Valentina Fogal

Cooperative nanomaterial system to sensitize, target, and treat tumors

A significant barrier to the clinical translation of systemically administered therapeutic nanoparticles is their tendency to be removed from circulation by the mononuclear phagocyte system. The addition of a targeting ligand that selectively interacts with cancer cells can improve the therapeutic efficacy of nanomaterials, although these systems have met with only limited success. Here, we present a cooperative nanosystem consisting of two discrete nanomaterials. The first component is gold nanorod (NR) “activators” that populate the porous tumor vessels and act as photothermal antennas to specify tumor heating via remote near-infrared laser irradiation. We find that local tumor heating accelerates the recruitment of the second component: a targeted nanoparticle consisting of either magnetic nanoworms (NW) or doxorubicin-loaded liposomes (LP). The targeting species employed in this work is a cyclic nine-amino acid peptide LyP-1 (Cys-Gly-Asn-Lys-Arg-Thr-Arg-Gly-Cys) that binds to the stress-related protein, p32, which we find to be upregulated on the surface of tumor-associated cells upon thermal treatment. Mice containing xenografted MDA-MB-435 tumors that are treated with the combined NR/LyP-1LP therapeutic system display significant reductions in tumor volume compared with individual nanoparticles or untargeted cooperative system.

Targeted Tumor-Penetrating siRNA Nanocomplexes for Credentialing the Ovarian Cancer Oncogene ID4

Tumor-penetrating siRNA nanocomplexes credential ID4 as a therapeutic oncogene target in human ovarian cancer. Nanotechnology Sets Sights on Ovarian Tumors In the world of anticancer research, targeting tumor cells is one challenge; penetrating the cells to deliver therapeutics is another. The combination of specific targeting and efficient delivery is the clinical holy grail, wherein optimization of this approach could lead to highly effective cancer therapy in humans. Ren et al. have now developed a nanotechnology platform that allows for just that: targeted intracellular delivery of RNA-based therapeutics to ovarian cancer cells, which halts the oncogenic activity of a potent gene, in this case ID4. In a screen of overexpressed and essential genes in human ovarian cancer, the authors first identified a potential oncogene, ID4. They then confirmed ID4 tumorigenicity and mechanism in vitro in cell lines. After confirming that ID4 was an oncogene, Ren et al. reasoned that “silencing” the gene using small interfering RNA (siRNA) would prevent tumor growth in vivo. The trick was to make sure the siRNA could cross the cell membrane to exert its silencing effects. To accomplish this, the authors designed a tumor-penetrating nanocomplex (TPN) that could not only bind a protein overexpressed on the surface of human cancer cells but also pass through the membrane via a cell-penetrating peptide. Once inside the cells, the TPN could release the siRNA directed against ID4. Tumor homing was confirmed in mouse models of human melanoma and ovarian cancer. In mice harboring subcutaneous ovarian tumors, TPN/siRNA decreased ID4 expression by up to 90% and suppressed tumor growth by 82%. In mice bearing disseminated intra-abdominal tumors, TPN/siRNA allowed 80% of the animals to live 60 or more days. Control treatments did not prevent tumor growth in either study, and the TPN/siRNA therapy did not elicit any immunogenic side effects. Locked and loaded with siRNA, these TPNs are ready to target and kill cancer cells. The authors envision this to be a platform for credentialing oncogenes and for validating RNA interference in preclinical models before development of therapeutics. However, before moving this TPN/siRNA approach to patients, some additional preclinical optimization is necessary, including pharmacokinetics, testing in human cancer models, and increasing siRNA efficiency at lower doses. The comprehensive characterization of a large number of cancer genomes will eventually lead to a compendium of genetic alterations in specific cancers. Unfortunately, the number and complexity of identified alterations complicate endeavors to identify biologically relevant mutations critical for tumor maintenance because many of these targets are not amenable to manipulation by small molecules or antibodies. RNA interference provides a direct way to study putative cancer targets; however, specific delivery of therapeutics to the tumor parenchyma remains an intractable problem. We describe a platform for the discovery and initial validation of cancer targets, composed of a systematic effort to identify amplified and essential genes in human cancer cell lines and tumors partnered with a novel modular delivery technology. We developed a tumor-penetrating nanocomplex (TPN) that comprised small interfering RNA (siRNA) complexed with a tandem tumor-penetrating and membrane-translocating peptide, which enabled the specific delivery of siRNA deep into the tumor parenchyma. We used TPN in vivo to evaluate inhibitor of DNA binding 4 (ID4) as a novel oncogene. Treatment of ovarian tumor–bearing mice with ID4-specific TPN suppressed growth of established tumors and significantly improved survival. These observations not only credential ID4 as an oncogene in 32% of high-grade ovarian cancers but also provide a framework for the identification, validation, and understanding of potential therapeutic cancer targets.

