Background: Overexpression of aspartate β-hydroxylase (ASPH) in human tumors contributes to their progression by stimulating cell proliferation, migration, and invasion. Several signaling pathways affected by ASPH have been identified, but the high number of potential targets of ASPH hydroxylation suggests that additional mechanisms may be involved. This study was performed to reveal new targets of ASPH signaling. Methods: The effect of ASPH on the oncogenicity of three mouse tumor cell lines was tested using proliferation assays, transwell assays, and spheroid invasion assays after inhibition of ASPH with the small molecule inhibitor MO-I-1151. ASPH was also deactivated with the CRISPR/Cas9 system. A transcriptomic analysis was then performed with bulk RNA sequencing and differential gene expression was evaluated. Expression data were verified by quantitative PCR and immunoblotting. Results: Inhibition or abrogation of ASPH reduced proliferation of the cell lines and their migration and invasiveness. Among the genes with differential expression in more than one cell line, two members of the lymphocyte antigen 6 (Ly6) family, Ly6a and Ly6c1, were found. Their downregulation was confirmed at the protein level by immunoblotting, which also showed their reduction after ASPH inhibition in other mouse cell lines. Reduced production of the Ly6D and Ly6K proteins was shown after ASPH inhibition in human tumor cell lines. Conclusions: Since increased expression of Ly6 genes is associated with the development and progression of both mouse and human tumors, these results suggest a novel mechanism of ASPH oncogenicity and support the utility of ASPH as a target for cancer therapy.

• Klíčová slova
• ASPH inhibitor, Ly6 family, RNA sequencing, Tumorigenesis,
• Publikační typ
• časopisecké články MeSH

The Czech Republic, a part of the former Czechoslovakia, has been at the forefront of several research directions in virology, genetics and physiology [...].

• MeSH
• virologie * MeSH
• Publikační typ
• práce podpořená grantem MeSH
• úvodníky MeSH
• Geografické názvy
• Česká republika MeSH

Tumor-associated macrophages (TAMs) plentifully infiltrate the tumor microenvironment (TME), but their role in anti-tumor immunity is controversial. Depending on the acquired polarization, they can either support tumor growth or participate in the elimination of neoplastic cells. In this study, we analyzed the TME by RNA-seq and flow cytometry and examined TAMs after ex vivo activation. Tumors with normal and either reversibly or irreversibly decreased expression of major histocompatibility complex class I (MHC-I) molecules were induced with TC-1, TC-1/A9, and TC-1/dB2m cells, respectively. We found that combined immunotherapy (IT), composed of DNA immunization and the CpG oligodeoxynucleotide (ODN) ODN1826, evoked immune reactions in the TME of TC-1- and TC-1/A9-induced tumors, while the TME of TC-1/dB2m tumors was mostly immunologically unresponsive. TAMs infiltrated both tumor types with MHC-I downregulation, but only TAMs from TC-1/A9 tumors acquired the M1 phenotype upon IT and were cytotoxic in in vitro assay. The anti-tumor effect of combined IT was markedly enhanced by a blockade of the colony-stimulating factor-1 receptor (CSF-1R), but only against TC-1/A9 tumors. Overall, TAMs from tumors with irreversible MHC-I downregulation were resistant to the stimulation of cytotoxic activity. These data suggest the dissimilarity of TAMs from different tumor types, which should be considered when utilizing TAMs in cancer IT.

• Klíčová slova
• colony-stimulating factor-1, immunotherapy, macrophages, major histocompatibility complex, repolarization, tumor,
• Publikační typ
• časopisecké články MeSH

Programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) blockade is a promising therapy for various cancer types, but most patients are still resistant. Therefore, a larger number of predictive biomarkers is necessary. In this study, we assessed whether a loss-of-function mutation of the interferon (IFN)-γ receptor 1 (IFNGR1) in tumor cells can interfere with anti-PD-L1 therapy. For this purpose, we used the mouse oncogenic TC-1 cell line expressing PD-L1 and major histocompatibility complex class I (MHC-I) molecules and its TC-1/A9 clone with reversibly downregulated PD-L1 and MHC-I expression. Using the CRISPR/Cas9 system, we generated cells with deactivated IFNGR1 (TC-1/dIfngr1 and TC-1/A9/dIfngr1). In tumors, IFNGR1 deactivation did not lead to PD-L1 or MHC-I reduction on tumor cells. From potential inducers, mainly IFN-α and IFN-β enhanced PD-L1 and MHC-I expression on TC-1/dIfngr1 and TC-1/A9/dIfngr1 cells in vitro. Neutralization of the IFN-α/IFN-β receptor confirmed the effect of these cytokines in vivo. Combined immunotherapy with PD-L1 blockade and DNA vaccination showed that IFNGR1 deactivation did not reduce tumor sensitivity to anti-PD-L1. Thus, the impairment of IFN-γ signaling may not be sufficient for PD-L1 and MHC-I reduction on tumor cells and resistance to PD-L1 blockade, and thus should not be used as a single predictive marker for anti-PD-1/PD-L1 cancer therapy.

• Klíčová slova
• IFN-α, IFN-β, IFNGR1, MHC class I, PD-1/PD-L1, cancer, immune checkpoint therapy,
• MeSH
• antigeny CD274 antagonisté a inhibitory   MeSH
• antigeny CD279 antagonisté a inhibitory   MeSH
• experimentální nádory farmakoterapie   imunologie   metabolismus   patologie   MeSH
• imunoterapie MeSH
• interferon gama antagonisté a inhibitory   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• protinádorové látky imunologicky aktivní farmakologie   MeSH
• transformované buněčné linie účinky léků   imunologie   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD274 MeSH
• antigeny CD279 MeSH
• Cd274 protein, mouse MeSH Prohlížeč
• interferon gama MeSH
• Pdcd1 protein, mouse MeSH Prohlížeč
• protinádorové látky imunologicky aktivní MeSH

Combined immunotherapy constitutes a novel, advanced strategy in cancer treatment. In this study, we investigated immunotherapy in the mouse TC-1/A9 model of human papillomavirus type 16 (HPV16)-associated tumors characterized by major histocompatibility complex class I (MHC-I) downregulation. We found that the induction of a significant anti-tumor response required a combination of DNA vaccination with the administration of an adjuvant, either the synthetic oligodeoxynucleotide ODN1826, carrying immunostimulatory CpG motifs, or α-galactosylceramide (α-GalCer). The most profound anti-tumor effect was achieved when these adjuvants were applied in a mix with a one-week delay relative to DNA immunization. Combined immunotherapy induced tumor infiltration with various subsets of immune cells contributing to tumor regression, of which cluster of differentiation (CD) 8⁺ T cells were the predominant subpopulation. In contrast, the numbers of tumor-associated macrophages (TAMs) were not markedly increased after immunotherapy but in vivo and in vitro results showed that they could be repolarized to an anti-tumor M1 phenotype. A blockade of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) immune checkpoint had a negligible effect on anti-tumor immunity and TAMs repolarization. Our results demonstrate a benefit of combined immunotherapy comprising the activation of both adaptive and innate immunity in the treatment of tumors with reduced MHC-I expression.

• Klíčová slova
• CpG ODN, DNA immunization, MHC-I, cancer immunotherapy, tumor-associated macrophages, α-galactosylceramide,
• MeSH
• adjuvancia imunologická terapeutické užití   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• down regulace MeSH
• experimentální nádory terapie   MeSH
• galaktosylceramidy imunologie   MeSH
• histokompatibilita - antigeny třídy I imunologie   MeSH
• imunoterapie metody   MeSH
• makrofágy imunologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oligodeoxyribonukleotidy imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adjuvancia imunologická MeSH
• galaktosylceramidy MeSH
• histokompatibilita - antigeny třídy I MeSH
• oligodeoxyribonukleotidy MeSH

