Correlation of the tumor escape phenotype with loss of PRELP expression in melanoma.

BACKGROUND: Despite immunotherapies having revolutionized the treatment of advanced cutaneous melanoma, effective and durable responses were only reported in a few patients. A better understanding of the interaction of melanoma cells with the microenvironment, including extracellular matrix (ECM) components, might provide novel therapeutic options. Although the ECM has been linked to several hallmarks of cancer, little information is available regarding the expression and function of the ECM protein purine-arginine-rich and leucine-rich protein (PRELP) in cancer, including melanoma. METHODS: The structural integrity, expression and function of PRELP, its correlation with the expression of immune modulatory molecules, immune cell infiltration and clinical parameters were determined using standard methods and/or bioinformatics. RESULTS: Bioinformatics analysis revealed a heterogeneous, but statistically significant reduced PRELP expression in available datasets of skin cutaneous melanoma when compared to adjacent normal tissues, which was associated with reduced patients' survival, low expression levels of components of the MHC class I antigen processing machinery (APM) and interferon (IFN)-γ signal transduction pathway, but increased expression of the transforming growth factor (TGF)-ß isoform 1 (TFGB1) and TGF-ß receptor 1 (TGFBR1). In addition, a high frequency of intra-tumoral T cells directly correlated with the expression of MHC class I and PRELP as well as the T cell attractant CCL5 in melanoma lesions. Marginal to low PRELP expression levels were found in the 47/49 human melanoma cell lines analysis. Transfection of PRELP into melanoma cell lines restored MHC class I surface expression due to transcriptional upregulation of major MHC class I APM and IFN-γ pathway components. In addition, PRELP overexpression is accompanied by high CCL5 secretion levels in cell supernatant, an impaired TGF-ß signaling as well as a reduced cell proliferation, migration and invasion of melanoma cells. CONCLUSIONS: Our findings suggest that PRELP induces the expression of MHC class I and CCL5 in melanoma, which might be involved in an enhanced T cell recruitment and immunogenicity associated with an improved patients' outcome. Therefore, PRELP might serve as a marker for predicting disease progression and its recovery could revert the tumorigenic phenotype, which represents a novel therapeutic option for melanoma.

Biglycan as a mediator of proinflammatory response and target for MDS and sAML therapy.

Myelodysplastic syndromes (MDS) and their progression to secondary acute myeloid leukemia (sAML) are associated with an altered protein expression including extracellular matrix (ECM) components thereby promoting an inflammatory environment. Since the role of the proteoglycan biglycan (BGN) as an inflammatory mediator has not yet been investigated in both diseases and might play a role in disease progression, its expression and/or function was determined in cell lines and bone marrow biopsies (BMBs) of MDS and sAML patients and subpopulations of MDS stem cells by Western blot and immunohistochemistry. The bone marrow (BM) microenvironment was analyzed by multispectral imaging, patients' survival by Cox regression. ROC curves were assessed for diagnostic value of BGN. All cell lines showed a strong BGN surface expression in contrast to only marginal expression levels in mononuclear cells and CD34+ cells from healthy donors. In the MDS-L cell line, CD34-CD33+ and CD34+CD33+ blast subpopulations exhibited a differential BGN surface detection. Increased BGN mediated inflammasome activity of CD34-CD33+TLR4+ cells was observed, which was inhibited by direct targeting of BGN or NLRP3. BGN was heterogeneously expressed in BMBs of MDS and sAML, but was not detected in control biopsies. BGN expression in BMBs positively correlated with MUM1+ and CD8+, but negatively with CD33+TLR4+ cell infiltration and was accompanied by a decreased progression-free survival of MDS patients. BGN-mediated inflammasome activation appears to be a crucial mechanism in MDS pathogenesis implicating its use as suitable biomarker and potential therapeutic target. Abbreviations: Ab, antibody; alloSCT, allogenic stem cell transplant; AML, acute myeloid leukemia; BGN, biglycan; BM, bone marrow; BMB, bone marrow biopsy; casp1, caspase 1; CTLA-4, cytotoxic T lymphocyte-associated protein 4; DAMP, danger-associated molecular pattern; ECM, extracellular matrix; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HD, healthy donor; HSPC, hematopoietic stem and progenitor cell; HSC, hematopoietic stem cell; IFN, interferon; IHC, immunohistochemistry; IL, interleukin; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; MSI, multispectral imaging; NGS, next-generation sequencing; NLRP3, NLR family pyrin domain containing 3; OS, overall survival; PBMC, peripheral blood mononuclear cell; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1, PFS, progression-free survival; PRR, pattern recognition receptor; SC, stem cell; SLRP, small leucine-rich proteoglycan; TGF, transforming growth factor; TIRAP, toll/interleukin 1 receptor domain-containing adapter protein; TLR, toll-like receptor; Treg, regulatory T cell.

