Marina Cardo Vila

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• Bearss, J., Bearss, J., Singh, N., Singh, N., Padi, S. K., Padi, S. K., Okumura, K., Okumura, K., Kraft, A. S., Kraft, A. S., Cardo-vila, M., & Cardo-Vila, M. (2021). Abstract 2296: Regulation of P-body dynamics and formation in tumors through EDC3 phosphorylation by PIM and AKT. Cancer Research, 81(13_Supplement), 2296-2296. doi:10.1158/1538-7445.am2021-2296
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  Abstract Processing bodies (P-bodies) are cytoplasmic mRNA granules that form a hub to regulate mRNA translation via controlling decapping, degradation, and storage. P-bodies markedly increase as part of a stress response for example during nutrient deprivation and hypoxia. Thus, P-bodies could regulate cell fate by tuning protein levels during the stress, and thereby contribute to tumor initiation, growth, and metastasis. However, it is unclear how P-body formation is regulated by these stress and what biologic pathways P-body controls. Dysregulation of PIM and AKT kinases has been found in multiple cancers, including prostate and breast cancers. We find that the protein Enhancer of mRNA-decapping Protein 3 (EDC3), a P-body component, binds with the PIM protein kinase and is phosphorylated on serine 161 by the PIM and AKT kinases, suggesting these kinases could regulate P-body formation and function. Thus, we hypothesize that these oncogenic kinases regulate the EDC3 activity and P-body function via the phosphorylation of EDC3. Suppression of EDC3 phosphorylation by inhibiting PIM and AKT activities with drugs leads to an increase in P-body number. A knock-in mutation substituting alanine for serine 161 in PC3-LN4 prostate cancer cell line inhibited growth, migration and invasion in vitro. Prostate cancer tumor growth in these cells containing EDC3 mutation was reduced in mouse xenograft model, suggesting that this single phosphorylation is essential for regulating cell growth. These results suggest that the level of specific RNAs and its translation is being controlled by this mutation. Indeed, integrin family proteins, ITGB1 and ITGA6, are shown to be decreased in these cells consistent with the inability of these cells to attach and invade. High levels of phosphorylated EDC3 were detected in the breast cancer cell lines tested, whereas non-transformed immortalized breast epithelial cell lines showed much lower EDC3 phosphorylation. IHC staining of human breast cancer samples with phospho-specific antibodies demonstrated that EDC3 was highly phosphorylated in contrast to normal breast tissue which demonstrated low level of phosphorylation. These data indicate that in tumors, breast and prostate cancer, with activated PIM and AKT, high phosphorylation of EDC3 would increase the translation of target mRNAs via reducing their storage and destruction and affecting tumor cell growth and motility. Therefore, the phosphorylation of EDC3 by Pim and AKT kinases can be a driver of malignancy by controlling mRNA levels in breast and prostate cancers. Citation Format: Jeremiah Bearss, Sathish K. Padi, Neha Singh, Marina Cardo-Vila, Andrew S. Kraft, Koichi Okumura. Regulation of P-body dynamics and formation in tumors through EDC3 phosphorylation by PIM and AKT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2296.
• Bearss, J., Padi, S., Singh, N., Cardo Vila, M., Song, J., Mouneimne, G., Fernandes, N., Li, Y., Harter, M., Gard, J., Peti, W., Cress, A. E., Nelson, A., Buchan, J. R., Kraft, A., & Okumura, K. (2021). EDC3 Phosphorylation Regulates Growth and Invasion through controlling P-body Formation and Dynamics. EMBO Report.
• Paluri, S. L., Burak, M., Senejani, A. G., Levinson, M., Rahim, T., Clairmont, K., Kashgarian, M., Alvarado-Cruz, I., Meas, R., Cardó-Vila, M., Zeiss, C., Maher, S., Bothwell, A. L., Coskun, E., Kant, M., Jaruga, P., Dizdaroglu, M., Stephen Lloyd, R., & Sweasy, J. B. (2021). DNA glycosylase deficiency leads to decreased severity of lupus in the Polb-Y265C mouse model. DNA repair, 105, 103152.
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  The Polb gene encodes DNA polymerase beta (Pol β), a DNA polymerase that functions in base excision repair (BER) and microhomology-mediated end-joining. The Pol β-Y265C protein exhibits low catalytic activity and fidelity, and is also deficient in microhomology-mediated end-joining. We have previously shown that the Polb and Polb mice develop lupus. These mice exhibit high levels of antinuclear antibodies and severe glomerulonephritis. We also demonstrated that the low catalytic activity of the Pol β-Y265C protein resulted in accumulation of BER intermediates that lead to cell death. Debris released from dying cells in our mice could drive development of lupus. We hypothesized that deletion of the Neil1 and Ogg1 DNA glycosylases that act upstream of Pol β during BER would result in accumulation of fewer BER intermediates, resulting in less severe lupus. We found that high levels of antinuclear antibodies are present in the sera of Polb mice deleted of Ogg1 and Neil1 DNA glycosylases. However, these mice develop significantly less severe renal disease, most likely due to high levels of IgM in their sera.
• Staquicini, D. I., Barbu, E. M., Zemans, R. L., Dray, B. K., Staquicini, F. I., Dogra, P., Cardo Vila, M., Miranti, C., Baze, W. B., Villa, L. L., Kalil, J., Sharma, G., Prossnitz, E. R., Wang, Z., Cristini, V., Sidman, R. L., Berman, A. R., Panettieri, Jr, R. A., Tuder, R. M., , Pasqualini, R., et al. (2021). Targeted Phage Display-based Pulmonary Vaccination in Mice and Non-human Primates. Med (Cell Press), 2(3), 321-342.
• Staquicini, F. I., Hajitou, A., Driessen, W. H., Proneth, B., Cardo Vila, M., Staquicini, D. I., Markosian, C., Hoh, M., Cortez, M., Hooda-Nehra, A., Jaloudi, M., Silva, I. T., Buttura, J., Nunes, D. N., Dias-Neto, E., Eckhardt, B., Ruiz-Ramírez, J., Dogra, P., Wang, Z., , Cristini, V., et al. (2021). An unrecognized functional interface among tumor-associated macrophages, a vitamin D receptor, and immune-response in triple-negative breast cancer. Elife.
• Nollet, E. A., Cardo-Vila, M., Ganguly, S. S., Tran, J. D., Schulz, V. V., Cress, A., Corey, E., & Miranti, C. K. (2020). Androgen receptor-induced integrin α6β1 and Bnip3 promote survival and resistance to PI3K inhibitors in castration-resistant prostate cancer. Oncogene, 39(31), 5390-5404.
