The tax-inducible actin-bundling protein fascin is crucial for release and cell-to-cell transmission of human T-cell leukemia virus type 1 (HTLV-1)

The delta-retrovirus Human T-cell leukemia virus type 1 (HTLV-1) preferentially infects CD4(+) T-cells via cell-to-cell transmission. Viruses are transmitted by polarized budding and by transfer of viral biofilms at the virological synapse (VS). Formation of the VS requires the viral Tax protein and polarization of the host cytoskeleton, however, molecular mechanisms of HTLV-1 cell-to-cell transmission remain incompletely understood. Recently, we could show Tax-dependent upregulation of the actin-bundling protein Fascin (FSCN-1) in HTLV-1-infected T-cells. Here, we report that Fascin contributes to HTLV-1 transmission. Using single-cycle replication-dependent HTLV-1 reporter vectors, we found that repression of endogenous Fascin by short hairpin RNAs and by Fascin-specific nanobodies impaired gag p19 release and cell-to-cell transmission in 293T cells. In Jurkat T-cells, Tax-induced Fascin expression enhanced virus release and Fascin-dependently augmented cell-to-cell transmission to Raji/CD4(+) B-cells. Repression of Fascin in HTLV-1-infected T-cells diminished virus release and gag p19 transfer to co-cultured T-cells. Spotting the mechanism, flow cytometry and automatic image analysis showed that Tax-induced T-cell conjugate formation occurred Fascin-independently. However, adhesion of HTLV-1-infected MT-2 cells in co-culture with Jurkat T-cells was reduced upon knockdown of Fascin, suggesting that Fascin contributes to dissemination of infected T-cells. Imaging of chronically infected MS9 T-cells in co-culture with Jurkat T-cells revealed that Fascin's localization at tight cell-cell contacts is accompanied by gag polarization suggesting that Fascin directly affects the distribution of gag to budding sites, and therefore, indirectly viral transmission. In detail, we found gag clusters that are interspersed with Fascin clusters, suggesting that Fascin makes room for gag in viral biofilms. Moreover, we observed short, Fascin-containing membrane extensions surrounding gag clusters and clutching uninfected T-cells. Finally, we detected Fascin and gag in long-distance cellular protrusions. Taken together, we show for the first time that HTLV-1 usurps the host cell factor Fascin to foster virus release and cell-to-cell transmission

The FDA-Approved Antiviral Raltegravir Inhibits Fascin1-Dependent Invasion of Colorectal Tumor Cells In Vitro and In Vivo

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Serrated adenocarcinoma (SAC) has been recently recognized by the WHO as a histological CRC with bad prognosis. Consistent with previous evidence, our group identified Fascin1 as a protein directly related to the invasiveness of tumor cells, overexpressed and positively correlated with worse survival in various carcinomas, including SAC. Therefore, Fascin1 has emerged as an ideal target for cancer treatment. In the present study, virtual screening has been carried out from a library of 9591 compounds, thus identifying the FDA-approved anti-retroviral raltegravir (RAL) as a potential Fascin1 blocker. In vitro and in vivo results show that RAL exhibits Fascin1-binding activity and Fascin1-dependent anti-invasive and anti-metastatic properties against CRC cells both in vitro and in vivo

A Panel of Eight miRNAs Is Deregulated in HTLV-2 Infected PBMCs and BJABGu Cell Line

Despite human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 being retroviruses closely related at a genomic level, HTLV-2 differs from HTLV-1 in terms of pathogenicity in both single infection and coinfection contexts. Moreover, the HTLV-2 association with clinical outcomes is still debated and several mechanisms underlying HTLV-2 infection remain unexplored as well. Cellular miRNAs are key factors in the post-transcriptional regulation of gene expression and they are known to be potential targets for several pathogens to control the host microenvironment and, in particular, escape immune responses. Here, we identified a HTLV-2-related signature of eight miRNAs (miR-125a-3p, miR-381-3p, miR-502-5p, miR-708-5p, miR-548d-5p, miR-548c-5p, miR-1-3p, and miR-511-5p) in both HTLV-2 infected PBMC and BJABGu cell lines. Altered miRNA expression patterns were correlated with the impairment of Th cell differentiation and signaling pathways driven by cytokines and transcriptional factors such as the Runt-related transcription factor (RUNX) family members. Specifically, we demonstrated that the RUNX2 protein was significantly more expressed in the presence of Tax-2 compared with Tax-1 in an in vitro cell model. To the best of our knowledge, these data represent the first contribution to elucidating the HTLV-2 mediated alteration of host cell miRNA profiles that may impact on HTLV-2 replication and persistent infection

