Adelaide Greco

Small interfering RNA-induced TLR3 activation inhibits blood and lymphatic vessel growth.

Neovascularization in response to tissue injury consists of the dual invasion of blood (hemangiogenesis) and lymphatic (lymphangiogenesis) vessels. We reported recently that 21-nt or longer small interfering RNAs (siRNAs) can suppress hemangiogenesis in mouse models of choroidal neovascularization and dermal wound healing independently of RNA interference by directly activating Toll-like receptor 3 (TLR3), a double-stranded RNA immune receptor, on the cell surface of blood endothelial cells. Here, we show that a 21-nt nontargeted siRNA suppresses both hemangiogenesis and lymphangiogenesis in mouse models of neovascularization induced by corneal sutures or hindlimb ischemia as efficiently as a 21-nt siRNA targeting vascular endothelial growth factor-A. In contrast, a 7-nt nontargeted siRNA, which is too short to activate TLR3, does not block hemangiogenesis or lymphangiogenesis in these models. Exposure to 21-nt siRNA, which we demonstrate is not internalized unless cell-permeating moieties are used, triggers phosphorylation of cell surface TLR3 on lymphatic endothelial cells and induces apoptosis. These findings introduce TLR3 activation as a method of jointly suppressing blood and lymphatic neovascularization and simultaneously raise new concerns about the undesirable effects of siRNAs on both circulatory systems.

Sphingosine Kinase 1 Overexpression Contributes to Cetuximab Resistance in Human Colorectal Cancer Models

Purpose: Although the anti–EGF receptor (EGFR) monoclonal antibody cetuximab is an effective strategy in colorectal cancer therapy, its clinical use is limited by intrinsic or acquired resistance. Alterations in the “sphingolipid rheostat”—the balance between the proapoptotic molecule ceramide and the mitogenic factor sphingosine-1-phosphate (S1P)—due to sphingosine kinase 1 (SphK1) overactivation have been involved in resistance to anticancer-targeted agents. Moreover, cross-talks between SphK1 and EGFR-dependent signaling pathways have been described. Experimental design: We investigated SphK1 contribution to cetuximab resistance in colorectal cancer, in preclinical in vitro/in vivo models, and in tumor specimens from patients. Results: SphK1 was found overexpressed and overactivated in colorectal cancer cells with intrinsic or acquired resistance to cetuximab. SphK1 contribution to resistance was supported by the demonstration that SphK1 inhibition by N,N-dimethyl-sphingosine or silencing via siRNA in resistant cells restores sensitivity to cetuximab, whereas exogenous SphK1 overexpression in sensitive cells confers resistance to these agents. Moreover, treatment of resistant cells with fingolimod (FTY720), a S1P receptor (S1PR) antagonist, resulted in resensitization to cetuximab both in vitro and in vivo, with inhibition of tumor growth, interference with signal transduction, induction of cancer cells apoptosis, and prolongation of mice survival. Finally, a correlation between SphK1 expression and cetuximab response was found in colorectal cancer patients. Clin Cancer Res; 19(1); 138–47. ©2012 AACR.

Eradication of Therapy-resistant Human Prostate Tumors Using an Ultrasound-guided Site-specific Cancer Terminator Virus Delivery Approach

Intratumoral injections of a replication-incompetent adenovirus (Ad) expressing melanoma differentiation-associated gene-7/interleukin-24 (Ad.mda-7), a secreted cytokine displaying cancer-selective, apoptosis-inducing properties, profoundly inhibits prostate cancer (PC) growth in immune-incompetent animals. In contrast, Ad.mda-7 is ineffective in PCs overexpressing antiapoptotic proteins such as Bcl-2 or Bcl-x(L). However, intratumoral injections of a conditionally replication-competent Ad (CRCA) in which expression of the adenoviral E1A gene is driven by the cancer-specific promoter of progression-elevated gene-3 (PEG-3) and which simultaneously expresses mda-7/interleukin (IL)-24 in the E3 region of the Ad (Ad.PEG-E1A-mda-7), a cancer terminator virus (CTV), is highly active in these cells. A major challenge for gene therapy is systemic delivery of nucleic acids directly into an affected tissue. Ultrasound (US) contrast agents (microbubbles-MBs) are viable candidates for gene delivery/therapy. Here, we show that MB/Ad.mda-7 complexes targeted to DU-145 cells using US dramatically reduced tumor burden in xenografted nude mice. Additionally, US-guided MB/CTV delivery completely eradicated not only targeted DU-145/Bcl-x(L)-therapy-resistant tumors, but also nontargeted distant tumors (established in the opposite flank), thereby implementing a cure. These findings highlight potential therapeutic applications of this novel image-guided gene therapy technology for advanced PC patients with metastatic disease.