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  The androgen receptor (AR) is the major driver of prostate cancer growth and survival. However, almost all patients relapse with castration-resistant disease (CRPC) when treated with anti-androgen therapy. In CRPC, AR is often aberrantly activated independent of androgen. Targeting survival pathways downstream of AR could be a viable strategy to overcome CRPC. Surprisingly, little is known about how AR drives prostate cancer survival. Furthermore, CRPC tumors in which Pten is lost are also resistant to eradication by PI3K inhibitors. We sought to identify the mechanism by which AR drives tumor survival in CRPC to identify ways to overcome resistance to PI3K inhibition. We found that integrins α6β1 and Bnip3 are selectively elevated in CRPC downstream of AR. While integrin α6 promotes survival and is a direct transcriptional target of AR, the ability of AR to induce Bnip3 is dependent on adhesion to laminin and integrin α6β1-dependent nuclear translocation of HIF1α. Integrins α6β1 and Bnip3 were found to promote survival of CRPC cells selectively on laminin through the induction of autophagy and mitophagy. Furthermore, blocking Bnip3 or integrin α6β1 restored sensitivity to PI3K inhibitors in Pten-negative CRPC. We identified an AR driven pathway that cooperates with laminin and hypoxia to drive resistance to PI3K inhibitors. These findings can help explain in part why PI3K inhibitors have failed in clinical trials to overcome AR-dependent CRPC.
• Padi, S. K., Singh, N., Bearss, J. J., Olive, V., Song, J. H., Cardó-Vila, M., Kraft, A. S., & Okumura, K. (2019). Phosphorylation of DEPDC5, a component of the GATOR1 complex, releases inhibition of mTORC1 and promotes tumor growth. Proceedings of the National Academy of Sciences of the United States of America, 116(41), 20505-20510.
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  The Pim and AKT serine/threonine protein kinases are implicated as drivers of cancer. Their regulation of tumor growth is closely tied to the ability of these enzymes to mainly stimulate protein synthesis by activating mTORC1 (mammalian target of rapamycin complex 1) signaling, although the exact mechanism is not completely understood. mTORC1 activity is normally suppressed by amino acid starvation through a cascade of multiple regulatory protein complexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP domain containing protein 5), a component of GATOR1 complex, contains Pim and AKT protein kinase phosphorylation consensus sequences. DEPDC5 phosphorylation by Pim and AKT kinases was confirmed in cancer cells through the use of phospho-specific antibodies and transfection of phospho-inactive DEPDC5 mutants. Consistent with these findings, during amino acid starvation the elevated expression of Pim1 overcame the amino acid inhibitory protein cascade and activated mTORC1. In contrast, the knockout of DEPDC5 partially blocked the ability of small molecule inhibitors against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. In animal experiments knocking in a glutamic acid (S1530E) in DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Our results indicate a phosphorylation-dependent regulatory mechanism targeting DEPDC5 through which Pim1 and AKT act as upstream effectors of mTORC1 to facilitate proliferation and survival of cancer cells.
• Bosseboeuf, A., Feron, D., Tallet, A., Rossi, C., Charlier, C., Garderet, L., Caillot, D., Moreau, P., Cardó-Vila, M., Pasqualini, R., Arap, W., Nelson, A. D., Wilson, B. S., Perreault, H., Piver, E., Weigel, P., Girodon, F., Harb, J., Bigot-Corbel, E., & Hermouet, S. (2017). Monoclonal IgG in MGUS and multiple myeloma targets infectious pathogens. JCI insight, 2(19).
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  Subsets of mature B cell neoplasms are linked to infection with intracellular pathogens such as Epstein-Barr virus (EBV), hepatitis C virus (HCV), or Helicobacter pylori. However, the association between infection and the immunoglobulin-secreting (Ig-secreting) B proliferative disorders remains largely unresolved. We investigated whether the monoclonal IgG (mc IgG) produced by patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM) targets infectious pathogens. Antigen specificity of purified mc IgG from a large patient cohort (n = 244) was determined using a multiplex infectious-antigen array (MIAA), which screens for reactivity to purified antigens or lysates from 9 pathogens. Purified mc IgG from 23.4% of patients (57 of 244) specifically recognized 1 pathogen in the MIAA. EBV was the most frequent target (15.6%), with 36 of 38 mc IgGs recognizing EBV nuclear antigen-1 (EBNA-1). MM patients with EBNA-1-specific mc IgG (14.0%) showed substantially greater bone marrow plasma cell infiltration and higher β2-microglobulin and inflammation/infection-linked cytokine levels compared with other smoldering myeloma/MM patients. Five other pathogens were the targets of mc IgG: herpes virus simplex-1 (2.9%), varicella zoster virus (1.6%), cytomegalovirus (0.8%), hepatitis C virus (1.2%), and H. pylori (1.2%). We conclude that a dysregulated immune response to infection may underlie disease onset and/or progression of MGUS and MM for subsets of patients.
• Lewis, V. O., Devarajan, E., Cardó-Vila, M., Thomas, D. G., Kleinerman, E. S., Marchiò, S., Sidman, R. L., Pasqualini, R., & Arap, W. (2017). BMTP-11 is active in preclinical models of human osteosarcoma and a candidate targeted drug for clinical translation. Proceedings of the National Academy of Sciences of the United States of America, 114(30), 8065-8070.
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  Osteosarcoma occurs predominantly in children and young adults. High-grade tumors require multidisciplinary treatment consisting of chemotherapy in the neoadjuvant and adjuvant settings, along with surgical intervention. Despite this approach, death from respiratory failure secondary to the development and progression of pulmonary metastases remains a significant problem. Here, we identify the IL-11 receptor α subunit (IL-11Rα) as a cell surface marker of tumor progression that correlates with poor prognosis in patients with osteosarcoma. We also show that both IL-11Rα and its ligand, IL-11, are specifically up-regulated in human metastatic osteosarcoma cell lines; engagement of this autocrine loop leads to tumor cell proliferation, invasion, and anchorage-independent growth in vitro. Consistently, IL-11Rα promotes lung colonization by human metastatic osteosarcoma cells in vivo in an orthotopic mouse model. Finally, we evaluate the IL-11Rα-targeted proapoptotic agent bone metastasis-targeting peptidomimetic (BMTP-11) in preclinical models of primary intratibial osteosarcomas, observing marked inhibition of both tumor growth and lung metastases. This effect was enhanced when BMTP-11 was combined with the chemotherapeutic drug gemcitabine. Our combined data support the development of approaches targeting IL-11Rα, and establish BMTP-11 as a leading drug candidate for clinical translation in patients with high-risk osteosarcoma.
