Katharina Auer

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.

Cyclin E1 (CCNE1) as independent positive prognostic factor in advanced stage serous ovarian cancer patients - A study of the OVCAD consortium

Cyclin E, coded by the genes CCNE1 and CCNE2, is the main regulator for transition from G1 to S phase determining cell division. CCNE1 and CCNE2 are known oncogenes in many cancer entities. Especially CCNE1 has frequently been associated with gene amplifications in various malignancies, emphasising its role as a putative oncogene. We determined gene expression and copy number of CCNE1 and CCNE2 by quantitative polymerase chain reaction (PCR) from 172 International Federation of Obstetrics and Gynecology (FIGO) II/III/IV stage serous epithelial ovarian cancer (EOC) tissues and analysed its impact on outcome. Furthermore, whole transcriptome gene expression changes correlating with CCNE1 expression were determined by microarray technology, interpreted by Signalling Pathway Impact Analysis (SPIA), Tool for Inferring Network of Genes (TINGe), and illustrated by hive plots. Protein-protein interaction (PPI) networks were also used for the interpretation. Interestingly, and contradictory to most reports and intuitive expectations, high CCNE1 expression correlated with better overall survival (p=0.005) if corrected for usual clinicopathologic parameters and a molecular subclassification. Using different grading systems or only high graded tumours had no impact on this correlation. Copy number of CCNE1 was increased in 25% of cases which correlated highly significantly with expression but showed no impact on outcome. CCNE2 had no impact on outcomes at all. Whole genome transcriptome analysis revealed 1872 differentially expressed genes correlated to CCNE1 expression, which were significantly enriched with genes from five pathways (e.g. cell cycle and viral carcinogenesis pathway were up-regulated and the Fanconi anaemia pathway was down-regulated). High CCNE1 gene expression is a significant and independent predictor for prolonged overall survival in FIGO III/IV EOC patients. This upside down impact of CCNE1 on survival probably reflects the special characteristic of EOC with tumour dissemination in the near anaerobic peritoneal cavity as the predominant cause of death, compared to other cancer entities where distant metastasis are predominantly lethal.

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.

Ambivalent role of pFAK-Y397 in serous ovarian cancer-a study of the OVCAD consortium

BackgroundFocal adhesion kinase (FAK) autophosphorylation seems to be a potential therapeutic target but little is known about the role and prognostic value of FAK and pFAK in epithelial ovarian cancer (EOC). Recently, we validated a gene signature classifying EOC patients into two subclasses and revealing genes of the focal adhesion pathway as significantly deregulated.MethodsFAK expression and pFAK-Y397 abundance were elucidated by immunohistochemistry and microarray analysis in 179 serous EOC patients. In particular the prognostic value of phosphorylated FAK (pFAK-Y397) and FAK in advanced stage EOC was investigated.ResultsMultiple Cox-regression analysis showed that high pFAK abundance was associated with improved overall survival (HR 0.54; p = 0.034). FAK was positive in a total of 92.2% (n = 165) and high pFAK abundance was found in 36.9% (n = 66). High pFAK abundance (36.9% ; n = 66) was associated with either nodal positivity and/or distant metastasis (p = 0.030). Whole genome gene expression data revealed a connection of the FAK-pFAK-Y397 axis and the mTOR-S6K1 pathway, shown to play a major role in carcinogenesis.ConclusionThe role of pFAK-Y397 remains controversial: although high pFAK-Y397 abundance is associated with distant and lymph node metastases, it is independently associated with improved overall survival.

Integrative Systemic and Local Metabolomics with Impact on Survival in High Grade Serous Ovarian Cancer.

