Nyamdelger Sukhbaatar

Correlation of circular RNA abundance with proliferation--exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues.

Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as miRNA sponges, thus part of the competing endogenous RNA network. We analysed the expression of circular and linear RNAs and proliferation in matched normal colon mucosa and tumour tissues. We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of global circular RNA abundance (the circRNA index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells.

Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.

mTORC1 and mTORC2 as regulators of cell metabolism in immunity

The mechanistic target of rapamycin (mTOR) pathway is an evolutionarily conserved signaling pathway that senses intra‐ and extracellular nutrients, growth factors, and pathogen‐associated molecular patterns to regulate the function of innate and adaptive immune cell populations. In this review, we focus on the role of the mTOR complex 1 (mTORC1) and mTORC2 in the regulation of the cellular energy metabolism of these immune cells to regulate and support immune responses. In this regard, mTORC1 and mTORC2 generally promote an anabolic response by stimulating protein synthesis, glycolysis, mitochondrial functions, and lipid synthesis to influence proliferation and survival, effector and memory responses, innate training and tolerance as well as hematopoietic stem cell maintenance and differentiation. Deactivation of mTOR restores cell homeostasis after immune activation and optimizes antigen presentation and memory T‐cell generation. These findings show that the mTOR pathway integrates spatiotemporal information of the environmental and cellular energy status by regulating cellular metabolic responses to guide immune cell activation. Elucidation of the metabolic control mechanisms of immune responses will help to generate a systemic understanding of the immune system.

mTOR-Mediated Regulation of Dendritic Cell Differentiation and Function

Dendritic cells (DCs) are essential antigen-presenting cells that sample the extra- and intracellular milieu to process antigens for the instruction of T cell responses. The mammalian target of rapamycin (mTOR) network senses environmental cues and is important for numerous cellular processes. This review discusses how DCs use mTOR complexes (mTORC1 and 2) to adapt their cellular metabolism, transcriptional responses, and translation machinery to control DC development, antigen processing, cytokine production, and T cell stimulation. We present a spatiotemporal model suggesting that the mTOR network integrates pattern recognition and growth factor receptor activation with nutritional information from the cell and surrounding tissue to support T cell stimulation and tolerance. mTOR develops into a central player that regulates DC differentiation and immune functions.

Role of the immune system in the peritoneal tumor spread of high grade serous ovarian cancer

The immune system plays a critical role in cancer progression and overall survival. Still, it is unclear if differences in the immune response are associated with different patterns of tumor spread apparent in high grade serous ovarian cancer patients and previously described by us. In this study we aimed to assess the role of the immune system in miliary (widespread, millet-sized lesions) and non-miliary (bigger, exophytically growing implants) tumor spread. To achieve this we comprehensively analyzed tumor tissues, blood, and ascites from 41 patients using immunofluorescence, flow cytometry, RNA sequencing, multiplexed immunoassays, and immunohistochemistry. Results showed that inflammation markers were systemically higher in miliary. In contrast, in non-miliary lymphocyte and monocyte/macrophage infiltration into the ascites was higher as well as the levels of PD-1 expression in tumor associated cytotoxic T-lymphocytes and PD-L1 expression in tumor cells. Furthermore, in ascites of miliary patients more epithelial tumor cells were present compared to non-miliary, possibly due to the active down-regulation of anti-tumor responses by B-cells and regulatory T-cells. Summarizing, adaptive immune responses prevailed in patients with non-miliary spread, whereas in patients with miliary spread a higher involvement of the innate immune system was apparent while adaptive responses were counteracted by immune suppressive cells and factors.

Two different, mutually exclusively distributed, TP53 mutations in ovarian and peritoneal tumor tissues of a serous ovarian cancer patient: indicative for tumor origin?

