Claudia Meier

DNp73 Exerts Function in Metastasis Initiation by Disconnecting the Inhibitory Role of EPLIN on IGF1R-AKT/STAT3 Signaling

Dissemination of cancer cells from primary tumors is the key event in metastasis, but specific determinants are widely unknown. Here, we show that DNp73, an inhibitor of the p53 tumor suppressor family, drives migration and invasion of nonmetastatic melanoma cells. Knockdown of endogenous DNp73 reduces this behavior in highly metastatic cell lines. Tumor xenografts expressing DNp73 show a higher ability to invade and metastasize, while growth remains unaffected. DNp73 facilitates an EMT-like phenotype with loss of E-cadherin and Slug upregulation. We provide mechanistic insight toward regulation of LIMA1/EPLIN by p73/DNp73 and demonstrate a direct link between the DNp73-EPLIN axis and IGF1R-AKT/STAT3 activation. These findings establish initiation of the invasion-metastasis cascade via EPLIN-dependent IGF1R regulation as major activity of DNp73.

Development of Adenoviral Delivery Systems to Target Hepatic Stellate Cells In Vivo

Hepatic stellate cells (HSCs) are known as initiator cells that induce liver fibrosis upon intoxication or other noxes. Deactivation of this ongoing remodeling process of liver parenchyma into fibrotic tissue induced by HSCs is an interesting goal to be achieved by targeted genetic modification of HSCs. The most widely applied approach in gene therapy is the utilization of specifically targeted vectors based on Adenovirus (Ad) serotype 5. To narrow down the otherwise ubiquitous tropism of parental Ad, two modifications are required: a) ablating the native tropism and b) redirecting the vector particles towards a specific entity solely present on the cells of interest. Therefore, we designed a peptide of the nerve growth factor (NGFp) with specific affinity for the p75 neurotrophin receptor (p75NTR) present on HSCs. Coupling of this NGFp to vector particles was done either via chemical conjugation using bifunctional polyethylene glycol (PEG) or, alternatively, by molecular bridging with a fusion protein specific for viral fiber knob and p75NTR. Both Ad vectors transmit the gene for the green fluorescent protein (GFP). GFP expression was monitored in vitro on primary murine HSCs as well as after systemic administration in mice with healthy and fibrotic livers using intravital fluorescence microscopy. Coupling of NGFp to Ad via S11 and/or PEGylation resulted in markedly reduced liver tropism and an enhanced adenoviral-mediated gene transfer to HSCs. Transduction efficiency of both specific Ads was uniformly higher in fibrotic livers, whereas Ad.GFP-S11-NGFp transduce activated HSCs better than Ad.GFP-PEG-NGFp. These experiments contribute to the development of a targeted gene transfer system to specifically deliver antifibrotic compounds into activated HSCs by systemically applied adenoviral vector modified with NGFp.

Association of RHAMM with E2F1 promotes tumour cell extravasation by transcriptional up-regulation of fibronectin.

Dissemination of cancer cells from primary to distant sites is a complex process; little is known about the genesis of metastatic changes during disease development. Here we show that the metastatic potential of E2F1‐dependent circulating tumour cells (CTCs) relies on a novel function of the hyaluronan‐mediated motility receptor RHAMM. E2F1 directly up‐regulates RHAMM, which in turn acts as a co‐activator of E2F1 to stimulate expression of the extracellular matrix protein fibronectin. Enhanced fibronectin secretion links E2F1/RHAMM transcriptional activity to integrin‐β1–FAK signalling associated with cytoskeletal remodelling and enhanced tumour cell motility. RHAMM depletion abolishes fibronectin expression and cell transmigration across the endothelial layer in E2F1‐activated cells. In a xenograft model, knock‐down of E2F1 or RHAMM in metastatic cells protects the liver parenchyma of mice against extravasation of CTCs, whereas the number of transmigrated cells increases in response to E2F1 induction. Expression data from clinical tissue samples reveals high E2F1 and RHAMM levels that closely correlate with malignant progression. These findings suggest a requirement for RHAMM in late‐stage metastasis by a mechanism involving cooperative stimulation of fibronectin, with a resultant tumourigenic microenvironment important for enhanced extravasation and distant organ colonization. Therefore, stimulation of the E2F1–RHAMM axis in aggressive cancer cells is of high clinical significance. Targeting RHAMM may represent a promising approach to avoid E2F1‐mediated metastatic dissemination. Copyright

MiR-182 promotes cancer invasion by linking RET oncogene activated NF-κB to loss of the HES1/Notch1 regulatory circuit

