Johannes von Burstin

E-Cadherin Regulates Metastasis of Pancreatic Cancer In Vivo and Is Suppressed by a SNAIL/HDAC1/HDAC2 Repressor Complex

BACKGROUND & AIMS Early metastasis is a hallmark of pancreatic ductal adenocarcinoma and responsible for >90% of pancreatic cancer death. Because little is known about the biology and genetics of the metastatic process, we desired to elucidate molecular pathways mediating pancreatic cancer metastasis in vivo by an unbiased forward genetic approach. METHODS Highly metastatic pancreatic cancer cell populations were selected by serial in vivo passaging of parental cells with low metastatic potential and characterized by global gene expression profiling, chromatin immunoprecipitation, and in vivo metastatic assay. RESULTS In vivo selection of highly metastatic pancreatic cancer cells induced epithelial-mesenchymal transition (EMT), loss of E-cadherin expression, and up-regulation of mesenchymal genes such as Snail. Genetic inactivation of E-cadherin in parental cells induced EMT and increased metastasis in vivo. Silencing of E-cadherin in highly metastatic cells is mediated by a transcriptional repressor complex containing Snail and histone deacetylase 1 (HDAC1) and HDAC2. In line, mesenchymal pancreatic cancer specimens and primary cell lines from genetically engineered Kras(G12D) mice showed HDAC-dependent down-regulation of E-cadherin and high metastatic potential. Finally, transforming growth factor beta-driven E-cadherin silencing and EMT of human pancreatic cancer cells depends on HDAC activity. CONCLUSIONS We provide the first in vivo evidence that HDACs and Snail play an essential role in silencing E-cadherin during the metastatic process of pancreatic cancer cells. These data link the epigenetic HDAC machinery to EMT and metastasis and provide preclinical evidence that HDACs are promising targets for antimetastatic therapy.

Highly sensitive detection of early‐stage pancreatic cancer by multimodal near‐infrared molecular imaging in living mice

Pancreatic cancer is a serious disease with poor patient outcome, often as a consequence of late diagnosis in advanced stages. This is in large part due to the lack of diagnostic tools for early detection. To address this deficiency, we have investigated novel molecular near‐infrared fluorescent (NIRF) in vivo imaging techniques in clinically relevant mouse models of pancreatic cancer. Genome wide gene expression profiling was used to identify cathepsin cystein proteases and matrix metalloproteinases (MMP) as targets for NIRF imaging. Appropriate protease activatable probes were evaluated for detection of early‐stage pancreatic cancer in mice with orthotopically implanted pancreatic cancer cell lines. Mice with pancreatitis served as controls. Whole body in vivo NIRF imaging using activatable cathepsin sensitive probes specifically detected pancreatic tumors as small as 1–2 mm diameter. Imaging of MMP activity demonstrated high specificity for MMP positive tumors. Intravital flexible confocal fluorescence lasermicroscopy of protease activity enabled specific detection of pancreatic tumors at the cellular level. Importantly, topical application of NIRF‐probes markedly reduced background without altering signal intensity. Taken together, macroscopic and confocal lasermicroscopic molecular in vivo imaging of protease activity is highly sensitive, specific and allows discrimination between normal pancreatic tissue, inflammation and pancreatic cancer. Translation of this approach to the clinic could significantly improve endoscopic and laparoscopic detection of early‐stage pancreatic cancer.

In vivo diagnosis of murine pancreatic intraepithelial neoplasia and early-stage pancreatic cancer by molecular imaging

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor patient outcome often resulting from late diagnosis in advanced stages. To date methods to diagnose early-stage PDAC are limited and in vivo detection of pancreatic intraepithelial neoplasia (PanIN), a preinvasive precursor of PDAC, is impossible. Using a cathepsin-activatable near-infrared probe in combination with flexible confocal fluorescence lasermicroscopy (CFL) in a genetically defined mouse model of PDAC we were able to detect and grade murine PanIN lesions in real time in vivo. Our diagnostic approach is highly sensitive and specific and proved superior to clinically established fluorescein-enhanced imaging. Translation of this endoscopic technique into the clinic should tremendously improve detection of pancreatic neoplasia, thus reforming management of patients at risk for PDAC.

