Elisa Espinet

Dependency of Colorectal Cancer on a TGF-β-Driven Program in Stromal Cells for Metastasis Initiation

A large proportion of colorectal cancers (CRCs) display mutational inactivation of the TGF-β pathway, yet, paradoxically, they are characterized by elevated TGF-β production. Here, we unveil a prometastatic program induced by TGF-β in the microenvironment that associates with a high risk of CRC relapse upon treatment. The activity of TGF-β on stromal cells increases the efficiency of organ colonization by CRC cells, whereas mice treated with a pharmacological inhibitor of TGFBR1 are resilient to metastasis formation. Secretion of IL11 by TGF-β-stimulated cancer-associated fibroblasts (CAFs) triggers GP130/STAT3 signaling in tumor cells. This crosstalk confers a survival advantage to metastatic cells. The dependency on the TGF-β stromal program for metastasis initiation could be exploited to improve the diagnosis and treatment of CRC.

CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell–autonomous detoxification mechanism that must be overcome to prevent drug resistance.

Endothelial Notch1 Activity Facilitates Metastasis

Endothelial cells (ECs) provide angiocrine factors orchestrating tumor progression. Here, we show that activated Notch1 receptors (N1ICD) are frequently observed in ECs of human carcinomas and melanoma, and in ECs of the pre-metastatic niche in mice. EC N1ICD expression in melanoma correlated with shorter progression-free survival. Sustained N1ICD activity induced EC senescence, expression of chemokines and the adhesion molecule VCAM1. This promoted neutrophil infiltration, tumor cell (TC) adhesion to the endothelium, intravasation, lung colonization, and postsurgical metastasis. Thus, sustained vascular Notch signaling facilitates metastasis by generating a senescent, pro-inflammatory endothelium. Consequently, treatment with Notch1 or VCAM1-blocking antibodies prevented Notch-driven metastasis, and genetic ablation of EC Notch signaling inhibited peritoneal neutrophil infiltration in an ovarian carcinoma mouse model.

Identification and Validation of Novel Subtype-specific Protein Biomarkers in Pancreatic Ductal Adenocarcinoma.

Objectives Pancreatic ductal adenocarcinoma (PDAC) has been subclassified into 3 molecular subtypes: classical, quasi-mesenchymal, and exocrine-like. These subtypes exhibit differences in patient survival and drug resistance to conventional therapies. The aim of the current study is to identify novel subtype-specific protein biomarkers facilitating subtype stratification of patients with PDAC and novel therapy development. Methods A set of 12 human patient-derived primary cell lines was used as a starting material for an advanced label-free proteomics approach leading to the identification of novel cell surface and secreted biomarkers. Cell surface protein identification was achieved by in vitro biotinylation, followed by mass spectrometric analysis of purified biotin-tagged proteins. Proteins secreted into a chemically defined serum-free cell culture medium were analyzed by shotgun proteomics. Results Of 3288 identified proteins, 2 pan-PDAC (protocadherin-1 and lipocalin-2) and 2 exocrine-like-specific (cadherin-17 and galectin-4) biomarker candidates have been validated. Proximity ligation assay analysis of the 2 exocrine-like biomarkers revealed their co-localization on the surface of exocrine-like cells. Conclusions The study reports the identification and validation of novel PDAC biomarkers relevant for the development of patient stratification tools. In addition, cadherin-17 and galectin-4 may serve as targets for bispecific antibodies as novel therapeutics in PDAC.

Saa3 is a key mediator of the protumorigenic properties of cancer-associated fibroblasts in pancreatic tumors

Significance Pancreatic ductal adenocarcinoma is one of the most malignant human tumors for which there are no efficacious therapeutic strategies. This tumor type is characterized by an abundant desmoplastic stroma that promotes tumor progression. Yet recent studies have shown that physical or genetic elimination of the stroma leads to more aggressive tumor development. Here, we decided to reprogram the stromal tissue by identifying and subsequently targeting genes responsible for their protumorigenic properties. Comparative transcriptome analysis revealed several genes overexpressed in cancer-associated fibroblasts compared with those present in normal pancreata. We provide genetic evidence that one of these genes, Saa3, plays a key role on the protumorigenic properties of the stroma, opening the door to the design of future therapeutic strategies. Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of abundant desmoplastic stroma primarily composed of cancer-associated fibroblasts (CAFs). It is generally accepted that CAFs stimulate tumor progression and might be implicated in drug resistance and immunosuppression. Here, we have compared the transcriptional profile of PDGFRα+ CAFs isolated from genetically engineered mouse PDAC tumors with that of normal pancreatic fibroblasts to identify genes potentially implicated in their protumorigenic properties. We report that the most differentially expressed gene, Saa3, a member of the serum amyloid A (SAA) apolipoprotein family, is a key mediator of the protumorigenic activity of PDGFRα+ CAFs. Whereas Saa3-competent CAFs stimulate the growth of tumor cells in an orthotopic model, Saa3-null CAFs inhibit tumor growth. Saa3 also plays a role in the cross talk between CAFs and tumor cells. Ablation of Saa3 in pancreatic tumor cells makes them insensitive to the inhibitory effect of Saa3-null CAFs. As a consequence, germline ablation of Saa3 does not prevent PDAC development in mice. The protumorigenic activity of Saa3 in CAFs is mediated by Mpp6, a member of the palmitoylated membrane protein subfamily of the peripheral membrane-associated guanylate kinases (MAGUK). Finally, we interrogated whether these observations could be translated to a human scenario. Indeed, SAA1, the ortholog of murine Saa3, is overexpressed in human CAFs. Moreover, high levels of SAA1 in the stromal component correlate with worse survival. These findings support the concept that selective inhibition of SAA1 in CAFs may provide potential therapeutic benefit to PDAC patients.

