SOX9 is a target of miR-134-3p and miR-224-3p in breast cancer cell lines.

The transcription factor SOX9 represents an important mediator of breast cancer progression. miRNAs are small non-coding RNAs inhibiting translation of target genes upon interaction with the 3'-UTR region of respective mRNA molecules. Deregulated miRNA expression is involved in hallmarks of cancer like sustained proliferation and inhibition of apoptosis. Here, we investigated the miRNA-mediated regulation of SOX9 expression in two breast cancer cell lines, thereby providing further insights into cellular mechanisms driving breast cancer progression. The modulating effects of miR-134-3p, miR-224-3p, and miR-6859-3p on SOX9 expression were analyzed by qPCR and Western blot in human MDA-MB-231 breast cancer cells. Direct binding of the above-mentioned miRNAs to the SOX9 3'-UTR was assessed by luciferase reporter assays and site-directed mutagenesis. Expression levels of the investigated miRNAs in tumor samples versus healthy tissues were analyzed in silico using publicly available databases. Transfection of miR-134-3p, miR-224-3p, or miR-6859-3p reduced SOX9 expression on mRNA and protein level. Reporter assays proved direct binding of miR-134-3p and miR-224-3p to the SOX9 3'-UTR in MDA-MB-231 and MCF-7 cells. Expression analysis performed in silico revealed reduced expression of both miRNAs in breast cancer tissues. We describe three novel miRNAs targeting SOX9 in human breast cancer cell lines. Among them miR-134-2p and miR-224-3p might act as tumor suppressors, whose down-regulation induces elevated SOX9 levels thereby promoting breast cancer progression.

Reprogramming of macrophages employing gene regulatory and metabolic network models.

Upon exposure to different stimuli, resting macrophages undergo classical or alternative polarization into distinct phenotypes that can cause fatal dysfunction in a large range of diseases, such as systemic infection leading to sepsis or the generation of an immunosuppressive tumor microenvironment. Investigating gene regulatory and metabolic networks, we observed two metabolic switches during polarization. Most prominently, anaerobic glycolysis was utilized by M1-polarized macrophages, while the biosynthesis of inosine monophosphate was upregulated in M2-polarized macrophages. Moreover, we observed a switch in the urea cycle. Gene regulatory network models revealed E2F1, MYC, PPARγ and STAT6 to be the major players in the distinct signatures of these polarization events. Employing functional assays targeting these regulators, we observed the repolarization of M2-like cells into M1-like cells, as evidenced by their specific gene expression signatures and cytokine secretion profiles. The predicted regulators are essential to maintaining the M2-like phenotype and function and thus represent potential targets for the therapeutic reprogramming of immunosuppressive M2-like macrophages.

Retraction Note to: cGMP-phosphodiesterase 6, transducin and Wnt5a/Frizzled-2-signaling control cGMP and Ca2+ homeostasis in melanoma cells.

The Editor-in-Chief has retracted this article [1] due to errors in Figs. 1b, c and 4.

Cost of decentralized CAR T-cell production in an academic nonprofit setting.

Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy with high acquisition costs, and it has raised concerns about affordability and sustainability in many countries. Furthermore, the current centralized production paradigm for the T cells is less than satisfactory. Therefore, several countries are exploring alternative T-cell production modes. Our study is based on the T-cell production experience in a nonprofit setting in Germany. We first identified the work steps and main activities in the production process. Then we determined the fixed costs and variable costs. Main cost components included personnel and technician salaries, expenditure on equipment, a clean room, as well as production materials. All costs were calculated in 2018 euros and converted into U.S. dollars. For a clean room with one machine for closed and automated manufacturing installed, annual fixed costs summed up to approximately €438 098 ($584 131). The variable cost per production was roughly €34 798 ($46 397). At the maximum capacity of one machine, total cost per product would be close to €60 000 ($78 849). As shown in the scenario analysis, if three machines were to be installed in the clean room, per production cost could be as low as €45 000 (roughly $59905). If a cheaper alternative to lentivirus was used, per production total cost could be further reduced to approximately €33 000 (roughly $44309). Decentralized T-cell production might be a less costly and more efficient alternative to the current centralized production mode that requires a high acquisition cost.

