Division of Functional Genome Analysis

Functional Genome Analysis (B070)
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.

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Functional Tumour Analyses / Pancreatic Cancer..
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Pancreatic Cancer		Other Tumour Entities
- Liquid biopsy for accurate IPMN stratification	- Novel therapy by drug-repositioning	See publication list
- TF binding to highly methylated promoter(s)	- Serum-based diagnosis: proteins
- TF FLI1 promotes cancer progression	- Serum-based diagnosis: microRNAs	Archive

Pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is the currently most deadly cancer. Mortality is close to incidence. Only about 5% of all patients survive longer than five years. Untreated, the average survival period after diagnosis is about five months. Surgical resection represents still the best curative treatment approach for pancreatic cancer. However, it can only be applied to 10% to 20% of patients. PDAC is currently the fourth and seventh most common cause of cancer-related death in the Western world (Europe and the USA) and China, respectively, although ranked only tenth in incidence. Numbers are continuously rising, however, while declining for many other tumour entities. In 2030, PDAC is expected to be the second most cause of cancer-related death in the Western world, surpassing colorectal and breast cancer.
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Joining forces with leading groups in basic and clinical pancreatic cancer research, we study various molecular aspects in order to improve the basic understanding of PDAC tumour biology and pathology. To this end, we look in much detail at the communication between different cell types within the tumour microenvironment, for example, with a special focus on proteins. All this is complemented by studies on the functional consequences of the observed molecular changes.
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Taking advantage of the gained knowledge, we are aiming at establishing novel approaches for early diagnosis, disease prognosis and risk stratification of pancreatic cancer. To this end, we pursue particularly non-invasive liquid biopsy processes based on blood samples. Applying and combining different analysis forms, molecular signatures at various levels are defined that could enable accurate diagnosis.
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Also, we are active in identifying new therapeutic routes of high potential so as to improve on the current prognosis by means of innovative therapeutic strategies. One possible therapeutic option is at a stage that requires a clinical trial as the next step in the evaluation of its clinical utility.
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All this is done in several collaborations with partners worldwide. We have participated repeatedly in international and national consortia that covered various aspects of pancreatic cancer research. Locally, we have a strong and continuous cooperation with the European Pancreas Center (EPZ) at the Surgery Department of the Heidelberg University Hospital.

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Image of a pancreatic cancer cell.
DNA in the nucleus is labelled blue; red signals show the cytoskeleton.


Njouendou et al. (2023) Mol. Cancer 22, 52.	Stoy et al. (2015) EMBO Mol. Med. 7, 1048-1062.	Youns et al. (2009) Biochem. Pharmacol. 78, 273-283.
Zhang et al. (2023) Clin. Cancer Res. 29, 1535-1545.	Jandaghi et al. (2015) Cell Cycle 14, 689-690.	Ketterer et al. (2009) Cancer Lett. 277, 72-81.
Heid et al. (2022) Cancer Metab. 10, 24.	Moskalev et al. (2015) Oncotarget 6, 4418-4427.	Börner et al. (2009) Biotechniques 46, 297-304.
Brindl et al. (2022) Cancers 14, 3562.	Haller et al. (2015) Int. J. Cancer 136, 1013-1023.	Bauer et al. (2009) Pancreatology 9, 34-44.
Wu et al. (2021) Cancers 13, 4569.	Keller et al. (2014) BMC Med. 12, 224.	Loos et al. (2007) Virchows Arch. 450, 719-726.
Al-Shaheri et al. (2021) Cancer Treat. Rev. 96, 102193.	Keleg et al. (2014) PLoS ONE 9, e100178.	Bauer et al. (2007) Grundl. Mol. Med., Springer, 346-362.
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Blood-based diagnosis and risk stratification of patients with intraductal papillary mucinous neoplasm (IPMN) to decide on surgical intervention

Intraductal papillary mucinous neoplasm (IPMN) is a precursor of PDAC. Patients with low-grade dysplasia have a relatively good prognosis and are kept under surveillance to monitor disease development, whereas high-grade dysplasia and IPMN invasive carcinoma require tumour resection. Diagnostic distinction of the two groups is difficult, however. We aimed to identify variations in protein concentration in peripheral blood for accurate discrimination. Sera from IPMN patients and healthy donors were analysed on microarrays made of 2,977 antibodies. For microRNA biomarkers, a PCR-based screen was performed and biomarker candidates confirmed by quantitative PCR.

A support vector machine (SVM) algorithm defined classifiers, which were validated on a separate sample set. A panel of five proteins and three miRNAs could distinguish high- and low-risk IPMN with an accuracy of 97%. This is substantially better than the accuracy obtained in the same patient cohort by using the guideline criteria for decision-making on performing surgery or not. The precise blood-based diagnosis and risk stratification will improve patient management and thus the prognosis of IPMN patients. In addition to the main finding, highly accurate discrimination was also achieved between other patient subgroups.

Zhang et al. (2023) Clin. Cancer Res. 29, 1535-1545.

Figure legend: (Left) Diagnostic performance of clinical parameters to discriminate high-risk from low-risk IPMN according to current guidelines. (Right) Much better results were obtained by a combined panel of 8 protein and miRNA biomarkers. The results are presented as ROC curves and corresponding AUC values as determined in the training and validation cohorts, respectively.