BackgroundDominant-activating mutations in the RET proto-oncogene, a receptor tyrosine kinase, are responsible for the development of medullary thyroid carcinoma (MTC) and causative for multiple endocrine neoplasia (MEN) type 2A and 2B. These tumors are highly aggressive with a high propensity for early metastasis and chemoresistance. This attribute makes this neoplasia an excellent model for probing mechanisms underlying cancer progression.MethodsThe expression level of miR-182 was measured in MTC tumor specimens and in TT cells by real-time RT-PCR. TT cells and modified NThy-ori 3.1 that stably express RETM918T were used to investigate RET-dependent regulation of miR-182. Identification and validation of miR-182 targets and pathways was accomplished with luciferase assays, qRT-PCR, Western blotting and immunofluorescence. In vitro, overexpression and knockdown experiments were carried out to examine the impact of miR-182 and HES1 on invasion and migration.ResultsWe found that miR-182 expression is significantly upregulated in MTC patient samples and tumor-derived cell lines harboring mutated RET. Inhibition of RET oncogenic signaling through a dominant-negative RET∆TK mutant in TT cells reduces miR-182, whereas overexpression of RETM918T in NThy-ori 3.1 cells increases miR-182 levels. We further show that overexpression of this miRNA in NThy.miR-182 cells promotes the invasive and migratory properties without affecting cell proliferation. MiR-182 is upregulated after RET induced NF-κB translocation into the nucleus via binding of NF-κB to the miR-182 promoter. Database analysis revealed that HES1, a repressor of the Notch pathway, is a target of miR-182, whose upregulation correlates with loss of HES1 transcription in MTC tissue samples and mutant RET cell lines. Moreover, we demonstrated that the 3′UTR of the HES1 mRNA bearing the targeting sequence for miR-182 clearly reduced luciferase reporter activity in cells expressing miR-182. Decreased expression of HES1 promotes migration by upregulating Notch1 inhibitor Deltex1 and consequent repression of Notch1.ConclusionWe demonstrate a novel mechanism for MTC aggressiveness in which mutated RET/NF-κB-driven expression of miR-182 impedes HES1 activation in a negative feedback loop. This observation might open new possibilities to treat RET oncogene associated metastatic cancer.

p73 and IGF1R Regulate Emergence of Aggressive Cancer Stem-like Features via miR-885-5p Control

Cancer stem-like cells (CSC) have been proposed to promote cancer progression by initiating tumor growth at distant sites, suggesting that stem-like cell features can support metastatic efficiency. Here, we demonstrate that oncogenic DNp73, a dominant-negative variant of the tumor-suppressor p73, confers cancer cells with enhanced stem-like properties. DNp73 overexpression in noninvasive melanoma and lung cancer cells increased anchorage-independent growth and elevated the expression of the pluripotency factors CD133, Nanog, and Oct4. Conversely, DNp73 depletion in metastatic cells downregulated stemness genes, attenuated sphere formation and reduced the tumor-initiating capability of spheroids in tumor xenograft models. Mechanistic investigations indicated that DNp73 acted by attenuating expression of miR-885-5p, a direct regulator of the IGF1 receptor (IGF1R) responsible for stemness marker expression. Modulating this pathway was sufficient to enhance chemosensitivity, overcoming DNp73-mediated drug resistance. Clinically, we established a correlation between low p73 function and high IGF1R/CD133/Nanog/Oct4 levels in melanoma specimens that associated with reduced patient survival. Our work shows how DNp73 promotes cancer stem-like features and provides a mechanistic rationale to target the DNp73-IGF1R cascade as a therapeutic strategy to eradicate CSC.

Novel subventricular zone early progenitor cell-specific adenovirus for in vivo therapy of central nervous system disorders reinforces brain stem cell heterogeneity

Neural stem/progenitor cells (NSPCs) have the potential to self-renew and to generate all neural lineages as well as to repopulate damaged areas in the brain. Our previous targeting strategies have indicated precursor cell heterogeneity between different brain regions that warrants the development of NSPC-specific delivery vehicles. Here, we demonstrate a target-specific adenoviral vector system for the in vivo manipulation of progenitor cells in the subventricular zone of the adult mouse brain. For this purpose, we identified a series of peptide ligands via phage display. The peptide with the highest affinity, SNQLPQQ, was expressed in conjunction with a bispecific adaptor molecule. To verify the targeting potential of the specific peptide, green fluorescent protein-expressing Ad vectors were coupled with the adaptor molecule and injected into the subventricular region of adult mice by stereotaxic surgery. An efficient and selective transduction of NSPCs in the SVZ was achieved, whereas hippocampal NSPCs were negative. Our results offer an expeditious and simple tool to produce retargeted viral vectors for a specific and direct in vivo manipulation of these progenitor cells. This powerful technique provides an opportunity to develop innovative strategies and express therapeutic genes in specific types of neural progenitor cells to allow success in treatment of brain disorders.