A vaccine targeting mutant IDH1 induces antitumour immunity.

Monoallelic point mutations of isocitrate dehydrogenase type 1 (IDH1) are an early and defining event in the development of a subgroup of gliomas and other types of tumour. They almost uniformly occur in the critical arginine residue (Arg 132) in the catalytic pocket, resulting in a neomorphic enzymatic function, production of the oncometabolite 2-hydroxyglutarate (2-HG), genomic hypermethylation, genetic instability and malignant transformation. More than 70% of diffuse grade II and grade III gliomas carry the most frequent mutation, IDH1(R132H) (ref. 3). From an immunological perspective, IDH1(R132H) represents a potential target for immunotherapy as it is a tumour-specific potential neoantigen with high uniformity and penetrance expressed in all tumour cells. Here we demonstrate that IDH1(R132H) contains an immunogenic epitope suitable for mutation-specific vaccination. Peptides encompassing the mutated region are presented on major histocompatibility complexes (MHC) class II and induce mutation-specific CD4(+) T-helper-1 (TH1) responses. CD4(+) TH1 cells and antibodies spontaneously occurring in patients with IDH1(R132H)-mutated gliomas specifically recognize IDH1(R132H). Peptide vaccination of mice devoid of mouse MHC and transgenic for human MHC class I and II with IDH1(R132H) p123-142 results in an effective MHC class II-restricted mutation-specific antitumour immune response and control of pre-established syngeneic IDH1(R132H)-expressing tumours in a CD4(+) T-cell-dependent manner. As IDH1(R132H) is present in all tumour cells of these slow-growing gliomas, a mutation-specific anti-IDH1(R132H) vaccine may represent a viable novel therapeutic strategy for IDH1(R132H)-mutated tumours.

A universal anti-cancer vaccine: Chimeric invariant chain potentiates the inhibition of melanoma progression and the improvement of survival.

For many years, clinicians and scientists attempt to develop methods to stimulate the immune system to target malignant cells. Recent data suggest that effective cancer vaccination requires combination immunotherapies to overcome tumor immune evasion. Through presentation of both MHC-I and II molecules, DCs-based vaccine platforms are effective in generating detectable CD4 and CD8 T cell responses against tumor-associated antigens. Several platforms include DC transfection with mRNA of the desired tumor antigen. These DCs are then delivered to the host and elicit an immune response against the antigen of interest. We have recently established an mRNA genetic platform which induced specific CD8+ cytotoxic T cell response by DC vaccination against melanoma. In our study, an MHC-II mRNA DCs vaccine platform was developed to activate CD4+ T cells and to enhance the anti-tumor response. The invariant chain (Ii) was modified and the semi-peptide CLIP was replaced with an MHC-II binding peptide sequences of melanoma antigens. These chimeric MHC-II constructs are presented by DCs and induce proliferation of tumor specific CD4+ T cells. When administered in combination with the MHC-I platform into tumor bearing mice, these constructs were able to inhibit tumor growth, and improve mouse survival. Deciphering the immunological mechanism of action, we observed an efficient CTLs killing in addition to higher levels of Th1 and Th2 subsets in the groups immunized with a combination of the MHC-I and MHC-II constructs. These universal constructs can be applied in multiple combinations and offer an attractive opportunity to improve cancer treatment.

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.

A transplantable tumor model allowing investigation of NY-BR-1-specific T cell responses in HLA-DRB1*0401 transgenic mice.