Biglycan as a potential regulator of tumorgenicity and immunogenicity in K-RAS-transformed cells.

The extracellular matrix component biglycan (BGN) plays an essential role in various physiological and pathophysiological processes. A deficient BGN expression associated with reduced immunogenicity was found in HER-2/neu-overexpressing cells. To determine whether BGN is suppressed by oncogene-driven regulatory networks, the expression and function of BGN was analyzed in murine and human BGNlow/BGNhigh K-RASG12V-transformed model systems as well as in different patients' datasets of colorectal carcinoma (CRC) lesions. K-RAS-mutated CRC tissues expressed low BGN mRNA and protein levels when compared to normal colon epithelial cells, which was associated with a reduced patients' survival. Transfection of BGN in murine and human BGNlow K-RAS-expressing cells resulted in a reduced growth and migration of BGNhigh vs BGNlow K-RAS cells. In addition, increased MHC class I surface antigens as a consequence of an enhanced antigen processing machinery component expression was found upon restoration of BGN, which was confirmed by RNA-sequencing of BGNlow vs. BGNhigh K-RAS models. Furthermore, a reduced tumor formation of BGNhigh versus BGNlow K-RAS-transformed fibroblasts associated with an enhanced MHC class I expression and an increased frequency of tumor-infiltrating lymphocytes in tumor lesions was found. Our data provide for the first time an inverse link between BGN and K-RAS expression in murine and human K-RAS-overexpressing models and CRC lesions associated with altered growth properties, reduced immunogenicity and worse patients' outcome. Therefore, reversion of BGN might be a novel therapeutic option for K-RAS-associated malignancies.

Immune Interaction Map of Human SARS-CoV-2 Target Genes: Implications for Therapeutic Avenues.

There exists increasing evidence that people with preceding medical conditions, such as diabetes and cancer, have a higher risk of infection with SARS-CoV-2 and are more vulnerable to severe disease. To get insights into the possible role of the immune system upon COVID-19 infection, 2811 genes of the gene ontology term "immune system process GO: 0002376" were selected for coexpression analysis of the human targets of SARS-CoV-2 (HT-SARS-CoV-2) ACE2, TMPRSS2, and FURIN in tissue samples from patients with cancer and diabetes mellitus. The network between HT-SARS-CoV-2 and immune system process genes was analyzed based on functional protein associations using STRING. In addition, STITCH was employed to determine druggable targets. DPP4 was the only immune system process gene, which was coexpressed with the three HT-SARS-CoV-2 genes, while eight other immune genes were at least coexpressed with two HT-SARS-CoV-2 genes. STRING analysis between immune and HT-SARS-CoV-2 genes plotted 19 associations of which there were eight common networking genes in mixed healthy (323) and pan-cancer (11003) tissues in addition to normal (87), cancer (90), and diabetic (128) pancreatic tissues. Using this approach, three commonly applicable druggable connections between HT-SARS-CoV-2 and immune system process genes were identified. These include positive associations of ACE2-DPP4 and TMPRSS2-SRC as well as a negative association of FURIN with ADAM17. Furthermore, 16 drugs were extracted from STITCH (score <0.8) with 32 target genes. Thus, an immunological network associated with HT-SARS-CoV-2 using bioinformatics tools was identified leading to novel therapeutic opportunities for COVID-19.

Expression and Clinical Significance of SARS-CoV-2 Human Targets in Neoplastic and Non-Neoplastic Lung Tissues.

