Upregulation and epigenetic modification of the creatine transporter SLC6A8 in non-small cell lung cancer.

INTRODUCTION: Lung cancer is a major cause of cancer-related death worldwide and effective therapies, besides surgery, are available only for a small proportion of patients. Since cellular respiration is known to be broadly altered in malignant tumors, the cellular processes of respiration can be a potential therapeutic target. One important element of cellular respiration is creatine and its transport by the creatine transporter SLC6A8. Here we describe the expression of SLC6A8 at the RNA and protein level, epigenetic modifications as well as survival analysis in NSCLC tissues and matched controls. MATERIALS AND METHODS: We analyzed epigenetic modifications of the SLC68A gene in 32 patients, of which 18 were additionally analyzed by transcriptome analysis. The expression of SLC6A8 at the protein level was assessed by immunohistochemistry using an independent cohort and correlated with clinicopathological data including survival. Kaplan-Meier analysis was performed to analyze the possible effects of the transcriptional levels of SLC6A8 in another separate cohort (n=1925). RESULTS: SLC6A8 loci are epigenetically modified in NSCLC compared with tumor-free controls. SLC6A8 is upregulated in NSCLC at the RNA and protein level. High mRNA expression of SLC6A8 was associated with an overall poor prognosis in lung adenocarcinoma patients and displayed the strongest adverse prognostic effect in male smokers with adenocarcinomas. Results of transcriptome analysis were partially confirmed at the protein level. CONCLUSIONS: Our results suggest an important role of creatine and its transport via SLC6A8 in NSCLC.

The Gene Expression Landscape of Prostate Cancer BM Reveals Close Interaction with the Bone Microenvironment.

Bone metastatic (BM) prostate cancer (PCa) belongs to the most lethal form of PCa, and therapeutic options are limited. Molecular profiling of metastases contributes to the understanding of mechanisms defining the bone metastatic niche. Our aim was to explore the transcriptional profile of PCa BM and to identify genes that drive progression. Paraffin-embedded tissues of 28 primary PCa and 30 BM were submitted to RNA extraction and analyzed by RNA sequencing using the Nanostring nCounter gene expression platform. A total of 770 cancer-related genes were measured using the Nanostring™ PanCancer progression panel. Gene Ontology (GO), KEGG, Reactome, STRING, Metascape, PANTHER, and Pubmed were used for data integration and gene annotation. We identified 116 differentially expressed genes (DEG) in BM compared to primaries. The most significant DEGs include CD36, FOXC2, CHAD, SPP1, MMPs, IBSP, and PTX3, which are more highly expressed in BM, and ACTG2, MYH11, CNN1, FGF2, SPOCK3, and CHRDL1, which have a lower expression. DEGs functionally relate to extracellular matrix (ECM) proteoglycans, ECM-receptors, cell-substrate adhesion, cell motility as well as receptor tyrosine kinase signaling and response to growth factors. Data integration and gene annotation of 116 DEGs were used to build a gene platform which we termed "Manually Annotated and Curated Nanostring-data Platform". In summary, our results highlight the significance of certain genes in PCa BM to which essential pro-metastatic functions could be ascribed. Data from this study provide a comprehensive platform of genes that are related to PCa BM and provide evidence for further investigations.

Analysis of tripartite motif (TRIM) family gene expression in prostate cancer bone metastases.

Tripartite motif (TRIM) family proteins are post-translational protein modifiers with E3-ubiquitin ligase activity, thereby involved in various biological processes. The molecular mechanisms driving prostate cancer (PCa) bone metastasis (BM) are incompletely understood, and targetable genetic alterations are lacking in the majority of cases. Therefore, we aimed to explore the expression and potential functional relevance of 71 TRIM members in bone metastatic PCa. We performed transcriptome analysis of all human TRIM family members and 770 cancer-related genes in 29 localized PCa and 30 PCa BM using Nanostring. KEGG, STRING and Ubibrowser were used for further bioinformatic gene correlation and pathway enrichment analyses. Compared to localized tumors, six TRIMs are under-expressed while nine TRIMs are over-expressed in BM. The differentially expressed TRIM proteins are linked to TNF-, TGFß-, PI3K/AKT- and HIF-1-signaling, and to features such as proteoglycans, platelet activation, adhesion and ECM-interaction based on correlation to cancer-related genes. The identification of TRIM-specific E3-ligase-substrates revealed insight into functional connections to oncogenes, tumor suppressors and cancer-related pathways including androgen receptor- and TGFß signaling, cell cycle regulation and splicing. In summary, this is the first study that comprehensively and systematically characterizes the expression of all TRIM members in PCa BM. Our results describe post-translational protein modification as an important regulatory mechanism of oncogenes, tumor suppressors, and pathway molecules in PCa progression. Therefore, this study may provide evidence for novel therapeutic targets, in particular for the treatment or prevention of BM.

Cracking it - successful mRNA extraction for digital gene expression analysis from decalcified, formalin-fixed and paraffin-embedded bone tissue.

With the advance of precision medicine, the availability of tumor tissue for molecular analysis has become a limiting factor. This is particularly the case for bone metastases which are frequently occurring in cancer types such as prostate cancer. Due to the necessary decalcification process it was long thought that transcriptome analysis will not be feasible from decalcified formalin-fixed, paraffin-embedded (DFFPE) in a large manner. Here we demonstrate that mRNA extraction from DFFPE is feasible, quick, robust and reproducible and that decalcification does not hamper subsequent gene expression analysis. This might assist in implementing transcriptome analysis from DFFPE into every day practice.

Performance of Different Diagnostic PD-L1 Clones in Head and Neck Squamous Cell Carcinoma.

Background: The approval of immune checkpoint inhibitors in combination with specific diagnostic biomarkers presents new challenges to pathologists as tumor tissue needs to be tested for expression of programmed death-ligand 1 (PD-L1) for a variety of indications. As there is currently no requirement to use companion diagnostic assays for PD-L1 testing in Germany different clones are used in daily routine. While the correlation of staining results has been tested in various entities, there is no data for head and neck squamous cell carcinomas (HNSCC) so far. Methods: We tested five different PD-L1 clones (SP263, SP142, E1L3N, 22-8, 22C3) on primary HNSCC tumor tissue of 75 patients in the form of tissue microarrays. Stainings of both immune and tumor cells were then assessed and quantified by pathologists to simulate real-world routine diagnostics. The results were analyzed descriptively and the resulting staining pattern across patients was further investigated by principal component analysis and non-negative matrix factorization clustering. Results: Percentages of positive immune and tumor cells varied greatly. Both the resulting combined positive score as well as the eligibility for certain checkpoint inhibitor regimens was therefore strongly dependent on the choice of the antibody. No relevant co-clustering and low similarity of relative staining patterns across patients was found for the different antibodies. Conclusions: Performance of different diagnostic anti PD-L1 antibody clones in HNSCC is less robust and interchangeable compared to reported data from other tumor entities. Determination of PD-L1 expression is critical for therapeutic decision making and may be aided by back-to-back testing of different PD-L1 clones.

Immunologic "Cold" Squamous Cell Carcinomas of the Head and Neck Are Associated With an Unfavorable Prognosis.

Background: Head and neck squamous cell carcinoma (HNSCC) represents a common cancer worldwide. Past therapeutic advances have not significantly improved HNSCC prognosis. Therefore, it is necessary to further stratify HNSCC, especially with recent advances in tumor immunology. Methods: Tissue microarrays were assembled from tumor tissue samples and were complemented with comprehensive clinicopathological data of n = 419 patients. H&E whole slides from resection specimen (n = 289) were categorized according to their immune cell infiltrate as "hot," "cold," or "excluded." Results: Investigating tumor immune cell patterns, we found significant differences in survival rates. Immunologic "hot" and "excluded" HNSCCs are associated with better overall survival than "cold" HNSCC patients (p < 0.05). Interestingly, the percentage of all three patterns is nearly identical in p16 positive and negative HNSCCs. Conclusions: Using a plain histological H&E approach to categorize HNSCC as being immunologic "hot," "cold," or "excluded" can offer a forecast of patients' prognosis and may thus aid as a potential prognostic tool in routine pathology reports. This "hot-cold-excluded" scheme needs to be applied to more HNSCC cohorts and possibly to other cancer types to determine prognostic meaning, e.g., regarding OS or DFS. Furthermore, our cohort reflects epidemiological data in the national, European, and international context. It may, therefore, be of use for future HNSCC characterization.