BACKGROUND: NY-BR-1 has been described as a breast cancer associated differentiation antigen with intrinsic immunogenicity giving rise to endogenous T and B cell responses. The current study presents the first murine tumor model allowing functional investigation of NY-BR-1-specific immune responses in vivo. METHODS: A NY-BR-1 expressing tumor model was established in DR4tg mice based on heterotopic transplantation of stable transfectant clones derived from the murine H2 compatible breast cancer cell line EO771. Composition and phenotype of tumor infiltrating immune cells were analyzed by qPCR and FACS. MHC I binding affinity of candidate CTL epitopes predicted in silico was determined by FACS using the mutant cell line RMA-S. Frequencies of NY-BR-1 specific CTLs among splenocytes of immunized mice were quantified by FACS with an epitope loaded Db-dextramer. Functional CTL activity was determined by IFNγ catch or IFNγ ELISpot assays and statistical analysis was done applying the Mann Whitney test. Tumor protection experiments were performed by immunization of DR4tg mice with replication deficient recombinant adenovirus followed by s.c. challenge with NY-BR-1 expressing breast cancer cells. RESULTS: Our results show spontaneous accumulation of CD8+ T cells and F4/80+ myeloid cells preferentially in NY-BR-1 expressing tumors. Upon NY-BR-1-specific immunization experiments combined with in silico prediction and in vitro binding assays, the first NY-BR-1-specific H2-Db-restricted T cell epitope could be identified. Consequently, flow cytometric analysis with fluorochrome conjugated multimers showed enhanced frequencies of CD8+ T cells specific for the newly identified epitope in spleens of immunized mice. Moreover, immunization with Ad.NY-BR-1 resulted in partial protection against outgrowth of NY-BR-1 expressing tumors and promoted intratumoral accumulation of macrophages. CONCLUSION: This study introduces the first H2-Db-resctricted CD8+ T cell epitope-specific for the human breast cancer associated tumor antigen NY-BR-1. Our novel, partially humanized tumor model enables investigation of the interplay between HLA-DR4-restricted T cell responses and CTLs within their joint attack of NY-BR-1 expressing tumors.

Clinical translation and regulatory aspects of CAR/TCR-based adoptive cell therapies-the German Cancer Consortium approach.

Adoptive transfer of T cells genetically modified by TCRs or CARs represents a highly attractive novel therapeutic strategy to treat malignant diseases. Various approaches for the development of such gene therapy medicinal products (GTMPs) have been initiated by scientists in recent years. To date, however, the number of clinical trials commenced in Germany and Europe is still low. Several hurdles may contribute to the delay in clinical translation of these therapeutic innovations including the significant complexity of manufacture and non-clinical testing of these novel medicinal products, the limited knowledge about the intricate regulatory requirements of the academic developers as well as limitations of funds for clinical testing. A suitable good manufacturing practice (GMP) environment is a key prerequisite and platform for the development, validation, and manufacture of such cell-based therapies, but may also represent a bottleneck for clinical translation. The German Cancer Consortium (DKTK) and the Paul-Ehrlich-Institut (PEI) have initiated joint efforts of researchers and regulators to facilitate and advance early phase, academia-driven clinical trials. Starting with a workshop held in 2016, stakeholders from academia and regulatory authorities in Germany have entered into continuing discussions on a diversity of scientific, manufacturing, and regulatory aspects, as well as the benefits and risks of clinical application of CAR/TCR-based cell therapies. This review summarizes the current state of discussions of this cooperative approach providing a basis for further policy-making and suitable modification of processes.

Identification of NY-BR-1-specific CD4(+) T cell epitopes using HLA-transgenic mice.

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer-related deaths among women. The differentiation antigen NY-BR-1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen-specific CD4(+) effector T cells, NY-BR-1 was screened for the presence of HLA-restricted CD4(+) T cell epitopes that could be included in immunological treatment approaches. Upon NY-BR-1-specific DNA immunization of HLA-transgenic mice and functional ex vivo analysis, a panel of NY-BR-1-derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY-BR-1-specific, HLA-DRB1*0301- or HLA-DRB1*0401-restricted CD4(+) T cell lines from splenocytes of peptide immunized HLA-transgenic mice. Notably, all four CD4(+) T cell lines recognized human HLA-DR-matched dendritic cells (DC) pulsed with lysates of NY-BR-1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4(+) T cells specific for all four CD4(+) T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4(+) T cell responses against the new epitopes are not deleted nor inactivated by self-tolerance mechanisms. Our results present the first NY-BR-1-specific HLA-DRB1*0301- and HLA-DRB1*0401-restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer.

