Dorina Veliceasa

Metronomic Low-Dose Chemotherapy Boosts CD95-Dependent Antiangiogenic Effect of the Thrombospondin Peptide ABT-510: A Complementation Antiangiogenic Strategy

Blocking angiogenesis is a promising approach in cancer therapy. Natural inhibitors of angiogenesis and derivatives induce receptor-mediated signals, which often result in the endothelial cell death. Low-dose chemotherapy, given at short regular intervals with no prolonged breaks (metronomic chemotherapy), also targets angiogenesis by obliterating proliferating endothelial cells and circulating endothelial cell precursors. ABT-510, a peptide derivative of thrombospondin, kills endothelial cell by increasing CD95L, a ligand for the CD95 death receptor. However, CD95 expression itself is unaffected by ABT-510 and limits its efficacy. We found that multiple chemotherapy agents, cyclophosphamide (cytoxan), cisplatin, and docetaxel, induced endothelial CD95 in vitro and in vivo at low doses that failed to kill endothelial cells (cytoxan > cisplatin > docetaxel). Thus, we concluded that some of these agents might complement each other and together block angiogenesis with maximal efficacy. As a proof of principle, we designed an antiangiogenic cocktail combining ABT-510 with cytoxan or cisplatin. Cyclophosphamide and cisplatin synergistically increased in vivo endothelial cell apoptosis and angiosuppression by ABT-510. This synergy required CD95, as it was reversible with the CD95 decoy receptor. In a mouse model, ABT-510 and cytoxan, applied together at low doses, acted in synergy to delay tumor take, to stabilize the growth of established tumors, and to cause a long-term progression delay of PC-3 prostate carcinoma. These antitumor effects were accompanied by major decreases in microvascular density and concomitant increases of the vascular CD95, CD95L, and apoptosis. Thus, our study shows a “complementation” design of an optimal cancer treatment with the antiangiogenic peptide and a metronomic chemotherapy.

miR-27b controls venous specification and tip cell fate

We discovered that miR-27b controls 2 critical vascular functions: it turns the angiogenic switch on by promoting endothelial tip cell fate and sprouting and it promotes venous differentiation. We have identified its targets, a Notch ligand Delta-like ligand 4 (Dll4) and Sprouty homologue 2 (Spry2). miR-27b knockdown in zebrafish and mouse tissues severely impaired vessel sprouting and filopodia formation. Moreover, miR-27b was necessary for the formation of the first embryonic vein in fish and controlled the expression of arterial and venous markers in human endothelium, including Ephrin B2 (EphB2), EphB4, FMS-related tyrosine kinase 1 (Flt1), and Flt4. In zebrafish, Dll4 inhibition caused increased sprouting and longer intersegmental vessels and exacerbated tip cell migration. Blocking Spry2 caused premature vessel branching. In contrast, Spry2 overexpression eliminated the tip cell branching in the intersegmental vessels. Blockade of Dll4 and Spry2 disrupted arterial specification and augmented the expression of venous markers. Blocking either Spry2 or Dll4 rescued the miR-27b knockdown phenotype in zebrafish and in mouse vascular explants, pointing to essential roles of these targets downstream of miR-27b. Our study identifies critical role of miR-27b in the control of endothelial tip cell fate, branching, and venous specification and determines Spry2 and Dll4 as its essential targets.

Biomimetic High Density Lipoprotein Nanoparticles For Nucleic Acid Delivery

We report a gold nanoparticle-templated high density lipoprotein (HDL AuNP) platform for gene therapy that combines lipid-based nucleic acid transfection strategies with HDL biomimicry. For proof-of-concept, HDL AuNPs are shown to adsorb antisense cholesterylated DNA. The conjugates are internalized by human cells, can be tracked within cells using transmission electron microscopy, and regulate target gene expression. Overall, the ability to directly image the AuNP core within cells, the chemical tailorability of the HDL AuNP platform, and the potential for cell-specific targeting afforded by HDL biomimicry make this platform appealing for nucleic acid delivery.

Transient potential receptor channel 4 controls thrombospondin-1 secretion and angiogenesis in renal cell carcinoma.