Purpose: Cancer metabolism is characterized by alterations including aerobic glycolysis, oxidative phosphorylation, and need of fuels and building blocks. Experimental Design: Targeted metabolomics of preoperative and follow-up sera, ascites, and tumor tissues, RNA sequencing of isolated tumor cells, local and systemic chemokine, and local immune cell infiltration data from up to 65 high-grade serous ovarian cancer patients and 62 healthy controls were correlated to overall survival and integrated in a Systems Medicine manner. Results: Forty-three mainly (poly)unsaturated glycerophospholipids and four essential amino acids (citrulline) were significantly reduced in patients with short compared with long survival and healthy controls. The glycerophospholipid fingerprint is identical to the fingerprint from isolated (very) low-density lipoproteins (vLDL), indicating that the source of glycerophospholipids consumed by tumors is (v)LDL. A glycerophospholipid-score (HR, 0.46; P = 0.007) and a 100-gene signature (HR, 0.65; P = 0.004) confirmed the independent impact on survival in training (n = 65) and validation (n = 165) cohorts. High concentrations of LDLs and glycerophospholipids were independently predictors for favorable survival. Patients with low glycerophospholipids presented with more systemic inflammation (C-reactive protein and fibrinogen negatively and albumin positively correlated) but less adaptive immune cell tumor infiltration (lower tumor and immune cell PD-L1 expression), less oxygenic respiration and increased triglyceride biosynthesis in tumor cells, and lower histone expressions, correlating with higher numbers of expressed genes and more transcriptional noise, a putative neo-pluripotent tumor cell phenotype. Conclusions: Low serum phospholipids and essential amino acids are correlated with worse outcome in ovarian cancer, accompanied by a specific tumor cell phenotype. Clin Cancer Res; 23(8); 2081–92. ©2016 AACR.

Absence of PD-L1 on tumor cells is associated with reduced MHC I expression and PD-L1 expression increases in recurrent serous ovarian cancer

Immune-evasion and immune checkpoints are promising new therapeutic targets for several cancer entities. In ovarian cancer, the clinical role of programmed cell death receptor ligand 1 (PD-L1) expression as mechanism to escape immune recognition has not been clarified yet. We analyzed PD-L1 expression of primary ovarian and peritoneal tumor tissues together with several other parameters (whole transcriptomes of isolated tumor cells, local and systemic immune cells, systemic cytokines and metabolites) and compared PD-L1 expression between primary tumor and tumor recurrences. All expressed major histocompatibility complex (MHC) I genes were negatively correlated to PD-L1 abundances on tumor tissues, indicating two mutually exclusive immune-evasion mechanisms in ovarian cancer: either down-regulation of T-cell mediated immunity by PD-L1 expression or silencing of self-antigen presentation by down-regulation of the MHC I complex. In our cohort and in most of published evidences in ovarian cancer, low PD-L1 expression is associated with unfavorable outcome. Differences in immune cell populations, cytokines, and metabolites strengthen this picture and suggest the existence of concurrent pathways for progression of this disease. Furthermore, recurrences showed significantly increased PD-L1 expression compared to the primary tumors, supporting trials of checkpoint inhibition in the recurrent setting.

Sequential activation of different pathway networks in ischemia-affected and non-affected myocardium, inducing intrinsic remote conditioning to prevent left ventricular remodeling

We have analyzed the pathway networks of ischemia-affected and remote myocardial areas after repetitive ischemia/reperfusion (r-I/R) injury without ensuing myocardial infarction (MI) to elaborate a spatial- and chronologic model of cardioprotective gene networks to prevent left ventricular (LV) adverse remodeling. Domestic pigs underwent three cycles of 10/10 min r-I/R by percutaneous intracoronary balloon inflation/deflation in the mid left anterior descending artery, without consecutive MI. Sham interventions (n = 8) served as controls. Hearts were explanted at 5 h (n = 6) and 24 h (n = 6), and transcriptomic profiling of the distal (ischemia-affected) and proximal (non-affected) anterior myocardial regions were analyzed by next generation sequencing (NGS) and post-processing with signaling pathway impact and pathway network analyses. In ischemic region, r-I/R induced early activation of Ca-, adipocytokine and insulin signaling pathways with key regulator STAT3, which was also upregulated in the remote areas together with clusterin (CLU) and TNF-alpha. During the late phase of cardioprotection, antigen immunomodulatory pathways were activated with upregulation of STAT1 and CASP3 and downregulation of neprilysin in both zones, suggesting r-I/R induced intrinsic remote conditioning. The temporo-spatially differently activated pathways revealed a global myocardial response, and neprilysin and the STAT family as key regulators of intrinsic remote conditioning for prevention of adverse remodeling.

