37 research outputs found
Novel microRNAs modulating ecto-5′-nucleotidase expression
IntroductionThe expression of immune checkpoint molecules (ICMs) by cancer cells is known to counteract tumor-reactive immune responses, thereby promoting tumor immune escape. For example, upregulated expression of ecto-5′-nucleotidase (NT5E), also designated as CD73, increases extracellular levels of immunosuppressive adenosine, which inhibits tumor attack by activated T cells. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Thus, the binding of miRNAs to the 3′-untranslated region of target mRNAs either blocks translation or induces degradation of the targeted mRNA. Cancer cells often exhibit aberrant miRNA expression profiles; hence, tumor-derived miRNAs have been used as biomarkers for early tumor detection.MethodsIn this study, we screened a human miRNA library and identified miRNAs affecting the expression of ICMs NT5E, ENTPD1, and CD274 in the human tumor cell lines SK-Mel-28 (melanoma) and MDA-MB-231 (breast cancer). Thereby, a set of potential tumor-suppressor miRNAs that decreased ICM expression in these cell lines was defined. Notably, this study also introduces a group of potential oncogenic miRNAs that cause increased ICM expression and presents the possible underlying mechanisms. The results of high-throughput screening of miRNAs affecting NT5E expression were validated in vitro in 12 cell lines of various tumor entities.ResultsAs result, miR-1285-5p, miR-155-5p, and miR-3134 were found to be the most potent inhibitors of NT5E expression, while miR-134-3p, miR-6859-3p, miR-6514-3p, and miR-224-3p were identified as miRNAs that strongly enhanced NT5E expression levels.DiscussionThe miRNAs identified might have clinical relevance as potential therapeutic agents and biomarkers or therapeutic targets, respectively
SOX5 is involved in balanced MITF regulation in human melanoma cells
Background: Melanoma is a cancer with rising incidence and new therapeutics are needed. For this, it is necessary to understand the molecular mechanisms of melanoma development and progression. Melanoma differs from other cancers by its ability to produce the pigment melanin via melanogenesis; this biosynthesis is essentially regulated by microphthalmia-associated transcription factor (MITF). MITF regulates various processes such as cell cycling and differentiation. MITF shows an ambivalent role, since high levels inhibit cell proliferation and low levels promote invasion. Hence, well-balanced MITF homeostasis is important for the progression and spread of melanoma. Therefore, it is difficult to use MITF itself for targeted therapy, but elucidating its complex regulation may lead to a promising melanoma-cell specific therapy. Method: We systematically analyzed the regulation of MITF with a novel established transcription factor based gene regulatory network model. Starting from comparative transcriptomics analysis using data from cells originating from nine different tumors and a melanoma cell dataset, we predicted the transcriptional regulators of MITF employing ChIP binding information from a comprehensive set of databases. The most striking regulators were experimentally validated by functional assays and an MITF-promoter reporter assay. Finally, we analyzed the impact of the expression of the identified regulators on clinically relevant parameters of melanoma, i.e. the thickness of primary tumors and patient overall survival. Results: Our model predictions identified SOX10 and SOX5 as regulators of MITF. We experimentally confirmed the role of the already well-known regulator SOX10. Additionally, we found that SOX5 knockdown led to MITF up-regulation in melanoma cells, while double knockdown with SOX10 showed a rescue effect; both effects were validated by reporter assays. Regarding clinical samples, SOX5 expression was distinctively up-regulated in metastatic compared to primary melanoma. In contrast, survival analysis of melanoma patients with predominantly metastatic disease revealed that low SOX5 levels were associated with a poor prognosis. Conclusion: MITF regulation by SOX5 has been shown only in murine cells, but not yet in human melanoma cells. SOX5 has a strong inhibitory effect on MITF expression and seems to have a decisive clinical impact on melanoma during tumor progression
Screening of Human Tumor Antigens for CD4+ T Cell Epitopes by Combination of HLA-Transgenic Mice, Recombinant Adenovirus and Antigen Peptide Libraries
BACKGROUND: As tumor antigen-specific CD4+ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4+ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients. METHODS AND FINDINGS: To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4+ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1) or TRP-2 (Ad5.TRP-2) were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4+ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4+ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4+ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-2(60-74) epitope and against the new epitope TRP-2(149-163). Importantly, human T cells specifically recognizing target cells loaded with the TRP-2(149-163)-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4+ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4+ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1(284-298) as a new HLA-DRB1*0301-restricted CD4+ T cell epitope. CONCLUSIONS: Our screening approach identified new HLA-DRB1*0301-restricted CD4+ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target antigens of other tumor entities and to different HLA class II molecules even without prior characterization of their peptide binding motives
Cognate Interaction With CD4+ T Cells Instructs Tumor-Associated Macrophages to Acquire M1-Like Phenotype