Angiogenic switch in renal cell carcinoma (RCC) is attributed to the inactivation of the von Hippel–Lindau tumor suppressor, stabilization of hypoxia inducible factor‐1 transcription factor and increased vascular endothelial growth factor. To evaluate the role of an angiogenesis inhibitor, thrombopsondin‐1 (TSP1), we compared TSP1 production in human RCC and normal tissue and secretion by the normal renal epithelium (human normal kidney, HNK) and RCC cells. Normal and RCC tissues stained positive for TSP1, and the levels of TSP1 mRNA and total protein were similar in RCC and HNK cells. However, HNK cells secreted high TSP1, which rendered them nonangiogenic, whereas RCC cells secreted little TSP1 and were angiogenic. Western blot and immunostaining revealed TSP1 in the cytoplasm of RCC cells on serum withdrawal, whereas, in HNK cells, it was rapidly exported. Seeking mechanisms of defective TSP1 secretion, we discovered impaired calcium uptake by RCC in response to vascular endothelial growth factor. In HNK cells, 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester, a calcium chelator, simulated TSP1 retention, mimicking the RCC phenotype. Further analysis revealed a profound decrease in transient receptor potential canonical ion channel 4 (TRPC4) Ca2+ channel expression in RCC cells. TRPC4 silencing in HNK cells caused TSP1 retention and impaired secretion. Double labeling of the secretory system components revealed TSP1 colocalization with coatomer protein II (COPII) anterograde vesicles in HNK cells. In contrast, in RCC cells, TSP1 colocalized with COPI vesicles, pointing to the retrograde transport to the endoplasmic reticulum caused by misfolding. Our study indicates that TRPC4 loss in RCC leads to impaired Ca2+ intake, misfolding, retrograde transport and diminished secretion of antiangiogenic TSP1, thus enabling angiogenic switch during RCC progression.

α1-antitrypsin inhibits angiogenesis and tumor growth

Disturbances of the ratio between angiogenic inducers and inhibitors in tumor microenvironment are the driving force behind angiogenic switch critical for tumor progression. Angiogenic inhibitors may vary depending on organismal age and the tissue of origin. We showed that α1‐antitrypsin (AAT), a serine protease inhibitor (serpin) is an inhibitor of angiogenesis, which induced apoptosis and inhibited chemotaxis of endothelial cells. S‐ and Z‐type mutations that cause abnormal folding and defective serpin activity abrogated AAT antiangiogenic activity. Removal of the C‐terminal reactive site loop had no effect on its angiostatic activity. Both native AAT and AAT truncated on C‐terminus (AATΔ) inhibited neovascularization in the rat cornea and delayed the growth of subcutaneous tumors in mice. Treatment with native AAT and truncated AATΔ, but not control vehicle reduced tumor microvessel density, while increasing apoptosis within tumor endothelium. Comparative analysis of the human tumors and normal tissues of origin showed correlation between reduced local α1‐antitrypsin expression and more aggressive tumor growth.

MiR-200b inhibits prostate cancer EMT, growth and metastasis

miRNA regulate gene expression at post-transcriptional level and fine-tune the key biological processes, including cancer progression. Here, we demonstrate the involvement of miR-200b in the metastatic spread of prostate cancer. We identified miR-200b as a downstream target of androgen receptor and linked its expression to decreased tumorigenicity and metastatic capacity of the prostate cancer cells. Overexpression of miR-200b in PC-3 cells significantly inhibited their proliferation and the formation of subcutaneous tumors. Moreover, in an orthotopic model, miR-200b blocked spontaneous metastasis and angiogenesis by PC-3 cells. This decreased metastatic potential was likely due to the reversal of the epithelial-to-mesenchymal transition, as was evidenced by increased pan-epithelial marker E-cadherin and specific markers of prostate epithelium, cytokeratins 8 and 18. In contrast, mesenchymal markers, fibronectin and vimentin, were significantly downregulated by miR-200b. Our results suggest an important role for miR-200b in prostate cancer progression and indicate its potential utility for prostate cancer therapy.

Androgen receptor targets NFκB and TSP1 to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti‐tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR‐expressing cells formed tumors in male mice at a much lower rate than the AR‐negative controls. Moreover, the AR‐expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti‐angiogenic protein, thrombospondin‐1. AR activation caused a decrease in RelA, a subunit of the pro‐survival transcription factor NFκB, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti‐apoptotic targets, Bcl‐2 and IL‐6. Increased apoptosis within AR‐expressing tumors was likely due to the NFκB suppression, since it was restricted to the cells lacking nuclear (active) NFκB. Thus we for the first time identified combined decrease of NFκB and increased TSP1 as molecular events underlying the AR anti‐tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early‐stage prostate cancer.

