Noncoding RNAs in Cancer Medicine

Several signalling proteins involved in cell growth and differentiation represent attractive candidate targets for cancer diagnosis and/or therapy since they can act as oncogenes. Because of their high specificity and low immunogeneicity, using artificial small noncoding RNA (ncRNAs) as therapeutics has recently become a highly promising and rapidly expanding field of interest. Indeed, ncRNAs may either interfere with RNA transcription, stability, translation or directly hamper the function of the targets by binding to their surface. The recent finding that the expression of several genes is under the control of small single-stranded regulatory RNAs, including miRNAs, makes these genes as appropriate targets for ncRNA gene silencing. Furthermore, another class of small ncRNA, aptamers, act as high-affinity ligands and potential antagonists of disease-associated proteins. We will review here the recent and innovative methods that have been developed and the possible applications of ncRNAs as inhibitors or tracers in cancer medicine

The Tumor Suppressive Role of PATZ1 in Thyroid Cancer: A Matter ofEpithelial-Mesenchymal Transition

PATZ1 is a chromatin-regulating factor with emerging roles in stemness and cancer. It has been suggested to play a dual oncogene/tumor suppressor role depending on the cellular context, but its function in human tumor biology is still far to be completely elucidated. We have recently identified its tumor suppressive role in thyroid carcinogenesis, possibly through the association between PATZ1 and p53 to oppose epithelial-mesenchymal transition and cell migration. These are major processes in tumor progression, local invasion, metastasis, and therapeutic resistance and play a recognized role in the development of thyroid cancer, particularly anaplastic thyroid carcinoma, but many questions about how they are orchestrated remain opened. Elucidation of the mechanisms regulating epithelial-mesenchymal transition and cell migration can suggest new candidates for antimetastatic drug development that could lead to more effective therapies for highly aggressive and lethal thyroid cancers

The Sso7d protein of Sulfolobus solfataricus

The physiological role of the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus is unknown. In vitro studies have shown that Sso7d promotes annealing of complementary DNA strands (Guagliardi et al. 1997), induces negative supercoiling (Lopez-Garcia et al. 1998), and chaperones the disassembly and renaturation of protein aggregates in an ATP hydrolysis-dependent manner (Guagliardi et al. 2000). In this study, we examined the relationships among the binding of Sso7d to double-stranded DNA, its interaction with protein aggregates, and its ATPase activity. Experiments with 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that exposed hydrophobic surfaces in Sso7d are responsible for interactions with protein aggregates and double-stranded DNA, whereas the site of ATPase activity has a non-hydrophobic character. The interactions of Sso7d with double-stranded DNA and with protein aggregates are mutually exclusive events, suggesting that the disassembly activity and the DNA-related activities of Sso7d may be competitive in vivo. In contrast, the hydrolysis of ATP by Sso7d is independent of the binding of Sso7d to double-stranded DNA or protein aggregates

distribution and bioactivity of the ret specific d4 aptamer in three dimensional collagen gel cultures

The success of tyrosine kinase inhibitors in cancer therapy prompted intensive research efforts addressed to the development of new specific diagnostics and therapeutics. Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacologic challenge. The D4 RNA-aptamer, isolated applying the Systematic Evolution of Ligand by Exponential Enrichment procedure on living cells, has been proven a specific inhibitor of the human receptor tyrosine kinase Ret. In our attempts to generate new powerful probes for in vivo applications, in the present study, we addressed the ability of D4 to preserve its biological activity in cells embedded in three-dimensional collagen gels. These matrices provide a microenvironment mimicking the cell organization as seen in vivo , thus representing a suitable tool to approach the use of the aptamer in vivo . By taking advantage of transformed fibroblasts expressing Ret as a model system, we showed that the cells maintain normal phenotype and growth patterns when cultured in three-dimensional matrices and that the D4 aptamer preserves its ability to inhibit Ret on the surface of the cells embedded in collagen. Because the biological activity of RNA aptamers is largely dictated by their folded structure, the results indicate that a folded conformation of D4 responsible of its inhibiting function is preserved in the three-dimensional constructs, thus supporting its use in tumors in vivo . [Mol Cancer Ther 2008;7(10):3381–8

Differential SELEX in Human Glioma Cell Lines

The hope of success of therapeutic interventions largely relies on the possibility to distinguish between even close tumor types with high accuracy. Indeed, in the last ten years a major challenge to predict the responsiveness to a given therapeutic plan has been the identification of tumor specific signatures, with the aim to reduce the frequency of unwanted side effects on oncologic patients not responding to therapy. Here, we developed an in vitro evolution-based approach, named differential whole cell SELEX, to generate a panel of high affinity nucleic acid ligands for cell surface epitopes. The ligands, named aptamers, were obtained through the iterative evolution of a random pool of sequences using as target human U87MG glioma cells. The selection was designed so as to distinguish U87MG from the less malignant cell line T98G. We isolated molecules that generate unique binding patterns sufficient to unequivocally identify any of the tested human glioma cell lines analyzed and to distinguish high from low or non-tumorigenic cell lines. Five of such aptamers act as inhibitors of specific intracellular pathways thus indicating that the putative target might be important surface signaling molecules. Differential whole cell SELEX reveals an exciting strategy widely applicable to cancer cells that permits generation of highly specific ligands for cancer biomarkers

