Person:

Zetter, Bruce

The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 or IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1(\alpha) and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies. Understanding the mechanisms that are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization.

Metastasis is a major cause of mortality and remains a hurdle in the search for a cure for cancer. Not much is known about metastatic cancer cells and endothelial cross-talk, which occurs at multiple stages during metastasis. Here we report a dynamic regulation of the endothelium by cancer cells through the formation of nanoscale intercellular membrane bridges, which act as physical conduits for transfer of microRNAs. The communication between the tumour cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits. These results lead us to define the notion of ‘metastatic hijack’: cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits. Pharmacological perturbation of these nanoscale membrane bridges decreases metastatic foci in vivo. Targeting these nanoscale membrane bridges may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer.

Background: Understanding the complex, multistep process of metastasis remains a major challenge in cancer research. Metastasis models can reveal insights in tumor development and progression and provide tools to test new intervention strategies. Methods: To develop a new cancer metastasis model, we used DU145 human prostate cancer cells and performed repeated rounds of orthotopic prostate injection and selection of subsequent lymph node metastases. Tumor growth, metastasis, cell migration and invasion were analyzed. Microarray analysis was used to identify cell migration- and cancer-related genes correlating with metastasis. Selected genes were silenced using siRNA, and their roles in cell migration and invasion were determined in transwell migration and Matrigel invasion assays. Results: Our in vivo cycling strategy created cell lines with dramatically increased tumorigenesis and increased ability to colonize lymph nodes (DU145LN1-LN4). Prostate tumor xenografts displayed increased vascularization, enlarged podoplanin-positive lymphatic vessels and invasive margins. Microarray analysis revealed gene expression profiles that correlated with metastatic potential. Using gene network analysis we selected 3 significantly upregulated cell movement and cancer related genes for further analysis: EPCAM (epithelial cell adhesion molecule), ITGB4 (integrin β4) and PLAU (urokinase-type plasminogen activator (uPA)). These genes all showed increased protein expression in the more metastatic DU145-LN4 cells compared to the parental DU145. SiRNA knockdown of EpCAM, integrin-β4 or uPA all significantly reduced cell migration in DU145-LN4 cells. In contrast, only uPA siRNA inhibited cell invasion into Matrigel. This role of uPA in cell invasion was confirmed using the uPA inhibitors, amiloride and UK122. Conclusions: Our approach has identified genes required for the migration and invasion of metastatic tumor cells, and we propose that our new in vivo model system will be a powerful tool to interrogate the metastatic cascade in prostate cancer.

Using an in vivo cycling strategy, we selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors. Repeated rounds of metastatic selection (LN1–LN4) progressively increased the epithelial phenotype, resulting in a new model of tumor cell mesenchymal-epithelial transition (MET). DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin. The MET in DU145-LN4 cells was accompanied by increased expression of the miR-200 family, and antimiRs to miR-200c and miR-141 induced an EMT. MET also correlated with the loss of miR-424. Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering. Our model provides evidence for spontaneous MET in vivo. We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

The development of controlled-release nanoparticle (NP) technologies has great potential to further improve the therapeutic efficacy of RNA interference (RNAi), by prolonging the release of small interfering RNA (siRNA) for sustained, long-term gene silencing. Herein, we present a NP platform with sustained siRNA-release properties, which can be self-assembled using biodegradable and biocompatible polymers and lipids. The hybrid lipid-polymer NPs showed excellent silencing efficacy, and the temporal release of siRNA from the NPs continued for over one month. When tested on luciferase-expressed HeLa cells and A549 lung carcinoma cells after short-term transfection, the siRNA NPs showed greater sustained silencing activity than lipofectamine 2000-siRNA complexes. More importantly, the NP-mediated sustained silencing of prohibitin 1 (PHB1) generates more effective tumor cell growth inhibition in vitro and in vivo than the lipofectamine complexes. We expect that this sustained-release siRNA NP platform could be of interest in both fundamental biological studies and clinical applications.

PURPOSE: It has been established that Sorsby's fundus dystrophy, a dominantly inherited form of blindness, is caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene. Because choroidal neovascularization is a prominent feature of Sorsby's fundus dystrophy, the authors have examined whether TIMP-3 protein plays a role in the regulation of angiogenesis. METHODS: Chemotaxis of endothelial cells toward vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was examined using a modified Boyden chamber assay. Endothelial cells placed in the upper chamber were allowed to migrate through a polycarbonate membrane with 8 microns pores toward VEGF or bFGF present in the lower chamber. Next, the ability of TIMP-3 to inhibit chemotaxis was studied by incubating the cells with varying amounts of TIMP-3 during the assay. Finally, an in vitro angiogenesis assay was performed on collagen gels. Endothelial cells were seeded onto three-dimensional collagen gels. Treatment with bFGF and VEGF induced invasion of the gel and the formation of tube-like structures. TIMPs (1, 2, and 3) were added to the cultures to determine their effect on invasion. An in vivo chorioallantoic membrane (CAM) assay was performed using methylcellulose discs containing bFGF with or without TIMP-3. Induction of new blood vessels was observed with a stereomicroscope. RESULTS: TIMP-3 inhibits chemotaxis of vascular endothelial cells toward VEGF and bFGF, inhibits collagen gel invasion and capillary morphogenesis in vitro, and inhibits bFGF-induced angiogenesis in the CAM assay in vivo. CONCLUSIONS: TIMP-3 has the potential to inhibit angiogenesis. These results allow us to speculate on a possible mechanism by which mutant TIMP-3 protein might contribute to the Sorsby fundus dystrophy phenotype.

Background Collagen XXIII is a transmembrane collagen previously shown to be upregulated in metastatic prostate cancer. This study’s purpose was to determine the protein expression of collagen XXIII in tumor tissues from a variety of cancers and to assess collagen XXIII’s utility as a biomarker for non small-cell lung cancer (NSCLC).

Methods A multi-cancer tissue microarray (TMA) was used for immunohistochemical examination of collagen XXIII protein expression in a variety of cancers. Subsequently, collagen XXIII expression was analyzed in three separate cohorts using TMAs with representative tumor and control lung tissues from NSCLC patients. In addition, NSCLC patient urine samples were analyzed for the presence of collagen XXIII via Western blot.

Results Collagen XXIII was present in tissue samples from a variety of cancers. Within lung cancer tissues, collagen XXIII staining was enriched in NSCLC subtypes. Collagen XXIII was present in 294 of 333 (88%) lung adenocarcinomas and 97 of 133 (73%) squamous cell carcinomas (SqCC). In urine, collagen XXIII was present in 23 of 29 (79%) NSCLC patient samples but only in 15 of 54 (28%) control samples. High collagen XXIII staining intensity correlated with shorter recurrence-free survival in NSCLC patients.

Conclusions We demonstrate the capability of collagen XXIII as a tissue and urinary biomarker for NSCLC, where positivity in tissue or urine significantly correlates with presence of NSCLC and high staining intensity is a significant recurrence predictor.

Impact Inclusion of collagen XXIII in a tissue or urine-based cancer biomarker panel could inform NSCLC patient treatment decisions.