Search

Chemotherapy has been reported to upregulate sphingomylinases and increase cellular ceramide, often linked to the induction to cell death. In this work, we show that sublethal doses of doxorubicin and vorinostat still increased cellular ceramide, which was located predominantly at the plasma membrane. To interrogate possible functions of this specific pool of ceramide, we used recombinant enzymes to mimic physiological levels of ceramide at the plasma membrane upon chemotherapy treatment. Using mass spectrometry and network analysis, followed by experimental confirmation, the results revealed that this pool of ceramide acutely regulates cell adhesion and cell migration pathways with weak connections to commonly established ceramide functions (eg, cell death). Neutral sphingomyelinase 2 (nSMase2) was identified as responsible for the generation of plasma membrane ceramide upon chemotherapy treatment, and both ceramide at the plasma membrane and nSMase2 were necessary and sufficient to mediate these “side” effects of chemotherapy on cell adhesion and migration. This is the first time a specific pool of ceramide is interrogated for acute signaling functions, and the results define plasma membrane ceramide as an acute signaling effector necessary and sufficient for regulation of cell adhesion and cell migration under chemotherapeutical stress. Fil: Canals, Daniel. Stony Brook University; State University of New York; Fil: Salamone, Silvia. Stony Brook University; State University of New York; Fil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Nemeth, Erika. Stony Brook University; State University of New York; Fil: Aguilar, Daniel. Biomedical Research Networking Center in Hepatic and Digestive Diseases; España Fil: Hernandez Corbacho, María José. Stony Brook University; State University of New York; Fil: Adada, Mohamad. Stony Brook University; State University of New York; Fil: Staquicini, Daniela I.. Rutgers Cancer Institute of New Jersey; Estados Unidos Fil: Arap, Wadih. Rutgers Cancer Institute of New Jersey; Estados Unidos Fil: Pasqualini, Renata. Rutgers Cancer Institute of New Jersey; Estados Unidos Fil: Haley, John. Stony Brook University; State University of New York; Fil: Obeid, Lina M.. Stony Brook University; State University of New York; Fil: Hannun, Yusuf A.. Stony Brook University; State University of New York

Here we review and discuss the link between regeneration capacity and tumor suppression comparing mammals (embryos versus adults) with highly regenerative vertebrates. Similar to mammal embryo morphogenesis, in amphibians (essentially newts and salamanders) the reparative process relies on a precise molecular and cellular machinery capable of sensing abnormal signals and actively reprograming or eliminating them. As the embryo's evil twin, tumor also retains common functional attributes. The immune system plays a pivotal role in maintaining a physiological balance to provide surveillance against tumor initiation or to support its initiation and progression. We speculate that susceptibility to cancer development in adult mammals may be determined by the loss of an advanced regenerative capability during evolution and believe that gaining mechanistic insights into how regenerative capacity linked to tumor suppression is postnatally lost in mammals might illuminate an as yet unrecognized route to cancer treatment.

BACKGROUND Receptors in tumor blood vessels are attractive targets for ligand-directed drug discovery and development. The authors have worked systematically to map human endothelial receptors (“vascular zip codes”) within tumors through direct peptide library selection in cancer patients. Previously, they selected a ligand-binding motif to the interleukin-11 receptor alpha (IL-11Rα) in the human vasculature. METHODS The authors generated a ligand-directed, peptidomimetic drug (bone metastasis-targeting peptidomimetic-11 [BMTP-11]) for IL-11Rα–based human tumor vascular targeting. Preclinical studies (efficacy/toxicity) included evaluating BMTP-11 in prostate cancer xenograft models, drug localization, targeted apoptotic effects, pharmacokinetic/pharmacodynamic analyses, and dose-range determination, including formal (good laboratory practice) toxicity across rodent and nonhuman primate species. The initial BMTP-11 clinical development also is reported based on a single-institution, open-label, first-in-class, first-in-man trial (National Clinical Trials number NCT00872157) in patients with metastatic, castrate-resistant prostate cancer. RESULTS BMTP-11 was preclinically promising and, thus, was chosen for clinical development in patients. Limited numbers of patients who had castrate-resistant prostate cancer with osteoblastic bone metastases were enrolled into a phase 0 trial with biology-driven endpoints. The authors demonstrated biopsy-verified localization of BMTP-11 to tumors in the bone marrow and drug-induced apoptosis in all patients. Moreover, the maximum tolerated dose was identified on a weekly schedule (20-30 mg/m2). Finally, a renal dose-limiting toxicity was determined, namely, dose-dependent, reversible nephrotoxicity with proteinuria and casts involving increased serum creatinine. CONCLUSIONS These biologic endpoints establish BMTP-11 as a targeted drug candidate in metastatic, castrate-resistant prostate cancer. Within a larger discovery context, the current findings indicate that functional tumor vascular ligand-receptor targeting systems may be identified through direct combinatorial selection of peptide libraries in cancer patients. Cancer 2015;121:2411–2421. © 2015 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