The many facets of PEDF in drug discovery and disease: a diamond in the rough or split personality disorder?

Introduction: Pigment epithelium-derived factor (PEDF) was discovered as a neurotrophic factor secreted by retinal pigment epithelial cells. A decade later, it re-emerged as a powerful angiogenesis inhibitor guarding ocular function. Since then, significant advances were made identifying PEDFs mechanisms, targets and biomedical applications. Areas covered: The authors review several methodologies that have generated significant new information about the potential of PEDF as a drug. Furthermore, the authors review and discuss mechanistic and structure–function analyses combined with the functional mapping of active fragments, which have yielded several short bioactive PEDF peptides. Additionally, the authors present functional studies in knockout animals and human correlates that have provided important information about conditions amenable to PEDF-based therapies. Expert opinion: Through its four known receptors, PEDF causes a wide range of cellular events vitally important for the organism, which include survival and differentiation, migration and invasion, lipid metabolism and stem cell maintenance. These processes are deregulated in multiple pathological conditions, including cancer, metabolic and cardiovascular disease. PEDF has been successfully used in countless preclinical models of these conditions and human correlates suggest a wide utility of PEDF-based drugs. The most significant clinical application of PEDF, to date, is its potential therapeutic use for age-related macular degeneration. Moreover, PEDF-based gene therapy has advanced to early stage clinical trials. PEDF active fragments have been mapped and used to design short peptide mimetics conferring distinct functions of PEDF, which may address specific clinical problems and become prototype drugs.

Androgen Receptor Drives Cellular Senescence

The accepted androgen receptor (AR) role is to promote proliferation and survival of prostate epithelium and thus prostate cancer progression. While growth-inhibitory, tumor-suppressive AR effects have also been documented, the underlying mechanisms are poorly understood. Here, we for the first time link AR anti-cancer action with cell senescence in vitro and in vivo. First, AR-driven senescence was p53-independent. Instead, AR induced p21, which subsequently reduced ΔN isoform of p63. Second, AR activation increased reactive oxygen species (ROS) and thereby suppressed Rb phosphorylation. Both pathways were critical for senescence as was proven by p21 and Rb knock-down and by quenching ROS with N-Acetyl cysteine and p63 silencing also mimicked AR-induced senescence. The two pathways engaged in a cross-talk, likely via PML tumor suppressor, whose localization to senescence-associated chromatin foci was increased by AR activation. All these pathways contributed to growth arrest, which resolved in senescence due to concomitant lack of p53 and high mTOR activity. This is the first demonstration of senescence response caused by a nuclear hormone receptor.

MCT-1 Oncogene Contributes to Increased In vivo Tumorigenicity of MCF7 Cells by Promotion of Angiogenesis and Inhibition of Apoptosis

Overexpression of a novel oncogene MCT-1 (multiple copies in a T cell malignancy) causes malignant transformation of murine fibroblasts. To establish its role in the pathogenesis of breast cancer in humans, we generated stable transfectants of MCF7 breast cancer cells negative for endogenous MCT-1 (MCF7-MCT-1). Overexpression of MCT-1 in these cells resulted in a slight elevation of estrogen receptor-alpha, and higher rates of DNA synthesis and growth in response to estradiol compared with the empty vector control (MCF7-EV). The pure antiestrogen fulvestrant inhibited the estradiol-stimulated proliferation of MCF7-MCT-1 cells. The MCF7-MCT-1 clones showed increased invasiveness in the presence of 50% serum compared with the MCF7-EV. In a tumor xenograft model, MCT-1-overexpressing cells showed higher take rates and formed significantly larger tumors than MCF7-EV controls. When we examined angiogenic phenotype and molecular mediators of angiogenesis in MCF7-MCT-1 tumors in vivo, we found greater microvascular density and lower apoptosis in the MCF7-MCT-1 tumors compared with MCF7-EV controls accompanied by a dramatic decline in the levels of angiogenesis inhibitor, thrombospondin-1 (TSP1). In vitro, blocking TSP1 in the medium conditioned by MCT-1-negative cells restored its angiogenic potential to that of the MCF7-MCT-1 cells. Conversely, despite an increase in mRNA encoding vascular endothelial growth factor upon MCT-1 overexpression, vascular endothelial growth factor protein levels have not been notably altered. Taken together, our results suggest that MCT-1 may contribute to the pathogenesis and progression of human breast cancer via at least two routes: promotion of angiogenesis through the decline of TSP1 and inhibition of apoptosis.