Optimization of Short RNA Aptamers for TNBC Cell Targeting

Triple-negative breast cancer (TNBC) is an aggressive cancer with limited targeted therapies. RNA aptamers, suitably chemically modified, work for therapeutic purposes in the same way as antibodies. We recently generated 2′ Fluoro-pyrimidines RNA-aptamers that act as effective recognition elements for functional surface signatures of TNBC cells. Here, we optimized three of them by shortening and proved the truncated aptamers as optimal candidates to enable active targeting to TNBC. By using prediction of secondary structure to guide truncation, we identified structural regions that account for the binding motifs of the full-length aptamers. Their chemical synthesis led to short aptamers with superb nuclease resistance, which specifically bind to TNBC target cells and rapidly internalize into acidic compartments. They interfere with the growth of TNBC cells as mammospheres, thus confirming their potential as anti-tumor agents. We propose sTN145, sTN58 and sTN29 aptamers as valuable tools for selective TNBC targeting and promising candidates for effective treatments, including therapeutic agents and targeted delivery nanovectors.Fil: Camorani, Simona. Institute Of Experimental Endocrinology And Oncology; ItaliaFil: D'Argenio, Annachiara. Institute Of Experimental Endocrinology And Oncology; ItaliaFil: Agnello, Lisa. Institute Of Experimental Endocrinology And Oncology; ItaliaFil: Nilo, Roberto. Institute Of Experimental Endocrinology And Oncology; ItaliaFil: Zanetti, Antonella. Institute Of Biostructures And Bioimaging; ItaliaFil: Ibarra, Luis Exequiel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Fedele, Monica. Institute Of Experimental Endocrinology And Oncology; ItaliaFil: Cerchia, Laura. Institute Of Experimental Endocrinology And Oncology; Itali

Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARλ 3 full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARλ 3, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARλ 3 provides a rationale for the different activation of the ligand towards PPARα and PPARλ 3, suggesting a novel basis for ligand design

PO-038 PDGFRβ as a new biomarker for metastatic triple-negative breast cancer: development of a theranostic anti-PDGFRβ aptamer for imaging and suppression of metastases

Introduction Triple-negative breast cancers (TNBCs) are a heterogeneous group of aggressive tumours lacking oestrogen and progesterone receptors and HER2 receptor, thus excluding the possibility of using targeted therapy against these proteins. Mesenchymal-like (ML) subtype, characterised by a stem-like, undifferentiated phenotype, is more invasive and metastatic than other TNBC subtypes and has a strong tendency to form vasculogenic mimicry (VM). Recently, platelet derived growth factor receptor β (PDGFRβ) has been shown to play a role in VM of TNBC. Regrettably, therapies targeting PDGFRβ with tyrosine kinase inhibitors are not effective in treating TNBCs, thus developing new strategies to target PDGFRβ in TNBC patients is crucial to improve their chances of survival. Here, we describe the characterisation of the Gint4.T anti-PDGFRβ nuclease-resistant RNA aptamer as high efficacious theranostic tool for imaging and suppression of ML TNBC metastases. Material and methods Immunohistochemical analyses on a human TNBC tissue microarray was performed to correlate PDGFRβ expression with clinical and molecular features of different subtypes. Functional assays were conducted on PDGFRβ-positive ML BT-549 and MDA-MB-231 cells to investigate the effect of Gint4.T in interfering with cell growth in 3D conditions, migration, invasion and VM formation. Gint4.T was conjugated with near-infrared (NIR) fluorescent VivoTag-S680 and its binding specificity to receptor was confirmed both in vitro (confocal microscopy and flow cytometry analyses of TNBC cells) and in vivo (fluorescence molecular tomography in mice bearing TNBC xenografts). MDA-MB-231 cells were i.v. injected in nude mice and Gint4.T-NIR was used to detect lung metastases in mice untreated or i.v. injected with Gint4.T or a scrambled aptamer. Results and discussions The expression of PDGFRβ was observed in human TNBC samples characterised by higher metastatic behaviour. Treatment of TNBC cell lines with Gint4.T aptamer blocked their invasive growth and vasculogenic properties in 3D culture conditions, and strongly reduced cell migration/invasion in vitro and metastases formation in vivo. The Gint4.T-NIR was able to specifically bind to TNBC xenografts and detect lung metastases in vivo. Therefore, the aptamer revealed a high efficacious theranostic tool for imaging and suppression of TNBC metastases. Conclusion These studies indicate PDGFRβ as a new biomarker for ML and metastatic TNBC subtype and propose a novel targeting agent for the diagnosis and treatment of metastatic TNBCs

Neutralizing Aptamers from Whole-Cell SELEX Inhibit the RET Receptor Tyrosine Kinase

Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance