Nicole Golob-Schwarzl

Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I–IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.

YAP-IL-6ST autoregulatory loop activated on APC loss controls colonic tumorigenesis.

Significance Current therapy for advanced colorectal cancer (CRC) is unsatisfactory and CRC remains a major cause of cancer-related deaths. Thus, novel and ubiquitously acting oncogenic mediators that are amenable to pharmacological targeting need to be identified. We found that loss of adenomatous polyposis coli (APC), which is mutated in the majority of human CRC, results in up-regulation of the signaling protein IL-6ST/gp130. This results in activation of Src family kinases (SFKs), YAP, Notch, and STAT3, which are simultaneously activated in 64% of human CRC. In addition to better explaining how APC loss initiates colorectal tumorigenesis, we show that combined treatment with SFK and JAK inhibitors results in regression of established colorectal tumors in mice. Loss of tumor suppressor adenomatous polyposis coli (APC) activates β-catenin to initiate colorectal tumorigenesis. However, β-catenin (CTNNB1) activating mutations rarely occur in human colorectal cancer (CRC). We found that APC loss also results in up-regulation of IL-6 signal transducer (IL-6ST/gp130), thereby activating Src family kinases (SFKs), YAP, and STAT3, which are simultaneously up-regulated in the majority of human CRC. Although, initial YAP activation, which stimulates IL6ST gene transcription, may be caused by reduced serine phosphorylation, sustained YAP activation depends on tyrosine phosphorylation by SFKs, whose inhibition, along with STAT3-activating JAK kinases, causes regression of established colorectal tumors. These results explain why APC loss is a more potent initiating event than the mere activation of CTNNB1.

Effects of formalin fixation and temperature on MR relaxation times in the human brain.

Post‐mortem MRI of the brain is increasingly applied in neuroscience for a better understanding of the contrast mechanisms of disease induced tissue changes. However, the influence of chemical processes caused by formalin fixation and differences in temperature may hamper the comparability with results from in vivo MRI.

Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage.

Elevated expression of the IGF2 mRNA binding protein 2 (IGF2BP2/IMP2) is linked to short survival and metastasis in esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) represents the sixth leading cause of cancer-related deaths and develops in Barrets esophagus affected tissues. The IGF2 mRNA binding protein IMP2/IGF2BP2/p62 was originally identified as an autoantigen in hepatocellular carcinoma. Aim of this study was to investigate the expression and prognostic role of IMP2 in EAC. Human EAC and Barrets esophagus tissue showed overexpression of IMP2, particularly in tumors of increased size and in metastatic tissues. Molecular classification based on published gene signatures of esophageal cancer revealed a specific subtype, in which the expression of IMP2 is high. According to GO and KEGG pathway analyses, genes showing highly correlated expression with IMP2 are associated with growth, proliferation, metabolism, inflammation, and cancerous processes. Clustering of EAC samples according to published survival marker genes strongly suggests that IMP2 overexpressing samples show poor survival. Finally, IMP2 expression correlated with short survival in patients with EAC or esophageal squamous carcinoma. Our data indicate that IMP2 might be a useful prognostic marker for Barrets esophagus and EAC.

Keratin 18-deficiency results in steatohepatitis and liver tumors in old mice: A model of steatohepatitis-associated liver carcinogenesis

Backround Steatohepatitis (SH)-associated liver carcinogenesis is an increasingly important issue in clinical medicine. SH is morphologically characterized by steatosis, hepatocyte injury, ballooning, hepatocytic cytoplasmic inclusions termed Mallory-Denk bodies (MDBs), inflammation and fibrosis. Results 17-20-months-old Krt18−/− and Krt18+/− mice in contrast to wt mice spontaneously developed liver lesions closely resembling the morphological spectrum of human SH as well as liver tumors. The pathologic alterations were more pronounced in Krt18−/− than in Krt18+/− mice. The frequency of liver tumors with male predominance was significantly higher in Krt18−/− compared to age-matched Krt18+/− and wt mice. Krt18-deficient tumors in contrast to wt animals displayed SH features and often pleomorphic morphology. aCGH analysis of tumors revealed chromosomal aberrations in Krt18−/− liver tumors, affecting loci of oncogenes and tumor suppressor genes. Materials and Methods Livers of 3-, 6-, 12- and 17-20-months-old aged wild type (wt), Krt18+/− and Krt18−/− (129P2/OlaHsd background) mice were analyzed by light and immunofluorescence microscopy as well as immunohistochemistry. Liver tumors arising in aged mice were analyzed by array comparative genomic hybridization (aCGH). Conclusions Our findings show that K18 deficiency of hepatocytes leads to steatosis, increasing with age, and finally to SH. K18 deficiency and age promote liver tumor development in mice, frequently on the basis of chromosomal instability, resembling human HCC with stemness features.