Rapid selection and proliferation of CD133+ cells from cancer cell lines: chemotherapeutic implications.

Cancer stem cells (CSCs) are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133(+)] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB) (Celdyne, Houston, TX). For comparison, another bioreactor, the rotary cell culture system (RCCS) manufactured by Synthecon (Houston, TX) was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133(+) cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a (+)15-fold proliferation of the CD133(+) cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (−)4.8-fold decrease in the CD133(+)cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133(+) cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.

FK506 binding protein 51 positively regulates melanoma stemness and metastatic potential

Melanoma is the most aggressive skin cancer; there is no cure in advanced stages. Identifying molecular participants in melanoma progression may provide useful diagnostic and therapeutic tools. FK506 binding protein 51 (FKBP51), an immunophilin with a relevant role in developmental stages, is highly expressed in melanoma and correlates with aggressiveness and therapy resistance. We hypothesized a role for FKBP51 in melanoma invasive behaviour. FKBP51 promoted activation of epithelial-to-mesenchymal transition (EMT) genes and improved melanoma cell migration and invasion. In addition, FKBP51 induced some melanoma stem cell (MCSC) genes. Purified MCSCs expressed high EMT genes levels, suggesting that genetic programs of EMT and MCSCs overlap. Immunohistochemistry of samples from patients showed intense FKBP51 nuclear signal and cytoplasmic positivity for the stem cell marker nestin in extravasating melanoma cells and metastatic brains. In addition, FKBP51 targeting by small interfering RNA (siRNA) prevented the massive metastatic substitution of liver and lung in a mouse model of experimental metastasis. The present study provides evidence that the genetic programs of cancer stemness and invasiveness overlap in melanoma, and that FKBP51 plays a pivotal role in sustaining such a program.

Ultrasound Biomicroscopy in Small Animal Research: Applications in Molecular and Preclinical Imaging

Ultrasound biomicroscopy (UBM) is a noninvasive multimodality technique that allows high-resolution imaging in mice. It is affordable, widely available, and portable. When it is coupled to Doppler ultrasound with color and power Doppler, it can be used to quantify blood flow and to image microcirculation as well as the response of tumor blood supply to cancer therapy. Target contrast ultrasound combines ultrasound with novel molecular targeted contrast agent to assess biological processes at molecular level. UBM is useful to investigate the growth and differentiation of tumors as well as to detect early molecular expression of cancer-related biomarkers in vivo and to monitor the effects of cancer therapies. It can be also used to visualize the embryological development of mice in uterus or to examine their cardiovascular development. The availability of real-time imaging of mice anatomy allows performing aspiration procedures under ultrasound guidance as well as the microinjection of cells, viruses, or other agents into precise locations. This paper will describe some basic principles of high-resolution imaging equipment, and the most important applications in molecular and preclinical imaging in small animal research.

The HMGA1-IGF-I/IGFBP System: A Novel Pathway for Modulating Glucose Uptake

We previously showed that loss of the high mobility group A1 (HMGA1) protein expression, induced in mice by disrupting the Hmga1 gene, considerably decreased insulin receptor expression in the major target tissues of insulin action, causing a type 2-like diabetic phenotype, in which, however, glucose intolerance was paradoxically associated with increased peripheral insulin sensitivity. Insulin hypersensitivity despite impairment of insulin action supports the existence of molecular adaptation mechanisms promoting glucose disposal via insulin-independent processes. Herein, we provide support for these compensatory pathways/circuits of glucose uptake in vivo, the activation of which under certain adverse metabolic conditions may protect against hyperglycemia. Using chromatin immunoprecipitation combined with protein-protein interaction studies of nuclear proteins in vivo, and transient transcription assays in living cells, we show that HMGA1 is required for gene activation of the IGF-binding proteins 1 (IGFBP1) and 3 (IGFBP3), two major members of the IGF-binding protein superfamily. Furthermore, by using positron emission tomography with (18)F-labeled 2-fluoro-2-deoxy-d-glucose, in combination with the euglycemic clamp with IGF-I, we demonstrated that IGF-Is bioactivity was increased in Hmga1-knockout mice, in which both skeletal muscle Glut4 protein expression and glucose uptake were enhanced compared with wild-type littermates. We propose that, by affecting the expression of both IGFBP protein species, HMGA1 can serve as a modulator of IGF-I activity, thus representing an important novel mediator of glucose disposal.