Prognostic significance of spontaneous antibody responses against tumor-associated antigens in malignant melanoma patients.

Distribution, patterns and prognostic impact of spontaneous antibody responses against different tumor-associated antigens (TAAs) in malignant melanoma patients are unknown so far and were investigated in this study for the first time in a large cohort at different stages of the disease, identifying new prognostic biomarkers for malignant melanoma. Serum samples from 365 melanoma patients (97 Stage I melanoma patients, 87 Stage II, 92 Stage III and 89 Stage IV) and 100 age and gender matched healthy control donors were analyzed. Samples were drawn at the time of diagnosis (Stages I-III) or at time of diagnosis of distant metastasis (Stage IV). Applying a novel multiplex assay, humoral immune responses against 29 TAAs were determined and the association between response and patient survival was investigated. Antibody responses were mainly found in melanoma patients and all tested antigens elicited immune responses in all disease stages. Antibody responses against single antigens were either associated with poor prognosis and/or shorter progression-free survival (PFS) or had no influence. While in Stages I-III significant associations were observed between an antibody response and overall survival or PFS, among Stage IV patients, no significant association was found. Multivariate analyses identified specific humoral immune responses as prognostic factors independently of age, chemotherapy and immunotherapy. Antibody responses against specific TAA in Stage I-III melanoma patients correlate with poor prognosis and/or shorter PFS. These results may help to design clinical studies in order to evaluate the potential of these responses as prognostic serological biomarkers.

T cell responses in early-stage melanoma patients occur frequently and are not associated with humoral response.

Endogenous tumor-specific T cells are detectable in patients with different tumor types including malignant melanoma (MM). They can control tumor growth, have impact on patient survival and correlate with improved clinical response to immune checkpoint therapy. Thus, they may represent a potent biomarker for respective treatment decisions. So far, major target antigens of endogenous MM-reactive T cells have not been determined systematically. Instead, autoantibodies are discussed as surrogate parameter for MM-specific T cells. Throughout a period of more than 60 days after tumor resection, we therefore determined in 38 non-metastasized primary MM patients and in healthy individuals by IFNγ ELISpot and bead-based fluorescent multiplex assay major target antigens of spontaneous T cell and humoral responses using a broad panel of MM antigens and assessed the presence and suppressive impact of MM-reactive regulatory T cells (Tregs). We show that MM-reactive T cells are frequent in MM patients, transiently increase after tumor removal and are mostly directed against Melan-A/MART-1, Tyrosinase, NA17-A and p53. MM-specific Tregs were only detected in few patients and inhibited MM-reactive T cells particularly early after tumor resection. Tumor-specific autoantibodies occurred in most patients, but did not correlate with T cell responses. Thus, endogenous antibodies may not be reliable surrogate parameters of MM-reactive T cells.

Estimating the activity of transcription factors by the effect on their target genes.

MOTIVATION: Understanding regulation of transcription is central for elucidating cellular regulation. Several statistical and mechanistic models have come up the last couple of years explaining gene transcription levels using information of potential transcriptional regulators as transcription factors (TFs) and information from epigenetic modifications. The activity of TFs is often inferred by their transcription levels, promoter binding and epigenetic effects. However, in principle, these methods do not take hard-to-measure influences such as post-transcriptional modifications into account. RESULTS: For TFs, we present a novel concept circumventing this problem. We estimate the regulatory activity of TFs using their cumulative effects on their target genes. We established our model using expression data of 59 cell lines from the National Cancer Institute. The trained model was applied to an independent expression dataset of melanoma cells yielding excellent expression predictions and elucidated regulation of melanogenesis. AVAILABILITY AND IMPLEMENTATION: Using mixed-integer linear programming, we implemented a switch-like optimization enabling a constrained but optimal selection of TFs and optimal model selection estimating their effects. The method is generic and can also be applied to further regulators of transcription. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

miR-137 inhibits proliferation of melanoma cells by targeting PAK2.