• Cardó-Vila, M., Marchiò, S., Sato, M., Staquicini, F. I., Smith, T. L., Bronk, J. K., Yin, G., Zurita, A. J., Sun, M., Behrens, C., Sidman, R. L., Lee, J. J., Hong, W. K., Wistuba, I. I., Arap, W., & Pasqualini, R. (2016). Interleukin-11 Receptor Is a Candidate Target for Ligand-Directed Therapy in Lung Cancer: Analysis of Clinical Samples and BMTP-11 Preclinical Activity. The American journal of pathology, 186(8), 2162-2170.
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  We previously isolated an IL-11-mimic motif (CGRRAGGSC) that binds to IL-11 receptor (IL-11R) in vitro and accumulates in IL-11R-expressing tumors in vivo. This synthetic peptide ligand was used as a tumor-targeting moiety in the rational design of BMTP-11, which is a drug candidate in clinical trials. Here, we investigated the specificity and accessibility of IL-11R as a target and the efficacy of BMTP-11 as a ligand-targeted drug in lung cancer. We observed high IL-11R expression levels in a large cohort of patients (n = 368). In matching surgical specimens (i.e., paired tumors and nonmalignant tissues), the cytoplasmic levels of IL-11R in tumor areas were significantly higher than in nonmalignant tissues (n = 36; P = 0.003). Notably, marked overexpression of IL-11R was observed in both tumor epithelial and vascular endothelial cell membranes (n = 301; P 
• Dobroff, A. S., D'Angelo, S., Eckhardt, B. L., Ferrara, F., Staquicini, D. I., Cardó-Vila, M., Staquicini, F. I., Nunes, D. N., Kim, K., Driessen, W. H., Hajitou, A., Lomo, L. C., Barry, M., Krishnamurthy, S., Sahin, A., Woodward, W. A., Prossnitz, E. R., Anderson, R. L., Dias-Neto, E., , Brown-Glaberman, U. A., et al. (2016). Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes. Proceedings of the National Academy of Sciences of the United States of America, 113(45), 12780-12785.
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  Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human virus thymidine kinase type-1 () transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.
• Dondossola, E., Dobroff, A. S., Marchiò, S., Cardó-Vila, M., Hosoya, H., Libutti, S. K., Corti, A., Sidman, R. L., Arap, W., & Pasqualini, R. (2016). Self-targeting of TNF-releasing cancer cells in preclinical models of primary and metastatic tumors. Proceedings of the National Academy of Sciences of the United States of America, 113(8), 2223-8.
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  Circulating cancer cells can putatively colonize distant organs to form metastases or to reinfiltrate primary tumors themselves through a process termed "tumor self-seeding." Here we exploit this biological attribute to deliver tumor necrosis factor alpha (TNF), a potent antitumor cytokine, directly to primary and metastatic tumors in a mechanism that we have defined as "tumor self-targeting." For this purpose, we genetically engineered mouse mammary adenocarcinoma (TSA), melanoma (B16-F10), and Lewis lung carcinoma cells to produce and release murine TNF. In a series of intervention trials, systemic administration of TNF-expressing tumor cells was associated with reduced growth of both primary tumors and metastatic colonies in immunocompetent mice. We show that these malignant cells home to tumors, locally release TNF, damage neovascular endothelium, and induce massive cancer cell apoptosis. We also demonstrate that such tumor-cell-mediated delivery avoids or minimizes common side effects often associated with TNF-based therapy, such as acute inflammation and weight loss. Our study provides proof of concept that genetically modified circulating tumor cells may serve as targeted vectors to deliver anticancer agents. In a clinical context, this unique paradigm represents a personalized approach to be translated into applications potentially using patient-derived circulating tumor cells as self-targeted vectors for drug delivery.
• Ferrara, F., Staquicini, D. I., Driessen, W. H., D'Angelo, S., Dobroff, A. S., Barry, M., Lomo, L. C., Staquicini, F. I., Cardó-Vila, M., Soghomonyan, S., Alauddin, M. M., Flores, L. G., Arap, M. A., Lauer, R. C., Mathew, P., Efstathiou, E., Aparicio, A. M., Troncoso, P., Navone, N. M., , Logothetis, C. J., et al. (2016). Targeted molecular-genetic imaging and ligand-directed therapy in aggressive variant prostate cancer. Proceedings of the National Academy of Sciences of the United States of America, 113(45), 12786-12791.
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  Aggressive variant prostate cancers (AVPC) are a clinically defined group of tumors of heterogeneous morphologies, characterized by poor patient survival and for which limited diagnostic and treatment options are currently available. We show that the cell surface 78-kDa glucose-regulated protein (GRP78), a receptor that binds to phage-display-selected ligands, such as the SNTRVAP motif, is a candidate target in AVPC. We report the presence and accessibility of this receptor in clinical specimens from index patients. We also demonstrate that human AVPC cells displaying GRP78 on their surface could be effectively targeted both in vitro and in vivo by SNTRVAP, which also enabled specific delivery of siRNA species to tumor xenografts in mice. Finally, we evaluated ligand-directed strategies based on SNTRVAP-displaying adeno-associated virus/phage (AAVP) particles in mice bearing MDA-PCa-118b, a patient-derived xenograft (PDX) of castration-resistant prostate cancer bone metastasis that we exploited as a model of AVPC. For theranostic (a merging of the terms therapeutic and diagnostic) studies, GRP78-targeting AAVP particles served to deliver the human virus thymidine kinase type-1 () gene, which has a dual function as a molecular-genetic sensor/reporter and a cell suicide-inducing transgene. We observed specific and simultaneous PET imaging and treatment of tumors in this preclinical model of AVPC. Our findings demonstrate the feasibility of GPR78-targeting, ligand-directed theranostics for translational applications in AVPC.
• Hosoya, H., Dobroff, A. S., Driessen, W. H., Cristini, V., Brinker, L. M., Staquicini, F. I., Cardó-Vila, M., D'Angelo, S., Ferrara, F., Proneth, B., Lin, Y. S., Dunphy, D. R., Dogra, P., Melancon, M. P., Stafford, R. J., Miyazono, K., Gelovani, J. G., Kataoka, K., Brinker, C. J., , Sidman, R. L., et al. (2016). Integrated nanotechnology platform for tumor-targeted multimodal imaging and therapeutic cargo release. Proceedings of the National Academy of Sciences of the United States of America, 113(7), 1877-82.