Mice Anesthesia, Analgesia, and Care, Part II: Anesthetic Considerations in Preclinical Imaging Studies

Animal experiments are necessary for a better understanding of diseases and for developing new therapeutic strategies. The mouse (Mus musculus) is currently the most popular laboratory animal in biomedical research. Mice imaging procedures are increasingly used in preclinical research because they allow in vivo monitoring and they are readily available for longitudinal and noninvasive studies as well as investigations into the evolution of diseases and the effects of new therapies. New imaging techniques and sophisticated laboratory animal imaging tools are currently producing a large body of evidence about the possible interference of anesthesia with different imaging methods that have the potential to compromise the results of in vivo studies. The purpose of this article is to review the existing literature on molecular imaging studies in mice, to describe the effects of different anesthetic protocols on their outcome, and to report our own experience with such studies.

FKBP51 increases the tumour-promoter potential of TGF-beta

BackgroundFKBP51 (FKBP5 Official Symbol) is a large molecular weight component of the family of FK506 binding proteins (FKBP). In recent years, research studies from our laboratory highlighted functions for FKBP51 in the control of apoptosis and melanoma progression. FKBP51 expression correlated with the invasiveness and aggressiveness of melanoma. Since a role for TGF-β in the enhanced tumorigenic potential of melanoma cells is widely described, we hypothesized a cooperative effect between FKBP51 and TGF-β in melanoma progression.MethodsSAN and A375 melanoma cell lines were utilized for this study. Balb/c IL2γ NOD SCID served to assess the ability to colonize organs and metastasize of different cell lines, which was evaluated by in vivo imaging. Realtime PCR and western blot served for measurement of mRNA and protein expression, respectively.ResultsBy comparing the metastatic potential of two melanoma cell lines, namely A375 and SAN, we confirmed that an increased capability to colonize murine organs was associated with increased levels of FKBP51. A375 melanoma cell line expressed FKBP51 mRNA levels 30-fold higher in comparison to the SAN mRNA level and appeared more aggressive than SAN melanoma cell line in an experimental metastasis model. In addition, A375 expressed, more abundantly than SAN, the TGF-β and the pro angiogenic TGF-β receptor type III (TβRIII) factors. FKBP51 silencing produced a reduction of TGF-β and TβRIII gene expression in A375 cell line, in accordance with previous studies. We found that the inducing effect of TGF-β on Sparc and Vimentin expression was impaired in condition of FKBP51 depletion, suggesting that FKBP51 is an important cofactor in the TGF-β signal. Such a hypothesis was supported by co immunoprecipitation assays, showing that FKBP51 interacted with either Smad2,3 and p300. In normal melanocytes, FKBP51 potentiated the effect of TGF-β on N-cadherin expression and conferred a mesenchymal-like morphology to such round-shaped cells.ConclusionsOverall, our findings show that FKBP51 enhances some pro oncogenic functions of TGF-β, suggesting that FKBP51-overexpression may help melanoma to take advantage of the tumor promoting activities of the cytokine.

PET/CT Imaging in Mouse Models of Myocardial Ischemia

Different species have been used to reproduce myocardial infarction models but in the last years mice became the animals of choice for the analysis of several diseases, due to their short life cycle and the possibility of genetic manipulation. Many techniques are currently used for cardiovascular imaging in mice, including X-ray computed tomography (CT), high-resolution ultrasound, magnetic resonance imaging, and nuclear medicine procedures. Cardiac positron emission tomography (PET) allows to examine noninvasively, on a molecular level and with high sensitivity, regional changes in myocardial perfusion, metabolism, apoptosis, inflammation, and gene expression or to measure changes in anatomical and functional parameters in heart diseases. Currently hybrid PET/CT scanners for small laboratory animals are available, where CT adds high-resolution anatomical information. This paper reviews mouse models of myocardial infarction and discusses the applications of dedicated PET/CT systems technology, including animal preparation, anesthesia, radiotracers, and images postprocessing.