BACKGROUND: A higher incidence of COVID-19 infection was demonstrated in cancer patients, including lung cancer patients. This study was conducted to get insights into the enhanced frequency of COVID-19 infection in cancer. METHODS: Using different bioinformatics tools, the expression and methylation patterns of ACE2 and TMPRSS2 were analyzed in healthy and malignant tissues, focusing on lung adenocarcinoma and data were correlated to clinical parameters and smoking history. RESULTS: ACE2 and TMPRSS2 were heterogeneously expressed across 36 healthy tissues with the highest expression levels in digestive, urinary and reproductive organs, while the overall analysis of 72 paired tissues demonstrated significantly lower expression levels of ACE2 in cancer tissues when compared to normal counterparts. In contrast, ACE2, but not TMPRSS2, was overexpressed in LUAD, which inversely correlated to the promoter methylation. This upregulation of ACE2 was age-dependent in LUAD, but not in normal lung tissues. TMPRSS2 expression in non-neoplastic lung tissues was heterogeneous and dependent on sex and smoking history, while it was downregulated in LUAD of smokers. Cancer progression was associated with a decreased TMPRSS2 but unaltered ACE2. In contrast, ACE2 and TMPRSS2 of lung metastases derived from different cancer subtypes was higher than organ metastases of other sites. TMPRSS2, but not ACE2, was associated with LUAD patients' survival. CONCLUSIONS: Comprehensive molecular analyses revealed a heterogeneous and distinct expression and/or methylation profile of ACE2 and TMPRSS2 in healthy lung vs. LUAD tissues across sex, age and smoking history and might have implications for COVID-19 disease.

Identification of microRNAs Targeting the Transporter Associated with Antigen Processing TAP1 in Melanoma.

The underlying molecular mechanisms of the aberrant expression of components of the HLA class I antigen processing and presentation machinery (APM) in tumors leading to evasion from T cell-mediated immune surveillance could be due to posttranscriptional regulation mediated by microRNAs (miRs). So far, some miRs controlling the expression of different APM components have been identified. Using in silico analysis and an miR enrichment protocol in combination with small RNA sequencing, miR-26b-5p and miR-21-3p were postulated to target the 3' untranslated region (UTR) of the peptide transporter TAP1, which was confirmed by high free binding energy and dual luciferase reporter assays. Overexpression of miR-26b-5p and miR-21-3p in melanoma cells downregulated the TAP1 protein and reduced expression of HLA class I cell surface antigens, which could be reverted by miR inhibitors. Moreover, miR-26b-5p overexpression induced a decreased T cell recognition. Furthermore, an inverse expression of miR-26b-5p and miR-21-3p with TAP1 was found in primary melanoma lesions, which was linked with the frequency of CD8+ T cell infiltration. Thus, miR-26-5p and miR-21-3p are involved in the HLA class I-mediated immune escape and might be used as biomarkers or therapeutic targets for HLA class Ilow melanoma cells.

Tumor-induced escape mechanisms and their association with resistance to checkpoint inhibitor therapy.

Immunotherapy aims to activate the immune system to fight cancer in a very specific and targeted manner. Despite the success of different immunotherapeutic strategies, in particular antibodies directed against checkpoints as well as adoptive T-cell therapy, the response of patients is limited in different types of cancers. This attributes to escape of the tumor from immune surveillance and development of acquired resistances during therapy. In this review, the different evasion and resistance mechanisms that limit the efficacy of immunotherapies targeting tumor-associated antigens presented by major histocompatibility complex molecules on the surface of the malignant cells are summarized. Overcoming these escape mechanisms is a great challenge, but might lead to a better clinical outcome of patients and is therefore currently a major focus of research.

Tumor-dependent Effects of Proteoglycans and Various Glycosaminoglycan Synthesizing Enzymes and Sulfotransferases on Patients' Outcome.

BACKGROUND: The small leucine-rich proteoglycans (SLRPs) biglycan (BGN) and decorin (DCN) linked with sulfated glycosaminoglycan (GAG) chains exhibit oncogenic or tumor suppressive potentials depending on the cellular context and association with GAGs. OBJECTIVE: We hypothesized that structural alterations and expression levels of BGN, DCN and their associated chondroitin sulfate (CS) polymerizing enzymes, dermatan sulfate (DS) epimerases and various sulfatases might be correlated with the tumor (sub)type and patients' survival. METHODS: We acquired breast cancer (BC) and glioma patients' datasets from cBioPortal and R2 Genomics. Structural alterations and the expression pattern of CS polymerizing enzymes, DS epimerases and carbohydrate sulfotransferases (CHST) were compared to that of BGN and DCN and correlated to their clinical relevance. RESULTS: In BC, no mutations, but amplifications (0.2 - 2.1 %) and deletions (0.05 - 0.4 %) were found in BGN, DCN and CS/DS enzymes. In contrast, missense and/or truncated mutations (0.1 - 0.5 %), but a reduced amplification rate (0 - 1.5 %) were found in glioma. When compared to BC, the structural abnormalities caused altered mRNA expression levels of BGN, DCN, GAG synthesizing enzymes and CHST. Mutations in SLPRs, CHSY1, CHST4 and CHSY3 were correlated with a poor prognosis in glioma, while lack of mutations and copy number variations in the SLRPs, CHSY3, CHST15 and DSE displayed an increased survival in BC. CONCLUSION: A distinct association of BGN and DCN with CHST, CS polymerizing enzymes and DS epimerases was found in BC and glioma. Thus, a unique pattern of structural alterations and expression, which has clinical relevance, was found for PGs and GAG synthesizing enzymes and CHST in BC and glioma, which might help to identify high-risk patients and to develop personalized therapeutics.

