Patricia Jaaks

Targeting hedgehog signaling reduces self-renewal in embryonal rhabdomyosarcoma.

Current treatment regimens for rhabdomyosarcoma (RMS), the most common pediatric soft tissue cancer, rely on conventional chemotherapy, and although they show clinical benefit, there is a significant risk of adverse side effects and secondary tumors later in life. Therefore, identifying and targeting sub-populations with higher tumorigenic potential and self-renewing capacity would offer improved patient management strategies. Hedgehog signaling has been linked to the development of embryonal RMS (ERMS) through mouse genetics and rare human syndromes. However, activating mutations in this pathway in sporadic RMS are rare and therefore the contribution of hedgehog signaling to oncogenesis remains unclear. Here, we show by genetic loss- and gain-of-function experiments and the use of clinically relevant small molecule modulators that hedgehog signaling is important for controlling self-renewal of a subpopulation of RMS cells in vitro and tumor initiation in vivo. In addition, hedgehog activity altered chemoresistance, motility and differentiation status. The core stem cell gene NANOG was determined to be important for ERMS self-renewal, possibly acting downstream of hedgehog signaling. Crucially, evaluating the presence of a subpopulation of tumor-propagating cells in patient biopsies identified by GLI1 and NANOG expression had prognostic significance. Hence, this work identifies novel functional aspects of hedgehog signaling in ERMS, redefines the rationale for its targeting as means to control ERMS self-renewal and underscores the importance of studying functional tumor heterogeneity in pediatric cancers.

The proprotein convertase furin in tumour progression

Proprotein convertases are proteases that have been implicated in the activation of a wide variety of proteins. These proteins are generally synthesised as precursor proteins and require limited proteolysis for conversion into their mature bioactive counterparts. Many of these proteins, including metalloproteases, growth factors and their receptors or adhesion molecules, have been shown to facilitate tumour formation and progression. Hence, this review will focus on the proprotein convertase furin and its role in cancer. The expression of furin has been confirmed in a large spectrum of cancers such as head and neck squamous cell carcinoma, breast cancer and rhabdomyosarcoma. Functional studies modulating furin activity uncovered its importance for the processing of many cancer‐related substrates and strongly indicate that high furin activity promotes the malignant phenotype of cancer cells. In this review, we summarise the expression and function of furin in different cancer types, discuss its role in processing cancer‐related proproteins and give examples of potential therapeutic approaches that take advantage of the proteolytic activity of furin in cancer cells.

Transcription Factor Activities Enhance Markers of Drug Sensitivity in Cancer

Transcriptional dysregulation induced by aberrant transcription factors (TF) is a key feature of cancer, but its global influence on drug sensitivity has not been examined. Here, we infer the transcriptional activity of 127 TFs through analysis of RNA-seq gene expression data newly generated for 448 cancer cell lines, combined with publicly available datasets to survey a total of 1,056 cancer cell lines and 9,250 primary tumors. Predicted TF activities are supported by their agreement with independent shRNA essentiality profiles and homozygous gene deletions, and recapitulate mutant-specific mechanisms of transcriptional dysregulation in cancer. By analyzing cell line responses to 265 compounds, we uncovered numerous TFs whose activity interacts with anticancer drugs. Importantly, combining existing pharmacogenomic markers with TF activities often improves the stratification of cell lines in response to drug treatment. Our results, which can be queried freely at dorothea.opentargets.io, offer a broad foundation for discovering opportunities to refine personalized cancer therapies.Significance: Systematic analysis of transcriptional dysregulation in cancer cell lines and patient tumor specimens offers a publicly searchable foundation to discover new opportunities to refine personalized cancer therapies. Cancer Res; 78(3); 769-80. ©2017 AACR.

The Proprotein Convertase Furin Contributes to Rhabdomyosarcoma Malignancy by Promoting Vascularization, Migration and Invasion.