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  A major challenge of targeted molecular imaging and drug delivery in cancer is establishing a functional combination of ligand-directed cargo with a triggered release system. Here we develop a hydrogel-based nanotechnology platform that integrates tumor targeting, photon-to-heat conversion, and triggered drug delivery within a single nanostructure to enable multimodal imaging and controlled release of therapeutic cargo. In proof-of-concept experiments, we show a broad range of ligand peptide-based applications with phage particles, heat-sensitive liposomes, or mesoporous silica nanoparticles that self-assemble into a hydrogel for tumor-targeted drug delivery. Because nanoparticles pack densely within the nanocarrier, their surface plasmon resonance shifts to near-infrared, thereby enabling a laser-mediated photothermal mechanism of cargo release. We demonstrate both noninvasive imaging and targeted drug delivery in preclinical mouse models of breast and prostate cancer. Finally, we applied mathematical modeling to predict and confirm tumor targeting and drug delivery. These results are meaningful steps toward the design and initial translation of an enabling nanotechnology platform with potential for broad clinical applications.
• Smith, T. L., Yuan, Z., Cardó-Vila, M., Sanchez Claros, C., Adem, A., Cui, M. H., Branch, C. A., Gelovani, J. G., Libutti, S. K., Sidman, R. L., Pasqualini, R., & Arap, W. (2016). AAVP displaying octreotide for ligand-directed therapeutic transgene delivery in neuroendocrine tumors of the pancreas. Proceedings of the National Academy of Sciences of the United States of America, 113(9), 2466-71.
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  Patients with inoperable or unresectable pancreatic neuroendocrine tumors (NETs) have limited treatment options. These rare human tumors often express somatostatin receptors (SSTRs) and thus are clinically responsive to certain relatively stable somatostatin analogs, such as octreotide. Unfortunately, however, this tumor response is generally short-lived. Here we designed a hybrid adeno-associated virus and phage (AAVP) vector displaying biologically active octreotide on the viral surface for ligand-directed delivery, cell internalization, and transduction of an apoptosis-promoting tumor necrosis factor (TNF) transgene specifically to NETs. These functional attributes of AAVP-TNF particles displaying the octreotide peptide motif (termed Oct-AAVP-TNF) were confirmed in vitro, in SSTR type 2-expressing NET cells, and in vivo using cohorts of pancreatic NET-bearing Men1 tumor-suppressor gene KO mice, a transgenic model of functioning (i.e., insulin-secreting) tumors that genetically and clinically recapitulates the human disease. Finally, preclinical imaging and therapeutic experiments with pancreatic NET-bearing mice demonstrated that Oct-AAVP-TNF lowered tumor metabolism and insulin secretion, reduced tumor size, and improved mouse survival. Taken together, these proof-of-concept results establish Oct-AAVP-TNF as a strong therapeutic candidate for patients with NETs of the pancreas. More broadly, the demonstration that a known, short, biologically active motif can direct tumor targeting and receptor-mediated internalization of AAVP particles may streamline the potential utility of myriad other short peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers and perhaps many nonmalignant diseases as well.
• Yao, V. J., Pasqualini, R., Ebaid, A., Cardo-vila, M., Arap, W., & Ahmadian, R. (2016). Decoding Tumor Zip Codes to Design Targeted Drugs to Treat Leukemia, Lymphoma, and Solid Tumors. The Hematologist, 13(2). doi:10.1182/hem.v13.2.5052
• Karjalainen, K., Jaalouk, D. E., Bueso-Ramos, C., Bover, L., Sun, Y., Kuniyasu, A., Driessen, W. H., Cardó-Vila, M., Rietz, C., Zurita, A. J., O'Brien, S., Kantarjian, H. M., Cortes, J. E., Calin, G. A., Koivunen, E., Arap, W., & Pasqualini, R. (2015). Targeting IL11 Receptor in Leukemia and Lymphoma: A Functional Ligand-Directed Study and Hematopathology Analysis of Patient-Derived Specimens. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(13), 3041-51.
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  The IL11 receptor (IL11R) is an established molecular target in primary tumors of bone, such as osteosarcoma, and in secondary bone metastases from solid tumors, such as prostate cancer. However, its potential role in management of hematopoietic malignancies has not yet been determined. Here, we evaluated the IL11R as a candidate therapeutic target in human leukemia and lymphoma.
• Mandelin, J., Cardó-Vila, M., Driessen, W. H., Mathew, P., Navone, N. M., Lin, S. H., Logothetis, C. J., Rietz, A. C., Dobroff, A. S., Proneth, B., Sidman, R. L., Pasqualini, R., & Arap, W. (2015). Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors. Proceedings of the National Academy of Sciences of the United States of America, 112(12), 3776-81.
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  We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand-receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer.
• Mintz, P. J., Rietz, A. C., Cardó-Vila, M., Ozawa, M. G., Dondossola, E., Do, K. A., Kim, J., Troncoso, P., Logothetis, C. J., Sidman, R. L., Pasqualini, R., & Arap, W. (2015). Discovery and horizontal follow-up of an autoantibody signature in human prostate cancer. Proceedings of the National Academy of Sciences of the United States of America, 112(8), 2515-20.
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  In response to an urgent need for improved diagnostic and predictive serum biomarkers for management of metastatic prostate cancer, we used phage display fingerprinting to analyze sequentially acquired serum samples from a patient with advancing prostate cancer. We identified a peptide ligand, CTFAGSSC, demonstrating an increased recovery frequency over time. Serum antibody reactivity to this peptide epitope increased in the index patient, in parallel with development of deteriorating symptoms. The antigen mimicking the peptide epitope was identified as alpha-2-Heremans-Schmid glycoprotein, also known as fetuin-A. Metastatic prostate cancer cell lines and bone metastasis samples displayed robust fetuin-A expression, and we demonstrated serum immune reactivity to fetuin-A with concomitant development of metastatic castrate-resistant disease in a large cohort of prostate cancer patients. Whereas fetuin-A is an established tumor antigen in several types of cancer, including breast cancer, glioblastoma, and pancreas cancer, this report is to our knowledge the first study implicating fetuin-A in prostate cancer and indicating that autoantibodies specific for fetuin-A show utility as a prognostic indicator for prostate cancer patients prone to progress to metastatic disease.
• Nunes, D. N., Dias-Neto, E., Cardó-Vila, M., Edwards, J. K., Dobroff, A. S., Giordano, R. J., Mandelin, J., Brentani, H. P., Hasselgren, C., Yao, V. J., Marchiò, S., Pereira, C. A., Passetti, F., Calin, G. A., Sidman, R. L., Arap, W., & Pasqualini, R. (2015). Synchronous down-modulation of miR-17 family members is an early causative event in the retinal angiogenic switch. Proceedings of the National Academy of Sciences of the United States of America, 112(12), 3770-5.
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  Six members of the microRNA-17 (miR-17) family were mapped to three different chromosomes, although they share the same seed sequence and are predicted to target common genes, among which are those encoding hypoxia-inducible factor-1α (HIF1A) and VEGFA. Here, we evaluated the in vivo expression profile of the miR-17 family in the murine retinopathy of prematurity (ROP) model, whereby Vegfa expression is highly enhanced at the early stage of retinal neovascularization, and we found simultaneous reduction of all miR-17 family members at this stage. Using gene reporter assays, we observed binding of these miRs to specific sites in the 3' UTRs of Hif1a and Vegfa. Furthermore, overexpression of these miRs decreased HIF1A and VEGFA expression in vitro. Our data indicate that this miR-17 family elicits a regulatory synergistic down-regulation of Hif1a and Vegfa expression in this biological model. We propose the existence of a coordinated regulatory network, in which diverse miRs are synchronously regulated to target the Hif1a transcription factor, which in turn, potentiates and reinforces the regulatory effects of the miRs on Vegfa to trigger and sustain a significant physiological response.
• Pasqualini, R., Millikan, R. E., Christianson, D. R., Cardó-Vila, M., Driessen, W. H., Giordano, R. J., Hajitou, A., Hoang, A. G., Wen, S., Barnhart, K. F., Baze, W. B., Marcott, V. D., Hawke, D. H., Do, K. A., Navone, N. M., Efstathiou, E., Troncoso, P., Lobb, R. R., Logothetis, C. J., & Arap, W. (2015). Targeting the interleukin-11 receptor α in metastatic prostate cancer: A first-in-man study. Cancer, 121(14), 2411-21.
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  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.
• Salameh, A., Lee, A. K., Cardó-Vila, M., Nunes, D. N., Efstathiou, E., Staquicini, F. I., Dobroff, A. S., Marchiò, S., Navone, N. M., Hosoya, H., Lauer, R. C., Wen, S., Salmeron, C. C., Hoang, A., Newsham, I., Lima, L. A., Carraro, D. M., Oliviero, S., Kolonin, M. G., , Sidman, R. L., et al. (2015). PRUNE2 is a human prostate cancer suppressor regulated by the intronic long noncoding RNA PCA3. Proceedings of the National Academy of Sciences of the United States of America, 112(27), 8403-8.
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  Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.
• Sidman, R. L., Li, J., Lawrence, M., Hu, W., Musso, G. F., Giordano, R. J., Cardó-Vila, M., Pasqualini, R., & Arap, W. (2015). The peptidomimetic Vasotide targets two retinal VEGF receptors and reduces pathological angiogenesis in murine and nonhuman primate models of retinal disease. Science translational medicine, 7(309), 309ra165.
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  Blood vessel growth from preexisting vessels (angiogenesis) underlies many severe diseases including major blinding retinal diseases such as retinopathy of prematurity (ROP) and aged macular degeneration (AMD). This observation has driven development of antibody inhibitors that block a central factor in AMD, vascular endothelial growth factor (VEGF), from binding to its receptors VEGFR-1 and mainly VEGFR-2. However, some patients are insensitive to current anti-VEGF drugs or develop resistance, and the required repeated intravitreal injection of these large molecules is costly and clinically problematic. We have evaluated a small cyclic retro-inverted peptidomimetic, D(Cys-Leu-Pro-Arg-Cys) [D(CLPRC)], and hereafter named Vasotide, that inhibits retinal angiogenesis by binding selectively to the VEGF receptors VEGFR-1 and neuropilin-1 (NRP-1). Delivery of Vasotide via either eye drops or intraperitoneal injection in a laser-induced monkey model of human wet AMD, a mouse genetic knockout model of the AMD subtype called retinal angiomatous proliferation (RAP), and a mouse oxygen-induced model of ROP decreased retinal angiogenesis in all three animal models. This prototype drug candidate is a promising new dual receptor inhibitor of the VEGF ligand with potential for translation into safer, less-invasive applications to combat pathological angiogenesis in retinal disorders.
• Stancevic, B., Zhang, J., Hua, G., Yin, X., Haimovitz-friedman, A., Kim, K., Qian, M., Cardo-vila, M., Fuks, Z., Pasqualini, R., Arap, W., Kolesnick, R., Rotolo, J. A., & Fuller, J. D. (2012). Anti-ceramide antibody prevents the radiation gastrointestinal syndrome in mice.. The Journal of Clinical Investigation, 122(5), 1786-90. doi:10.1172/jci59920
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  Radiation gastrointestinal (GI) syndrome is a major lethal toxicity that may occur after a radiation/nuclear incident. Currently, there are no prophylactic countermeasures against radiation GI syndrome lethality for first responders, military personnel, or remediation workers entering a contaminated area. The pathophysiology of this syndrome requires depletion of stem cell clonogens (SCCs) within the crypts of Lieberkühn, which are a subset of cells necessary for postinjury regeneration of gut epithelium. Recent evidence indicates that SCC depletion is not exclusively a result of DNA damage but is critically coupled to ceramide-induced endothelial cell apoptosis within the mucosal microvascular network. Here we show that ceramide generated on the surface of endothelium coalesces to form ceramide-rich platforms that transmit an apoptotic signal. Moreover, we report the generation of 2A2, an anti-ceramide monoclonal antibody that binds to ceramide to prevent platform formation on the surface of irradiated endothelial cells of the murine GI tract. Consequently, we found that 2A2 protected against endothelial apoptosis in the small intestinal lamina propria and facilitated recovery of crypt SCCs, preventing the death of mice from radiation GI syndrome after high radiation doses. As such, we suggest that 2A2 represents a prototype of a new class of anti-ceramide therapeutics and an effective countermeasure against radiation GI syndrome mortality.