BACKGROUND The authors developed an ex vivo methodology to perform drug library screening against human leukemia. METHODS The strategy for this screening relied on human blood or bone marrow cultures under hypoxia; under these conditions, leukemia cells deplete oxygen faster than normal cells, causing a hemoglobin oxygenation shift. Several advantages were observed: 1) partial recapitulation of the leukemia microenvironment, 2) use of native hemoglobin oxygenation as a real-time sensor/reporter, 3) cost-effectiveness, 4) species specificity, and 5) a format that enables high-throughput screening. RESULTS For a proof of concept, a chemical library (size, approximately 20,000 compounds) was screened against human leukemia cells. Seventy compounds were identified (“hit” rate, 0.35%; Z-factor = 0.71) that had activity, and 20 compounds were examined to identify 18 true-positive compounds (90%). Finally, the results demonstrated that carbonohydraxonic diamide group-containing compounds are potent antileukemia agents that induce cell death in leukemia cells and in patient-derived samples. CONCLUSIONS The current results indicated that this unique functional assay can identify novel drug candidates and can help with the development of future applications in personalized drug selection for patients with leukemia. Cancer 2014;120:589–602. © 2013 American Cancer Society.

We discuss a case study that highlights the features and limitations of a principled Bayesian decision theoretic approach to massive multiple comparisons. We consider inference for a mouse phage display experiment with three stages. The data are tripeptide counts by tissue and stage. The primary aim of the experiment is to identify ligands that bind with high affinity to a given tissue. The inference goal is to select from a large list of peptide and tissue pairs those with significant increase over stages. The desired inference summary involves a massive multiplicity problem. We consider two alternative approaches to address this multiplicity issue. First we propose an approach based on the control of the posterior expected false discovery rate. We notice that the implied solution ignores the relative size of the increase. This motivates a second approach based on a utility function that includes explicit weights for the size of the increase.

We discuss inference for a human phage display experiment with three stages. The data are tripeptide counts by tissue and stage. The primary aim of the experiment is to identify ligands that bind with high affinity to a given tissue. We formalize the research question as inference about the monotonicity of mean counts over stages. The inference goal is then to identify a list of peptide-tissue pairs with significant increase over stages. We use a semi-parametric Dirichlet process mixture of Poisson model. The posterior distribution under this model allows the desired inference about the monotonicity of mean counts. However, the desired inference summary as a list of peptide-tissue pairs with significant increase involves a massive multiplicity problem. We consider two alternative approaches to address this multiplicity issue. First we propose an approach based on the control of the posterior expected false discovery rate. We notice that the implied solution ignores the relative size of the increase. This motivates a second approach based on a utility function that includes explicit weights for the size of the increase.

Delivery and penetration of chemotherapeutic drugs into tumors are limited by a number of factors related to abnormal vasculature and altered stroma composition in neoplastic tissues. Coupling of chemotherapeutic drugs with tumor vasculature-homing peptides or administration of drugs in combination with biological agents that affect the integrity of the endothelial lining of tumor vasculature is an appealing strategy to improve drug delivery to tumor cells. Promising approaches to achieve this goal are based on the use of Asn-Gly-Arg (NGR)-containing peptides as ligands for drug delivery and of NGR-TNF, a peptide-tumor necrosis factor-α fusion protein that selectively alters drug penetration barriers and that is currently tested in a randomized Phase III trial in patients with malignant pleural mesothelioma.

BACKGROUND: Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. In this study, the authors used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS: Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo that were designed to test the extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS: In the extracellular environment, the results confirmed that soluble osteopontin is required for its prometastatic effects; this phenomenon is specific, arginine-glycine-aspartic acid (RGD)-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induced rapid tyrosine 418 (Tyr-418) dephosphorylation of the cellular homolog of the Rous sarcoma virus (c-Src), with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation was followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSIONS: The results of this study revealed a complex molecular interaction as well as a dual role for osteopontin in metastasis that depends on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to antimetastasis strategies. Cancer 2009. © 2009 American Cancer Society.

BACKGROUND: Recently, considerable efforts have been directed toward antivascular therapy as a new modality to treat human cancers. However, targeting a therapeutic gene of interest to the tumor vasculature with minimal toxicity to other tissues remains the objective of antivascular gene therapy. Tumor necrosis factor-α (TNF-α) is a potent antivascular agent but has limited clinical utility because of significant systemic toxicity. At the maximum tolerated doses of systemic TNF-α, there is no meaningful antitumor activity. Hence, the objective of this study was to deliver TNF-α targeted to tumor vasculature by systemic delivery to examine its antitumor activity. METHODS: A hybrid adeno-associated virus phage vector (AAVP) was used that targets tumor endothelium to express TNF-α (AAVP-TNF-α). The activity of AAVP-TNF-α was analyzed in various in vitro and in vivo settings using a human melanoma tumor model. RESULTS: In vitro, AAVP-TNF-α infection of human melanoma cells resulted in high levels of TNF-α expression. Systemic administration of targeted AAVP-TNF-α to melanoma xenografts in mice produced the specific delivery of virus to tumor vasculature. In contrast, the nontargeted vector did not target to tumor vasculature. Targeted AAVP delivery resulted in expression of TNF-α, induction of apoptosis in tumor vessels, and significant inhibition of tumor growth. No systemic toxicity to normal organs was observed. CONCLUSIONS: Targeted AAVP vectors can be used to deliver TNF-α specifically to tumor vasculature, potentially reducing its systemic toxicity. Because TNF-α is a promising antivascular agent that currently is limited by its toxicity, the current results suggest the potential for clinical translation of this strategy. Cancer 2009. Published 2008 by the American Cancer Society.

BACKGROUND Previous studies of the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model vasculature suggest that, as tumors develop, vessels invade the glandular epithelium. However, changes in the vasculature are difficult to study in conventional thin tissue sections. The authors used a new approach to characterize morphologic and architectural changes of blood vessels and pericytes during tumor development in TRAMP mice. METHODS Eighty-micron cryostat sections of normal prostate and three histopathologic stages of TRAMP tumor sections, classified by epithelial cell E-cadherin immunoreactivity, were immunostained with vascular endothelial cell and pericyte receptor antibodies and evaluated by confocal microscopy. RESULTS In the normal mouse prostate, capillaries were most abundant in the fibromuscular tunica between the epithelium and smooth muscle of the ductules. In the prostatic intraepithelial neoplasia (PIN) stage, vessels accompanied epithelial cell protrusions into the ductule lumen but remained in the connective tissue at the basal side of the epithelium. Well differentiated tissues had extensive angiogenesis with five times the normal mean vascularity outside ductules. Vessels were of variable diameter, were associated with an increased number of pericytes, and some had endothelial sprouts. Angiogenic blood vessels from poorly differentiated adenocarcinomas were tortuous, variable in caliber, and lacked the normal hierarchy. Pericytes on these vessels had an abnormal phenotype manifested by α-smooth muscle actin expression and loose association with endothelial cells. Angiogenesis and loss of vascular hierarchy were also found in human prostate carcinoma. CONCLUSIONS Vascular abnormalities, which begin at the PIN stage and intensify in well differentiated and poorly differentiated tumors, may be useful readouts for early detection and treatment assessment in prostate carcinoma. Cancer 2005. © 2005 American Cancer Society.

Purpose: To locally increase the relative biological effectives (RBE) of proton therapy by introducing phage‐targeted‐gold‐nanoscaffolds into cancer cells. Methods: Prostate‐cancer cells were treated with a ligand‐directed nanoscaffold, a non‐targeted nanoscaffold, goldnanoparticles or no treatment. After incubating, the cells were irradiated in a clinical 160MeV proton beam to doses of 1, 2, 3, 4 and 6CGE. Two configurations were tested: with the cells at a depth of 2cm and spread‐out Bragg peak (SOBP) of 4cm and the other at a depth of 9cm with a 10cm SOBP. The clonogenic assay was performed post‐irradiation to determine the effects of the treatments on cell survival. We fit the surviving fraction (SF) data to the linear‐quadratic model for analysis. A control irradiation of cells in a Co‐60 unit was also performed. Results: Our analysis showed that all protontreatments resulted in lower SF than the Co‐60 treatment over the measured doses. For proton irradiations, the actively targeted nanoscaffold achieved the lowest SF compared to all of the control treatments. In the range of typical clinical fractionation, 1.8–4 CGE/fraction, the nanoscaffold treatment reduced the SF by 22%–42% more than the untreated cells for proton irradiation at 2cm. For the 9cm depth, the reduction in cancer cell SF was 8–33% for the targeted nanoscaffold versus the untreated cells. RBE calculated at 50% and 10% SF showed 27 and 28% enhancement at the 2cm depth and 7 and 18% at 9cm. Conclusions: The observed decrease in SF of cells treated with the targeted nanoscaffold prior to irradiation confirms that the nanoscaffold enhances the RBE of the radiation. The enhanced RBE is likely due to the specific internalization of the nanoscaffolds into cells. With further investigation, we believe that targeted nanoscaffolds will be a clinically viable platform to enhance the sensitivity of localized tumors to protonradiation. Conflict of Interest: Drs. Pasqualini and Arap have shared interest in Alvos Therapeutics.