The proprotein convertase (PC) furin cleaves precursor proteins, an important step in the activation of many cancer-associated proteins. Substrates of furin and furin-like PCs play a role in proliferation, metastasis and invasion. Some of them are involved in the progression of the pediatric soft tissue sarcoma rhabdomyosarcoma (RMS). In this study, we show that PCs, and in particular furin, are expressed in RMS cell lines. To investigate the functional role of furin, we generated RMS cell lines with modulated furin activity. Silencing or stable inhibition of furin delayed tumor growth in Rh30 and RD xenografts in vivo, and was correlated with lower microvessel density. Reduced furin activity also decreased migration and invasion abilities in vitro, and inhibition of furin in RMS cells diminished processing of IGF1R, VEGF-C, PDGF-B and MT1-MMP, leading to lower levels of mature proteins. Furthermore, we found that furin activity is required for proper IGF signaling in RMS cells, as furin silencing resulted in reduced phosphorylation of Akt upon IGF1 stimulation. Taken together, our results suggest that furin plays an important role in the malignant phenotype of RMS cells by activating proteins involved in tumor growth and vascularization, metastasis and invasion.

The catalytic domain of inositol-1,4,5-trisphosphate 3-kinase-a contributes to ITPKA-induced modulation of F-actin

Inositol‐1,4,5‐trisphosphate‐3‐kinase‐A (ITPKA) has been considered as an actin bundling protein because its N‐terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross‐linked actin filaments, here we analyzed if full length‐ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F‐actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C‐terminal ABD, we exclude that ITPKA cross‐links actin filaments by simultaneous F‐actin binding with two different ABDs. Instead, stimulated‐emission‐depletion‐microscopy and measurement of InsP3Kinase activity give evidence that that N‐terminal ABD‐homodimers of ITPKA bind to F‐actin while the monomeric C‐termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3Kinase activity is doubled and the product of InsP3Kinase activity, Ins(1,3,4,5)P4, inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3Kinase domain substantially contributes to this process.

The proprotein convertase furin is required to maintain viability of alveolar rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Success of current therapies is still limited and outcome is particularly poor for metastatic alveolar rhabdomyosarcoma (aRMS). We previously identified the proprotein convertase furin as potential target for specific drug delivery with RMS-homing peptides. Furin is a protease that converts inactive precursor proteins into bioactive proteins and peptides. In this study, we investigate the biological role of furin in aRMS progression in vitro and in vivo. Furin expression was confirmed in over 86% RMS biopsies in a tissue microarray (n=89). Inducible furin silencing in vitro led to significant impairment of cell viability and proliferation in all investigated aRMS cell lines, but not in MRC5 fibroblasts. Furthermore, the aRMS cell lines Rh3 and Rh4 revealed to be very sensitive to furin silencing, undergoing caspase-dependent cell death. Notably, furin silencing in vivo led to complete remission of established Rh4 tumors and to delayed growth in Rh30 tumors. Taken together, these findings identify furin as an important factor for aRMS progression and survival. Thus, we propose furin as a novel therapeutic target for treatment of aRMS.

Prolonged circulation and increased tumor accumulation of liposomal vincristine in a mouse model of rhabdomyosarcoma

AIM Our goal was to improve vincristine (VCR) based rhabdomyosarcoma (RMS) therapy by encapsulating the drug into liposomes. A targeting strategy was attempted to enhance tumor accumulation. MATERIALS & METHODS VCR was loaded in control and peptide-decorated liposomes via an active method. The interaction of an RMS-specific peptide with the presumed target furin and the cellular uptake of both liposomal groups were studied in vitro. Pharmacokinetics and biodistribution of VCR-containing liposomes were assessed in an RMS xenograft mouse model. RESULTS Liposomes ensured high VCR concentration in plasma and in the tumor. Peptide-decorated liposomes showed modest uptake in RMS cells. CONCLUSION The investigated peptide-modified liposomal formulation may not be optimal for furin-mediated RMS targeting. Nevertheless, VCR-loaded liposomes could serve as a delivery platform for experimental RMS.

