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The processes of neurite extension and remodeling require a close coordination between the cytoskeleton and the cell membranes. The small GTPase ARF6 (ADP-ribosylation factor 6) has a central role in regulating membrane traffic and actin dynamics, and its activity has been demonstrated to be involved in neurite elaboration. EFA6A has been shown to act as a guanine nucleotide exchange factor (GEF) for ARF6. Here, we report that two distinct isoforms of the EFA6A gene are expressed in murine neural tissue: a long isoform of 1025 amino acids (EFA6A), and a short isoform of 393 amino acids (EFA6As). EFA6A encompasses proline-rich regions, a Sec7 domain (mediating GEF activity on ARF6), a PH domain, and a C-terminal region with coiled-coil motifs. EFA6As lacks the Sec7 domain, and it comprises the PH domain and the C-terminal region. The transcript encoding EFA6As is the result of alternative promoter usage. EFA6A and EFA6As have distinct biological activities: upon overexpression in HeLa cells, EFA6A induces membrane ruffles, whereas EFA6As gives rise to cell elongation; in primary cortical neurons EFA6A promotes neurite extension, whereas EFA6As induces dendrite branching. Our findings suggest that EFA6A could participate in neuronal morphogenesis through the regulated expression of two functionally distinct isoforms.

Blood-brain barrier (BBB) is a major barrier to delivering drugs to the brain parenchyma. Affinity carriers able to cross BBB would allow delivery drugs for the treatment of neurological diseases such as multiple sclerosis, Huntington's, Parkinson's and Alzheimer's. In the presence of brain tumors the BBB is locally disrupted, however, the infiltrative guerilla glioma cells distant from the tumor core hide behind intact BBB beyond the reach of anticancer therapies. Thus, the activity of brain cancer drugs would be potentiated by carriers that facilitate BBB penetration. Here, we used in vivo T7 phage display of cyclic CX7C peptide libraries on healthy Balb/C mice to identify peptides that home to brain upon systemic administration. In vivo biopanning in combination with deep sequencing of peptide-encoding phage genomic DNA and bioinformatics analyses led to identification of a peptide-phage clone that selectively homes to normal brain. Tested as an individual phage clone, the phage displaying the brain-homing peptide, KT1, showed about 50 times higher brain tropism compared to insertless control phage. In vivo alanine scan was used to map the residues that are critical for KT1 brain homing. Our ongoing work deals with elucidating the mechanism of KT1 recruitment to brain microvessels and evaluation of its potential as a BBB penetrating agent. We are also evaluating KT1 as a ligand for targeting drugs and imaging agents to glioblastomas, especially to the infiltrative "guerilla" glioma cells.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain malignancy. Even when subjected to multimodal treatment using surgery, chemo- and radiotherapy, the majority of GBM patients die within a year after diagnosis. Affinity targeting of anti-glioma drugs to malignant lesions, in particular to disseminated cells hiding behind the blood-brain-barrier, may dramatically improve efficacy of antiglioma therapies. Upon intravenous administration, tumor penetrating peptides (TPP-s) home to malignant lesions and extravasate into tumor parenchyma. Interestingly, these peptides retain their activity to penetrate excised tumor explants in short-term ex vivo assays. Here, we report development of an in vitro 3D penetration assay of homing peptide-functionalized silver nanoparticles (AgNP) in U87 glioma spheroids. U87 spheroids were incubated with fluorescent silver nanoparticles functionalized with prototypic tumor penetrating peptides, iRGD (CRGDKGPDC) and RPARPAR, or control peptides. Subsequently, spheroids were incubated with a non-toxic etching solution that selectively removes the extracellular AgNP with no effect on internalized AgNP. Fluorescence imaging and Matlab -based quantitative analysis of spheroid cryosections and of live spheroids was used to assess distribution of AgNP with and without etching treatment. Compared to control nanoparticles, iRGD and RPARPAR functionalized AgNP showed dramatically increased binding and penetration of the particles in the U87 spheroids. The binding was inhibited by peptide receptor-blocking anti NRP-1 and anti-av-integrin antibodies (but not by control antibodies), suggesting specific effect of AgNP surface peptides. Establishment and validation of 3D in vitro tumor spheroid penetration assay may provide a standardized system for evaluation tumor homing and penetration ability of homing peptides and to allow medium- throughput mechanistic and experimental therapy studies.