Cell Surface Markers in HTLV-1 Pathogenesis

The phenotype of HTLV-1-transformed CD4+ T lymphocytes largely depends on defined viral effector molecules such as the viral oncoprotein Tax. In this review, we exemplify the expression pattern of characteristic lineage markers, costimulatory receptors and ligands of the tumor necrosis factor superfamily, cytokine receptors, and adhesion molecules on HTLV-1-transformed cells. These molecules may provide survival signals for the transformed cells. Expression of characteristic surface markers might therefore contribute to persistence of HTLV-1-transformed lymphocytes and to the development of HTLV-1-associated disease

Fascin in Gynecological Cancers: An Update of the Literature

Fascin is an actin-binding protein that is encoded by the gene (located on chromosome 7). It triggers membrane projections and stimulates cell motility in cancer cells. Fascin overexpression has been described in different types of human cancers in which its expression correlated with tumor growth, migration, invasion, and metastasis. Moreover, overexpression of fascin was found in oncovirus-infected cells, such as human papillomaviruses (HPVs) and Epstein-Barr virus (EBV), disrupting the cell-cell adhesion and enhancing cancer progression. Based on these findings, several studies reported fascin as a potential biomarker and a therapeutic target in various cancers. This review provides a brief overview of the FSCN1 role in various cancers with emphasis on gynecological malignancies. We also discuss fascin interactions with other genes and oncoviruses through which it might induce cancer development and progression

Viral Carcinogenesis Beyond Malignant Transformation: EBV in the Progression of Human Cancers

Cancer progression begins when malignant cells colonize adjacent sites, and it is characterized by increasing tumor heterogeneity, invasion and dissemination of cancer cells. Clinically, progression is the most relevant stage in the natural history of cancers. A given virus is usually regarded as oncogenic because of its ability to induce malignant transformation of cells. Nonetheless, oncogenic viruses may also be important for the progression of infection-associated cancers. Recently this hypothesis has been addressed because of studies on the contribution of the Epstein–Barr virus (EBV) to the aggressiveness of nasopharyngeal carcinoma (NPC). Several EBV products modulate cancer progression phenomena, such as the epithelial–mesenchymal transition, cell motility, invasiveness, angiogenesis, and metastasis. In this regard, there are compelling data about the effects of EBV latent membrane proteins (LMPs) and EBV nuclear antigens (EBNAs), as well as nontranslated viral RNAs, such as the EBV-encoded small nonpolyadenylated RNAs (EBERs) and viral microRNAs, notably EBV miR-BARTs. The available data on the mechanisms and players involved in the contribution of EBV infection to the aggressiveness of NPC are discussed in this review. Overall, this conceptual framework may be valuable for the understanding of the contribution of some infectious agents in the progression of cancers

Functional interactions of the Tax and p13 proteins of Human T-cell Leukemia Virus Type I

Human T-cell leukemia virus type 1 (HTLV-1) establishes a lifelong persistent infection in humans. Approximately 3% of the infected individuals will develop adult T-cell leukemia/lymphoma (ATLL), an aggressive malignancy of mature CD4+ T-cells. The viral protein Tax plays a major role in HTLV-1 pathogenicity by activating the NF-κB pathway. Tax activates both the canonical and non-canonical NF-κB pathways, promoting NF-κB translocation to the nucleus and transcription of genes that favour T-cell proliferation and survival. Our previous studies showed that the p13 protein of HTLV-1 enhances mitochondrial ROS production, resulting in activation of normal T-cells. ROS constitute a homeostatic rheostat that controls the activity of several key pathways, including the NF-κB pathway.Thus, we hypothesized that the effects of p13 on ROS production could affect the activation of the NF-κB pathway by Tax in primary T-cells. The work described in the present thesis was aimed at testing the hypothesis that Tax and p13 might act in concert to activate the NF-κB signal transduction pathway in primary T-cells. To this end, we optimized a transfection protocol for primary T-cells using an innovative approach based on the electroporation of in vitro-transcribed RNA. Activation of the NF-κB pathway was then analysed by measuring expression of the NF-κB target genes CD25 and 4-1BB. Results showed that the co-transfection of Tax and p13 resulted in a synergistic activation of the NF-κB pathway in primary T-cells measured as an increase in the expression levels of both CD25 and 4-1BB. In addition to being a transcriptional target of NF-κB, CD25 is also an early marker of T-cell activation. To further test the effects of Tax and p13 on cell activation, we measured CD38 expression by flow cytometry. Jurkat T-cells, which exhibit a constitutively activated CD38 positive phenotype, were used as a control. Results of this analysis confirmed the synergy of Tax and p13, although the effect was not so prominent as that observed for the CD25 marker, suggesting that, within the time frame of our experiments, Tax and p13 drove T-cells to an early-intermediate stage of activation. Taken together, these findings suggest that, in contrast to the well-established role of Tax as an activator of the NF-κB pathway in tumor cell lines, in the context of normal T-cells, the induction of NF-κB target genes requires the concerted action of Tax and p13. Current studies are aimed at verifying the ROS-dependence of this effect and testing the functional interaction of Tax and p13 in the context of the complete HTLV-1 genome using wild type HTLV-1 and a p13-knock-out HTLV-1 molecular clone. These experiments will be carried out in primary T-cells as well as in dendritic cells, which have recently emerged as an important target of the virus in vivo

