-circulating miR-375 serves as a surrogate marker for tumor burden in patients with advanced MCC, its function within MCC cells has not been established. Nearly complete miR-375 knockdown in MCC cell lines was achieved using antagomiRs via nucleofection. The cell viability, growth characteristics, and morphology were not altered by this knockdown. miR-375 target genes and related signaling pathways were determined using Encyclopedia of RNA Interactomes (ENCORI) revealing Hippo signaling and epithelial to mesenchymal transition (EMT)-related genes likely to be regulated. Therefore, their expression was analyzed by multiplexed qRT-PCR after miR-375 knockdown, demonstrating only a limited change in expression. In summary, highly effective miR-375 knockdown in classical MCC cell lines did not significantly change the cell viability, morphology, or oncogenic signaling pathways. These observations render miR-375 an unlikely intracellular oncogene in MCC cells, thus suggesting that likely functions of miR-375 for the intercellular communication of MCC should be addressed.

MCPyV Large T Antigen-Induced Atonal Homolog 1 Is a Lineage-Dependency Oncogene in Merkel Cell Carcinoma.

Despite the fact that the transcription factor ATOH1 is a master regulator of Merkel cell development, its role in Merkel cell carcinoma (MCC) carcinogenesis remains controversial. Here, we provide several lines of evidence that ATOH1 is a lineage-dependent oncogene in MCC. Luciferase assays revealed binding of ATOH1 and subsequent activation to the promoter of miR-375, which is one of the most abundant microRNAs in MCCs. Overexpression of ATOH1 in variant MCC cell lines and fibroblasts induced miR-375 expression, whereas ATOH1 knockdown in classical MCC cell lines reduced miR-375 expression. Moreover, ATOH1 overexpression in these cells changed their growth characteristics from adherent to suspension and/orspheroidal growth, that is, resembling the neuroendocrine growth pattern of classical MCC cell lines. Notably, ectopic expression of different Merkel cell polyomavirus (MCPyV)-derived truncated large T antigens induced ATOH1 expression in fibroblasts, which was paralleled by miR-375 expression and similar morphologic changes. In summary, MCPyV-associated carcinogenesis is likely to induce the characteristic neuroendocrine features of MCC via induction of ATOH1; thus, ATOH1 can be regarded as a lineage-dependent oncogene in MCC.

Pharmacological Inhibition of Serine Palmitoyl Transferase and Sphingosine Kinase-1/-2 Inhibits Merkel Cell Carcinoma Cell Proliferation.

The majority of Merkel cell carcinoma, a highly aggressive neuroendocrine cancer of the skin, is associated with Merkel cell polyomavirus infection. Polyomavirus binding, internalization, and infection are mediated by glycosphingolipids. Besides receptor function, bioactive sphingolipids are increasingly recognized as potent regulators of several hallmarks of cancer. Merkel cell polyomavirus+ and Merkel cell polyomavirus- cells express serine palmitoyl transferase subunits and sphingosine kinase (SK) 1/2 mRNA. Induced expression of Merkel cell polyomavirus-large tumor antigen in human lung fibroblasts resulted in upregulation of SPTLC1-3 and SK 1/2 expression. Therefore, we exploited pharmacological inhibition of sphingolipid metabolism as an option to interfere with proliferation of Merkel cell polyomavirus+ Merkel cell carcinoma cell lines. We used myriocin (a serine palmitoyl transferase antagonist) and two SK inhibitors (SKI-II and ABC294640). In MKL-1 and WaGa cells myriocin decreased cellular ceramide, sphingomyelin, and sphingosine-1-phosphate content. SKI-II increased ceramide species but decreased sphingomyelin and sphingosine-1-phosphate concentrations. Aberrant sphingolipid homeostasis was associated with reduced cell viability, increased necrosis, procaspase-3 and PARP processing, caspase-3 activity, and decreased AKTS473 phosphorylation. Myriocin and SKI-II decreased tumor size and Ki-67 staining of xenografted MKL-1 and WaGa tumors on the chorioallantoic membrane. Our data suggest that pharmacological inhibition of sphingolipid synthesis could represent a potential therapeutic approach in Merkel cell carcinoma.