A major limiting factor in the development of cancer therapy, especially for solid tumors, is the delivery of therapeutic agents into target cells in vivo to reach the site of action. Major barriers to the efficient delivery of drugs, especially macromolecules and nanomaterials, include the vascular walls, extravascular tissue, and membrane of cells and intracellular organelles. Peptides with high affinity to signature surface receptors displayed on tumor vasculature have been attractive carriers of therapeutic and diagnostic agents into tumors. Major efforts have been devoted to complexing these peptides with drug carriers of various physical and chemical properties, while little is known about either the cellular machinery that mediates the intake of these agents, or the underlying regulatory mechanism for delivery efficiency. We tackle this problem by characterizing the cell entry and tissue penetration process of a novel class of tumor-penetrating peptides. These peptides contain a carboxy (C)-terminal, basic sequence R/KXXR/K motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) bind to neuropilin-1 (NRP1) on the cell surface and initiate an endocytic process into cells. Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development, and their expression is often upregulated on tumor vasculature and tumor cells. Many growth factors and other signalling molecules bind to NRPs through the CendR motif. To achieve a tumor-specific homing in vivo, the CendR motif is rendered cryptic in the middle of the peptide. Tumor-homing CendR peptides recognize different primary receptors to achieve the initial homing to the target tissues. The notable example is iRGD (CRGD(K/R)GP(D/E)C; the terminal Cys residues form a disulfide bond; CendR motif underlined), which use the RGD motif to first recognize αv integrins highly expressed on tumor endothelium. The peptide is then proteolytically cleaved to expose the CendR motif at the C-terminus. The activated CendR peptides thus bind to NRP1, which initiate an active penetration process across the blood vessels and throughout the extravascular tumor tissue. This tumor homing and penetration effect has been shown in a wide range of tumor types, and displays a good compatibility with various drug types. Most interestingly, the cargo does not need to be chemically conjugated to the peptide; co-injection with iRGD can also enhance the transport of diverse therapeutic payloads from the circulation into the tumor parenchyma (bystander activity), leading to an improved antitumor efficacy. To decipher the molecular machinery of CendR-mediated cell entry, we performed a genome-wide RNAi screening to identify important genes for the cellular uptake of a prototypic CendR peptide displayed on silver-based nanoparticles. Silver-based platform is advantageous here since it allows the exclusive tracing of the internalized particles while the extracellular ones are effectively removed by a mild etching procedure. The genome screen and subsequent validation studies demonstrated that NRP1-mediated endocytosis of CendR peptides is a novel cell entry mechanism distinct from known endocytic pathways. CendR endocytosis depends little on critical genes of the other pathways, and is resistance to the treatment of the established endocytic inhibitors. CendR cargo also exhibits little colocalization with structural components of classic endocytic vesicles (e.g. clathrin-coated pits and caveolae) over time. Ultrastructurally, CendR endocytosis resembles macropinocytosis. But they are mechanistically different, especially in the receptor (NRP1) dependence of CendR endocytosis. The uniqueness of CendR pathway also lies in the regulatory mechanism of its activity. The genome screen surprisingly showed that nutrient-sensing networks, such as mTOR signaling, rank the highest among the canonical pathways regulating the activity of CendR endocytosis. Nutrient deprivation was shown to enhance the penetration of CendR cargo into cells in vitro, live tumor slices ex vivo and tumor tissue in vivo. Moreover, we developed assays to observe the intercellular transport of CendR cargo, which is also stimulated by nutrient deprivation. We are carrying on subsequent studies to elucidate the structural and molecular basis of CendR transport pathway. A methodology has been developed to synchronize the peptide entry, and ultrastructural imaging pinpoints a step-wise roadmap for the subcellular transport of CendR cargo. We have also performed magnetic isolation of intracellular vesicles containing CendR cargo, and identified signature molecules associated with these vesicles based on proteomic analysis. Moreover, we are able to observe the engulfment of bystander cargo in vitro, and molecular and ultrastructural characterization is undergoing to elucidate this intriguing cellular process. Using zebrafish and mouse models, we are monitoring the vascular penetration in vivo and characterizing the dynamic profile. Meanwhile, we are continuing to decode how mTOR regulates the activity of CendR endocytosis. A transcription factor, Sp1, has been found to serve as a link between mTOR and cell surface NRP1 level. Overall, we have discovered a novel pathway for peptide-functionalized payloads to enter tumor cells and tissue after receptor engagement. The ultrastructural studies of CendR endocytic structures supports the notion that CendR peptides activate a bulk transport system sweeping along bystander compounds, such as drugs and imaging agents present within tumor vessels. The platform to monitor the intercellular transfer of CendR cargo in vitro, together with the in vivo models, enables us to further optimize the transport efficiency of therapeutic agents across the vascular barriers. Particularly, the nutrient regulation of CendR transport pathway is of great significance by linking cancer metabolism with drug delivery. Due to dysfunctional angiogenesis, the nutrient conditions vary significantly in solid tumors. Some regions (e.g. hypoxia) are well known to undergo molecular changes and thus adapt to the nutrient-deprived environment. As a bulk transport pathway, our results suggest a role for CendR pathway in nutrient transport. Moreover, our studies point out a new direction to apply CendR-enhanced drug delivery into nutrient-deprived pathological tissues. These results shed new light in the fields of cell biology, cancer metabolism and drug delivery, while they also highlight the importance to bridge drug delivery with mechanistic studies. Citation Format: Hongbo Pang, Gary B. Braun, Tomas Friman, Pedro Aza-Blanc, Manuel E. Ruidiaz, Kazuki N. Sugahara, Tambet Teesalu, Erkki Ruoslahti. A novel endocytic and intercellular transport pathway for drug delivery across blood vessels and into nutrient-deprived tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG03. doi:10.1158/1538-7445.AM2015-NG03

