Karen A. Olson

Endogenous angiogenin in endothelial cells is a general requirement for cell proliferation and angiogenesis.

Angiogenin is an angiogenic protein that undergoes nuclear translocation in endothelial cells where it accumulates in the nucleolus and stimulates rRNA transcription, a rate-limiting step in ribosome biogenesis, protein translation, and cell growth. Here, we report that angiogenin is required for cell proliferation induced by various other angiogenic proteins including acidic and basic fibroblast growth factors (aFGF and bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF). Downregulation of angiogenin in endothelial cells by small interfering RNA (siRNA) and antisense results in a decrease in rRNA transcription, ribosome biogenesis, and cell proliferation induced by these angiogenic factors. Inhibitors of the nuclear translocation of angiogenin abolish the angiogenic activities of these factors. Stable angiogenin antisense transfection in HeLa cells reduces tumor angiogenesis in athymic mice despite the elevated expression level of bFGF and VEGF. Thus, nuclear angiogenin assumes an essential role in endothelial cell proliferation and is necessary for angiogenesis induced by other angiogenic factors. Angiogenin-stimulated rRNA transcription in endothelial cells may thus serve as a crossroad in the process of angiogenesis induced by various angiogenic factors.

Angiogenin is translocated to the nucleus of HeLa cells and is involved in ribosomal RNA transcription and cell proliferation

Angiogenin is an angiogenic protein known to play a role in rRNA transcription in endothelial cells. Nuclear translocation of angiogenin in endothelial cells decreases as cell density increases and ceases when cells are confluent. Here we report that angiogenin is constantly translocated to the nucleus of HeLa cells in a cell density-independent manner. Down-regulation of angiogenin expression by antisense and RNA interference results in a decrease in rRNA transcription, ribosome biogenesis, proliferation, and tumorigenesis both in vitro and in vivo. Exogenous angiogenin rescues the cells from antisense and RNA interference inhibition. The results showed that angiogenin is constitutively translocated into the nucleus of HeLa cells where it stimulates rRNA transcription. Thus, besides its angiogenic activity, angiogenin also plays a role in cancer cell proliferation.

A small-molecule inhibitor of the ribonucleolytic activity of human angiogenin that possesses antitumor activity

The results of previous preclinical and clinical studies have identified angiogenin (ANG) as a potentially important target for anticancer therapy. Here we report the design and implementation of a high-throughput screening assay to identify small molecules that bind to the ribonucleolytic active site of ANG, which is critically involved in the induction of angiogenesis by this protein. Screening of 18,310 compounds from the National Cancer Institute (NCI) Diversity Set and ChemBridge DIVERSet yielded 15 hits that inhibit the enzymatic activity of ANG with Ki values <100 μM. One of these, NCI compound 65828 [8-amino-5-(4′-hydroxybiphenyl-4ylazo)naphthalene-2-sulfonate; Ki = 81 μM], was selected for more detailed studies. Minor changes in ANG or ligand structure markedly reduced potency, demonstrating that inhibition reflects active-site rather than nonspecific binding; these observations are consistent with a computationally generated model of the ANG⋅65828 complex. Local treatment with modest doses of 65828 significantly delayed the formation of s.c. tumors from two distinct human cancer cell types in athymic mice. ANG is the likely target involved because (i) a 65828 analogue with much lower potency against the enzymatic activity of ANG failed to exert any antitumor effect, (ii) tumors from 65828-treated mice had fewer interior blood vessels than those from control mice, and (iii) 65828 appears to have no direct effect on the tumor cells. Our findings provide considerable support for the targeting of the enzymatic active site of ANG as a strategy for developing new anticancer drugs.

Inhibition of prostate carcinoma establishment and metastatic growth in mice by an antiangiogenin monoclonal antibody.

A neutralizing monoclonal antibody (MAb) 26‐2F to human angiogenin, a potent inducer of neovascularization, has been shown previously to prevent or delay the appearance of angiogenin‐secreting human colon, fibrosarcoma and lung tumor cell xenografts implanted subcutaneously (s.c.) into athymic mice. In an analogous model system, we report here that the antibody also prevents the establishment of PC‐3 androgen‐independent human prostate cancer tumors in, on average, 40% of treated mice (p < 0.0001, survivor analysis). Intriguingly, combining MAb 26‐2F together with cisplatin and suramin, 2 therapeutic agents that together showed little antitumor activity in the aforementioned model, resulted in an even greater degree of protection (71% protected, p = 0.009 compared to antibody treatment alone). This protective effect persisted several weeks after cessation of treatment. Additionally, prophylactic systemic administration of MAb 26‐2F dramatically reduced by 50% the formation of spontaneous regional metastasis originating from primary growth in the prostate gland of PC‐3M cells, highly metastatic variants of PC‐3. Protection from metastasis was still significant when treatment with MAb 26‐2F was delayed until after the primary tumor was well established. The antibody is not directly cytotoxic to either cell type, both of which secrete angiogenin in vitro and when growing as tumors in vivo, but changes the pattern of vascularity in primary tumors growing orthotopically. These findings, together with the observation that angiogenin protein and mRNA are apparently overexpressed in cancerous vs. normal human prostate tissues, demonstrate that angiogenin antagonism represents a promising new approach for preventing progression and metastasis of clinical prostate cancer.