Krt19+/Lgr5− Cells Are Radioresistant Cancer-Initiating Stem Cells in the Colon and Intestine

Epithelium of the colon and intestine are renewed every 3 days. In the intestine there are at least two principal stem cell pools. The first contains rapid cycling crypt-based columnar (CBC) Lgr5(+) cells, and the second is composed of slower cycling Bmi1-expressing cells at the +4 position above the crypt base. In the colon, however, the identification of Lgr5(-) stem cell pools has proven more challenging. Here, we demonstrate that the intermediate filament keratin-19 (Krt19) marks long-lived, radiation-resistant cells above the crypt base that generate Lgr5(+) CBCs in the colon and intestine. In colorectal cancer models, Krt19(+) cancer-initiating cells are also radioresistant, while Lgr5(+) stem cells are radiosensitive. Moreover, Lgr5(+) stem cells are dispensable in both the normal and neoplastic colonic epithelium, as ablation of Lgr5(+) stem cells results in their regeneration from Krt19-expressing cells. Thus, Krt19(+) stem cells are a discrete target relevant for cancer therapy.

The Prrx1 homeodomain transcription factor plays a central role in pancreatic regeneration and carcinogenesis

Pancreatic exocrine cell plasticity can be observed during development, pancreatitis with subsequent regeneration, and also transformation. For example, acinar-ductal metaplasia (ADM) occurs during acute pancreatitis and might be viewed as a prelude to pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC) development. To elucidate regulatory processes that overlap ductal development, ADM, and the progression of normal cells to PanIN lesions, we undertook a systematic approach to identify the Prrx1 paired homeodomain Prrx1 transcriptional factor as a highly regulated gene in these processes. Prrx1 annotates a subset of pancreatic ductal epithelial cells in Prrx1creER(T2)-IRES-GFP mice. Furthermore, sorted Prrx1(+) cells have the capacity to self-renew and expand during chronic pancreatitis. The two isoforms, Prrx1a and Prrx1b, regulate migration and invasion, respectively, in pancreatic cancer cells. In addition, Prrx1b is enriched in circulating pancreatic cells (Pdx1cre;LSL-Kras(G12D/+);p53(fl/+);R26YFP). Intriguingly, the Prrx1b isoform, which is also induced in ADM, binds the Sox9 promoter and positively regulates Sox9 expression. This suggests a new hierarchical scheme whereby a Prrx1-Sox9 axis may influence the emergence of acinar-ductal metaplasia and regeneration. Furthermore, our data provide a possible explanation of why pancreatic cancer is skewed toward a ductal fate.

Pancreatic ductal morphogenesis and the Pdx1 homeodomain transcription factor.

Embryonic development of the pancreas is marked by an early phase of dramatic morphogenesis, in which pluripotent progenitor cells of the developing pancreatic epithelium give rise to the full array of mature exocrine and endocrine cell types. The genetic determinants of acinar and islet cell lineages are somewhat well defined; however, the molecular mechanisms directing ductal formation and differentiation remain to be elucidated. The complex ductal architecture of the pancreas is established by a reiterative program of progenitor cell expansion and migration known as branching morphogenesis, or tubulogenesis, which proceeds in mouse development concomitantly with peak Pdx1 transcription factor expression. We therefore evaluated Pdx1 expression with respect to lineage-specific markers in embryonic sections of the pancreas spanning this critical period of duct formation and discovered an unexpected population of nonislet Pdx1-positive cells displaying physical traits of branching. We then established a 3D cell culture model of branching morphogenesis using primary pancreatic duct cells and identified a transient surge of Pdx1 expression exclusive to branching cells. From these observations we propose that Pdx1 might be involved temporally in a program of gene expression sufficient to facilitate the biochemical and morphological changes necessary for branching morphogenesis.

