Helene Damhofer

Hedgehog-Stimulated Chemotaxis Is Mediated by Smoothened Located Outside the Primary Cilium

Hedgehog stimulates a chemotactic or transcriptional response depending on the intracellular trafficking of the co-receptor Smoothened. Signaling Move from Outside the Cilium Hedgehog (Hh) can trigger transcriptional responses that control differentiation or it can stimulate chemotaxis or axonal pathfinding. The transcriptional response to Hh requires trafficking of the receptor Smoothened (Smo) to the primary cilium. Bijlsma et al. showed that cells expressing mutants of Smo that could not traffic to the primary cilium or cells lacking components needed for maintenance of the primary cilium exhibited a robust Smo-mediated chemotactic response, but failed to exhibit a transcriptional response. Cells expressing mutants that could not traffic to the primary cilium showed an enhanced chemotactic response. The intracellular trafficking of Smo was altered in the absence of the primary cilium or for Smo mutants without the ability to target to the cilium. Experiments with pharmacological inhibitors indicated that pertussis toxin–sensitive G proteins and autocrine leukotriene signaling contributed to the chemotactic response. Thus, Hh can stimulate a chemotactic or transcriptional response, which depends on the intracellular trafficking of Smo and involves distinct signaling pathways. Regulation of the Hedgehog (Hh) pathway relies on an interaction of two receptors. In the absence of Hh, Patched1 (Ptch1) inhibits the pathway. Binding of the ligand Hh to Ptch1 stimulates the localization of the activating receptor Smoothened (Smo) to the primary cilium, which is required for the transcriptional Hh response. Hh can also induce chemotaxis through a nontranscriptional pathway. We assessed the effects of defective ciliary localization of Smo on its subcellular trafficking and ability to mediate chemotactic signaling. Cells expressing mutants of Smo that could not localize to the primary cilium or cells lacking the primary cilium showed altered intracellular trafficking of Smo and, in response to Hh or Smo agonists, decreased transcriptional signaling and enhanced chemotactic responsiveness. Thus, the ciliary localization machinery appears to transport Smo to subcellular sites where it can mediate transcriptional signaling and away from locations where it can mediate chemotactic signaling. The subcellular localization of Smo is thus a crucial determinant of its signaling characteristics and implies the existence of a pool of Smo dedicated to chemotaxis.

Establishment of patient-derived xenograft models and cell lines for malignancies of the upper gastrointestinal tract

BackgroundThe upper gastrointestinal tract is home to some of most notorious cancers like esophagogastric and pancreatic cancer. Several factors contribute to the lethality of these tumors, but one that stands out for both tumor types is the strong inter- as well as intratumor heterogeneity. Unfortunately, genetic tumor models do not match this heterogeneity, and for esophageal cancer no adequate genetic models exist. To allow for an improved understanding of these diseases, tissue banks with sufficient amount of samples to cover the extent of diversity of human cancers are required. Additionally, xenograft models that faithfully mimic and span the breadth of human disease are essential to perform meaningful functional experiments.MethodsWe describe here the establishment of a tissue biobank, patient derived xenografts (PDXs) and cell line models of esophagogastric and pancreatic cancer patients. Biopsy material was grafted into immunocompromised mice and PDXs were used to establish primary cell cultures to perform functional studies. Expression of Hedgehog ligands in patient tumor and matching PDX was assessed by immunohistochemical staining, and quantitative real-time PCR as well as flow cytometry was used for cultured cells. Cocultures with Hedgehog reporter cells were performed to study paracrine signaling potency. Furthermore, SHH expression was modulated in primary cultures using lentiviral mediated knockdown.ResultsWe have established a panel of 29 PDXs from esophagogastric and pancreatic cancers, and demonstrate that these PDXs mirror several of the (immuno)histological and biochemical characteristics of the original tumors. Derived cell lines can be genetically manipulated and used to further study tumor biology and signaling capacity. In addition, we demonstrate an active (paracrine) Hedgehog signaling mode by both tumor types, the magnitude of which has not been compared directly in previous studies.ConclusionsOur established PDXs and their matching primary cell lines retain important characteristics seen in the original tumors, and this should enable future studies to address the responses of these tumors to different treatment modalities, but also help in gaining mechanistic insight in how some tumors respond to certain regimens and others do not.

Assessment of the stromal contribution to Sonic Hedgehog-dependent pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non‐tumor cells that together provide a tumor‐promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH‐driven stromal response in PDAC. For this a novel mixed‐species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next‐generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH‐dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non‐microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH‐dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment options are still inadequate.

ADAM10-mediated release of heregulin confers resistance to trastuzumab by activating HER3.