SummaryTissue plasminogen activator (tPA) is increasingly recognized to play important roles in various physiological and pathological processes in the central nervous system (CNS). Much of the data on the involvement of plasminogen activators in neurophysiology and -pathology have been derived from studies on experimental animals. We have now performed a systematic characterization of the expression of tPA and its inhibitor, neuroserpin, in normal human CNS. Brain and spinal cord samples from 30-36 anatomic locations covering all major brain regions were collected at 9 autopsies of donors with no neurological disease. Tissues were embedded in paraffin and tissue arrays were constructed. In two cases parallel samples were snap-frozen for biochemical analysis. Expression and activity profiling of tPA and neuroserpin were performed by immunohistochemistry, in situ hybridization, immunocapture and zymography assays. In the adult CNS, tPA was expressed at the mRNA and protein levels in many types of neurons, in particular in thalamus, cortex of cerebellum, pontine nuclei, neocortex, limbic system, and medulla oblongata. Interestingly, tPA was often co-expressed with its CNS inhibitor, neuroserpin. Despite overlapping expression of tPA and neuroserpin, zymography and immunocapture assays demonstrated that human neural tissue is a rich source of active tPA. Our analysis documents a detailed map of expression of tPA and its inhibitor in the human CNS and is compatible with the view that tPA is a key player in CNS physiology and pathology.

This is a report of a workshop held on the establishment of human research tissue banking which was held in Levi, Finland 21-24 March 2002. There were 21 participants from 7 European countries. This meeting was attended by representatives from academia, research tissue banks and from the Biotech and Pharmaceutical Industries. The principal aim of the workshop was to find a way to progress the recommendations from ECVAM workshop 44 (ATLA 29, 125-134, 2001) and ECVAM workshop 32 (ATLA 26, 763-777, 1998). The workshop represented the first unofficial meeting of the European Network of Research Tissue Banks (ENRTB) steering group. It is expected that in the period preceding the next workshop the ENRTB steering group will co-ordinate the ethical, legislative and organisational aspects of research tissue banking. Key issues dealt with by the Levi workshop included the practical aspects of sharing expertise and experiences across the different European members. Such collaboration between research tissue banks and end users of such material seeks to ultimately enable shared access to human tissue for medical and pharmaco-toxicological research while maintaining strict adherence to differences in legal and ethical aspects related to the use of human tissue in individual countries.