Specific penetration and accumulation of a homing peptide within atherosclerotic plaques of apolipoprotein E-deficient mice

The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1+ cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1–coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[18F]fluorobenzoic acid ([18F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [18F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [18F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions.

Cell surface nucleolin antagonist causes endothelial cell apoptosis and normalization of tumor vasculature

Nucleolin is specifically transported to the surface of proliferating endothelial cells in vitro and in vivo. In contrast to its well defined functions in the nucleus and cytoplasm, the function of cell surface nucleolin is poorly defined. We have previously identified the nucleolin-binding antibody NCL3 that specifically binds to cell surface nucleolin on angiogenic blood vessels in vivo and is internalized into the cell. Here, we show that NCL3 inhibits endothelial tube formation in vitro as well as angiogenesis in the matrigel plaque assay and subcutaneous tumor models in vivo. Intriguingly, the specific targeting of proliferating endothelial cells by NCL3 in subcutaneous tumor models leads to the normalization of the tumor vasculature and as a result to an increase in tumor oxygenation. Treatment of endothelial cells with anti-nucleolin antibody NCL3 leads to a decrease of mRNA levels of the anti-apoptotic molecule Bcl-2 and as a consequence induces endothelial cell apoptosis as evidenced by PARP cleavage. These data reveal a novel mode of action for anti-angiogenic therapy and identify cell surface nucleolin as a novel target for combinatorial chemotherapy.

Novel anti-glioblastoma agents and therapeutic combinations identified from a collection of FDA approved drugs

BackgroundGlioblastoma (GBM) is a therapeutic challenge, associated with high mortality. More effective GBM therapeutic options are urgently needed. Hence, we screened a large multi-class drug panel comprising the NIH clinical collection (NCC) that includes 446 FDA-approved drugs, with the goal of identifying new GBM therapeutics for rapid entry into clinical trials for GBM.MethodsScreens using human GBM cell lines revealed 22 drugs with potent anti-GBM activity, including serotonergic blockers, cholesterol-lowering agents (statins), antineoplastics, anti-infective, anti-inflammatories, and hormonal modulators. We tested the 8 most potent drugs using patient-derived GBM cancer stem cell-like lines. Notably, the statins were active in vitro; they inhibited GBM cell proliferation and induced cellular autophagy. Moreover, the statins enhanced, by 40-70 fold, the pro-apoptotic activity of irinotecan, a topoisomerase 1 inhibitor currently used to treat a variety of cancers including GBM. Our data suggest that the mechanism of action of statins was prevention of multi-drug resistance protein MDR-1 glycosylation. This drug combination was synergistic in inhibiting tumor growth in vivo. Compared to animals treated with high dose irinotecan, the drug combination showed significantly less toxicity.ResultsOur data identifies a novel combination from among FDA-approved drugs. In addition, this combination is safer and well tolerated compared to single agent irinotecan.ConclusionsOur study newly identifies several FDA-approved compounds that may potentially be useful in GBM treatment. Our findings provide the basis for the rational combination of statins and topoisomerase inhibitors in GBM.