• Staquicini, F. I., Cardo-vila, M., Kolonin, M. G., Trepel, M., Edwards, J. K., Nunes, D. N., Sergeeva, A., Efstathiou, E., Sun, J., Almeida, N. F., Botz, G. H., Wallace, M. J., O'connell, D. J., Krajewski, S., Gershenwald, J. E., Molldrem, J. J., Flamm, A. L., Koivunen, E., Pentz, R. D., , Dias-neto, E., et al. (2011). Vascular ligand-receptor mapping by direct combinatorial selection in cancer patients.. Proceedings of the National Academy of Sciences of the United States of America, 108(46), 18637-42. doi:10.1073/pnas.1114503108
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  Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ~2.35 x 10(6) motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications.
• Cardo-vila, M., Giordano, R. J., Sidman, R. L., Bronk, L. F., Fan, Z., Mendelsohn, J., Arap, W., & Pasqualini, R. (2010). From combinatorial peptide selection to drug prototype (II): targeting the epidermal growth factor receptor pathway.. Proceedings of the National Academy of Sciences of the United States of America, 107(11), 5118-23. doi:10.1073/pnas.0915146107
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  The epidermal growth factor receptor (EGFR), a tyrosine kinase, is central to human tumorigenesis. Typically, three classes of drugs inhibit tyrosine kinase pathways: blocking antibodies, small kinase inhibitors, and soluble ligand receptor traps/decoys. Only the first two types of EGFR-binding inhibitory drugs are clinically available; notably, no EGFR decoy has yet been developed. Here we identify small molecules mimicking EGFR and that functionally behave as soluble decoys for EGF and TGFalpha, ligands that would otherwise activate downstream signaling. After combinatorial library selection on EGFR ligands, a panel of binding peptides was narrowed by structure-function analysis. The most active motif was CVRAC (EGFR 283-287), which is necessary and sufficient for specific EGFR ligand binding. Finally, a synthetic retro-inverted derivative, (D)(CARVC), became our preclinical prototype of choice. This study reveals an EGFR-decoy drug candidate with translational potential.
• Giordano, R. J., Cardo-vila, M., Salameh, A., Anobom, C. D., Zeitlin, B. D., Hawke, D. H., Valente, A. P., Almeida, F. C., Nor, J. E., Sidman, R. L., Pasqualini, R., & Arap, W. (2010). From combinatorial peptide selection to drug prototype (I): targeting the vascular endothelial growth factor receptor pathway.. Proceedings of the National Academy of Sciences of the United States of America, 107(11), 5112-7. doi:10.1073/pnas.0915141107
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  Inhibition of blood vessel formation is a viable therapeutic approach in angiogenesis-dependent diseases. We previously used a combinatorial screening on vascular endothelial growth factor (VEGF)-activated endothelial cells to select the sequence CPQPRPLC and showed that the motif Arg-Pro-Leu targets VEGF receptor-1 and neuropilin-1. Here, we evaluated and validated (D)(LPR), a derivative molecule with strong antiangiogenesis attributes. This prototype drug markedly inhibits neovascularization in three mouse models: Matrigel-based assay, functional human/murine blood vessel formation, and retinopathy of prematurity. In addition to its systemic activity, (D)(LPR) also inhibits retinal angiogenesis when administered in an eye-drop formulation. Finally, in preliminary studies, we have showed targeted drug activity in an experimental tumor-bearing mouse model. These results show that drugs targeting extracellular domains of VEGF receptors are active, affect signal transduction, and have potential for clinical application. On a larger context, this study illustrates the power of ligand-directed selection plus retro-inversion for rapid drug discovery and development.
• Ozawa, M. G., Cardo-vila, M., Mintz, P. J., Arap, W., & Pasqualini, R. (2010). Cracking the code for compartment-specific dual functionality proteins in cancer: the case for CRKL.. Cell cycle (Georgetown, Tex.), 9(1), 8-9. doi:10.4161/cc.9.1.10288
• Mintz, P. J., Cardo-vila, M., Ozawa, M. G., Hajitou, A., Rangel, R., Guzman-rojas, L., Christianson, D. R., Arap, M. A., Giordano, R. J., Souza, G. R., Easley, J., Salameh, A., Oliviero, S., Brentani, R. R., Koivunen, E., Arap, W., & Pasqualini, R. (2009). An unrecognized extracellular function for an intracellular adapter protein released from the cytoplasm into the tumor microenvironment.. Proceedings of the National Academy of Sciences of the United States of America, 106(7), 2182-7. doi:10.1073/pnas.0807543105
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  Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of beta(1) integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein-protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.
• Cardo-vila, M., Zurita, A. J., Giordano, R. J., Sun, J., Rangel, R., Guzman-rojas, L., Anobom, C. D., Valente, A. P., Almeida, F. C., Lahdenranta, J., Kolonin, M. G., Arap, W., & Pasqualini, R. (2008). A ligand peptide motif selected from a cancer patient is a receptor-interacting site within human interleukin-11.. PloS one, 3(10), e3452. doi:10.1371/journal.pone.0003452
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  Interleukin-11 (IL-11) is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC). Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i) the peptide mimics a receptor-binding site within IL-11, (ii) the binding of CGRRAGGSC to the IL-11R alpha is functionally relevant, (iii) Arg4 and Ser8 are the key residues mediating the interaction, and (iv) the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11R alpha has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs.
