Steffen Heeg

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.

Creating oral squamous cancer cells: A cellular model of oral–esophageal carcinogenesis

Immortalization and malignant transformation are important steps in tumor development. The ability to induce these processes from normal human epithelial cells with genetic alterations frequently found in the corresponding human cancer would significantly enhance our understanding of tumor development. Alterations in several key intracellular regulatory pathways (the pRB, p53, and mitogenic signaling pathways and the telomere maintenance system) appear to be sufficient for the neoplastic transformation of normal human cells. Nevertheless, in vitro transformation models to date depend on viral oncogenes, most prominently the simian virus 40 early region, to induce immortalization and malignant transformation of normal human epithelial cells. Here, we demonstrate a transformation model creating oral–esophageal cancer cells by using a limited set of genetic alterations frequently observed in the corresponding human cancer. In a stepwise model, cyclin D1 overexpression and p53 inactivation led to immortalization of oral keratinocytes. Additional ectopic epithelial growth factor receptor overexpression followed by c-myc overexpression as well as consecutive reactivation of telomerase induced by epithelial growth factor receptor sufficed to transform oral epithelial cells, truly recapitulating the development of the corresponding human disease.

EGFR overexpression induces activation of telomerase via PI3K/AKT-mediated phosphorylation and transcriptional regulation through Hif1-alpha in a cellular model of oral–esophageal carcinogenesis

Telomerase plays an important role during immortalization and malignant transformation as crucial steps in the development of human cancer. In a cellular model of oral–esophageal carcinogenesis, recapitulating the human disease, immortalization occurred independent of the activation of telomerase but through the recombination‐based alternative lengthening of telomeres (ALT). In this stepwise model, additional overexpression of EGFR led to in vitro transformation and activation of telomerase with homogeneous telomere elongation in already immortalized oral squamous epithelial cells (OKF6‐D1_dnp53). More interestingly, EGFR overexpression activated the PI3K/AKT pathway. This strongly suggested a role for telomerase in tumor progression in addition to just elongating telomeres and inferring an immortalized state. Therefore, we sought to identify the regulatory mechanisms involved in this activation of telomerase and in vitro transformation induced by EGFR. In the present study we demonstrate that telomerase expression and activity are induced through both direct phosphorylation of hTERT by phospho‐AKT as well as PI3K‐dependent transcriptional regulation involving Hif1‐alpha as a key transcription factor. Furthermore, EGFR overexpression enhanced cell cycle progression and proliferation via phosphorylation and translocation of p21. Whereas immortalization was induced by ALT, in vitro transformation was associated with telomerase activation, supporting an additional role for telomerase in tumor progression besides elongating telomeres. (Cancer Sci 2011; 102: 351–360)

Isolation, culture and genetic manipulation of mouse pancreatic ductal cells

The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles duct cells morphologically and, to some extent, at a molecular level. Recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and ductlike cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. This approach is fast (∼4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas.

Inhibition of telomerase induces alternative lengthening of telomeres during human esophageal carcinogenesis

Immortalization is an important step toward the malignant transformation of human cells and is critically dependent upon telomere maintenance. Two mechanisms are known to maintain human telomeres. The process of telomere maintenance is either mediated through activation of the enzyme telomerase or through an alternative mechanism of telomere lengthening called alternative lengthening of telomeres (ALT). Whereas 85% of all human tumors show reactivation of telomerase, the remaining 15% are able to maintain telomeres via ALT. Telomerase inhibitors are already investigated in clinical trials, although the regulation as well as potential coexistence and redundancy of both telomere maintenance mechanisms during distinct steps of carcinogenesis are poorly understood. Herein, we demonstrate that telomerase activity and ALT alternate in a cell cycle dependent fashion in human esophageal epithelial cells, and can coexist in a genetically defined model of oral-esophageal squamous carcinogenesis. Moreover, we show that immortalized premalignant cells as well as cancer cells are able to switch from telomerase activation to ALT upon inhibition of telomerase. This indicates that cancer cells treated with telomerase inhibitors can use alternative and adaptive ways to maintain their telomeres and thereby escape telomere-based therapeutic strategies.