Recurrent HNSCC Harbor an Immunosuppressive Tumor Immune Microenvironment Suggesting Successful Tumor Immune Evasion.

PURPOSE: Recurrent tumors (RT) of head and neck squamous cell carcinoma (HNSCC) occur in up to 60%, with poor therapeutic response and detrimental prognosis. We hypothesized that HNSCC RTs successfully evade antitumor immune response and aimed to reveal tumor immune microenvironment (TIME) changes of primary tumors (PT) and corresponding RTs. EXPERIMENTAL DESIGN: Tumor-infiltrating leukocytes (TIL) of 300 PTs and 108 RTs from two large independent and clinically well-characterized HNSCC cohorts [discovery cohort (DC), validation cohort (VD)] were compared by IHC. mRNA expression analysis of 730 immune-related genes was performed for 18 PTs and RTs after adjuvant chemoradiotherapy (CRT). The effect of chemotherapy and radiation resistance was assessed with an in vitro spheroid/immunocyte coculture model. RESULTS: TIME analysis revealed overall decrease of TILs with significant loss of CD8+ T cells (DC P = 0.045/VC P < 0.0001) and B lymphocytes (DC P = 0.036/VC P < 0.0001) in RTs compared with PTs in both cohorts. Decrease predominantly occurred in RTs after CRT. Gene expression analysis confirmed loss of TILs (P = 0.0004) and B lymphocytes (P < 0.0001) and showed relative increase of neutrophils (P = 0.018), macrophages (P < 0.0001), dendritic cells (P = 0.0002), and mast cells (P = 0.0057) as well as lower overall expression of immune-related genes (P = 0.018) in RTs after CRT. Genes involved in B-lymphocyte functions and number of tertiary lymphoid structures showed the strongest decrease. SPP1 and MAPK1 were upregulated in vivo and in vitro, indicating their potential suitability as therapeutic targets in CRT resistance. CONCLUSIONS: HNSCC RTs have an immunosuppressive TIME, which is particularly apparent after adjuvant CRT and might substantially contribute to poor therapeutic response and prognosis.

In silico analysis reveals EP300 as a panCancer inhibitor of anti-tumor immune response via metabolic modulation.

The tumor immune microenvironment (TIME) of head and neck squamous cell carcinomas (HNSCC) and other solid malignancies is a key determinant of therapy response and prognosis. Among other factors, it is shaped by the tumor mutational burden and defects in DNA repair enzymes. Based on the TCGA database we aimed to define specific, altered genes associated with different TIME types, which might represent new predictive markers or targets for immuno-therapeutic approaches. The HNSCC cohort of the TCGA database was used to define 3 TIME types (immune-activated, immune-suppressed, immune-absent) according to expression of immune-related genes. Mutation frequencies were correlated to the 3 TIME types. Overall survival was best in the immune-activated group. 9 genes were significantly differentially mutated in the 3 TIME types with strongest differences for TP53 and the histone-acetyltransferase EP300. Mutations in EP300 correlated with an immune-activated TIME. In panCancer analyses anti-tumor immune activity was increased in EP300 mutated esophageal, stomach and prostate cancers. Downregulation of EP300 gene expression was associated with higher anti-tumor immunity in most solid malignancies. Since EP300 is a promoter of glycolysis, which negatively affects anti-tumor immune response, we analyzed the association of EP300 with tumor metabolism. PanCancer tumor metabolism was strongly shifted towards oxidative phosphorylation in EP300 downregulated tumors. In silico analyses of of publicly available in vitro data showed a decrease of glycolysis-associated genes after treatment with the EP300 inhibitor C646. Our study reveals associations of specific gene alterations with different TIME types. In detail, we defined EP300 as a panCancer inhibitor of the TIME most likely via metabolic modulation. In this context EP300 represents a promising predictive biomarker and an immuno-therapeutic target.