The present study aimed to elucidate the role of cluster of differentiation (CD)8+, CD4+, natural killer (NK), and myeloid (CD11b+) cells in the course of the growth and rejection of experimental major histocompatibility complex (MHC) class I-deficient, HPV16 E6/E7-associated TC-1/A9 tumors in mice. Stable mouse lines (F30) generated by inbreeding of Balb/c and C57BL/6 strains, which were characterized by H-2Db+d-NK1.1neg (B6-neg) and H-2Db-d+NK1.1high (Balb-high) phenotypes, were used for the present study. The novel strains spontaneously regressed tumors in 70-90% of cases. Ex vivo histological analysis of the tumor microenvironment in cryosections showed an indirect correlation between the growth of the transplanted tumor (progressor vs. regressor mice) and the proportion of immunocompetent cell infiltration in the tumors. The regressor mice exhibited a higher infiltration of tumors with CD4+ and CD8+ cells, and in Balb-high with NK cells as well, compared with the progressors. All tumor transplants also indicated a huge infiltration of CD11b+ cells, but this infiltration was not dependent on the stage of the TC-1/A9 tumor development. Depletion of individual cell subpopulations in vivo exhibited different effects on the tumor development in the two strains. Elimination of CD8-positive cells enhanced growth of TC-1/A9 tumor transplants in both hybrid stains, whereas CD4+ cell depletion affected rejection of TC-1/A9 tumors in the B6-neg mice only. Depletion of NK cells with anti-asialo GM1 antibody in the Balb-high strain led to enhancement of tumor growth, which was more pronounced after depletion of the NK1.1+ subpopulation. On the other hand, depletion of NK cells with anti-asialo GM1 in B6-neg mice did not affect the regression of TC-1/A9 tumor transplants, but increased the CD11b+ cell infiltration. In summary, these results indicate that co-operation of particular subsets of immunocompetent cells is essential for the rejection of TC-1/A9 tumor transplants. In B6-neg mice, the co-operative action of CD8+ and CD4+ cells is required, whereas in Balb-high mice, the synergy of CD8+ and NK1.1+ cells is of major importance.

• Klíčová slova
• B6-neg and Balb-high mice, TC-1/A9 tumor, depletion in vivo, novel mouse strains, spontaneous tumor rejection, tumor-infiltrating cells,
• Publikační typ
• časopisecké články MeSH

Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFNγ. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFNγ treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFNγ or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.

• MeSH
• down regulace MeSH
• epigeneze genetická MeSH
• fibrosarkom genetika   imunologie   metabolismus   MeSH
• geny MHC třídy I * MeSH
• interferon gama genetika   imunologie   metabolismus   MeSH
• metylace DNA * MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• prezentace antigenu genetika   MeSH
• regulace genové exprese u nádorů MeSH
• signální transdukce MeSH
• transfekce MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• interferon gama MeSH

BACKGROUND: Epigenetic mechanisms have important roles in the tumour escape from immune responses, such as in MHC class I downregulation or altered expression of other components involved in antigen presentation. Chemotherapy with DNA methyltransferase inhibitors (DNMTi) can thus influence the tumour cell interactions with the immune system and their sensitivity to immunotherapy. METHODS: We evaluated the therapeutic effects of the DNMTi 5-azacytidine (5AC) against experimental MHC class I-deficient and -positive tumours. The 5AC therapy was combined with immunotherapy, using a murine model for HPV16-associated tumours. RESULTS: We have demonstrated 5AC additive effects against MHC class I-positive and -deficient tumours when combined with unmethylated CpG oligodeoxynucleotides or with IL-12-producing cellular vaccine. The efficacy of the combined chemoimmunotherapy against originally MHC class I-deficient tumours was partially dependent on the CD8(+)-mediated immune responses. Increased cell surface expression of MHC class I cell molecules, associated with upregulation of the antigen-presenting machinery-related genes, as well as of genes encoding selected components of the IFNγ-signalling pathway in tumours explanted from 5AC-treated animals, were observed. CONCLUSION: Our data suggest that chemotherapy of MHC class I-deficient tumours with 5AC combined with immunotherapy is an attractive setting in the treatment of MHC class I-deficient tumours.