Neamine inhibits xenografic human tumor growth and angiogenesis in athymic mice.

Purpose: We have previously shown that the aminoglycoside antibiotic neomycin blocks the nuclear translocation of angiogenin and inhibits its angiogenic activity. However, neomycin has not been considered as a favorable drug candidate for clinical development because of its known nephrotoxicity and ototoxicity. The aim of this study is to determine whether neamine, a nontoxic derivative of neomycin, possesses antitumor activity. Experimental Design: The effect of neamine on the nuclear translocation of angiogenin was examined by means of immunofluorescence and Western blotting. The antitumor activity of neamine was determined with three different animal models. Results: Neamine effectively blocked the nuclear translocation of angiogenin in endothelial cells and inhibited angiogenin-induced cell proliferation. It inhibited the establishment of human tumor xenografts in athymic mice in both ectopic and orthotopic tumor models. It also inhibited the progression of established human tumor transplants, whereas the structurally related antibiotic paromomycin had no effect. Immunohistochemical staining showed that both angiogenesis and cancer cell proliferation are inhibited by neamine. Conclusion: These results suggest that the nontoxic aminoglycoside antibiotic neamine is an effective inhibitor of nuclear translocation of angiogenin and may serve as an inhibitor for angiogenin-induced angiogenesis and cancer progression.

The Crystal Structure of Human Angiogenin in Complex with an Antitumor Neutralizing Antibody

The murine monoclonal antibody 26-2F neutralizes the angiogenic and ribonucleolytic activities of human angiogenin (ANG) and is highly effective in preventing the establishment and metastatic dissemination of human tumors in athymic mice. Here we report a 2.0 A resolution crystal structure for the complex of ANG with the Fab fragment of 26-2F that reveals the detailed interactions between ANG and the complementarity-determining regions (CDRs) of the antibody. Surprisingly, Fab binding induces a dramatic conformational change in the cell binding region of ANG at the opposite end of the molecule from the combining site; crosslinking experiments indicate that this rearrangement also occurs in solution. The ANG-Fab complex structure should be invaluable for designing maximally humanized versions of 26-2F for potential clinical use.

Abstract LB-304: ATSP-9172, a novel Stapled Peptide inhibitor of HIF-dependent transcriptional activity with in vivo antitumor efficacy in a preclinical model of prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Hypoxia-inducible transcription factors (HIF) are key regulators of cellular adaptation to hypoxia in solid tumors, and HIF-1α controls the expression of genes involved in anaerobic metabolism, angiogenesis, cell growth and survival. Hydrocarbon cross-linked alpha helical peptides (Stapled Peptides) are a breakthrough approach to create new class of drugs that modulate intracellular protein-protein interactions. Here, we have mimicked the structure and function of the CITED2 protein, an endogenous negative regulator of the interaction between HIF-1α and p300 proteins, by designing a Stapled Peptide derived from CITED2 to generate the first example of a potent and selective Stapled Peptide inhibitor of HIF-1α-dependent transcription. ATSP-9172 bound to the CH1 domain of p300 and disrupted the HIF-1α C-TAD/CH1 interaction in biochemical assays and inhibited HIF-dependent reporter gene activity in ME-180 cells. Examination of endogenous transcript levels in ME-180 cells revealed that ATSP-9172 down-regulated the transcription of HIF-1α target genes, such as adolase C, angiopoietin-like 4, and carbonic anhydrase 9 in a dose-dependent manner, but did not affect the expression of non-HIF target genes, verifying a specific and on-target mechanism of action. ATSP-9172 exhibited a dramatic improvement in solubility and plasma stability profile relative to the linear peptide, and demonstrated favorable pharmacokinetic properties in mice by providing high systemic exposure, low plasma clearance and long elimination half-life. Finally, intravenous administration of ATSP-9172 on an every other day schedule significantly inhibited tumor growth in a PC-3 human prostate tumor xenograft model (p < 0.05); this inhibition was found to be dose-dependent. Our results demonstrate that a Stapled Peptide mimicking the HIF inhibitory function of the native CITED2 protein provides a novel and specific strategy to suppress HIF-1α-dependent gene expression for cancer therapy. Citation Format: Kaiming Sun, Steven J. DeMarco, Vincent Guerlavais, Aditi Mukherjee, Sean Irwin, Eric Shi, Hongliang Cai, Krzysztof Darlak, Solimar Santiago, Jessica Pero, Karen A. Olson, Huw M. Nash, Yong Chang. ATSP-9172, a novel Stapled Peptide inhibitor of HIF-dependent transcriptional activity with in vivo antitumor efficacy in a preclinical model of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-304. doi:10.1158/1538-7445.AM2013-LB-304