Circulating Cell-Free miR-375 as Surrogate Marker of Tumor Burden in Merkel Cell Carcinoma.

PURPOSE: Merkel cell carcinoma (MCC) is an aggressive skin cancer with neuroendocrine differentiation. There is an unmet need for MCC-specific blood-based surrogate biomarkers of tumor burden; circulating cell-free miRNA may serve this purpose. EXPERIMENTAL DESIGN: Expression of miR-375 was quantified in 24 MCC and 23 non-MCC cell lines, 67 MCC and 58 non-MCC tumor tissues, sera of 2 preclinical MCC models, and sera of 109 patients with MCC and 30 healthy controls by nCounter human-v2-miRNA expression or miR-375-specific real-time PCR assays. The patients' sera consisted of two retrospective (discovery and training) and two prospective (validation) cohorts. RESULTS: miR-375 expression was high in MCC cell lines and tissues compared with non-MCCs. It was readily detected in MCC-conditioned medium and sera of preclinical models bearing MCC xenografts. miR-375 levels were higher in sera from tumor-bearing patients with MCC than in tumor-free patients or healthy controls (P < 0.0005). Moreover, miR-375 serum levels correlated with tumor stage in tumor-bearing (P = 0.037) but not in tumor-free (P = 0.372) patients with MCC. miR-375 serum level showed high diagnostic accuracy to discriminate tumor-bearing and tumor-free patients with MCC as demonstrated by ROC curve analysis in the retrospective cohorts (AUC = 0.954 and 0.800) as well as in the prospective cohorts (AUC = 0.929 and 0.959). miR-375 serum level reflected dynamic changes in tumor burden of patients with MCC during therapeutic interventions. CONCLUSIONS: Circulating cell-free miR-375 proved as a surrogate marker for tumor burden in MCC without restriction to polyomavirus positivity; it thus appears to be useful for therapy monitoring and the follow-up of patients with MCC.

Epigenetic priming restores the HLA class-I antigen processing machinery expression in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive, yet highly immunogenic skin cancer. The latter is due to its viral or UV-associated carcinogenesis. For tumor progression MCC has to escape the host's immuno-surveillance, e.g. by loss of HLA class-I expression. Indeed, a reduced HLA class-I expression was observed in MCC tumor tissues and MCC cell lines. This reduced HLA class-I surface expression is caused by an impaired expression of key components of the antigen processing machinery (APM), including LMP2 and LMP7 as well as TAP1 and TAP2. Notably, experimental provisions of HLA class-I binding peptides restored HLA class-I surface expression on MCC cells. Silencing of the HLA class-I APM is due to histone deacetylation as inhibition of histone deacetylases (HDACs) not only induced acetylation of histones in the respective promoter regions but also re-expression of APM components. Thus, HDAC inhibition restored HLA class-I surface expression in vitro and in a mouse xenotransplantation model. In contrast to re-induction of HLA class-I by interferons, HDAC inhibitors did not interfere with the expression of immuno-dominant viral proteins. In summary, restoration of HLA class-I expression on MCC cells by epigenetic priming is an attractive approach to enhance therapies boosting adaptive immune responses.

Reversal of epigenetic silencing of MHC class I chain-related protein A and B improves immune recognition of Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a virally associated cancer characterized by its aggressive behavior and strong immunogenicity. Both viral infection and malignant transformation induce expression of MHC class I chain-related protein (MIC) A and B, which signal stress to cells of the immune system via Natural Killer group 2D (NKG2D) resulting in elimination of target cells. However, despite transformation and the continued presence of virally-encoded proteins, MICs are only expressed in a minority of MCC tumors in situ and are completely absent on MCC cell lines in vitro. This lack of MIC expression was due to epigenetic silencing via MIC promoter hypo-acetylation; indeed, MIC expression was re-induced by pharmacological inhibition of histone deacetylases (HDACs) both in vitro and in vivo. This re-induction of MICs rendered MCC cells more sensitive to immune-mediated lysis. Thus, epigenetic silencing of MICs is an important immune escape mechanism of MCCs.

Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-ß signalling pathway.

The transforming growth factor-ß (TGF-ß) signalling pathway participates in various biological processes. Dysregulation of Smad4, a central cellular transducer of TGF-ß signalling, is implicated in a wide range of human diseases and developmental disorders. However, the mechanisms underlying Smad4 dysregulation are not fully understood. Using a functional screening approach based on luciferase reporter assays, we identified 39 microRNAs (miRNAs) as potential regulators of Smad4 from an expression library of 388 human miRNAs. The screening was supported by bioinformatic analysis, as 24 of 39 identified miRNAs were also predicted to target Smad4. MiR-199a, one of the identified miRNAs, was inversely correlated with Smad4 expression in various human cancer cell lines and gastric cancer tissues, and repressed Smad4 expression and blocked canonical TGF-ß transcriptional responses in cell lines. These effects were dependent on the presence of a conserved, but not perfect seed paired, miR-199a-binding site in the Smad4 3'-untranslated region (UTR). Overexpression of miR-199a significantly inhibited the ability of TGF-ß to induce gastric cancer cell growth arrest and apoptosis in vitro, and promoted anchorage-independent growth in soft agar, suggesting that miR-199a plays an oncogenic role in human gastric tumourigenesis. In conclusion, our functional screening uncovers multiple miRNAs that regulate the cellular responsiveness to TGF-ß signalling and reveals important roles of miR-199a in gastric cancer by directly targeting Smad4.

Geft is dispensable for the development of the second heart field.

Geft is a guanine nucleotide exchange factor, which can specifically activate Rho family of small GTPase by catalyzing the exchange of bound GDP for GTP. Geft is highly expressed in the excitable tissue as heart and skeletal muscle and plays important roles in many cellular processes, such as cell proliferation, migration, and cell fate decision. However, the in vivo role of Geft remains unknown. Here, we generated a Geft conditional knockout mouse by flanking exons 5-17 of Geft with loxP sites. Cre-mediated deletion of the Geft gene in heart using Mef2c-Cre transgenic mice resulted in a dramatic decrease of Geft expression. Geft knockout mice develop normally and exhibit no discernable phenotype, suggesting Geft is dispensable for the development of the second heart field in mouse. The Geft conditional knockout mouse will be a valuable genetic tool for uncovering the in vivo roles of Geft during development and in adult homeostasis.

[Cardiomycyte overexpression of miR-27b resulted in cardiac fibrosis and mitochondria injury in mice].

Previous microRNA (miRNA) array results have shown that the expression of miR-27b is upregulated in heart tissues from human cardiomyopathy and pressure-overloaded hypertrophic mouse model, implying that miR-27b might play an important role in heart diseases. To study the in vivo function of miR-27b, we generated a transgenic mouse line overexpressing miR-27b under the control of the 5.5 kb promoter of a-myosin heavy chain (a-MHC). Real-time PCR results demonstrated that miR-27b precursor and mature miR-27b were significantly increased in the heart tissues of miR-27b transgenic mice. miR-27b transgenic mice not only displayed cardiac hypertrophy, but also exhibited significant cardiac fibrosis. Further study showed that matrix metalloproteinase 13 (MMP13), a key regulator involved in cardiac fibrosis, was the target of miR-27b. The expression of MMP13 was decreased and the expression of Col I and III was increased in miR-27b transgenic mice.. In addition, defects in ultrastructral architecture were also found in miR-27b trans-genic mice. The above results demonstrated that miR-27b might promote cardiac fibrosis through inhibiting MMP13.

Cardiomyocyte overexpression of miR-27b induces cardiac hypertrophy and dysfunction in mice.