In vitro and in vivo anticancer effects of mevalonate pathway modulation on human cancer cells

Background:The increasing usage of statins (the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) has revealed a number of unexpected beneficial effects, including a reduction in cancer risk.Methods:We investigated the direct anticancer effects of different statins approved for clinical use on human breast and brain cancer cells. We also explored the effects of statins on cancer cells using in silico simulations.Results:In vitro studies showed that cerivastatin, pitavastatin, and fluvastatin were the most potent anti-proliferative, autophagy inducing agents in human cancer cells including stem cell-like primary glioblastoma cell lines. Consistently, pitavastatin was more effective than fluvastatin in inhibiting U87 tumour growth in vivo. Intraperitoneal injection was much better than oral administration in delaying glioblastoma growth. Following statin treatment, tumour cells were rescued by adding mevalonate and geranylgeranyl pyrophosphate. Knockdown of geranylgeranyl pyrophosphate synthetase-1 also induced strong cell autophagy and cell death in vitro and reduced U87 tumour growth in vivo. These data demonstrate that statins main effect is via targeting the mevalonate synthesis pathway in tumour cells.Conclusions:Our study demonstrates the potent anticancer effects of statins. These safe and well-tolerated drugs need to be further investigated as cancer chemotherapeutics in comprehensive clinical studies.

High-efficiency liposomal encapsulation of a tyrosine kinase inhibitor leads to improved in vivo toxicity and tumor response profile

Staurosporine (STS) is a potent pan-kinase inhibitor with marked activity against several chemotherapy-resistant tumor types in vitro. The translational progress of this compound has been hindered by poor pharmacokinetics and toxicity. We sought to determine whether liposomal encapsulation of STS would enhance antitumor efficacy and reduce toxicity, thereby supporting the feasibility of further preclinical development. We developed a novel reverse pH gradient liposomal loading method for STS, with an optimal buffer type and drug-to-lipid ratio. Our approach produced 70% loading efficiency with good retention, and we provide, for the first time, an assessment of the in vivo antitumor activity of STS. A low intravenous dose (0.8 mg/kg) inhibited U87 tumors in a murine flank model. Biodistribution showed preferential tumor accumulation, and body weight data, a sensitive index of STS toxicity, was unaffected by liposomal STS, but did decline with the free compound. In vitro experiments revealed that liposomal STS blocked Akt phosphorylation, induced poly(ADP-ribose) polymerase cleavage, and produced cell death via apoptosis. This study provides a basis to explore further the feasibility of liposomally encapsulated STS, and potentially related compounds for the management of resistant solid tumors.

A novel small molecule inhibitor of p32 mitochondrial protein overexpressed in glioma

BackgroundThe mitochondrial protein p32 is a validated therapeutic target of cancer overexpressed in glioma. Therapeutic targeting of p32 with monoclonal antibody or p32-binding LyP-1 tumor-homing peptide can limit tumor growth. However, these agents do not specifically target mitochondrial-localized p32 and would not readily cross the blood–brain barrier to target p32-overexpressing gliomas. Identifying small molecule inhibitors of p32 overexpressed in cancer is a more rational therapeutic strategy. Thus, in this study we employed a pharmacophore modeling strategy to identify small molecules that could bind and inhibit mitochondrial p32.MethodsA pharmacophore model of C1q and LyP-1 peptide association with p32 was used to screen a virtual compound library. A primary screening assay for inhibitors of p32 was developed to identify compounds that could rescue p32-dependent glutamine-addicted glioma cells from glutamine withdrawal. Inhibitors from this screen were analyzed for direct binding to p32 by fluorescence polarization assay and protein thermal shift. Affect of the p32 inhibitor on glioma cell proliferation was assessed by Alamar Blue assay, and affect on metabolism was examined by measuring lactate secretion.ResultsIdentification of a hit compound (M36) validates the pharmacophore model. M36 binds directly to p32 and inhibits LyP-1 tumor homing peptide association with p32 in vitro. M36 effectively inhibits the growth of p32 overexpressing glioma cells, and sensitizes the cells to glucose depletion.ConclusionsThis study demonstrates a novel screening strategy to identify potential inhibitors of mitochondrial p32 protein overexpressed in glioma. High throughput screening employing this strategy has potential to identify highly selective, potent, brain-penetrant small molecules amenable for further drug development.

Abstract 1856: Preclinical study for statins as anticancer drug.