IMP2/IGF2BP2 expression, but not IMP1 and IMP3, predicts poor outcome in patients and high tumor growth rate in xenograft models of gallbladder cancer

Overexpression of the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2/IGF2BP2) has been described in different cancer types. Gallbladder carcinoma (GBC) is a rare but highly aggressive cancer entity with late clinical detection and poor prognosis. The aim of this study was to investigate the role of IMP2 in human GBC. Tissue microarrays (TMAs) of an international multi-center GBC sample collection from n = 483 patients were analyzed by immunohistochemistry. IMP2 immunoreactivity was found in 74.3% of the tumor samples on TMA, of which 14.0% showed strong and 86.0% low staining intensity. 72.4% of the tumor samples were IMP1 positive, but IMP1 showed lower expression in tumor tissue compared to control tissues. IMP3 immunoreactivity was observed in 92.7% of all tumors, of which 53.6% revealed strong IMP3 expression. Kaplan-Meier analysis linked high IMP2 expression to shorter survival time (p = 0.033), whereas neither IMP1 nor IMP3 expression was linked to a decreased survival time. Eight different human biliary tract cancer (BTC) cell lines were evaluated for tumor growth kinetics in mouse xenografts. Cell lines with high IMP2 expression levels showed the fastest increase in tumor volumes in murine xenografts. Furthermore, IMP2 expression in these cells correlated with the generation of reactive oxygen species (ROS) and RAC1 expression in BTC cells, suggesting RAC1-induced ROS generation as a potential mechanism of IMP2-promoted progression of GBC. In conclusion, IMP2 is frequently overexpressed in GBC and significantly associated with poor prognosis and growth rates in vivo. IMP2 might therefore represent a new target for the treatment of advanced GBC.

Separation of low and high grade colon and rectum carcinoma by eukaryotic translation initiation factors 1, 5 and 6

Colorectal cancer (CRC) is the third most common cause of cancer related death worldwide. Furthermore, with more than 1.2 million cases registered per year, it constitutes the third most frequent diagnosed cancer entity worldwide. Deregulation of protein synthesis has received considerable attention as a major step in cancer development and progression. Eukaryotic translation initiation factors (eIFs) are involved in the regulation of protein synthesis and are functionally linked to the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. The identification of factors accounting for colorectal carcinoma (CRC) development is a major gap in the field. Besides the importance of eIF3 subunits and the eIF4 complex, eIF1, eIF5 and eIF6 were found to be altered in primary and metastatic CRC. We observed significant difference in the expression profile between low and high grade CRC. eIF1, eIF5 and eIF6 are involved in translational control in CRC. Our findings also indicate a probable clinical impact when separating them into low and high grade colon and rectum carcinoma. eIF and mTOR expression were analysed on protein and mRNA level in primary low and high grade colon carcinoma (CC) and rectum carcinoma (RC) samples in comparison to non-neoplastic tissue without any disease-related pathology. To assess the therapeutic potential of targeting eIF1, eIF5 and eIF6 siRNA knockdown in HCT116 and HT29 cells was performed. We evaluated the eIF knockdown efficacy on protein and mRNA level and investigated proliferation, apoptosis, invasion, as well as colony forming and polysome associated fractions. These results indicate that eIFs, in particular eIF1, eIF5 and eIF6 play a major role in translational control in colon and rectum cancer.

Influence of eukaryotic translation initiation factor 6 on non–small cell lung cancer development and progression

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Dysregulation of protein synthesis plays a major role in carcinogenesis, a process regulated at multiple levels, including translation of mRNA into proteins. Ribosome assembly requires correct association of ribosome subunits, which is ensured by eukaryotic translation initiation factors (eIFs). eIFs have become targets in cancer therapy studies, and promising data on eIF6 in various cancer entities have been reported. Therefore, we hypothesised that eIF6 represents a crossroad for pulmonary carcinogenesis. High levels of eIF6 are associated with shorter patient overall survival in adenocarcinoma (ADC), but not in squamous cell carcinoma (SQC) of the lung. We demonstrate significantly higher protein expression of eIF6 in ADC and SQC than in healthy lung tissue based on immunohistochemical data from tissue microarrays (TMAs) and on fresh frozen lung tissue. Depletion of eIF6 in ADC and SQC lung cancer cell lines inhibited cell proliferation and induced apoptosis. Knockdown of eIF6 led to pre-rRNA processing and ribosomal 60S maturation defects. Our data indicate that eIF6 is upregulated in NSCLC, suggesting an important contribution of eIF6 to the development and progression of NSCLC and a potential for new treatment strategies against NSCLC.

STAT5 deficiency in hepatocytes reduces diethylnitrosamine-induced liver tumorigenesis in mice

Chronic liver diseases and the development of hepatocellular carcinoma are closely linked and pose a major medical challenge as treatment options are limited. Animal studies have shown that genetic deletion of the signal transducer and activator of transcription (STAT) 5 in liver is associated with higher susceptibility to fatty liver disease, fibrosis and cancer, indicating a protective role of hepatic STAT5 in mouse models of chronic liver disease. To investigate the role of STAT5 in the etiology of liver cancer in more detail, we applied the chemical carcinogen diethylnitrosamine (DEN) to mice harboring a hepatocyte-specific deletion of Stat5 (S5KO). At 8 months after DEN injections, tumor formation in S5KO was significantly reduced. This was associated with diminished tumor frequency and less aggressive liver cancer progression. Apoptosis and inflammation markers were not changed in S5KO livers suggesting that the reduced tumor burden was not due to impaired inflammatory response. Despite reduced mRNA expression of the DEN bio-activator cytochrome P450 2e1 (Cyp2e1) in S5KO livers, protein levels were similar. Yet, delayed tumor formation in S5KO mice coincided with decreased activation of c-Jun N-terminal Kinase (JNK). Taken together, while STAT5 has a protective role in fatty liver-associated liver cancer, it exerts oncogenic functions in DEN-induced liver cancer.