Investigating the role of fascin in murine models of inflammatory bowel disease and tumourigenesis

Recent evidence suggests that stem cells are important for cancer metastasis and that the epithelial-to-mesenchymal transition also involves a transition toward stemness. Current thinking suggests that lgr5, a 7-transmembrane spanning G-protein, also marks a certain population of stem cells capable of regenerating an intestinal crypt and that the specialised immune secretory paneth cell, is important for maintenance of the stem cell niche. We implicate fascin in regulating the balance of lgr5 stem cells during acute intestinal inflammation and in regenerating intestinal tissue. Fascin is an actin bundling protein that drives the assembly of filopodia through the cross-linking of actin filaments into straight bundles. Conserved from amoebas to man, fascin was originally purified from extracts of sea urchin oocytes and coelomocytes and later found in Drosophila as the singed gene product. It is involved in the invasion and metastasis of multiple epithelial cancer types through stabilisation of actin in invadopodia, finger like protrusions used by cancer cells to invade into and degrade the extra-cellular matrix. Fascin, whilst normally low or absent from epithelia, localises to the leading edges of migratory cells and is over-expressed in many cancers of the same epithelial origin including lung, colorectal, pancreatic and liver. Fascin has also recently been shown to increase during inflammatory bowel disease (IBD) conditions such as diverticulitis, Crohn’s disease and ulcerative colitis. In this thesis I have investigated the role of fascin in murine models of IBD and have demonstrated that fascin is required for the haematopoietic production of leucocytes, in response to inflammation and that the loss of fascin, in the presence of high Wnt levels, results in enhanced proliferation of intestinal epithelial cells. One of the serious consequences of IBD is the increased lifetime risk of the patient developing an intestinal malignancy secondary to the disease. The exact mechanism underlying the increase in malignancies has not yet been fully established, however it is postulated that chronic inflammation and the effect this has on the major molecular pathways involved in carcinogenesis underlies the transformation from benign to malignant disease. Highest fascin expression has been shown in the dysplastic, pre-malignant cells in human IBD tissues indicating an important role of fascin in the transformation of benign to malignant cells. In this thesis, I have demonstrated that loss of fascin impairs tumour initiation in inflammatory driven and spontaneous intestinal tumourigenesis models, which is likely, in part, to be as a consequence of reduced leucocytes, in particular neutrophils, which may be CXCL2 mediated

NEUROPILIN-1 IS UPREGULATED BY HTLV-1 bZIP FACTOR AND INHIBITS CELL-TO-CELL TRANSMISSION OF HTLV-1

Human T-cell Leukemia Virus Type 1 (HTLV-1) is the etiologic agent of devastating diseases, including adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 relies heavily on cell-to-cell transmission as free virions are poorly infectious. Although cell-to-cell transmission is critical for efficient spread of HTLV-1, much is unknown about the impact of extracellular proteins on viral transmission. Infection studies have been predominantly focused on HTLV-1 Transactivator protein (Tax), a viral protein with many roles in infection. HTLV-1 basic leucine zipper factor (HBZ) has recently been implicated infection, but relatively little is known about the role of HBZ in HTLV-1 viral spread. In this study, we found that HBZ upregulates expression of neuropilin-1 (NRP1). Neuropilin-1 is a ubiquitously expressed transmembrane receptor and an HTLV-1 receptor. HBZ is known to interact with a variety of cellular transcription factors, including AP-1 basic leucine zipper (bZIP) factors and cAMP response element binding protein (CBP)/p300 coactivator proteins. Our results indicate that HBZ interacts with certain AP-1 bZIP factors and CBP/p300 at a putative enhancer site downstream of NRP1. We propose a model in which HBZ upregulates NRP1 expression by forming an HBZ/AP-1 bZIP factor heterodimer, which interacts with the putative enhancer site with CBP/p300 coactivators and basal transcription machinery to upregulate expression of NRP1. Intriguingly, we discovered that NRP1 expression on HTLV-1-infected T-cells inhibits cell-to-cell transmission of HTLV-1. Furthermore, NRP1 expression does not alter virion release from infected cells, suggesting that NRP1 doesn’t inhibit transmission through virion retention. We also provide evidence that NRP1 is incorporated into viral particles, resulting in a reduction in virion infectivity. Together, these results indicate that HBZ upregulates expression of NRP1, which reduces infection efficiency

Diseño y caracterización de inhibidores de la fascina para el bloqueo de la invasión y la metástasis en cáncer colorrectal.