High-grade serous ovarian cancer (HGSOC) is characterized by a TP53 mutation rate of up to 96.7% and associated with a more aggressive tumor biology. The origin of HGSOC is thought to arise either from fallopian tube secretory cells or the ovarian surface epithelium/inclusion cysts, the former with more evidence. Peritoneal tumor spread is heterogeneous, either excessive in the peritoneum (with miliary appearance) or more confined to the ovaries with only few (bigger and exophytically growing) peritoneal implants. Using RNA sequencing and DNA digital droplet polymerase chain reaction (PCR), we identified two different functional TP53 mutations in one HGSOC patient: one exclusively in the ovarian tumor mass and the other exclusively in ascites tumor cells, peritoneal tumor masses, and a lymph node metastasis. In blood, both mutations could be detected, the one from the peritoneal tumors with much higher frequency, presumably because of the higher tumor load. We conclude that this mutually exclusive distribution of two different TP53 mutations in different tumor tissues indicates the development of two independent carcinomas in the peritoneal cavity, probably one originating from a precancerous lesion in the fallopian tube and the other from the ovaries. In addition, in the patients ascites CD45 and EpCAM, double-positive cells were found—proliferating but testing negative for the above-mentioned TP53 mutations. This mutually exclusive distribution of two TP53 mutations is probably further evidence that HGSOC can originate either from the fallopian tube or (more seldom) the ovaries, the former more prone for excessive peritoneal tumor spread.

Abstract B55: Peritoneal tumor spread in high-grade serous ovarian cancer: Differences in the immune response.

Ovarian Cancer is the most lethal gynecological malignancy in the western world. Despite efforts to find biomarkers for early diagnosis, patients often present with advanced disease with widespread peritoneal tumor load and malignant ascites. Recently, we described two different modes of peritoneal tumor spread in high grade serous ovarian cancer patients (HGSOC): miliary, characterized by small, millet-like lesions, scattered all over the peritoneal surface and leading to a worse overall survival compared to non-miliary, defined by only few but bigger metastases in the peritoneum (Auer et al. Oncotarget (2015) 6:17261). Although we discovered differences in the tumor cell transcriptome between miliary and non-miliary, the impact of the immune system on the tumor development remained unclear. Therefore, we collected peripheral blood, ascites and tumor tissues from 41 HGSOC patients and analyzed them with respect to tumor spread types. The local and global immune system was assessed by flow cytometry. Activated NK cells and naive B-cells were significantly elevated in miliary blood. In ascites, reduced levels of cytotoxic T-cells and activated NKT cells as well as higher frequencies of NK cells and B-cells could be observed. As monocytes could not be assessed from cryopreserved specimen, CD14+ and/or CD16+ monocytes in ascites were measured by fluorescence staining and quantification. CD14+ monocytes (FDR 0.03) as well as the pro-inflammatory subset of CD14+CD16+ monocytes (trend) were enriched in non-miliary ascites. Transcriptomes of immune cells were assessed by RNA-sequencing of CD45 enriched floating ascites cells and digested primary and metastatic tumors. In ascites, 26 genes were significantly differentially expressed (FDR Cytokines were measured in serum and ascites with a multiplexed immunoassay. In accordance with elevated numbers of B-cells, the B-cell attractant CXCL13 was enriched in miliary ascites, as well as the anti-inflammatory cytokine IL-10. In non-miliary ascites prominent pro-angiogenic factors (FGFbasic, MIF, sVEGFR1), the hormone prolactin, and CCL25, shown to play a role in chemotaxis of thymocytes and ovarian carcinoma cells, were elevated significantly. Taking together these comprehensive results shed some light on the impact of the immune system on the tumor spread in HGSOC: patients with only few, big metastases (non-miliary type) show a more inflamed situation with higher levels of monocytes, cytotoxic T-cells, thymocyte attractants and pro-inflammatory cytokines in the peritoneal cavity as compared to the miliary type. It is conceivable that tumor cells that survive the specific milieu of the ascites (hypoxia, inflammation) or use other routes to metastasize (i.e. the haematolymphatic system) are capable of forming new lesions on the peritoneal surface only in limited numbers and these grow into big, vascularized tumors. On the other hand miliary is characterized by less inflammation in the peritoneal cavity, elevated levels of naive B-cells and IL-10 in the ascites and inhibited WNT and cell cycle pathways of immune cells. The tumor cells of this subtype probably adhere to the peritoneal wall more easily and grow into small nodules with impaired ability to induce neoangiogenesis. We conclude that these two types of tumor spread are not only driven by the tumor itself but rather by a complex interplay with the microenvironment, especially the local immune system. Citation Format: Katharina Auer, Anna Bachmayr-Heyda, Nyamdelger Sukhbaatar, Stefanie Aust, Christoph Grimm, Reinhard Horvat, Dietmar Pils. Peritoneal tumor spread in high-grade serous ovarian cancer: Differences in the immune response. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B55.