Abstract IA22: CYP3A5 mediates resistance to small molecule inhibitors in a subtype of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is clinically still treated as a single disease. We have generated patient-derived models representing the recently identified quasi-mesenchymal, classical and exocrine-like PDAC subtypes, and report a two-marker set facilitating patient stratification by immunohistochemistry. The subtypes show significant differences in overall survival and drug sensitivity, with the exocrine-like subtype being resistant to the tyrosine kinase inhibitors erlotinib, dasatinib, as well as the chemotherapeutic paclitaxel. Highly expressed cytochrome P450 3A5 (CYP3A5) actively metabolizes these compounds in the exocrine-like subtype, and pharmacological or shRNA-mediated CYP3A5 inhibition sensitizes tumor cells in vivo. Additionally, we investigated the transcriptional network underlying the subtype-specific CYP3A5 expression. Hence, these data show that exocrine-like PDAC adopts a highly effective detoxification mechanism akin to that of hepatocytes. High expression of CYP3A5 in other tumor entities suggests this pathway as an important target to overcome drug resistance and to predict response to therapy with small molecule drugs. Citation Format: Elisa M. Noll, Elisa M. Noll, Christian Eisen, Christian Eisen, Elisa Espinet, Elisa Espinet, Albrecht Stenzinger, Wilko Weichert, Martin R. Sprick, Andreas Trumpp, Andreas Trumpp. CYP3A5 mediates resistance to small molecule inhibitors in a subtype of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA22.

Abstract A61: Exploring the PDAC-subtype-associated microenvironment in PDX models and patients

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with dismal prognosis (1, 2). Despite extensive research and the discovery of several promising drug candidates, little progress in PDAC treatment has been reported in the last years (3, 4). Two facts can be behind these disappointing results. In one hand, although PDAC is still clinically treated as a single disease, three distinct molecular subtypes (classical, quasi-mesenchymal and exocrine) were recently described based on expression profiling of microdissected epithelial tumor cells of PDAC samples (5). Interestingly, in vitro studies revealed differences in drug response of two of the proposed subtypes for which cell lines were available. These results highlight the importance of considering PDAC as a heterogeneous disease and point to the stratification of patients as a possible way to improve PDAC treatment response. On another hand, an additional reason behind the limited efficacy of PDAC treatment might be the tumor microenvironment. PDAC is the solid tumor with the highest stromal content, which can account for up to 90% of the total tumor mass. The PDAC microenvironment is known to actively affect tumorigenesis (6) and to impair drug delivery (7). Thus, rendering the PDAC microenvironment as an appealing therapeutic target to improve PDAC care (8). We have developed a novel workflow to efficiently generate patient-derived orthotopic xenografts (PDX) and serum-free cell cultures from primary resected PDAC tumors. The established primary cell lines comprise for first time all three described PDAC subtypes. Additionally, when re-injected into immunodeficient mice, these cells generate xenografts with high pathological similarity to the original patient tumor, including a prominent stromal presence. To explore the differences in the microenvironment associated to the individual PDAC subtypes we have now generated gene expression profiles for the stroma of a number of xenografts from our PDX model representing all three subtypes. Besides, RNA sequencing from different sub-populations isolated from fresh primary human PDAC tumors (as separated by fluorescent activated cell sorting according to surface markers) may reveal interesting interactions between the different tumor compartments. We have developed a set of immunohistochemical markers to identify the PDAC-subtypes that can be used in patient paraffin sections. Hence, the RNAseq data of the different tumor populations can be also easily studied in the context of the different subtypes. We believe that these approaches will shed some light on how different stromal expression patterns are interconnected with different epithelial expression profiles and vice versa, and how this information can be ultimately exploited for patient stratification and therapy. 1. Hidalgo M. Pancreatic cancer. N Engl J Med 2010 2. Vincent A et al. Pancreatic cancer. Lancet, 2011 3. Werner J et al. Advanced-stage pancreatic cancer: therapy options. Nature Reviews Clinical Oncology, 2013 4. Hidalgo M et al. Translational therapeutic opportunities in ductal adenocarcinoma of the pancreas. Clin Cancer Res, 2012 5. Collisson EA et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nat Med, 2011 6. Feig C et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblastssynergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci USA, 2013 7. Provenzano PP et al. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell, 2012 8. Neesse A et al. Emerging concepts in pancreatic cancer medicine: targeting the tumor stroma. Onco Targets Ther, 2013 Citation Format: Elisa Espinet Espinet, Christian Eisen, Elisa M. Noll, Vanessa Vogel, Corinna Klein, Zuguang Gu, Matthias Schlesner, Tobias Bauer, Nathalia Giese, Roland Eils, Jens Werner, Wilko Weichert, Martin R. Sprick, Andreas Trumpp. Exploring the PDAC-subtype-associated microenvironment in PDX models and patients. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A61.