The Pancreatic and Duodenal Homeobox Protein PDX-1 Regulates the Ductal Specific Keratin 19 through the Degradation of MEIS1 and DNA Binding

Background Pancreas organogenesis is the result of well-orchestrated and balanced activities of transcription factors. The homeobox transcription factor PDX-1 plays a crucial role in the development and function of the pancreas, both in the maintenance of progenitor cells and in determination and maintenance of differentiated endocrine cells. However, the activity of homeobox transcription factors requires coordination with co-factors, such as PBX and MEIS proteins. PBX and MEIS proteins belong to the family of three amino acid loop extension (TALE) homeodomain proteins. In a previous study we found that PDX-1 negatively regulates the transcriptional activity of the ductal specific keratin 19 (Krt19). In this study, we investigate the role of different domains of PDX-1 and elucidate the functional interplay of PDX-1 and MEIS1 necessary for Krt19 regulation. Methodology/Principal Findings Here, we demonstrate that PDX-1 exerts a dual manner of regulation of Krt19 transcriptional activity. Deletion studies highlight that the NH2-terminus of PDX-1 is functionally relevant for the down-regulation of Krt19, as it is required for DNA binding of PDX-1 to the Krt19 promoter. Moreover, this effect occurs independently of PBX. Second, we provide insight on how PDX-1 regulates the Hox co-factor MEIS1 post-transcriptionally. We find specific binding of MEIS1 and MEIS2 to the Krt19 promoter using IP-EMSA, and siRNA mediated silencing of Meis1, but not Meis2, reduces transcriptional activation of Krt19 in primary pancreatic ductal cells. Over-expression of PDX-1 leads to a decreased level of MEIS1 protein, and this decrease is prevented by inhibition of the proteasome. Conclusions/Significance Taken together, our data provide evidence for a dual mechanism of how PDX-1 negatively regulates Krt19 ductal specific gene expression. These findings imply that transcription factors may efficiently regulate target gene expression through diverse, non-redundant mechanisms.

Detection of Tumor Suppressor Genes in Cancer Development by a Novel shRNA-Based Method

Pancreatic cancer is one of the deadliest cancers with poor survival rates and limited therapeutic options. To improve the understanding of this diseases biology, a prerequisite for the generation of novel therapeutics, new platforms for rapid and efficient genetic and therapeutic screening are needed. Therefore, a combined in vitro/in vivo hybrid shRNA assay was developed using isolated murine primary pancreatic ductal cells (PDCs), in which oncogenic KrasG12D could be activated in vitro by genomic recombination through 4OH-tamoxifen–induced nuclear translocation of Cre-ERT2 expressed under control of the ROSA26 promoter. Further genetic manipulation was achieved through selective and stable RNAi against the tumor suppressors p16Ink4a (CDKN2A) or Trp53 (TP53) using lentiviral gene delivery. Treatment of PDCs with 4OH-tamoxifen increased phosphorylation of ERK downstream of KRAS, and subsequent lentiviral transduction resulted in sustained target gene repression. Double-mutant PDCs were then reintroduced into the pancreata of NOD-SCID-gamma (NSG) mice and monitored for tumor growth. Orthotopic implantation of PDCs carrying the activated KrasG12D-allele and shRNA against p16Ink4a or Trp53 resulted in tumor growth, metastasis, and reduced survival of NSG mice. In contrast, KrasG12D alone was not sufficient to induce tumor growth. Implications: The combinatory in vitro/in vivo approach described in this study allows for rapid and efficient identification of genes involved in carcinogenesis and opens new avenues for the development of therapeutic strategies to improve cancer treatment. Mol Cancer Res; 13(5); 863–9. ©2015 AACR.