Receptor tyrosine kinases of the HER-family are involved in the development and progression of multiple epithelial tumors, and have consequently become widely used targets for new anti-cancer therapies. Trastuzumab, an antibody against HER2, has shown potent growth inhibitory effects on HER2 overexpressing tumors, including gastro-esophageal cancer, however, resistance to this therapy is inevitable. Unfortunately, a paucity of data on the cellular mechanisms of resistance to targeted therapeutic agents exists in esophageal adenocarcinoma. Using primary established HER2-overexpressing cultures and patient-derived xenograft models, we now reveal a novel resistance mechanism to trastuzumab in esophageal cancer: In response to trastuzumab, both HER3 and the metalloprotease ADAM10 are simultaneously upregulated. The proteolytic activity of the latter then releases the HER3 ligand heregulin from the cell surface to activate HER3 and confer resistance to trastuzumab by inducing compensatory growth factor receptor signaling. Blocking either HER3 or ADAM10 effectively reverts the acquired resistance to trastuzumab. Our data thus provide strategies to inhibit this signaling and circumvent resistance to trastuzumab.

Blocking Hedgehog release from pancreatic cancer cells increases paracrine signaling potency

ABSTRACT Members of the Hedgehog (Hh) family of morphogens play crucial roles in development but are also involved in the progression of certain types of cancer. Despite being synthesized as hydrophobic dually lipid-modified molecules, and thus being strongly membrane-associated, Hh ligands are able to spread through tissues and act on target cells several cell diameters away. Various mechanisms that mediate Hh release have been discussed in recent years; however, little is known about dispersion of this ligand from cancer cells. Using co-culture models in conjunction with a newly developed reporter system, we were able to show that different members of the ADAM family of metalloproteinases strongly contribute to the release of endogenous bioactive Hh from pancreatic cancer cells, but that this solubilization decreases the potency of cancer cells to signal to adjacent stromal cells in direct co-culture models. These findings imply that under certain conditions, cancer-cell-tethered Hh molecules are the more potent signaling activators and that retaining Hh on the surface of cancer cells can unexpectedly increase the effective signaling range of this ligand, depending on tissue context.

Stromal SPOCK1 supports invasive pancreatic cancer growth

Pancreatic ductal adenocarcinoma (PDAC) is marked by an abundant stromal deposition. This stroma is suspected to harbor both tumor‐promoting and tumor‐suppressing properties. This is underscored by the disappointing results of stroma targeting in clinical studies. Given the complexity of tumor–stroma interaction in PDAC, there is a need to identify the stromal proteins that are predominantly tumor‐promoting. One possible candidate is SPOCK1 that we previously identified in a screening effort in PDAC. We extensively mined PDAC gene expression datasets, and used species‐specific transcript analysis in mixed‐species models for PDAC to study the patterns and driver mechanisms of SPOCK1 expression in PDAC. Advanced organotypic coculture models with primary patient‐derived tumor cells were used to further characterize the function of this protein. We found SPOCK1 expression to be predominantly stromal. Expression of SPOCK1 was associated with poor disease outcome. Coculture and ligand stimulation experiments revealed that SPOCK1 is expressed in response to tumor cell‐derived transforming growth factor‐beta. Functional assessment in cocultures demonstrated that SPOCK1 strongly affects the composition of the extracellular collagen matrix and by doing so, enables invasive tumor cell growth in PDAC. By defining the expression pattern and functional properties of SPOCK1 in pancreatic cancer, we have identified a stromal mediator of extracellular matrix remodeling that indirectly affects the aggressive behavior of PDAC cells. The recognition that stromal proteins actively contribute to the protumorigenic remodeling of the tumor microenvironment should aid the design of future clinical studies to target specific stromal targets.

Patched-2 functions to limit Patched-1 deficient skin cancer growth

PurposeBasal cell carcinoma (BCC) is one of the most common skin cancers, and is typically driven by an aberrantly activated Hedgehog (Hh) pathway. The Hh pathway is regulated by interactions between the Patched-1 (Ptch1) and Smoothened (Smo) receptors. Smo is an activating receptor and is subject to inhibition by Ptch1. Following ligand binding to Ptch1, its inhibitory action is relieved and pathway activation occurs. This receptor interaction is pivotal to restraining uncontrolled cellular growth. Both receptors have been found to be frequently mutated in BCCs. Ptch2 is a Ptch1 paralog that exhibits overlapping functions in both normal development and tissue homeostasis. As yet, its contribution to cancer growth is poorly defined. Here we set out to assess how Ptch2 inhibits BCC growth.MethodsWe used several in vitro readouts for transcriptional and chemotactic Hh signaling in BCC-derived ASZ001 cells, and a novel xenograft model to assess in vivo BCC tumor growth. Gene editing by TALEN was used to untangle the different Ptch2-dependent responses to its ligand sonic hedgehog (Shh).ResultsWe first defined the signaling competence of Ptch2 in Ptch1-deficient ASZ001 cells in vitro, and found that Ptch2 ligand binding drives their migration rather than eliciting a transcriptional response. We found that subsequent targeting of Ptch2 abrogated the chemotaxic effect. Next, we tested the contribution of Ptch2 to in vivo tumor growth using a xenograft model and found that reduced Ptch function results in increased tumor growth, but that selective pressure appatently acts against complete Ptch2 ablation.ConclusionsWe conclude that like Ptch1, Ptch2 exerts a tumor-suppressive function in BCC cells, and that after targeting of both paralogs, ligand-independent activation of the Hh pathway contributes to tumor growth.