El adenocarcinoma serrado (ACS) es un subtipo histológico de cáncer colorrectal reconocido por la Organización Mundial de la Salud (OMS) y que representa alrededor del 9% de los carcinomas colorrectales. En comparación con el carcinoma convencional (CC), el ACS tiene peor pronóstico y supervivencia. Estas características son debidas a que el ACS presenta un débil desarrollo de la respuesta inmune, un frente invasor muy activo y no responde de manera eficiente a terapias dirigidas anti-EGFR debido a la alta frecuencia de mutaciones en KRAS/BRAF. No obstante, recientemente, se han identificado posibles dianas moleculares que podrían favorecer la búsqueda de nuevos tratamientos específicos para el ACS. En esta línea se ha identificado la fascina, una proteína que se encuentra sobre-expresada en este subtipo histológico de cáncer colorrectal y que es una proteína clave en el reordenamiento del citoesqueleto de actina ya que participa en la formación de filopodios y lamelipodios implicados en el movimiento celular. Debido al papel fundamental de la fascina en el empaquetamiento de haces de actina, esta proteína se convierte en una diana para el bloqueo de la invasión tumoral. Objetivos: El principal objetivo de de la presente Tesis ha sido buscar nuevos inhibidores contra la fascina en cáncer colorrectal mediante cribado farmacológico. Posteriormente, hemos evaluado, mediante técnicas in vitro, el efecto de estos inhibidores en la proliferación, movilidad e invasión en líneas celulares de cáncer colorrectal con distintos niveles de expresión basal de la fascina. Igualmente, se analizó la capacidad anti-invasora y anti-metastásica de los inhibidores en modelos in vivo de pez cebra y murinos. Resultados: Se realizó una búsqueda, mediante modelado molecular, de potenciales compuestos anti-fascina de una librería de 9591 compuestos (que incluían 2037 aprobados por la Food and Drug Administration (FDA). Fruto de estudios in silico, identificamos varios compuestos como potenciales inhibidores de la fascina entre los que caben destacar: la imipramina (IMIP), el raltegravir (RAL) y el monastrol (MON). Previamente, el grupo del Dr. HY Huang descubrió el compuesto G2 mediante screening farmacológico y lo caracterizó como un inhibidor de la fascina capaz de bloquear la migración celular, la invasión y la metástasis en cáncer de mama. . Así pues, en esta Tesis, hemos testado las propiedades anti-fascina del compuesto G2, la IMIP, el RAL y el MON mediante ensayos in vitro e in vivo en los que se ha empleado la migrastatina (MGS) como fármaco control anti-fascina. En todos los casos, se observó un efecto antimigratorio y anti-invasor al tratar líneas celulares de cáncer de colon como HCT-116 y DLD-1 con los compuestos anti-fascina en los ensayos in vitro. También se ha observado la capacidad anti-metastásica de los inhibidores in vivo (pez cebra) y hemos estudiado el efecto que tienen la IMIP y el RAL en un modelo murino de tumor primario. Conclusiones: El cribado de fármacos juega un papel fundamental en la investigación traslacional. Hemos encontrado nuevas propiedades antimigratorias y anti-invasoras de los compuestos G2, IMIP, RAL y MON en células de cáncer colorrectal. Además, existe un efecto dosis dependiente de estos compuestos que ha sido demostrado mediante un modelo in vivo de invasión y metástasis tumoral en pez cebra. Los resultados del modelo murino obtenidos en la presente Tesis son aún preliminares y no fueron significativos en comparación con los grupos control. Ello apunta a la necesidad de evaluar en el futuro, el efecto sinérgico de quimioterapéuticos junto con los inhibidores de la fascina descritos en este trabajo. Los presentes resultados abalan el reposicionamiento de fármacos en la investigación clínica, así como la utilidad terapéutica de estos fármacos para el tratamiento de las células tumorales metastásicas más agresivas, como las que se desarrollan en el ACS.Medicin