Statins are a class of drug that inhibits 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase that is the rate-limiting enzyme of cholesterols synthesis. Now, statins are among the most prescribed drug used for treatment of hypercholesterolemia by lower down the serum cholesterol concentration and it is also a major means to preventing and reducing the risk for cardiovascular diseases. In addition to prevent and treat cardiovascular diseases, the emerging evidences indicated the statins usage has a number of beneficial effect such as anti-inflammation and lowering down the risk of cancer. To investigate the anti-cancer effect statins drugs that have been approved to clinical usage we evaluated the inhibition of breast cancer and GBM cell line growth. Statins are also potent inhibitors to stem cell-like primary GBM cells freshly isolated from patients. Unexpected, statins treatment induces strong cell autophagy death signals as weak if not at all cellular apoptosis. Tumor cells can be rescued after statin treatment by adding intermediated product of cholesterol synthesis (mevalonate and GGPP), it indicates that the target of the statins is specifically related the cholesterols synthesis pathway. Knock-down the expression of geranylgeranyl pyrophosphate synthetase-1 (GGPS-1), another key enzyme in cholesterol synthesis pathway, also stimulated strong cell autophagy and cell death in vitro, also dramatically reduced U87 tumor growth in vivo. In vivo data also showed that directly injection of statin is better than oral administrate to delay GBM tumor growth. This study showed statins are potent anti-cancer drug in vitro and in animal model. These safe and well-tolerate drugs are good candidates for clinical test as cancer chemotherapy reagents. Citation Format: Pengfei Jiang, Rajesh Mukthavavam, Ying Chao, Natsuko Nomura, Valentina Fogal, Sandra Pastorino, Ila Summit, Santosh Kesari. Preclinical study for statins as anticancer drug. [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 1856. doi:10.1158/1538-7445.AM2013-1856 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5203: Lineage-specific mechanisms regulating PDGFRα oncogenic activity in glioblastoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The highly heterogeneous nature of human tumors is a confounding feature which hinders understanding of the biology of cancer as well as design of effective therapies. Although a number of oncogenes and tumor suppressors, such as PI3K, Ras, TP53, CDKN2A, are frequently altrered across cancers of different origin, it is now apparent that cell lineage exerts a substantial effect on the distribution of genetic alterations in tumors. Consistent with the more recent notion that normal tissue stem cells or partially differentiated progenitor cells may serve as the cells of origin that undergo oncogenic transformation, it is conceivable to think that the cellular programs governing normal lineage development do also underlie the tumorigenic mechanisms. Therefore, the recognition of the tumor dependencies to oncogene functions in a lineage-specific context may offer more meaningful approaches for targeted cancer therapy. PDGF/PDGFRA signaling plays a particularly important role in the development of the brain, as a potent mitogen of oligodendrocyte precursor cells; it is also expressed in the type B neural stem cell population in the subventricular zone of adult brain. PDGF signaling has also been widely implicated in the formation of brain tumors. The Cancer Genome Atlas effort has revealed that overexpression and/or amplification of PDGFRA is the key feature of the proneural subtype of Glioblastomas (GBM), which constitutes 20-30% of all GBMs, occurs in younger patients and is extremely resistant to chemo- and radio-therapy. In proneural GBMs, PDGFRA expression clusters together with other proneural developmental genes such as Olig2 and Sox2. The targeting of PDGFRα has shown limited success in the setting of randomized clinical trials and in in vitro studies employing stable tumor cell lines. However, using a clinically relevant model, our data show that fresh patient-derived GBM stem like cells (Pt-GSLCs) belonging to the proneural subtype are particularly vulnerable to the inhibition of PDGFRα, both in vitro and in vivo, indicating a possible state of addiction to PDGFRα signaling. Interestingly, we observed that the neural transcription factor Olig2, might play a role in PDGFRα addiction. Inhibition of PDGFRα decreased phosphorylation of Olig2, while PDGFRα-inhibitor resistant lines upregulate the levels of Olig2 protein. Moreover Olig2 inhibition potentiates the effects of PDGFRα inhibition in proneural GBMs. Finally, PDGFRα and Olig2 inhibitors sensitize proneural GBM to radiation. These results provide an indication that oncogene deregulation may be “conditioned” by tumor lineage. We are now further investigating the lineage-specific mechanisms regulating addiction to PDGFRα of the proneural subtype of GBM with the hope to establish new pharmacological targets for the treatment of Glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5203. doi:1538-7445.AM2012-5203