The immunosuppressive tumor microenvironment (TME) established by tumor cells, stromal cells and inhibitory immune cells counteracts the function of tumor reactive T cells. Tumor associated macrophages (TAMs) showing functional plasticity contribute to this process as so called M2-like macrophages can suppress the function of effector T cells and promote their differentiation into regulatory T cells (Tregs). Furthermore, tumor antigen specific CD4+ T effector cells can essentially sustain anti-tumoral immune responses as shown for various tumor entities, thus suggesting that cognate interaction between tumor antigen-specific CD4+ Th1 cells and TAMs might shift the intra-tumoral M1/M2 ratio toward M1. This study demonstrates repolarization of M2-like PECs upon MHC II-restricted interaction with tumor specific CD4+ Th1 cells in vitro as shown by extensive gene and protein expression analyses. Moreover, adoptive transfer of OVA-specific OT-II cells into C57BL/6 mice bearing OVA expressing IAb−/− tumors resulted in increased accumulation of M1-like TAMs with enhanced M1 associated gene and protein expression profiles. Thus, this paper highlights a so far underestimated function of the CD4+ Th1/TAM axis in re-conditioning the immunosuppressive tumor microenvironment
Patterns of antibody responses to nonviral cancer antigens in head and neck squamous cell carcinoma patients differ by human papillomavirus status
There have been hints that nonviral cancer antigens are differentially expressed in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC). Antibody responses (AR) to cancer antigens may be used to indirectly determine cancer antigen expression in the tumor using a noninvasive and tissue-saving liquid biopsy. Here, we set out to characterize AR to a panel of nonviral cancer antigens in HPV-positive and HPV-negative HNSCC patients. A fluorescent microbead multiplex serology to 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens and 8 oncogenes) and 29 HPV-antigens was performed in 382 HNSCC patients from five independent cohorts (153 HPV-positive and 209 HPV-negative). AR to any of the cancer antigens were found in 272/382 patients (72%). The ten most frequent AR were CT47, cTAGE5a, c-myc, LAGE-1, MAGE-A1, -A3, -A4, NY-ESO-1, SpanX-a1 and p53. AR to MAGE-A3, MAGE-A9 and p53 were found at significantly different prevalences by HPV status. An analysis of AR mean fluorescent intensity values uncovered remarkably different AR clusters by HPV status. To identify optimal antigen selections covering a maximum of patients with ≤10 AR, multiobjective optimization revealed distinct antigen selections by HPV status. We identified that AR to nonviral antigens differ by HPV status indicating differential antigen expression. Multiplex serology may be used to characterize antigen expression using serum or plasma as a tissue-sparing liquid biopsy. Cancer antigen panels should address the distinct antigen repertoire of HPV-positive and HPV-negative HNSCC
Controlling the Immune Suppressor: Transcription Factors and MicroRNAs Regulating CD73/NT5E
Controlling the Immune Suppressor: Transcription Factors and MicroRNAs Regulating CD73/NT5E
The NT5E (CD73) molecule represents an ecto-5′-nucleotidase expressed on the cell surface of various cell types. Hydrolyzing extracellular adenosine monophosphate into adenosine and inorganic phosphate, NT5E performs numerous homeostatic functions in healthy organs and tissues. Importantly, NT5E can act as inhibitory immune checkpoint molecule, since free adenosine generated by NT5E inhibits cellular immune responses, thereby promoting immune escape of tumor cells. MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression on posttranscriptional level through binding to mRNAs, resulting in translational repression or degradation of the targeted mRNA molecule. In tumor cells, miRNA expression patterns are often altered which in turn might affect NT5E surface expression and eventually influence the efficacy of antitumor immune responses. This review describes the diverse roles of NT5E, summarizes current knowledge about transcription factors controlling NT5E expression, and highlights the significance of miRNAs involved in the posttranscriptional regulation of NT5E expression
Expression and activity of alcohol and aldehyde dehydrogenases in melanoma cells and in melanocytes
Rare Drosha Splice Variants Are Deficient in MicroRNA Processing but Do Not Affect General MicroRNA Expression in Cancer Cells12
Drosha is a key enzyme in microRNA biogenesis, generating the precursor miRNA (pre-miRNA) by excising the stem-loop embedded in the primary transcripts (pri-miRNA). The specificity for the pri-miRNAs and determination of the cleavage site are provided by its binding partner DGCR8, which is necessary for efficient processing. The crucial Drosha domains for pri-miRNA cleavage are the middle part, the two enzymatic RNase III domains (RIIID), and the dsRNA binding domain (dsRBD) in the C-terminus. Here, we identify alternatively spliced transcripts in human melanoma and NT2 cell lines, encoding C-terminally truncated Drosha proteins lacking part of the RIIIDb and the entire dsRBD. Proteins generated from these alternative splice variants fail to bind to DGCR8 but still interact with Ewing sarcoma protein (EWS). In vitro as well as in vivo, the Drosha splice variants are deficient in pri-miRNA processing. However, the aberrant transcripts in melanoma cells do not consistently reduce mature miRNA levels compared with melanoma cell lines lacking those splice variants, possibly owing to their limited abundance. Our findings show that alternative processing-deficient Drosha splice variants exist in melanoma cells. In elevated amounts, these alternatively spliced transcripts could provide one potential mechanism accounting for the deregulation of miRNAs in cancer cells. On the basis of our results, the search for alternative inactive splice variants might be fruitful in different tumor entities to unravel the molecular basis of the previously observed decreased microRNA processing efficiency in cancer