Promoter methylation promotes the binding of transcrption factor NFATc1, triggering oncogenic gene activation in pancreatic cancer

Studies have indicated that some genes involved in carcinogenesis are highly methylated in their promoter regions but nevertheless strongly transcribed. It has been proposed that transcription factors could bind specifically to methylated promoters and trigger transcription. We looked at this rather comprehensively for pancreatic ductal adenocarcinoma (PDAC) and studied some cases in more detail. Some 2% of regulated genes in PDAC exhibited higher transcription coupled to promoter hypermethylation in comparison to healthy tissue.

Screening 661 transcription factors, several were found to bind specifically to methylated promoters, in particular molecules of the NFAT family. One of them - NFATc1 - was substantially more expressed in PDAC than control tissue and exhibited a strong oncogenic role. Functional studies combined with computational analyses allowed determining affected genes. A prominent one was gene ALDH1A3, which accelerates PDAC metastasis and correlates with a bad prognosis. Further studies confirmed the direct up-regulation of ALDH1A3 transcription by NFATc1 promoter binding in a methylation-dependent process, providing insights into the oncogenic role of transcription activation in PDAC that is promoted by DNA methylation.

Wu et al. (2021) Cancers 13, 4569.

Figure legend: Identification of transcription factors that bind preferentially to the mutant hTERT promoter. (A) A protein microarray is shown that presents 667 transcription factor DNA binding domains. Proteins were immunostained with fluorescently labelled antibodies that target terminal tags. Incubation of a 35 bp fragment resembling (B) the mutated or (C) the wildtype version of the promoter sequence identified specific binding; in (D) a merger of the images is shown. (E) The ratio, resulting from four independent experiments, is presented of the normalized binding signals. Transcription factors are highlighted in red, which exhibited stronger binding to the mutated sequence. (F) The results of few transcription factors are presented in more detail (left panel). In addition, the relative protein level was deduced from the antibody labelling of their terminal tags (right panel). Green and orange bars indicate the signal intensities obtained at the N- and C-termini, respectively (G) Sequence recognition motifs of the three best binding transcription factors are depicted in comparison to the binding motif generated by the mutations (leftmost panel).

Transcription factor FLI1 promotes cancer progression by affecting cell cycle regulation

Binding of transcription factors to mutated DNA sequences is a likely regulator of cancer progression. Noncoding regulatory mutations such as those on the core promoter of the gene encoding human telomerase reverse transcriptase have been shown to affect gene expression in cancer. Using a protein microarray of 667 transcription factor DNA-binding domains and subsequent functional assays, we looked for transcription factors that preferentially bind the mutant hTERT promoter and characterized their downstream effects.

One of them, friend leukemia integration 1 (FLI1), exhibited particularly strong effects with respect to regulating hTERT expression, while the even better binding ELK3 did not. Depletion of FLI1 decreased expression of the genes for cyclin D1 (CCND1) and E2F transcription factor 2 (E2F2) resulting in a G1/S cell cycle arrest and in consequence a reduction of cell proliferation. FLI1 also affected CMTM7, another gene involved in G1/S transition, although by another process that suggests a balanced regulation of the tumour suppressor gene’s activity via opposing regulation processes.

FLI1 expression was found upregulated and correlated with an increase in CCND1 expression in pancreatic cancer and brain tumours. In non-neoplastic lung cells, however, FLI1 depletion led to rapid progression through the cell cycle. This coincides with the fact that FLI1 is down-regulated in lung tumours. Taken together, our data indicate a cell cycle regulatory hub involving FLI1, hTERT, CCND1 and E2F2 in a tissue- and context-dependent manner.

Miao et al. (2020) Int. J. Cancer 147, 189-201.

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Novel Therapy Option:
DRD2 is critical for pancreatic cancer and promises pharmacological therapy by already established antagonistsr

Incidence and mortality of pancreatic ductal adenocarcinoma (PDAC) are almost equivalent, urging for the development of better therapeutic strategies. We investigated novel potential therapeutic targets for PDAC by performing global gene expression profiling in 195 PDAC and 41 normal pancreatic tissue samples. Superimposing the pathway context and interaction networks of aberrantly expressed genes, we identified factors with central roles in PDAC pathways. Next, tissue microarray analysis was used to verify the expression of the candidate targets in an independent set of 152 samples comprising 40 normal pancreatic tissues, 63 PDAC sections and 49 samples of chronic pancreatitis.

We identified dopamine receptor D2 (DRD2) as a key modulator of cancer pathways in PDAC. DRD2 up-regulation at the protein level was validated in a large independent sample cohort. Most importantly, we found that blockade of DRD2, through RNAi or pharmacological inhibition using FDA-approved antagonists, such as Haloperidol, hampers the proliferative and invasive capacities of pancreatic cancer cells in vitro and in vivo while modulating cAMP and endoplasmic reticulum stress pathways. Our findings demonstrate that inhibiting DRD2 represents a novel therapeutic approach for PDAC. Given that DRD2 antagonists are currently routinely used for the management of schizophrenia or other mental diseases, a drug-repositioning strategy could facilitate the clinical use of these agents for treating pancreatic cancer.

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Bakadlag et al. (2019) Expert Opin. Ther. Tar. 23, 365-367.

Jandaghi et al. (2016) Gastroenterology 151, 1218-1231.

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