Oncogenic human papillomavirus (HPV) plays a possible aetiological role in a subset of head and neck cancers, particularly in tonsillar carcinomas. For establishing a model to study mechanisms involved in HPV-associated tonsillar carcinogenesis, normal human tonsillar epithelial (HTE) cells were transfected with full-length HPV-16 DNA. The transfections produced four immortalised cell lines, designated HTE-114/K1, HTE-114/K2, HTE-114/K3 and HTE-114/B. All transfected HTE cell lines were cytogenetically abnormal. They exhibited altered morphology and impaired expression of cytokeratins in organotypic cultures. They failed to form colonies in soft agarose and formed no tumours in nude mice within 6 months. Each of them contained integrated viral DNA in a distinctive pattern as shown by Southern blot hybridisation. Early viral transcripts containing the E7 gene were detected by northern blot hybridisation. In conclusion, primary HTE cells can be immortalised following transfection with full-length HPV-16 DNA; the immortalised cell lines had partially retained epithelial characteristics in their morphology and function. They seem to represent early stages of premalignant epithelial cells and thus provide a useful model for studying further the multistep molecular events of HPV-16-associated tonsillar carcinogenesis.

Extracellular proteolysis represents a potent and irreversible mechanism of modulating cell-cell and cell-matrix interactions and remodeling structural components of tissues. Understanding of the role of extracellular proteolysis has evolved during the last decade from a somewhat simplistic concept of proteases being important for breaking down/ remodeling mechanical tissue barriers of the extracellular matrix (ECM) into a more complex view of proteases affecting indirectly many aspects of cell physiology, from cell division and differentiation to programmed cell death (Basbaum and Werb, 1996; Werb, 1997). Not surprisingly, proteases and their regulatory molecules are involved in a wide range of physiological processes of tissue growth and remodeling, as well as in pathological conditions such as tumorigenesis and tissue-destructive diseases (Werb et al., 1999; Andreasen et al., 2000).

Transport of molecules across the vascular wall, through tissue, and into target cells plays an important role in various physiological and pathological processes. We previously described a novel class of tumor-targeting peptides (CendR peptides) that penetrate into cells and tumor tissue through an as yet incompletely characterized transport pathway initiated by peptide binding to neuropilin-1 (NRP1). This pathway has shown promise in enhancing drug delivery into tumors. Here we perform a genome-wide RNAi screen to identify components responsible for cell penetration of CendR peptides. We show that the binding of CendR peptides to NRP1 initiates a novel endocytic process that depends on a subset of genes and a transport route distinct from those of known endocytic pathways. Strikingly, we found the internalization of CendR peptides to be controlled by nutrient-sensing and cell growth-regulatory pathways. Nutrient deprivation stimulated the cell penetration and intercellular transport of CendR peptides, both in vitro and in vivo. These data suggest a physiological role for CendR pathway in nutrient sensing and transport. The ability to activate this pathway with peptides provides immediate applications to drug delivery. Citation Format: Hong-Bo Pang, Gary B. Braun, Tomas Friman, Pedro Aza-Blanc, Manuel E. Ruidiaz, Kazuki N. Sugahara, Tambet Teesalu, Erkki Ruoslahti. The CendR pathway: A novel cell penetration and transcytosis pathway regulated by nutrient availability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5406. doi:10.1158/1538-7445.AM2014-5406

Breast cancer patients' outcome and survival depends on the timely diagnosis of early malignant lesions. The lack of molecular understanding of the early changes in breast tissue that facilitate tumor progression has limited the development of tools to non-invasively distinguish early stage disease from normal breast tissue. Therefore, probes to target and better understand early breast tumors are much needed. Our laboratory has pioneered in vivo screening of phage libraries to identify peptides that specifically recognize tumor vessels, including breast cancer vasculature. These peptides have been employed to specifically deliver drugs, diagnostic agents, and nanoparticles to breast tumors. Breast cancer progression constitutes a multistep process through a series of intermediate hyperplastic and neoplastic stages to invasive carcinoma. In this study, we aimed to identify peptides that specifically recognize premalignant lesions in the mammary tissue. To achieve this goal, we utilized the power of phage display to probe hyperplastic lesions associated with premalignant disease in a transgenic MMTV-PyMT animal model. After multiple ex-vivo and in-vivo rounds of selection, we identified a peptide, Prem-1, that on intravenous administration, specifically homed to premalignant mammary lesions. Prem-1 also homed to fully developed breast tumors in the same animal model, suggesting that the putative receptor for Prem-1 is expressed throughout the progression of the disease. Interestingly, Prem-1 did not show any affinity to normal breast tissue. Furthermore, we also identified 2 other candidate peptides that showed significant homing to premalignant lesions with a very different binding pattern as compared to Prem-1. We hypothesized that all three peptides recognize early changes in the breast tissue microenvironment but each bind a different target receptor in the tissue. We are currently investigating these receptors and analyzing their expression in breast cancer progression. Secondly, we are testing the peptides identified herein for the delivery of therapeutic nanoparticles as a mode of early intervention in breast cancer progression. This project utilized the natural environment in the early breast tumor to probe for new markers for detection of early disease. Methods to detect and study early premalignant mammary lesions will expand our understanding of the natural history of the disease, especially the changes in breast tissue that likely lead to tumor development. Hence, the knowledge gained from this study would provide a basis for therapies aimed at suppressing or eradicating premalignant breast lesions. This would be particularly beneficial for women at high-risk of breast cancer based on their genetic predisposition (mutations in BRCA genes). Citation Format: Aman P. Mann, Ramana Kotamraju, Tambet Teesalu, Erkki Ruoslahti. Targeting premalignant lesions for early breast cancer detection and intervention. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3258. doi:10.1158/1538-7445.AM2014-3258

