New Therapeutic Approach for Targeting Hippo Signalling Pathway

Nuclear localization signals are short amino acid sequences that target proteins for nuclear import. In this manuscript, we have generated a chimeric tri-functional peptide composed of a cell penetrating peptide (CPP), a nuclear localization sequence and an interfering peptide blocking the interaction between TEAD and YAP, two transcription factors involved in the Hippo signalling pathway, whose deregulation is related to several types of cancer. We have validated the cell penetration and nuclear localization by flow cytometry and fluorescence microscopy and shown that the new generated peptide displays an apoptotic effect in tumor cell lines thanks to the specific nuclear delivery of the cargo, which targets a protein/protein interaction in the nucleus. In addition, the peptide has an anti-tumoral effect in vivo in xenograft models of breast cancer. The chimeric peptide designed in the current study shows encouraging prospects for developing nuclear anti- neoplastic drugs.Facultad de Ciencias Médica

Presentation of Integrins on Leukocyte Microvilli: A Role for the Extracellular Domain in Determining Membrane Localization

Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins α4β7, αLβ2, and αMβ2 were heterologously expressed in K562 cells. α4β7 initiated adhesion under flow and localized to microvilli, whereas β2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the α4β7 cytoplasmic and/or transmembrane domains with the homologous domains of αLβ2 or αMβ2. Unexpectedly, these chimeras efficiently mediated adhesion to the α4β7 ligand mucosal addressin cell adhesion molecule–1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of α4 and β2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins α4β1, α5β1, α6Aβ1, αvβ3, and αEβ7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli

Beneficial autoimmunity at body surfaces – immune surveillance and rapid type 2 immunity regulate tissue homeostasis and cancer

Epithelial cells line body surface tissues and provide a physicochemical barrier to the external environment. Frequent microbial and non-microbial challenges such as those imposed by mechanical disruption, injury or exposure to noxious environmental substances including chemicals, carcinogens, ultraviolet-irradiation or toxins cause activation of epithelial cells with release of cytokines and chemokines as well as alterations in the expression of cell surface ligands. Such display of epithelial stress is rapidly sensed by tissue resident immunocytes, which can directly interact with self-moieties on epithelial cells and initiate both local and systemic immune responses. Epithelial cells are thus key drivers of immune surveillance at body surface tissues. However, epithelial cells have a propensity to drive type 2 immunity (rather than type 1) upon non-invasive challenge or stress – a type of immunity whose regulation and function still remain enigmatic. Here we review the induction and possible role of type 2 immunity in epithelial tissues and propose that rapid immune surveillance and type 2 immunity are key regulators of tissue homeostasis and carcinogenesis

Global identification of genes and pathways regulated by Akt during activation of T helper cells

We previously demonstrated that Akt differentially modulated a subset of NF-kB target genes during T cell activation. In the current study, we further explored the broader effects of Akt inhibition on T cell gene induction. Global microarray analysis was used to characterize T helper cell transcriptional responses following antigen receptor stimulation in the absence or presence of Akti1/2 (an allosteric inhibitor which targets Akt1 and Akt2), to identify novel targets dependent upon Akt and obtain a more comprehensive view of Akt-sensitive genes in Th2 helper T cells. Pathway analysis of microarray data from a CD4+ Th2 T cell line revealed effects on gene networks involving ribosomal and T cell receptor signaling pathways associated with Akti1/2 treatment. Using real-time PCR analysis, we validated the differential regulation of several genes in these pathways, including Ier3, Il13, Egr1, Ccl1 and Ccl4, among others. Additionally, transcription factor target gene (TFactS) analysis revealed that NF-kB and Myc were the most significantly enriched transcription factors among Akt-dependent genes after T cell receptor and CD28 stimulation. Akt activation elicited increases in the enrichment of NF-kB- and Myc-targeted genes. The present study has identified a diverse set of genes, and possible mechanisms for their regulation, that are dependent on Akt during T cell activation