Abstract B56: Peritoneal tumor spread in high grade serous ovarian cancer: An effect of the competing endogenous RNA network?

Epithelial ovarian cancer (EOC) is still one of the deadliest malignancies in women frequently involving peritoneal tumor spread and ascites. Despite the heterogeneity of EOC, patients are mostly treated with standard therapy (cytoreductive surgery and platinum based chemotherapy). Understanding molecular mechanisms of EOC and its peritoneal metastasis is essential to develop new targeted therapies. In this study, we extended the transcriptome analysis of large RNA (Auer et al. Oncotarget 2015, 6:17261) to small RNA including micro RNAs (miRNAs) and piwi protein interacting RNAs (piRNAs). The transcriptome was analyzed concerning peritoneal tumor spread as introduced in our previous study: miliary – i.e. numerous small millet-like tumor seedings - versus non-miliary – i.e. big, bulky implants. Small RNA-sequencing (sRNA-seq) was performed with a total of 43 tumor cell enriched samples isolated from ascites and solid tumors from 23 chemo-naive high grade serous EOC (HGSOC) patients and analyzed together with corresponding RNA-seq data from our previous study. qPCR was applied for validation of sRNA-seq data (n=48). Expression differences were determined (I) between miliary and non-miliary tumor spread in (Ia) ascitic and (Ib) solid tumor cells and (II) between ascitic and solid tumor cells in (IIa) miliary and (IIb) non-miliary on three levels: single miRNAs, gene-miR sets, and general types of RNAs (especially those of the competing endogenous RNA network, ceRNA). sRNA-seq yielded evidence for expression of 649 known and 2,306 newly predicted miRNAs and 22,987 known and 783 newly predicted piRNAs. qPCR validation rate was 90% for 48 selected sRNAs. (Ia) 401 miRNAs, but no gene-miR set and (Ib) one miRNA, but >19,000 gene-miR sets were differentially expressed between miliary and non-miliary in ascitic and solid tumor cells, respectively. 72% of these deregulated gene-miR sets were characterized by down-regulated coding genes and up-regulated miRNAs. (IIa) 109 miRNAs and 49 gene-miR sets and (IIb) 55 miRNAs and no gene-miR set were differentially expressed between ascitic and solid tumor cells in miliary and non-miliary, respectively. These numbers indicate varying strategies for differential expression between miliary and non-miliary, one the one hand, and between e. g. ascitic and solid tumor cells, on the other hand: thousands of small changes in miRNA and gene expression versus few, but greater changes in individual miRNA and gene expression. Global analyses of coding, long non-coding (lncRNAs), circular (circRNAs) RNAs, and miRNAs revealed a down-regulated ceRNA network (i.e. less circRNAs and lncRNAs compared to coding RNAs), but an unchanged total amount of miRNAs. Given the sponge function of the ceRNA network, this indicates more free miRNAs available for regulation in miliary compared to non-miliary. In addition, a 13 sRNA signature was developed to predict the peritoneal tumor spread type from FFPE tissues. It was used for validation of the impact on overall survival (OS) in an independent cohort of 32 HGSOC patients. In accordance with our previous study, miliary tumor spread had a negative impact on OS independent of age and FIGO stage. We are the first to perform an integrative analysis of large and small RNA-seq data of ascitic and solid tumor cells from 23 HGSOC patients. We found evidence for hundreds of known mi- and piRNAs and thousands of newly predicted mi- and piRNAs. We could show a negative prognostic impact of the miliary tumor spread in a validation cohort. Solid tumors of the miliary type seem to be more influenced by miRNA regulation than solid tumors of the non-miliary type which implies different spread strategies. This fundamentally different mechanism of gene regulation and the different prognosis of these two spread types underline the need for alternative treatment regiments. Citation Format: Anna Bachmayr-Heyda, Katharina Auer, Nyamdelger Sukhbaatar, Stefanie Aust, Simon Deycmar, Agnes T. Reiner, Stephan Polterauer, Sabine Dekan, Dietmar Pils. Peritoneal tumor spread in high grade serous ovarian cancer: An effect of the competing endogenous RNA network? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B56.