The TALE homeodomain transcription factor MEIS1 activates the pro-metastatic melanoma cell adhesion molecule Mcam to promote migration of pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most deadly types of cancer, and the majority of pancreatic cancer deaths is caused by metastasis. Therapeutic options for systemic disease are limited, in particular due to the heterogeneous events leading to tumor dissemination. Previous studies highlighted an association of the homeodomain transcription factor MEIS1 with a ductal phenotype in pancreatic tissue architecture. Using immunohistochemistry, we demonstrate that MEIS1 is expressed in aberrant duct structures of Ela‐TGFα transgenic mice as well as in pancreatic intraepithelial neoplasia (PanINs), primary PDAC, and metastatic disease in Ptf1aCre/+;LSL‐KrasG12D/+ mice. To assess a putative role of MEIS1 in the progression of PDAC, MEIS1 overexpressing pancreatic cancer cell lines were generated by retroviral gene delivery and assessed for proliferation and migration. MEIS1 did not affect proliferation but increased migration in a subset of cell lines tested. Subsequent genome wide expression analysis identified upregulation of the pro‐metastatic gene melanoma cell adhesion molecule (Mcam) in migrating cells. Employing DNA‐pulldown and chromatin immunoprecipitation (ChIP) assays we reveal interaction of MEIS1 with the enhancer‐DNA of Mcam and its transcriptional activation to facilitate migration of pancreatic cancer cells in vitro. Activation of Mcam through MEIS1 occurs in a cell type dependent fashion, reflecting the different routes that lead to metastasis in vivo. To our knowledge, these results show for the first time a pro‐metastatic function of Mcam in pancreatic cancer.

Abstract 3301: Multiple cytokeratin-19 positive stem cells contribute to the clonal origin of colonic tumors in mice

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: The clonal origin of tumors is not well understood. Previous studies using chimeric mice have suggested that the origin of tumors in the mammalian small intestine may be polyclonal, with 2 or more progenitor cells contributing to intestinal adenomas. In the intestine, tissue stem cells are believed to be present in small numbers, and either quiescent or actively dividing, as previously shown with Lgr-5+ cells. Cytokeratin 19 (K19) is a potential progenitor marker expressed in the intestinal isthmus. Using the K19 gene promoter to drive Cre recombinase expression, we tracked the lineage of K19+ cells in an inducible fashion. Here, we used K19-CreERT2;ROSA26rLacZ mice to examine whether K19+ cells contribute to colonic tumor formation. Methods: In order to recapitulate the endogenous expression pattern of K19, we used a BAC strategy and generated a transgenic mouse with a Tamoxifen inducible Cre under the control of the K19 promoter (K19-BAC-Cre-ERT2). K19CreERT2 mice were crossed to ROSA26rLacZ (or GFP) reporter mice and treated with tamoxifen (6 mg/ day p.o. for 3 days) to assess K19+ lineage tracing by X-gal or GFP staining. To examine the contribution of the K19+ cells to colonic tumorigenesis, we used the AOM/DSS inflammation-associated model of colonic carcinogenesis. Mice were sacrificed 20 weeks following AOM induction and X-gal staining performed to stain for the K19+ cell lineage. Results: Treatment of control K19CreERT2/Rosa26r mice with tamoxifen resulted in the stochastic labelling of about 20-50% of colonic and intestinal glands, respectively. Consistent with the labeling of stem cells, marked glands stained positive beyond 52 weeks following tamoxifen induction. K19CreERT2/Rosa26r mice treated with AOM or DSS alone following tamoxifen labelling of K19+ glands displayed evidence of K19+ cell expansion and crypt fission, as determined by increased labelling of contiguous X-gal positive glands. K19CreERT2/Rosa26r mice were then treated with a single dose of AOM followed by DSS. When K19+ cells were labelled by tamoxifen administration prior to AOM, we observed that ∼10% tumors were entirely X-gal positive, suggesting derivation from a single K19+ cell. Interestingly, when K19+ cells were labelled by tamoxifen shortly after tumor initiation with AOM, some tumors were derived from both K19 recombined and non-recombined cells in a clonal fashion, suggesting a model whereby a K19+ cell gives rise to multiple K19+ daughter cells that each contribute to the tumor. Conclusions. Using K19CreERT2/Rosa26r mice, we show that K19 marks stem cells within the intestinal isthmus of the small intestine and colon. Using a carcinogen induced model of inflammation-associated carcinogenesis, we demonstrate that K19 expressing cells contribute to tumor formation in a manner consistent with tumor initiation following division of K19+ cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3301. doi:1538-7445.AM2012-3301