CXCR4/CXCL12 expression and signalling in kidney cancer

CXCL12 (SDF-1), a CXC-chemokine, and its specific receptor, CXCR4, have recently been shown to be involved in tumourgenesis, proliferation and angiogenesis. Therefore, we analysed CXCL12α/CXCR4 expression and function in four human kidney cancer cell lines (A-498, CAKI-1, CAKI-2, HA-7), 10 freshly harvested human tumour samples and corresponding normal kidney tissue. While none of the analysed tumour cell lines expressed CXCL12α, A-498 cells were found to express CXCR4. More importantly, real-time RT–PCR analysis of 10 tumour samples and respective adjacent normal kidney tissue disclosed a distinct and divergent downregulation of CXCL12α and upregulation of CXCR4 in primary tumour tissue. To prove that the CXCR4 protein is functionally active, rhCXCL12α was investigated for its ability to induce changes of intracellular calcium levels in A-498 cells. Moreover, we used cDNA expression arrays to evaluate the biological influence of CXCL12α. Comparing gene expression profiles in rhCXCL12α stimulated vs unstimulated A-498 kidney cancer cells revealed specific regulation of 31 out of 1176 genes tested on a selected human cancer array, with a prominent stimulation of genes involved in cell-cycle regulation and apoptosis. The genetic changes reported here should provide new insights into the developmental paths leading to tumour progression and may also aid the design of new approaches to therapeutic intervention

Function and Dynamics of Tetraspanins during Antigen Recognition and Immunological Synapse Formation

Tetraspanin-enriched microdomains (TEMs) are specialized membrane platforms driven by protein protein interactions that integrate membrane receptors and adhesion molecules. Tetraspanins participate in antigen recognition and presentation by antigen-presenting cells (APCs) through the organization of pattern-recognition receptors (PRRs) and their downstream induced signaling, as well as the regulation of MHC-II-peptide trafficking. T lymphocyte activation is triggered upon specific recognition of antigens present on the APC surface during immunological synapse (IS) formation. This dynamic process is characterized by a defined spatial organization involving the compartmentalization of receptors and adhesion molecules in specialized membrane domains that are connected to the underlying cytoskeleton and signaling molecules. Tetraspanins contribute to the spatial organization and maturation of the IS by controlling receptor clustering and local accumulation of adhesion receptors and integrins, their downstream signaling, and linkage to the actin cytoskeleton. This review offers a perspective on the important role of TEMs in the regulation of antigen recognition and presentation and in the dynamics of IS architectural organization.The cost of this publication has been paid in part by FEDER funds.S

Expression of KAI1 in Paraffin-Embedded Normal, Hyperplastic and Neoplastic Prostate and Prostate Carcinoma Cell Lines

Expression of KAI1, a tumor metastasis suppressor gene, was studied with different fixatives in frozen and paraffin-embedded sections of human and rat prostate carcinoma cell lines and human prostate lesions by Immunohisto-chemistry. Immunoreactivity of the membrane antigen in cell lines was associated with known expression levels in these lines and the fixative used. Formalin and paraformaldehyde helped maintain the immunoreactivity of cells. In human prostate, frozen sections revealed diffuse reactivity of the antigen in normal and neoplastic tissues while paraffin-embedded tissues usually showed focal reactivity, although more than 50% of cases with normal epithelium and adenocarcinomas were reactive. In some cases, pretreatment with trypsln enhanced immunoreactivity. Benign prostatic hyperplasia (BPH) showed the most intense diffuse immunoreactivity, which suggested enhanced expression. Prostatic intraepithelial neoplasia (PIN) also often expressed high levels of KAI1. Three of five metastases were reactive but two primaries and their metastases were not. Lymphocytes in primary carcinomas and lymphocytes and germinal center cells in lymph nodes were immunoreactive, while adjacent primary or metastatic prostate adenocarcinoma epithelium was not immunoreactive. Although paraffin-embedded human tissues were not optimal for determining levels of expression of KAI1, they did show immunoreactivity that could have prognostic value and showed the specific cytoplasmlc localization of the protein in cells