MicroRNAs (miRNA) are key players in a variety of cancers including malignant melanoma. miR-137 has been reported to be a tumor suppressor in melanoma and several targets have been identified for this miRNA. We previously developed a novel proteomics technology, (35) S in vivo/vitro labelling analysis for dynamic proteomics (SiLAD). Because of its high sensitivity in analysing protein expression rates, SiLAD has the potential to unravel miRNA effects on mRNAs coding for proteins with long half-lives or high abundance. Using SiLAD, we discovered that miR-137 significantly downregulated the expression rate of p21-activated kinase 2 (PAK2) in melanoma cells. Bioinformatics analysis predicted PAK2 as a direct target of miR-137, which was confirmed by luciferase reporter assay and Western blot analysis. We found that overexpression of miR-137 inhibited the proliferation of melanoma cells, which could be phenocopied by knockdown of PAK2 using siRNAs. Furthermore, overexpression of PAK2 restored miR-137-mediated suppression of cell proliferation. These findings indicate that miR-137 could inhibit proliferation through targeting PAK2 in melanoma cells.

LRAT overexpression diminishes intracellular levels of biologically active retinoids and reduces retinoid antitumor efficacy in the murine melanoma B16F10 cell line.

BACKGROUND/AIM: Vitamin A (all- trans -retinol, ATRol) serves as a precursor for all- trans -retinoic acid (ATRA), a ligand for the retinoic acid receptor (RAR), representing a potent regulator for many physiological processes. While murine melanoma cells are highly sensitive to retinoid treatment, human melanoma cells have developed still unidentified mechanisms that mediate cellular retinoid resistance. One of the key retinoid metabolizing enzymes is lecithin retinol acyltransferase (LRAT), which catalyzes the transformation of ATRol into inactive retinyl esters. LRAT is highly expressed in human melanoma cells. The aim of this study was to identify the mechanisms in retinol metabolism that are responsible for cellular retinoid sensitivity in the murine melanoma cell line B16F10. METHODS: mRNA expression analysis, cell viability assessment and determination of intracellular retinoid levels using HPLC analysis of a generated LRAT-overexpressing B16F10 cell line compared to the control B16F10 cell line. RESULTS: We found that the murine retinoid-sensitive B16F10 cell line does not express the enzyme LRAT. LRAT overexpression decreased the antiproliferative effects of retinoid treatment in these melanoma cells. The RAR-regulated enzyme Cyp26a1 showed a significantly lower expression in LRAT-overexpressing B16F10 cells. Cyp26a1 expression was restored after ATRA incubation. HPLC analysis revealed that the level of inactive retinyl ester increased after ATRol treatment, and levels of the substrate ATRol and biologically active ATRA significantly decreased in LRAT-overexpressing murine melanoma. Consistently with this, levels of 4-oxoretinoic acid, an ATRA metabolite and Cyp26a1 product, were also decreased in LRAT-overexpressing cells. CONCLUSION: Our results revealed a direct link between LRAT expression and regulation of ATRA levels indicating that the absence of LRAT-catalyzed retinol esterification is important for mediating retinoid sensitivity in murine melanoma cells. Thus, our data suggest that LRAT overexpression represents a novel mechanism by which tumor cells can escape high supplementary ATRA levels that mediate tumor-suppressive RAR signaling.

Survivin blockade sensitizes rhabdomyosarcoma cells for lysis by fetal acetylcholine receptor-redirected T cells.