Abstract A78: Serum metabolomics, cytokine measurements, and tumor RNA-seq identified phospholipids correlated with a molecular subclass as strong predictor for outcome in high-grade serous ovarian cancer.

Background: Currently, the field of ‘omics’ is still growing, giving step by step deeper insight into the biological system of cancer. Increasing evidence suggests that alterations in cancer metabolism might have a tremendous impact on survival and could guide new treatment strategies. Therefore, including metabolic phenotyping in a study design would allow better understanding of the biological function and status of tumor cells and their hosts. Epithelial ovarian cancer (EOC) is very heterogeneous in both, local tumor spread and survival. Molecular subtypes presenting with distinct whole-genome expression profiles and significant survival differences have been described and awareness of the molecular alterations is required to identify new targets for therapy. In this study, serum metabolite changes were identified and evaluated as potential prognostic markers in a test set of 65 high grade serous EOC (HGSOC) patients. The prognostic impact was validated in an independent cohort of 165 EOC patients. Methods: Targeted metabolomics was performed on serum using the AbsoluteIDQ p180 kit (Biocrates Life Sciences), measuring 188 metabolites. In addition to a test and validation set of 65 HGSOC and 165 serous advanced stage EOC patients, the metabolomics profile of 62 healthy women was determined. In a subset of patients 16 cancer biomarkers and 40 chemokines were measured using Luminex bead-based assays and whole genome transcriptomes were assessed from different tumor tissues (primary, metastatic, and ascitic) by RNA sequencing. Results: 53 metabolites were significantly negatively correlated to overall survival (OS). Thereof, the majority (43) constituted glycerophospholipids (GPhL), followed by five acylcarnitines, four sphingolipids and one biogenic amine. A non-linear dimensionality reduction approach was used to reduce the information of the 43 GPhLs to three dimensions. The first showed the strongest impact on OS and revealed, as GPhL predictor, a significant impact on OS in uni- and multivariate (HR 0.38; p To provide more profound survival analysis, a validation was performed using expression data initially used for validation of a molecular subclassification with a tremendous impact on OS in 165 serous advanced stage EOC patients (Pils et al. Cancer Sci. 103:1334). Using the 50 most significantly up- and the 50 down-regulated genes a robust predictor was built. This gene predictor represents the GPhL-predictor and had a significant independent impact on OS (HR 0.66; p=0.004). In addition to this validation of the survival impact, a very strong correlation between the observed GPhL changes (via the gene predictor) and the molecular subclass was found, indicating a functional connection between these molecular subclasses and the serum lipidome. Conclusion: Targetd metabolomics revealed a strong independent impact of the serum concentrations of glycerophospholipids on HGSOC outcome in the test and a validation cohort. Transcriptomics data indicate changes in the energy metabolism of the tumor. Interestingly, the lipidome-changes were strongly correlated to a previously described molecular subclass in serous EOC. Citation Format: Stefanie Aust, Anna Bachmayr-Heyda, Katharina Auer, Nyamdelger Sukhbaatar, Samuel M. Meier, Christopher Gerner, Dietmar Pils. Serum metabolomics, cytokine measurements, and tumor RNA-seq identified phospholipids correlated with a molecular subclass as strong predictor for outcome in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A78.