• MeSH
• antimetabolity antitumorózní farmakologie   MeSH
• azacytidin farmakologie   MeSH
• DNA primery MeSH
• ELISA MeSH
• experimentální nádory farmakoterapie   imunologie   terapie   virologie   MeSH
• histokompatibilita - antigeny třídy I imunologie   MeSH
• imunoterapie * MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidský papilomavirus 16 izolace a purifikace   MeSH
• metylace MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• sekvence nukleotidů MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antimetabolity antitumorózní MeSH
• azacytidin MeSH
• DNA primery MeSH
• histokompatibilita - antigeny třídy I MeSH

DNA vaccines showed great promise in preclinical models of infectious and malignant diseases, but their potency was insufficient in clinical trials and is needed to be improved. In this study, we tested systemic administration of two conventional adjuvants, synthetic oligodeoxynucleotide carrying immunostimulatory CpG motifs (CpG-ODN) and levamisole (LMS), and evaluated their effect on immune reactions induced by DNA vaccines delivered by a gene gun. DNA vaccination was directed either against the E7 oncoprotein of human papillomavirus type 16 or against the BCR-ABL1 oncoprotein characteristic for chronic myeloid leukemia. High doses of both adjuvants reduced activation of mouse splenic CD8(+) T lymphocytes, but the overall antitumor effect was enhanced in both tumor models. High-dose CpG-ODN exhibited a superior adjuvant effect in comparison with any combination of CpG-ODN with LMS. In summary, our results demonstrate the benefit of combined therapy with gene-gun-delivered antitumor DNA vaccines and systemic administration of CpG-ODN or LMS.

• MeSH
• adjuvancia imunologická aplikace a dávkování   MeSH
• bcr-abl fúzové proteiny genetika   imunologie   metabolismus   MeSH
• biolistika MeSH
• CD8-pozitivní T-lymfocyty účinky léků   metabolismus   patologie   MeSH
• DNA vakcíny MeSH
• experimentální nádory imunologie   patologie   terapie   MeSH
• imunita účinky léků   MeSH
• levamisol aplikace a dávkování   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oligodeoxyribonukleotidy aplikace a dávkování   MeSH
• Papillomavirus E7 - proteiny genetika   imunologie   metabolismus   MeSH
• protinádorové vakcíny * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adjuvancia imunologická MeSH
• bcr-abl fúzové proteiny MeSH
• CPG-oligonucleotide MeSH Prohlížeč
• DNA vakcíny MeSH
• levamisol MeSH
• oligodeoxyribonukleotidy MeSH
• Papillomavirus E7 - proteiny MeSH
• protinádorové vakcíny * MeSH

Infection with high-risk types of human papillomavirus (HPV) can cause the development of malignant tumors. To study mechanisms responsible for immune escape of tumor cells infected with HPV16, we previously used mouse oncogenic TC-1 cells producing HPV16 E6 and E7 oncoproteins to derive TC-1 clones resistant to immunization against E7. We have found immunoresistance of the clones to correlate with the point mutation in the E7 oncogene, which resulted in the N53S substitution in the immunodominant epitope RAHYNIVTF (aa 49-57). Here, we have shown that this mutation reduced stabilization of H-2D(b) molecules on RMA-S cells and eliminated immunogenicity of E7. The resistance of TC-1 clones was E7-specific as immunization against E6 inhibited tumor growth. Transduction of the TC-1/F9 clone carrying the mutated epitope with the wild-type E7 gene restored susceptibility to immunization against E7. Our results suggest that mutagenesis of tumor antigens can lead to the escape of malignant cells and should be considered in the development and evaluation of cancer immunotherapy.

• MeSH
• antigeny nádorové chemie   MeSH
• bodová mutace MeSH
• epitopy chemie   MeSH
• H-2 antigeny chemie   MeSH
• histokompatibilní antigen H-2D MeSH
• imunodominantní epitopy chemie   MeSH
• imunoterapie metody   MeSH
• lidé MeSH
• mutace MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádory imunologie   metabolismus   terapie   MeSH
• onkogenní proteiny virové chemie   genetika   MeSH
• onkogenní proteiny chemie   MeSH
• Papillomavirus E7 - proteiny MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny nádorové MeSH
• epitopy MeSH
• H-2 antigeny MeSH
• histokompatibilní antigen H-2D MeSH
• imunodominantní epitopy MeSH
• oncogene protein E7, Human papillomavirus type 16 MeSH Prohlížeč
• onkogenní proteiny virové MeSH
• onkogenní proteiny MeSH
• Papillomavirus E7 - proteiny MeSH