EV-Associated MMP9 in High-Grade Serous Ovarian Cancer Is Preferentially Localized to Annexin V-Binding EVs

High-grade serous ovarian cancer (HGSOC) is the most aggressive type of ovarian cancer and is responsible for most deaths caused by gynecological cancers. Numerous candidate biomarkers were identified for this disease in the last decades, but most were not sensitive or specific enough for clinical applications. Hence, new biomarkers for HGSOC are urgently required. This study aimed to identify new markers by isolating different extracellular vesicle (EV) types from the ascites of ovarian cancer patients according to their affinities for lipid-binding proteins and analyzing their protein cargo. This approach circumvents the low signal-to-noise ratio when using biological fluids for biomarker discovery and the issue of contamination by large non-EV complexes. We isolated and analyzed three distinct EV populations from the ascites of patients with ovarian cancer or cirrhosis and observed that Annexin V-binding EVs have higher levels of matrix metalloproteinase 9 in malignant compared to portal-hypertensive ascites. As this protein was not detected in other EV populations, this study validates our approach of using different EV types for optimal biomarker discovery. Furthermore, MMP9 in Annexin V-binding EVs could be a HGSOC biomarker with enhanced specificity, because its identification requires detection of two distinct components, that is, lipid and protein.

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.

Intrinsic remote conditioning of the myocardium as a comprehensive cardiac response to ischemia and reperfusion

We have previously shown that distal anterior wall ischemia/reperfusion induces gene expression changes in the proximal anterior myocardial area, involving genes responsible for cardiac remodeling. Here we investigated the molecular signals of the ischemia non-affected remote lateral and posterior regions and present gene expression profiles of the entire left ventricle by using our novel and straightforward method of 2D and 3D image reconstruction. Five or 24h after repetitive 10min ischemia/reperfusion without subsequent infarction, pig hearts were explanted and myocardial samples from 52 equally distributed locations of the left ventricle were collected. Expressional changes of seven genes of interest (HIF-1α; caspase-3, transcription factor GATA4; myocyte enhancer factor 2C /MEF2c/; hexokinase 2 /HK2/; clusterin /CLU/ and excision repair cross-complementation group 4 /ERCC4/) were measured by qPCR. 2D and 3D gene expression maps were constructed by projecting the fold changes on the NOGA anatomical mapping coordinates. Caspase-3, GATA4, HK2, CLU, and ERCC4 were up-regulated region-specifically in the ischemic zone at 5 h post ischemia/reperfusion injury. Overexpression of GATA4, clusterin and ERCC4 persisted after 24 h. HK2 showed strong up-regulation in the ischemic zone and down-regulation in remote areas at 5 h, and was severely reduced in all heart regions at 24 h. These results indicate a quick onset of regulation of apoptosis-related genes, which is partially reversed in the late phase of ischemia/reperfusion cardioprotection, and highlight variations between ischemic and unaffected myocardium over time. The NOGA 2D and 3D construction system is an attractive method to visualize expressional variations in the myocardium.We have previously shown that distal anterior wall ischemia/reperfusion induces gene expression changes in the proximal anterior myocardial area, involving genes responsible for cardiac remodeling. Here we investigated the molecular signals of the ischemia non-affected remote lateral and posterior regions and present gene expression profiles of the entire left ventricle by using our novel and straightforward method of 2D and 3D image reconstruction. Five or 24h after repetitive 10min ischemia/reperfusion without subsequent infarction, pig hearts were explanted and myocardial samples from 52 equally distributed locations of the left ventricle were collected. Expressional changes of seven genes of interest (HIF-1α; caspase-3, transcription factor GATA4; myocyte enhancer factor 2C /MEF2c/; hexokinase 2 /HK2/; clusterin /CLU/ and excision repair cross-complementation group 4 /ERCC4/) were measured by qPCR. 2D and 3D gene expression maps were constructed by projecting the fold changes on the NOGA anatomical mapping coordinates. Caspase-3, GATA4, HK2, CLU, and ERCC4 were up-regulated region-specifically in the ischemic zone at 5 h post ischemia/reperfusion injury. Overexpression of GATA4, clusterin and ERCC4 persisted after 24 h. HK2 showed strong up-regulation in the ischemic zone and down-regulation in remote areas at 5 h, and was severely reduced in all heart regions at 24 h. These results indicate a quick onset of regulation of apoptosis-related genes, which is partially reversed in the late phase of ischemia/reperfusion cardioprotection, and highlight variations between ischemic and unaffected myocardium over time. The NOGA 2D and 3D construction system is an attractive method to visualize expressional variations in the myocardium.