Prrx1 isoform switching regulates pancreatic cancer invasion and metastatic colonization

The two major isoforms of the paired-related homeodomain transcription factor 1 (Prrx1), Prrx1a and Prrx1b, are involved in pancreatic development, pancreatitis, and carcinogenesis, although the biological role that these isoforms serve in the systemic dissemination of pancreatic ductal adenocarcinoma (PDAC) has not been investigated. An epithelial-mesenchymal transition (EMT) is believed to be important for primary tumor progression and dissemination, whereas a mesenchymal-epithelial transition (MET) appears crucial for metastatic colonization. Here, we describe novel roles for both isoforms in the metastatic cascade using complementary in vitro and in vivo models. Prrx1b promotes invasion, tumor dedifferentiation, and EMT. In contrast, Prrx1a stimulates metastatic outgrowth in the liver, tumor differentiation, and MET. We further demonstrate that the switch from Prrx1b to Prrx1a governs EMT plasticity in both mouse models of PDAC and human PDAC. Last, we identify hepatocyte growth factor ( HGF) as a novel transcriptional target of Prrx1b. Targeted therapy of HGF in combination with gemcitabine in a preclinical model of PDAC reduces primary tumor volume and eliminates metastatic disease. Overall, we provide new insights into the isoform-specific roles of Prrx1a and Prrx1b in primary PDAC formation, dissemination, and metastatic colonization, allowing for novel therapeutic strategies targeting EMT plasticity.

A Mouse Model of Oral-Esophageal Carcinogenesis

Squamous cancers of the oral cavity and esophagus are common worldwide. A number of environmental factors as well as genetic alterations have been identified. However, the specific combination of genetic events and their interplay with environmental carcinogens are largely unknown. Furthermore, no good animal model existed to study the molecular changes important in the induction and progression of the disease. Here we summarize the efforts made to establish a mouse model of oralesophageal carcinogenesis. Cyclin D1 overexpressing (L2D1+) mice were generated using an EBV promoter to specifically target the oral cavity and the esophageal squamous epithelium. Besides analyzing different environmental factors, such as nitrosamines and zinc deficiency, cyclin D1 transgenic mice were crossbred with p53-deficient mice. While L2D1+ mice exhibited a phenotype of dysplasia, different combinations of mice resulted in invasive oral-esophageal cancer. This mouse model provides a well-defined and reproducible model of oralesophageal cancer that should be useful for testing chemopreventive, diagnostic, and therapeutic strategies.

Personalized Clinical Decision Making Through Implementation of a Molecular Tumor Board: A German Single-Center Experience

Purpose Dramatic advances in our understanding of the molecular pathophysiology of cancer, along with a rapidly expanding portfolio of molecular targeted drugs, have led to a paradigm shift toward personalized, biomarker-driven cancer treatment. Here, we report the 2-year experience of the Comprehensive Cancer Center Freiburg Molecular Tumor Board (MTB), one of the first interdisciplinary molecular tumor conferences established in Europe. The role of the MTB is to recommend personalized therapy for patients with cancer beyond standard-of-care treatment. Methods This retrospective case series includes 198 patients discussed from March 2015 through February 2017. The MTB guided individual molecular diagnostics, assessed evidence of actionability of molecular alterations, and provided therapy recommendations, including approved and off-label treatments as well as available matched clinical trials. Results The majority of patients had metastatic solid tumors (73.7%), mostly progressive (77.3%) after a mean of 2.0 lines of standard treatment. Diagnostic recommendations resulted in 867 molecular diagnostic tests for 172 patients (five per case), including exome analysis in 36 cases (18.2%). With a median turnaround time of 28 days, treatment recommendations were given to 104 patients (52.5%). These included single-agent targeted therapies (42.3%), checkpoint inhibitors (37.5%), and combination therapies (18.3%). Treatment recommendations were implemented in 33 of 104 patients (31.7%), of whom 19 (57.6%) showed stable disease or partial response, including 14 patients (7.1% of the entire population) receiving off-label treatments. Conclusion Personalized extended molecular-guided patient care is effective for a small but clinically meaningful proportion of patients in challenging clinical situations. Limited access to targeted drugs, lack of trials, and submission at late disease stage prevents broader applicability, whereas genome-wide analyses are not a strict requirement for predictive molecular testing.