Cellular immunotherapy may provide a strategy to overcome the poor prognosis of metastatic and recurrent rhabdomyosarcoma (RMS) under the current regimen of polychemotherapy. Because little is known about resistance mechanisms of RMS to cytotoxic T cells, we investigated RMS cell lines and biopsy specimens for expression and function of immune costimulatory receptors and anti-apoptotic molecules by RT-PCR, Western blot analysis, IHC, and cytotoxicity assays using siRNA or transfection-modified RMS cell lines, together with engineered RMS-directed cytotoxic T cells specific for the fetal acetylcholine receptor. We found that costimulatory CD80 and CD86 were consistently absent from all RMSs tested, whereas inducible T-cell co-stimulator ligand (ICOS-L; alias B7H2) was expressed by a subset of RMSs and was inducible by tumor necrosis factor α in two of five RMS cell lines. Anti-apoptotic survivin, along with other inhibitor of apoptosis (IAP) family members (cIAP1, cIAP2, and X-linked inhibitor of apoptosis protein), was overexpressed by RMS cell lines and biopsy specimens. Down-regulation of survivin by siRNA or pharmacologically in RMS cells increased their susceptibility toward a T-cell attack, whereas induction of ICOS-L did not. Treatment of RMS-bearing Rag(-/-) mice with fetal acetylcholine receptor-specific chimeric T cells delayed xenograft growth; however, this happened without definitive tumor eradication. Combined blockade of survivin and application of chimeric T cells in vivo suppressed tumor proliferation during survivin inhibition. In conclusion, survivin blockade provides a strategy to sensitize RMS cells for T-cell-based therapy.

Knockdown of lecithin retinol acyltransferase increases all-trans retinoic acid levels and restores retinoid sensitivity in malignant melanoma cells.

Retinoids such as all-trans retinoic acid (ATRA) influence cell growth, differentiation and apoptosis and may play decisive roles in tumor development and progression. An essential retinoid-metabolizing enzyme known as lecithin retinol acyltransferase (LRAT) is expressed in melanoma cells but not in melanocytes catalysing the esterification of all-trans retinol (ATRol). In this study, we show that a stable LRAT knockdown (KD) in the human melanoma cell line SkMel23 leads to significantly increased levels of the substrate ATRol and biologically active ATRA. LRAT KD restored cellular sensitivity to retinoids analysed in cell culture assays and melanoma 3D skin models. Furthermore, ATRA-induced gene regulatory mechanisms drive depletion of added ATRol in LRAT KD cells. PCR analysis revealed a significant upregulation of retinoid-regulated genes such as CYP26A1 and STRA6 in LRAT KD cells, suggesting their possible involvement in mediating retinoid resistance in melanoma cells. In conclusion, LRAT seems to be important for melanoma progression. We propose that reduction in ATRol levels in melanoma cells by LRAT leads to a disturbance in cellular retinoid level. Balanced LRAT expression and activity may provide protection against melanoma development and progression. Pharmacological inhibition of LRAT activity could be a promising strategy for overcoming retinoid insensitivity in human melanoma cells.

Lecithin retinol acyltransferase as a potential prognostic marker for malignant melanoma.

Metabolism inside cells differs between cancer and normal cells. Because disturbance of vitamin A metabolism might be important, we investigated expression of the enzymes lecithin retinol acyltransferase (LRAT) and RPE65 by immunohistochemistry in melanoma metastases and melanocytic nevi. Semiquantitative evaluation of this expression revealed downregulated expression of RPE65 in malignant melanoma compared with benign melanocytic nevi (P < 0.001). In contrast, expression of LRAT was not significantly different (P = 0.339). High LRAT expression in melanoma metastases was inversely correlated with patient survival; Kaplan-Meier analysis revealed earlier melanoma-related death (P = 0.003). Expression of LRAT might, therefore, be a prognostic marker of the clinical course of melanoma.

Novel microRNAs modulating ecto-5'-nucleotidase expression.

Introduction: The 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. Methods: In 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. Results: As 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. Discussion: The miRNAs identified might have clinical relevance as potential therapeutic agents and biomarkers or therapeutic targets, respectively.