Patient-specific logic models of signaling pathways from screenings on cancer biopsies to prioritize personalized combination therapies

Mechanistic modeling of signaling pathways mediating patient-specific response to therapy can help to unveil resistance mechanisms and improve therapeutic strategies. Yet, creating such models for patients, in particular for solid malignancies, is challenging. A major hurdle to build these models is the limited material available, that precludes the generation of large-scale perturbation data. Here, we present an approach that couples ex vivo high-throughput screenings of cancer biopsies using microfluidics with logic-based modeling to generate patient-specific dynamic models of extrinsic and intrinsic apoptosis signaling pathways. We used the resulting models to investigate heterogeneity in pancreatic cancer patients, showing dissimilarities especially in the PI3K-Akt pathway. Variation in model parameters reflected well the different tumor stages. Finally, we used our dynamic models to efficaciously predict new personalized combinatorial treatments. Our results suggest our combination of microfluidic experiments and mathematical model can be a novel tool toward cancer precision medicine.

Transcription factor activities enhance markers of drug response in cancer

Transcriptional dysregulation is a key feature of cancer. Transcription factors (TFs) are the main link between signalling pathways and the transcriptional regulatory machinery of the cell, positioning them as key oncogenic inductors and therefore potential targets of therapeutic intervention. We implemented a computational pipeline to infer TF regulatory activities from basal gene expression and applied it to publicly available and newly generated RNA-seq data from a collection of 1,010 cancer cell lines and 9,250 primary tumors. We show that the predicted TF activities recapitulate known mechanisms of transcriptional dysregulation in cancer and dissect mutant-specific effects in driver genes. Importantly, we show the potential for predicted TF activities to be used as markers of sensitivity to the inhibition of their upstream regulators. Furthermore, combining these inferred activities with existing pharmacogenomic markers significantly improves the stratification of sensitive and resistant cell lines for several compounds. Our approach provides a framework to link driver genomic alterations with transcriptional dysregulation that helps to predict drug sensitivity in cancer and to dissect its mechanistic determinants.

Abstract 3571: Furin activity: A driver of rhabdomyosarcoma progression

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The prognosis and survival rate is often very poor and therefore new therapy approaches are required. Thus, we focus on 1) employing RMS-specific peptides for targeted delivery of chemotherapeutics to the tumor site and 2) identifying novel therapeutic targets. In a previous study we discovered that RMS-homing peptides bind preferably to the proprotein convertase furin. Initial investigations confirmed a high expression of furin throughout different pediatric soft tissue sarcoma types and hinted that furin promotes the tumorigenic phenotype of RMS cells in vitro as well as in corresponding xenografts in vivo. Here we present a novel approach of tetracycline-induced shRNA-based silencing of furin in vitro and in vivo in order to investigate in depth the role of furin in RMS progression. Furin depletion was confirmed at mRNA, protein and activity level and led to impaired maturation furin substrates insulin like growth factor 1 receptor (IGF1R) and transforming growth factor beta 1 (TGFβ-1). We found that loss of furin activity suppresses the malignant phenotype of Rh30 cells by decreasing proliferation and affecting formation of colonies. Furthermore, we observed increased caspase 3/7 activity and enrichment of nucleosomes in the cytoplasm upon furin depletion, thus hinting initiation of apoptotic processes. Induction of furin silencing in RMS xenografts in NOD/SCID mice delayed tumour growth, indicating a crucial role of furin in early phases of tumour growth. Taken together, our data underscore the importance of furin for RMS progression and therefore targeting the activity of furin represents a promising tool for treatment of RMS. Citation Format: Patricia AIM Jaaks, Gianmarco Meier, Beat W. Schafer, Michele Bernasconi. Furin activity: A driver of rhabdomyosarcoma progression. [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 3571. doi:10.1158/1538-7445.AM2015-3571