106. Novel Subventricular Zone Early Progenitor Cell Specific Adenovirus for In Situ Brain Stem Cell Bioengineering Unveils Cellular Heterogeneity

Neural stem/progenitor cells (NSPC) have the potential to self-renew and to generate all neural lineages as well as to repopulate damaged areas in the brain. Our recent endeavors in the generation of Ad vectors that specifically target SPC in situ have focused on linking selective peptides to viral particles that direct the vector to yet unknown receptors on the surface of stem cells. Using an Ad-PEGylation approach, we demonstrated that a short artificial peptide, which specifically binds neural precursor cells established from the hippocampal area of adult mice or rats, was highly effective in targeting NSPC after vector administration into the brain. These studies also revealed that long-term transgene expression was restricted to precursor cells located in dentate gyrus, and was not detected in other brain regions such as the SVZ and the ependyma of the lateral ventricle. This new indirect evidence for precursor cell heterogeneity in different brain regions that warrants the development of NSPC-specific delivery vehicles was further investigated. Here, we demonstrate a target-specific adenoviral vector system for in vivo manipulation of stem cells in the subventricular zone of the adult mouse brain. For this purpose, we identified a series of peptide ligands via phage display. The peptide with the highest affinity was expressed in conjunction with a bispecific adaptor molecule. In order to verify the targeting potential of the specific peptide, green fluorescent protein-expressing Ad vectors were coupled with the adaptor molecule and injected into the subventricular region of adult mice by stereotaxic surgery. An efficient and selective transduction of NSPC in the SVZ was achieved, whereas hippocampal NSPC were negative. Our results offer an expeditious and simple tool to produce target-specific viral vectors for selective and direct bioengineering of stem cells in situ. This powerful technique provides an excellent opportunity to gain new insight into the fundamental mechanisms that control stem cell fate and to develop innovative strategies for a successfull treatment of brain disorders.This work was supported by the German Research Foundation (DFG) grant PU 188/8-1, WR 20/2-1 and Research funding provided by the Medical Faculty of Rostock University (FORUN program).

Abstract 4099: IGF1R signaling connects DNp73-mediated EMT with the acquisition of cancer stem cell properties

Metastatic spread is the major cause of cancer-associated mortality and induced in primary tumor cells by the acquisition of mesenchymal features. Emerging evidence suggests that metastasis-initiating cancer cells undergoing epithelial-mesenchymal transition (EMT) possess stem cell-like traits. In this study, we demonstrate that oncogenic DNp73, an inhibitor of p53 tumor suppressor family members, has a fundamental role in driving both, transition into a mesenchymal state and providing cancer cells with stem cell-like properties through constitutive activation of IGF1R signaling. In patient melanoma tissues, DNp73 expression correlates with increased Breslow depth and is enriched in skin cancer metastases. Knockdown of endogenous DNp73 in highly metastatic cell lines abolishes the aggressive phenotype, whereas tumor xenografts overexpressing DNp73 possess significantly greater capacity to invade the surrounding tissue and form metastatic lesions. Transition from an epithelial to a motile mesenchymal phenotype is accompanied by a drastic reorganization of the cytoskeleton and modulation of cell-cell adhesion molecules. Importantly, regulation of EMT by DNp73 is induced through activation of IGF1R-AKT signaling followed by an increase of Slug and loss of E-cadherin. Additionally, we identified a novel interplay between DNp73 and IGF1R in the development of cancer stemness. Tumor spheres derived from metastatic melanoma cells with endogenously high DNp73 display a strong capacity for self-renewal and tumor growth after subcutaneous injection in mice compared to DNp73-depleted cancer stem cells. Our results clearly demonstrate that DNp73-driven stemness involves IGF1R activation. Selective inhibition of IGF1R blocks DNp73-mediated upregulation of Nanog, Oct4 and CD133, which is accompanied by a decrease in spheroid formation and tumor invasiveness. Taken together, our findings show for the first time that DNp73 is crucial to endow cancer cells with mesenchymal properties and the ability to self-renew, which is responsible for malignant progression and tumor dissemination. Moreover, we establish IGF1R as driving force for a DNp73-induced aggressive cancer phenotype, highlighting the DNp73-IGF1R cascade as promising therapeutic target to selectively prevent metastasis initiation. Citation Format: Claudia Meier, Marc Steder, Philip Hardtstock, Vijay Alla, Brigitte M. Putzer. IGF1R signaling connects DNp73-mediated EMT with the acquisition of cancer stem cell properties. [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 4099. doi:10.1158/1538-7445.AM2015-4099