• Bover, L. C., Cardo-vila, M., Kuniyasu, A., Sun, J., Rangel, R., Takeya, M., Aggarwal, B. B., Arap, W., & Pasqualini, R. (2007). A previously unrecognized protein-protein interaction between TWEAK and CD163: potential biological implications.. Journal of immunology (Baltimore, Md. : 1950), 178(12), 8183-94. doi:10.4049/jimmunol.178.12.8183
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  TWEAK (TNF-like weak inducer of apoptosis) is a TNF superfamily member implicated in several mechanisms. Although fibroblast growth factor inducible 14 (Fn14)/TweakR has been reported as its receptor, an as yet unrecognized surface molecule(s) might modulate TWEAK function(s). Thus, we set out to identify TWEAK-binding proteins by screening a combinatorial peptide library. Cyclic peptides containing a consensus motif (WXDDG) bound to TWEAK specifically. These peptides were similar to CD163, a scavenger receptor cysteine-rich domain family member, restricted to the monocyte/macrophage lineage and responsible for the uptake of circulating haptoglobin-hemoglobin (Hp-Hb) complexes. Sequence profile analysis suggested that TWEAK mimicked the CD163 natural ligand (Hp-Hb). Consistently, we show dose-dependent TWEAK binding to CD163 and blockade by an anti-CD163 Ab. In a competition assay, both soluble CD163 and Fn14/TweakR were able to compete off TWEAK binding to coated Fn14/TweakR or CD163, respectively. Flow-cytometry and immunofluorescence assays showed that human monocytes (Fn14/TweakR negative and CD163 positive) bind TWEAK, thus blocking the recognition of CD163 and reducing the activation mediated by a specific mAb in these cells. We demonstrate that monocytes can sequester TWEAK from supernatants, thus preventing tumor cell apoptosis; this effect was reverted by preincubation with the peptide mimicking CD163 or with a mAb anti-CD163, indicating specificity. Finally, we show that recombinant human TWEAK binding to CD163-transfected Chinese hamster ovary cells is inhibited by the presence of either unlabeled TWEAK or the Hp-Hb complex. Together, these data are consistent with the hypothesis that CD163 either acts as a TWEAK scavenger in pathological conditions or serves as an alternate receptor for TWEAK in cells lacking Fn14/TweakR.
• Kolonin, M. G., Bover, L., Sun, J., Zurita, A. J., Lahdenranta, J., Cardo-vila, M., Giordano, R. J., Jaalouk, D. E., Ozawa, M. G., Moya, C. A., Souza, G. R., Staquicini, F. I., Kunyiasu, A., Scudiero, D. A., Holbeck, S. L., Sausville, E. A., Arap, W., Pasqualini, R., & Do, K. A. (2006). Ligand-directed surface profiling of human cancer cells with combinatorial peptide libraries.. Cancer research, 66(1), 34-40. doi:10.1158/0008-5472.can-05-2748
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  A collection of 60 cell lines derived from human tumors (NCI-60) has been widely explored as a tool for anticancer drug discovery. Here, we profiled the cell surface of the NCI-60 by high-throughput screening of a phage-displayed random peptide library and classified the cell lines according to the binding selectivity of 26,031 recovered tripeptide motifs. By analyzing selected cell-homing peptide motifs and their NCI-60 recognition patterns, we established that some of these motifs (a) are similar to domains of human proteins known as ligands for tumor cell receptors and (b) segregate among the NCI-60 in a pattern correlating with expression profiles of the corresponding receptors. We biochemically validated some of the motifs as mimic peptides of native ligands for the epidermal growth factor receptor. Our results indicate that ligand-directed profiling of tumor cell lines can select functional peptides from combinatorial libraries based on the expression of tumor cell surface molecules, which in turn could be exploited as "druggable" receptors in specific types of cancer.
• Barker, T. H., Baneyx, G., Cardo-vila, M., Workman, G. A., Menon, P. M., Dedhar, S., Rempel, S. A., Arap, W., Pasqualini, R., Vogel, V., Sage, E. H., & Weaver, M. S. (2005). SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.. The Journal of biological chemistry, 280(43), 36483-93. doi:10.1074/jbc.m504663200
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  SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.
• Giordano, R. J., Anobom, C. D., Cardo-vila, M., Kalil, J., Valente, A. P., Pasqualini, R., Almeida, F. C., & Arap, W. (2005). Structural basis for the interaction of a vascular endothelial growth factor mimic peptide motif and its corresponding receptors.. Chemistry & biology, 12(10), 1075-83. doi:10.1016/j.chembiol.2005.07.008
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  Vascular endothelial growth factor (VEGF) is central to the survival and development of the vascular and nervous systems. We screened phage display libraries and built a peptide-based ligand-receptor map of binding sites within the VEGF family. We then validated a cyclic peptide, CPQPRPLC, as a VEGF-mimic that binds specifically to neuropilin-1 and VEGF receptor-1. Here, we use NMR spectroscopy to understand the structural basis of the interaction between our mimic peptide and the VEGF receptors. We show that: (1) CPQPRPLC has multiple interactive conformations; (2) receptor binding is mediated by the motif Arg-Pro-Leu; and (3) the Pro residue within Arg-Pro-Leu participates in binding to neuropilin-1 but not to VEGF receptor-1, perhaps representing an evolutionary gain-of-function. Therefore, Arg-Pro-Leu is a differential ligand motif to VEGF receptors and a candidate peptidomimetic lead for VEGF pathway modulation.
• Zurita, A. J., Hajitou, A., Cardo-vila, M., Troncoso, P., Logothetis, C. J., Pasqualini, R., & Arap, W. (2004). Preclinical development of an interleukin-11 receptor-targeted pro-apoptotic peptide against advanced prostate cancer. Journal of Clinical Oncology, 22(14_suppl), 3190-3190. doi:10.1200/jco.2004.22.90140.3190
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  3190 Background: Direct screening of combinatorial peptide libraries in patients allows the identification of ligands that target biochemical differences in the endothelium of blood vessels. In a screening performed on a patient, we selected and isolated a mimic motif of interleukin 11 (IL-11) from prostate biopsies after an intravenous administration of a phage display peptide library. We also demonstrated that the IL-11 phage mimic bound specifically to a corresponding IL-11 receptor (IL-11Rα). More recently we showed that IL-11Rα is a potential target for intervention in human prostate cancer (PCa) through morphologic and functional analyses (Zurita et al., Cancer Res 2004, in press). We observed stage-specific up-regulation of IL-11Rα during disease progression, particularly in androgen-independent and metastatic PCa and their associated blood vessels. Moreover, a pro-apoptotic peptide was specifically targeted and internalized through IL-11Rα in PCa cell lines. Here we evaluate antitumor efficacy, se...