Recent studies have begun to reveal critical roles of microRNAs (miRNAs) in the pathogenesis of cardiac hypertrophy and dysfunction. In this study, we tested whether a transforming growth factor-ß (TGF-ß)-regulated miRNA played a pivotal role in the development of cardiac hypertrophy and heart failure (HF). We observed that miR-27b was upregulated in hearts of cardiomyocyte-specific Smad4 knockout mice, which developed cardiac hypertrophy. In vitro experiments showed that the miR-27b expression could be inhibited by TGF-ß1 and that its overexpression promoted hypertrophic cell growth, while the miR-27b suppression led to inhibition of the hypertrophic cell growth caused by phenylephrine (PE) treatment. Furthermore, the analysis of transgenic mice with cardiomyocyte-specific overexpression of miR-27b revealed that miR-27b overexpression was sufficient to induce cardiac hypertrophy and dysfunction. We validated the peroxisome proliferator-activated receptor-γ (PPAR-γ) as a direct target of miR-27b in cardiomyocyte. Consistently, the miR-27b transgenic mice displayed significantly lower levels of PPAR-γ than the control mice. Furthermore, in vivo silencing of miR-27b using a specific antagomir in a pressure-overload-induced mouse model of HF increased cardiac PPAR-γ expression, attenuated cardiac hypertrophy and dysfunction. The results of our study demonstrate that TGF-ß1-regulated miR-27b is involved in the regulation of cardiac hypertrophy, and validate miR-27b as an efficient therapeutic target for cardiac diseases.

miR-21 promotes keratinocyte migration and re-epithelialization during wound healing.

MicroRNAs involved in keratinocyte migration and wound healing are largely unknown. Here, we revealed the indispensable role of miR-21 in keratinocyte migration and in re-epithelialization during wound healing in mice. In HaCaT cell, miR-21 could be upregulated by TGF-ß1. Similar to the effect of TGF-ß1, miR-21 overexpression promoted keratinocyte migration. Conversely, miR-21 knockdown attenuated TGF-ß1-induced keratinocyte migration, suggesting that miR-21 was essential for TGF-ß-driven keratinocyte migration. Furthermore, we found that miR-21 was upregulated during wound healing, coincident with the temporal expression pattern of TGF-ß1. Consistently, knockdown of endogenous miR-21 using a specific antagomir dramatically delayed re-epithelialization possibly due to the reduced keratinocyte migration. TIMP3 and TIAM1, direct targets of miR-21, were verified to be regulated by miR-21 in vitro and in vivo, indicating that these two molecules might contribute to miR-21-induced keratinocyte migration. Taken together, our results demonstrate that miR-21 promotes keratinocyte migration and boosts re-epithelialization during skin wound healing.

miR-148a promoted cell proliferation by targeting p27 in gastric cancer cells.

Accumulating evidence has shown that miRNAs are aberrantly expressed in human gastric cancer and crucial to tumorigenesis. Herein, we identified the role of miR-148a in gastric cell proliferation. miR-148a knockdown inhibited cell proliferation in gastric cancer cell lines. Conversely, miR-148a overexpression promoted cell proliferation and cell cycle progression. p27, a key inhibitor of cell cycle, was verified as the target of miR-148a, indicating miR-148a might downregulate p27 expression to promote gastric cell proliferation. Moreover, we confirmed that miR-148a expression was frequently and dramatically downregulated in human advanced gastric cancer tissues, and observed a good inverse correlation between miR-148a and p27 expression in tumor samples. Thus, our results demonstrated that miR-148a downregulation might exert some sort of antagonistic function in cell proliferation, rather than promote cell proliferation in gastric cancer.

[Preparation of polyclonal antibody against human Fkbp19].

AIM: To prepare the polyclonal antibody against human Fkbp19 for future functional studies of Fkbp19. METHODS: Prokaryotic expression vector pET21a-Fkbp19 was transformed into E.coli BL21-DE3, and then induced by IPTG. The recombinant protein was purified by Ni-NTA resin and used to immunize rabbits. Antiserums were used for Western blot and immunofluorescence to detect Fkbp19 in breast tumor cell. RESULTS: His-Fkbp19 fusion protein was successfully expressed in E.coli. High titer polyclonal antiserum was obtained by immunizing rabbits with purified His-Fkbp19 fusion protein. The antiserum could detect endogenous Fkbp19 protein in breast tumor cell very well by Western blot. CONCLUSION: The polyclonal antiserum against Fkbp19 was successfully generated.