In vivo peptide phage display has been widely used to explore tumor vascular heterogeneity and to develop targeting ligands for the delivery of anticancer drugs and imaging agents. Numerous tumor homing peptides with various targeting specificities have been identified. To select a suitable targeting ligand for a particular tumor from an existing peptide panel, it is crucial to perform unbiased quantitative assessment of in vivo peptide homing. Here we describe an approach for parallel evaluation of in vivo tumor homing of peptide-displaying phage clones. Mice bearing orthotopic xenografts of gliomas, breast tumors, and prostate tumors were intravenously injected with equimolar mix of phage clones displaying 12 different tumor homing or control peptides. After 30 min circulation and removal of blood by perfusion, representation of each phage clone in tumors and control organs was determined by high throughput sequencing (HTS) and quantitative PCR (qPCR). The ratiometric analysis demonstrated a clear and reproducible homing signature for each tumor model. Calibrated copy number data obtained by qPCR correlated well with the HTS analysis. Selected phage clones were validated for tumor homing by conventional single-phage homing studies using immunofluorescence, phage titration, and qPCR. Our study shows that the ratiometric in vivo phage biodistribution analysis can be used to rapidly identify peptides that efficiently deliver payloads to particular tumor types. As the peptides are evaluated side-by-side in a same animal, inter-animal variability is eliminated and the number of animals required is dramatically decreased. The choice of analysis (HTS vs qPCR) depends on the complexity of phage pool to be audited. HTS enables the analysis of highly complex phage pools, but introduces a potential bias owing to multiple DNA amplification steps. On the other hand, qPCR allows precise quantification of the copy number of the target sequences, and is suitable for low complexity pools. Narrowing down a complex pool with HTS followed by precise evaluation with qPCR can be a potential combination of the two analytical methods. Citation Format: Kadri Toome, Tarmo Mölder, Kuldar Kõiv, Pille Säälik, Kazuki N. Sugahara, Erkki Ruoslahti, Tambet Teesalu. In vivo audition of tumor homing peptides using high-throughput sequencing and q-PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 705. doi:10.1158/1538-7445.AM2014-705

Activity of tumor penetrating peptides depends on C-end Rule (CendR) peptide motif (R/KXXR/K) that must be C-terminally exposed for tissue and cell penetration. We describe here a cryptic CendR peptide (uCendR; amino acid sequence: RPARSGR↓SAGGSVA) that is proteolytically activated by urokinase-type plasminogen activator (uPA), a protease overexpressed in human tumors. uPA cleavage unmasks the cryptic CendR element of uCendR peptide and triggers its binding and internalization to the neuropilin-expressing cultured cancer cells. Intradermally injected post-cleavage mimic of uCendR peptide (RPARSGR) increases permeability of skin microvessels to tracer compounds. Phage and silver nanoparticles functionalized with uCendR peptide in combination with angiogenic integrin-recruitment motif (CRGDC) home to tumor blood vessels, extravasate, and trigger vascular exit of co-injected tracer compounds. This study shows that modular tumor penetrating peptides that are specifically activated by a tumor derived protease can be developed for tumor-targeted drug delivery. Citation Format: Tambet Teesalu, Kazuki N. Sugahara, Gary B. Braun, Venkata Ramana Kotamraju, Erkki Ruoslahti. Urokinase plasminogen activator-dependent tumor penetrating peptide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4504. doi:10.1158/1538-7445.AM2014-4504