ETV5 regulates ductal morphogenesis with Sox9 and is critical for regeneration from pancreatitis: ETV5 in Pancreatic Regeneration

Background: The plasticity of pancreatic acinar cells to undergo acinar to ductal metaplasia (ADM) has been demonstrated to contribute to the regeneration of the pancreas in response to injury. Sox9 is critical for ductal cell fate and important in the formation of ADM, most likely in concert with a complex hierarchy of, as yet, not fully elucidated transcription factors. Results: By using a mouse model of acute pancreatitis and three dimensional organoid culture of primary pancreatic ductal cells, we herein characterize the Ets‐transcription factor Etv5 as a pivotal regulator of ductal cell identity and ADM that acts upstream of Sox9 and is essential for Sox9 expression in ADM. Loss of Etv5 is associated with increased severity of acute pancreatitis and impaired ADM formation leading to delayed tissue regeneration and recovery in response to injury. Conclusions: Our data provide new insights in the regulation of ADM with implications in our understanding of pancreatic homeostasis, pancreatitis and epithelial plasticity. Developmental Dynamics 247:854–866, 2018.

Radiofrequency ablation is a treatment option for early stages of verrucous esophageal carcinoma

Figure 1. Upper endoscopic view before operation, showing a thickened, contact-vulnerable, exophytic, and lumen-constricting verrucous mass in the esophagus over a distance of 15 cm (asterisk Z esophageal lumen). (From Kulemann B, Fischer A, Hoeppner J. Esophageal stenosis caused by a rare entity. Gastroenterology 2014;146:618-871. Used with permission.) A 47-year-old man with stenosing verrucous carcinoma of the mid and distal esophagus (Figs. 1 and 2) underwent an abdominothoracic esophagectomy. The resection was complicated by severely hardened periesophageal inflammation tissue. Therefore, the initially intended total esophagectomy with a collar anastomosis was abandoned intraoperatively in favor of a subtotal resection with an intrathoracic anastomosis 27 cm from the incisors. Staging and grading showed G1pT2pN0(0/15) L0V0Pn0R0 status (Figs. 3 and 4). At day 15, the patient was discharged in good clinical condition after an uneventful postoperative course. During the follow-up period, an endoscopy initially showed a regular postoperative result with normal esophageal mucosa and an unsuggestive anastomosis (Video 1, available online at www. VideoGIE.org). However, 22 months after the operation, another endoscopy revealed suggestive, disseminated, white spot-like lesions affecting the entire remaining esophageal mucosa (Fig. 5 and Video 1, available online at www. VideoGIE.org). As suspected, pathologic examination confirmed the recurrence of verrucous carcinoma in the entire remaining esophagus. A penetration depth of up to 500 mm (pT1a) was determined on the basis of biopsy specimens (taken by Jumbo Biopsy Forceps, Endo-Flex, Voerde, Germany); the histologic evaluation was conducted by a calibrated eyepiece. Subsequently, the interdisciplinary consensus with the attending surgeons was to attempt an interventional endoscopic approach to postpone or even avoid a second tumor resection. Given that the mucosa was affected only superficially (500 mm), radiofrequency ablation (RFA), with a known penetration depth of 800 mm to 1000 mm, seemed to be a promising option. Within an interval of 3 months, 2 consecutive RFA sessions (Fig. 6 and Video 1, available online at www.VideoGIE.org) were performed by use of the Barrx 90 and the Barrx 60 RFA Focal Catheter (Covidien, Mansfield, Mass). The standard ablation regimen was chosen, 2 applications of energy (15 J/cm) were given, the ablation zone was cleaned, and 2 additional applications of energy were given according to the Van Vilsteren simplified ablation method.