Abstract A69: A novel mechanism mediates drug resistance in the exocrine-like pancreatic ductal adenocarcinoma (PDAC) subtype

PDAC is a highly aggressive disease with dismal prognosis [1, 2]. Despite extensive research and the discovery of several drug candidates, little progress has been reported since the approval of gemcitabine and erlotinib [1]. Moreover, recent trials with targeted therapies have shown only limited or no benefit [1, 2]. For a number of other carcinomas, tumor subclasses have been uncovered that allow the use of targeted therapies. The mutational landscape of PDAC is complex and heterogeneous, raising the question whether subclasses also exist in PDAC [3]. Collisson et al. described three PDAC subtypes that were identified based on their gene-expression profiles: The classical, the quasi-mesenchymal and the exocrine-like subtype [4]. However, not all subtypes could be identified in the previously available model systems. We have established a novel patient-derived model system that allows the analysis of these three human PDAC subtypes in vitro and in vivo. Hence, we provide a systematic workflow to propagate human PDAC in orthotopic xenografts and to derive tumor-initiating primary cell lines of all three PDAC subtypes. HNF-1 and Keratin 81 were identified as markers for subtype stratification by immunohistochemistry. Application of this two-marker set on a 258 large patient cohort confirmed a predominantly non-overlapping staining and revealed a significant difference in overall survival across the three subtypes. Furthermore, a drug screen uncovered subtype-specific drug sensitivities towards a number of drugs, including gemcitabine and dasatinib. Notably, the exocrine-like subtype was resistant towards all compounds tested. Thus, we aimed to identify the underlying cause of the observed drug resistance. Molecular analysis including gene set enrichment analysis (GSEA) allowed us to identify a putative novel mechanism of drug resistance. Analysis by qRT-PCR and Western blot demonstrated the enhanced expression of several genes mediating this mechanism particularly in the exocrine-like subtype in vitro and in vivo. These findings led to the identification of a novel protein target central to this mechanism. Additionally, retrospective immunohistochemical analysis of a large patient cohort confirmed that this target is predominantly found in those patient tumors classified as exocrine-like. Hence, we hypothesized that the observed strong activation of this mechanism in the exocrine-like PDAC subtype could be responsible for the drug resistance observed in this subclass. In line with this, functional inhibition of this mechanism resulted in increased drug sensitivity in the exocrine-like subtype. Hence, our findings may ultimately advance personalized treatment by applying novel marker-based patient selection strategies in combination with tailored drug use, a strategy which will be presented in more detail at the conference. [1] Hidalgo, M. Pancreatic cancer. The New England journal of medicine. 362, 1605-1617, doi:10.1056/NEJMra0901557 (2010). [2] Vincent, A., Herman, J., Schulick, R., Hruban, R. H. & Goggins, M. Pancreatic cancer. Lancet. 378, 607-620, doi:10.1016/S0140-6736(10)62307-0 (2011). [3] Jones, S. et al. Core signalling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 321, 1801-1806, doi:10.1126/science.1164368 (2008). [4] Collisson, E. A. et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nature medicine. 17, 500-503, doi:10.1038/nm.2344 (2011). Citation Format: Elisa M. Noll, Christian Eisen, Elisa Espinet, Vanessa Vogel, Corinna Klein, Albrecht Stenzinger, Franziska Zickgraf, Peter Neuhaus, Marcus Bahra, Bruno V. Sinn, Christian Lutz, Michael Kulke, Andreas Pahl, Nathalia A. Giese, Oliver Strobel, Jens Werner, Wilko Weichert, Andreas Trumpp, Martin R. Sprick. A novel mechanism mediates drug resistance in the exocrine-like pancreatic ductal adenocarcinoma (PDAC) subtype. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A69.