Neuropilin-binding tumor-penetrating peptides increase vascular and tissue permeability in tumors, allowing drugs conjugated to the peptide and even free drugs co-injected with the peptide to widely distribute within the tumor tissue (Sugahara KN et al, Cancer Cell 16:510, 2009; Science 328:1031, 2010). Thus, the peptides provide an elegant way to enhance therapeutic index of anti-cancer drugs. However, the ability of the peptides to increase vascular permeability has raised concerns that the peptides might enhance metastasis by promoting cell seeding from the primary tumor. Here, we show a recent unexpected finding that a prototypic tumor-penetrating peptide, iRGD (amino acid sequence: CRGDK/RGPD/EC), is a potent metastasis inhibitor. iRGD did not cause systemic tumor cell dissemination or metastasis in mice bearing breast tumors with low metastatic potential. iRGD treatment of mice bearing aggressive orthotopic pancreatic tumors resulted in significant decrease in spontaneous metastasis formation. Primary tumor growth was not affected by the treatment. Another tumor-penetrating peptide, iNGR, also inhibited metastasis, while conventional RGD peptides or a scrambled iRGD peptide that lack neuropilin affinity failed to do. These results indicate that the anti-metastatic effect of iRGD was dependent on its neuropilin-binding peptide motif. In vitro migration assays demonstrated that iRGD potently inhibits tumor cell migration and serves as a chemorepellent of tumor cells in a neuropilin-dependent manner. Chemorepulsion assays based on live cell imaging confirmed the findings, and further demonstrated that iRGD dramatically collapses tumor cell protrusions leading to an instantaneous retraction of tumor cells from iRGD-bound surfaces. Our finding refutes the hypothesis that iRGD may cause enhanced metastasis. The mechanism of the anti-metastatic effect may involve neuropilin-mediated chemorepulsion of tumor cells. Collectively, iRGD may simultaneously achieve tumor-specific drug delivery and metastasis inhibition when utilized for cancer treatment. Citation Format: Kazuki N. Sugahara, Gary B. Braun, Tatiana H. de Mendoza, Venkata R. Kotamraju, Tambet Teesalu, Erkki Ruoslahti, Randall P. French, Andrew M. Lowy. A tumor-specific tissue penetrating peptide inhibits metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-102. doi:10.1158/1538-7445.AM2014-LB-102

Mouse midlate placental development involves extensive tissue remodeling and cell invasion, processes which could be mediated by extracellular proteolytic enzymes. We have performed in situ expression analysis of urokinase type plasminogen activator (uPA), as well as functionally related molecules (uPA receptor, low density lipoprotein receptor-related protein, plasminogen activator inhibitor type-1) in day 10.5 to 18.5 post coitum (p.c.) murine placentas. In situ hybridization demonstrated the presence of uPA transcripts in the invasive trophoblast cells, in particular in glycogen-rich trophoblasts, a cell population that between embryonic days 12.5 and 15.5 infiltrates the maternal decidual tissue. In addition, we observed high uPA expression in the cells of uterine epithelium. Enzymatically active uPA was detected in both sites of uPA mRNA expression by in situ zymography. Expression and activity data suggest a role for this protease in the processes of cell invasion and uterine epithelial remodeling. Only low levels of uPA receptor (uPAR) transcripts were found in trophoblasts and decidual tissue at days 10.5 and 11.5 p.c. At the same stages, a prominent expression of plasminogen activator inhibitor type-1 (PAI-1) by spongiotrophoblasts and giant trophoblasts, as well as of LDL receptor-related protein (LRP) by spongiotrophoblasts and decidual cells could be detected, suggesting a role in regulating extracellular proteolysis in the area of fetomaternal interface. Analysis of uPA null placentas showed the presence of decidual extravascular fibrin deposits, which were not detected in wild type placentas. At the same time, the extent of infiltration of trophoblast cells in maternal decidual tissue, evaluated by anti-cytokeratin immunostaining, was similar in wild type and uPA null placentas. Our studies show that in murine hemochorial placentation, uPA has an essential role in the maintenance of the fibrinogenic/fibrinolytic balance in the decidua. The function of uPA in trophoblast invasion appears not to be indispensable, and its absence can be overcome by redundant or compensatory mechanisms.