Abstract 5177: Heterogeneity of pancreatic ductal adenocarcinoma visualized

The outcome of pancreatic ductal adenocarcinoma (PDAC) remains dismal. Factors that contribute to the lethality of this disease include its intrinsic resistance to therapeutics, an aggressive growth pattern, and a large degree of both inter- as well as intratumor heterogeneity. All these features have been attributed (at least in part) to the presence and function of a population of pluripotent cells called cancer stem cells. Despite the alleged importance of these cells for clinical outcome, the methods that have been used to study these cells in PDAC are troubled by some serious caveats. Defining important quantitative parameters for these cells in an unbiased, marker-agnostic, way is called for and we aim to achieve this by unbiased tracing methods in patient derived xenografts. A panel of PDX-derived lines generated in our institute was labeled using the LeGO (Lentiviral Gene Ontology; Weber et al. 2012 Nat. Prot) system to enable tracking of single cell clones by color. The system utilizes three vectors coding for red, green, and blue fluorophores much like a television uses these colors to generate an almost infinite range of colors in the visible range. Nuances in color and intensity are generated by the many possible combinations of fluorophores, the different integration numbers of the genes coding for them, and the variations in expression levels determined by for instance the integration sites. The end result is a population of cells in which each cell is endowed with a unique color. This will allow detection of the offspring of such a cell by identifying clones within a structural unit in for instance a tumor, or in a culture dish, of similar color. The ability to give rise to such a structure is then, depending on the experimental context and outcome, considered a proxy for stem cell function. Several primary lines have proven amenable to LeGO-marking. These lines showed stable color patterns during culturing in vitro as analyzed by continuous FACS measurements, and tumors derived from marked cells did not show a difference to umarked tumors as assessed by gross histology and growth rate. We are currently testing the ability of these cells to faithfully occupy and mark structural units in PDAC, much like intestinal stem cells would in a lineage tracing experiment in the gut, and derive numbers for stem cell number as well as replacement rate. Experiments are currently being performed to assess the system9s potential to measure parameters associated with stemness, and possibly also differentiation status of the original tumor, as the cells grow in vitro. (Radio)chemotherapy regimens are included to formally address the purported resistance of cancer stem cells to these treatments without having to rely on an a priori chosen marker. Furthermore, more detailed comparison should reveal use of LeGO-derived data to measure the intra- and intertumor heterogeneity in PDAC tumor biology in our panel of PDXs. Citation Format: Veronique Veenstra, Helene Damhofer, Tom van Leusden, Jan Kessler, Jan Paul Medema, Hanneke van Laarhoven, Louis Vermeulen, Maarten Bijlsma. Heterogeneity of pancreatic ductal adenocarcinoma visualized. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2015-5177

Abstract B71: Elucidation of the stromal contribution to sonic hedgehog-dependent pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies with a dismal 5-year survival rate of less than 5%, urging the discovery of new therapeutic strategies. Although numerous signaling pathways are misregulated in PDAC, the developmental Hedgehog pathway has proven to be particularly crucial to the progression of these tumors. It was recently established that the ligand sonic hedgehog (Shh), aberrantly expressed in the tumor cells, signals through a paracrine loop to neighboring nontumor/stromal cells, which in turn produce factors that support tumor growth. We aim to elucidate the nature of these stromal factors and their crosstalk with the tumor cells to improve our understanding of the processes implicated in cancer development and progression. In order to identify the reciprocal signals from the stromal compartment, we employed a mixed-species in vitro model for Shh-dependent tumor-stroma interaction that mimics the growth conditions used for embryoid bodies. The species difference allows us to genetically dissect the interaction between the human tumor- and the mouse stromal cells by performing deep transcriptome sequencing (RNA-Seq). This experimental approach revealed numerous interesting extracellular proteins upregulated in response to Shh including several matrix metalloproteinases, tumor necrosis factor family members, growth factors, and chemokines. Validation of these factors in vitro, but also in vivo in an orthotopic mouse model of PDAC and on resected patient material should help to develop new potential leads for treatment strategies. Citation Format: Helene Damhofer, Maarten F. Bijlsma, Jan Paul Medema. Elucidation of the stromal contribution to sonic hedgehog-dependent pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B71.