• Zurita, A. J., Troncoso, P., Cardo-vila, M., Logothetis, C. J., Pasqualini, R., & Arap, W. (2004). Combinatorial screenings in patients: the interleukin-11 receptor alpha as a candidate target in the progression of human prostate cancer.. Cancer research, 64(2), 435-9. doi:10.1158/0008-5472.can-03-2675
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  Direct screening of combinatorial peptide libraries in patients may allow the identification of ligands that target biochemical differences in the endothelium of blood vessels. In a screening performed in a patient, we selected and isolated a mimic motif of interleukin 11 (IL-11) from prostate biopsies after an i.v. administration of a phage display peptide library. We also demonstrated that the IL-11 phage mimic (displaying the cyclic nonapeptide CGRRAGGSC) bound specifically to a corresponding IL-11 receptor (IL-11Ralpha). Here we show that IL-11Ralpha is a potential target for intervention in human prostate cancer through morphological and functional analyses. First, a comprehensive serial immunohistochemical analysis of primary and metastatic prostate cancer samples showed increased stage-specific expression of IL-11Ralpha during disease progression. Second, a proapoptotic peptide was specifically targeted and internalized through this functional IL-11Ralpha-based ligand-receptor pair: treatment of prostate cancer cells in vitro with a proapoptotic peptide guided by the CGRRAGGSC peptide to the IL-11Ralpha resulted in dose-dependent apoptosis. Together, these data indicate that the IL-11Ralpha is a candidate target for translational clinical trials against advanced and metastatic prostate cancer. Moreover, our results illustrate the ability of direct combinatorial screening systems in cancer patients for identification of relevant targets in the context of human disease.
• Cardo-vila, M., Arap, W., & Pasqualini, R. (2003). Alpha v beta 5 integrin-dependent programmed cell death triggered by a peptide mimic of annexin V.. Molecular cell, 11(5), 1151-62. doi:10.1016/s1097-2765(03)00138-2
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  The diverse cytoplasmic domain sequences within the various integrin subunits are critical for integrin-mediated signaling into the cell (outside-in signaling) and for activation of ligand binding affinity (inside-out signaling). Here we introduce an approach based on phage display technology to identify molecules that specifically interact with the cytoplasmic domain of the beta 5 integrin subunit. We show that a peptide selected for binding specifically to the beta 5 cytoplasmic domain (VVISYSMPD) induces apoptosis upon internalization. The cell death process induced by VVISYSMPD is sensitive to modulation by growth factors and by protein kinase C (PKC), and it cannot be triggered in beta 5 null cells. Finally, we show that the VVISYSMPD peptide is a mimic of annexin V. Our results suggest a functional link between the alpha v beta 5 integrin, annexin V, and programmed cell death. We propose the term "endothanatos" to designate this phenomenon.
• Tamm, I., Trepel, M., Cardo-vila, M., Sun, Y., Welsh, K., Cabezas, E., Swatterthwait, A., Arap, W., Reed, J. C., & Pasqualini, R. (2003). Peptides targeting caspase inhibitors.. The Journal of biological chemistry, 278(16), 14401-5. doi:10.1074/jbc.m210133200
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  Here we report on the identification of peptides targeting the X-inhibitor of apoptosis protein (XIAP). XIAP functions as a caspase inhibitor and is a member of the inhibitors of apoptosis (IAP) family of proteins. IAPs are often overexpressed in cancers and leukemias and are associated with an unfavorable clinical prognosis. We have selected peptides from a phage library by using recombinant full-length human XIAP or a fragment containing only the baculovirus IAP repeat 2 (BIR2) domain. A consensus motif, C(D/E/P)(W/F/Y)-acid/basic-XC, was recovered from two independent screenings by using different libraries. Phage-displaying variations of the consensus sequence bound specifically to the BIR2 domain of XIAP but not to other IAPs. The interaction was specific as it could be blocked by the cognate synthetic peptides in a dose-dependent manner. Phage displaying the XIAP-binding motif CEFESC bound to the BIR2 domain of XIAP with an estimated dissociation constant of 1.8 nm as determined by surface plasmon resonance. Protein-protein interaction assays revealed that caspase-3 and caspase-7 (but not caspase-8) blocked the binding of the CEFESC phage to XIAP, indicating that this peptide targets a domain within XIAP that is related to the caspase-binding site. In fact, the sequence EFES is homologous to a loop unique to the executioner caspase-3 and caspase-7 that are targeted by XIAP. Finally, we demonstrated that an internalizing version of the XIAP-binding peptide identified in our screenings (PFKQ) can induce programmed cell death in leukemia cells. Peptides interacting with XIAP could serve as prototypes for the design of low molecular weight modulators of apoptosis.
• Cardó-Vila, M., Arden, K. C., Cavenee, W. K., Pasqualini, R., & Arap, W. (2001). Is annexin 7 a tumor suppressor gene in prostate cancer?. Pharmacogenomics J., 1(2), 92-4. doi:10.1038/sj.tpj.6500028
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  Two features make the prostate and its tumors unusual. First, the prostate gland continues to grow throughout adult life, even doubling in size between the second and fourth decades.1 As a result, benign prostate hypertrophy affects most aging men to some degree. Second, and much more serious, cancer of the prostate is the most frequent malignant tumor and second leading cause of cancer-related deaths among men in the United States and Europe.2 While one out of every 11 men will develop prostate cancer during their lifetime, many tumors remain clinically quiescent. Without entirely reliable ways of predicting which tumors will progress, many prostate cancer patients are treated aggressively with radical prostatectomy or radiation therapy on the chance of cure, but often at the price of devastating treatment side-effects such as urinary incontinence and sexual impotence. Thus, there is a clear need for markers of cellular growth potential as new diagnostic and therapeutic targets in prostate cancer
• Giordano, R. J., Cardo-vila, M., Lahdenranta, J., Pasqualini, R., & Arap, W. (2001). Biopanning and rapid analysis of selective interactive ligands.. Nature medicine, 7(11), 1249-53. doi:10.1038/nm1101-1249
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  Here we introduce a new approach for the screening, selection and sorting of cell-surface-binding peptides from phage libraries. Biopanning and rapid analysis of selective interactive ligands (termed BRASIL) is based on differential centrifugation in which a cell suspension incubated with phage in an aqueous upper phase is centrifuged through a non-miscible organic lower phase. This single-step organic phase separation is faster, more sensitive and more specific than current methods that rely on washing steps or limiting dilution. As a proof-of-principle, we screened human endothelial cells stimulated with vascular endothelial growth factor (VEGF) and constructed a peptide-based ligand-receptor map of the VEGF family. Next, we validated the motif PQPRPL as a novel chimeric ligand mimic that binds specifically to VEGF receptor-1 and to neuropilin-1. BRASIL may prove itself a superior method for probing target cell surfaces with a broad range of potential applications.

Proceedings Publications

• Yin, J., Wang, L., Cardo-vila, M., Arap, W., Pasqualini, R., Tuder, R. M., & Kuebler, W. M. (2012). Endothelial Apoptosis Promotes Lung Tissue Remodeling And Emphysema Via Matrix Metalloproteinase 9. In D29. STICKY SITUATION: MULTI-FATE OF CELL AND MATRIX INTERACTIONS.