Bit1 is a proapoptotic mitochondrial protein associated with anoikis, cell death resulting from the loss of cell attachment to extracellular matrix. Upon cell detachment, Bit1 is released into the cytoplasm, where it complexes with the Groucho family protein amino-terminal enhancer of split, and triggers cell death through a caspase-independent pathway. Bit1 consists of 179 amino acids; the N-terminal 62 amino acid mitochondrial localization domain, a transmembrane domain, and the C-terminal peptidyl-tRNA hydrolase domain. In the present study, we localized the Bit1 cell death domain (BCDD) to the N-terminal 62 amino acids of Bit1 by introducing truncated Bit1 proteins into the cytoplasm with cDNAtransfection and protein transduction. BCDD was more potent than the full-length Bit1 protein, when equivalent amounts of cDNA was transfected. Treatment of cultured prostate and breast cancer cells with recombinant proteins in which BCDD was fused with two cell-internalizing peptides, one of which is tumor specific, also caused cell death. Bit1 and BCDD shared the characteristics previously attributed to Bit1-induced cell death. In addition, we found that the cells exhibited the signs of autophagy. These results define the cell death domain in Bit1 and reveal autophagy as the main mechanism of Bit1-induced cell death. Tumor-targeted exogenous BCDD may have potential as an anticancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C33.

We have cloned and characterized the HSP150 gene of Saccharomyces cerevisiae, which encodes a glycoprotein (hsp150) that is secreted into the growth medium. Unexpectedly, the HSP150 gene was found to be regulated by heat shock and nitrogen starvation. Shifting the cells from 24 degrees C to 37 degrees C resulted in an abrupt increase in the steady-state level of the HSP150 mRNA, and de novo synthesized hsp150 protein. Returning the cells to 24 degrees C caused a rapid decrease in mRNA and protein synthesis to basal levels. The HSP150 5'-flanking region contains several heat shock element-like sequences (HSE). To study the function of these sequences, a strain bearing a disrupted copy of the HSP150 gene was transformed with plasmids in which the coding region of HSP150, or a HSP150-lacZ fusion gene, was preceded by 5' deletion derivatives of the HSP150 promoter. Site-directed mutagenesis of one HSE-like element, located between the TATA box and transcription initiation sites, abolished heat activation of transcription. In addition to heat shock, the HSP150 gene is regulated by the availability of nutrients in the growth medium. The HSP150 mRNA level was increased by nitrogen limitation at 24 degrees C, even when under the control of a HSP150 promoter region of 137 bp carrying the mutagenized HSE.

e13590 Background: Poor drug penetration is a major problem in cancer treatment that leads to ineffective therapy and drug resistance. We have discovered a tumor-specific tissue penetration system ...

Tumor-selective drug conjugates can potentially improve the prognosis for patients affected by glioblastoma (GBM) - the most common and malignant type of brain cancer with no effective cure. Here we evaluated a novel tumor penetrating peptide that targets cell surface p32, LinTT1 (AKRGARSTA), as a GBM targeting ligand for systemically-administered nanoparticles. LinTT1-functionalization increased tumor homing of iron oxide nanoworms (NWs) across a panel of five GBM models ranging from infiltratively-disseminating to angiogenic phenotypes. LinTT1-NWs homed to CD31-positive tumor blood vessels, including to transdifferentiated endothelial cells, and showed co-localization with tumor macrophages and lymphatic vessels. LinTT1 functionalization also resulted in increased GBM delivery of other types of systemically-administered nanoparticles: silver nanoparticles and albumin-paclitaxel nanoparticles. Finally, LinTT1-guided proapoptotic NWs exerted strong anti-glioma activity in two models of GBM, including doubling the lifespan of the mice in an aggressive orthotopic stem cell-like GBM that recapitulates the histological hallmarks of human GBM. Our study suggests that LinTT1 targeting strategy can be used to increase GBM uptake of systemic nanoparticles for improved imaging and therapy. ispartof: JOURNAL OF CONTROLLED RELEASE vol:308 pages:109-118 ispartof: location:Netherlands status: published