Biglycan-mediated upregulation of MHC class I expression in HER-2/neu-transformed cells.

The extracellular matrix protein biglycan (BGN) has oncogenic or tumor suppressive potential depending on the cellular origin. HER-2/neu overexpression in murine fibroblasts and human model systems is inversely correlated with BGN expression. Upon its restoration BGNhigh HER-2/neu+ fibroblasts were less tumorigenic in immune competent mice when compared to BGNlow/neg HER-2/neu+ cells, which was associated with enhanced immune cell responses and higher frequencies of immune effector cells in tumors and peripheral blood. The increased immunogenicity of BGNhigh HER-2/neu+ fibroblasts appears to be due to upregulated MHC class I surface antigens and reduced expression levels of transforming growth factor (TGF)-ß isoforms and the TGF-ß receptor 1 suggesting a link between BGN, TGF-ß pathway and HER-2/neu-mediated downregulation of MHC class I antigens. Treatment of BGNlow/neg HER-2/neu+ cells with recombinant BGN or an inhibitor of TGF-ß enhanced MHC class I surface antigens in BGNlow/neg HER-2/neu-overexpressing murine fibroblasts, which was mediated by a transcriptional upregulation of major MHC class I antigen processing components. Furthermore, BGN expression in HER-2/neu+ cells was accompanied by an increased expression of the proteoglycan decorin (DCN). Since recombinant DCN also elevated MHC class I surface expression in BGNlow/neg HER-2/neu+ cells, both proteoglycans might act synergistically. This was in accordance with in silico analyses of mRNA data obtained from The Cancer Genome Atlas (TCGA) dataset available for breast cancer (BC) patients. Thus, our data provide for the first time evidence that proteoglycan signatures are modulated by HER-2/neu and linked to MHC class I-mediated immune escape associated with an altered TGF-ß pathway.

Influence of oxygen deficiency and the role of specific amino acids in cryopreservation of garlic shoot tips.

BACKGROUND: Garlic has lost its ability to form seeds in the course of its domestication. Therefore, the germplasm storage via cryopreservation is increasingly applied. The progression of the various steps within the cryopreservation procedure is accompanied by declining survival rates of the explants. Much of the recent work on cryo-stress has been focussed on osmotic and cold stress components. However, two decades after invention of garlic cryopreservation, the function of metabolites and oxygen in and around the cryopreserved tissues is still largely obscure. METHODS: In this study, hypoxia was characterized in cryopreservation of garlic with oxygen sensors and amino acid metabolism. Furthermore, malondialdehyde, soluble sugars and ammonium were quantified to demonstrate the influence of cryo-stress in declining survival rates. RESULTS: To better understand the possible reasons for a reduction in the survival rate at the subsequent steps of cryopreservation, the concentration of amino acids, ammonium, γ-aminobutyric acid (GABA), soluble sugars, malondialdehyde (MDA), and oxygen were measured in garlic shoot tips undergoing cryopreservation. Using microsensors, a very low oxygen concentration (<0.1 µM) was detected within the central meristem region of the shoot apex. When apices were immersed in cryoprotectant solution, the well-oxygenated peripheral regions (foliage leaf bases) became likewise hypoxic within a few minutes, probably resulting from strongly restricted gaseous diffusion. CONCLUSIONS: Tissue level oxygen measurements supported the occurrence of hypoxia while biochemical analysis indicated adaptive responses, in particular the modulation in alanine and glutamate metabolism. The possible role of serine and glycine metabolism during cryopreservation is also discussed.