Sticky siRNAs targeting survivin and cyclin B1 exert an antitumoral effect on melanoma subcutaneous xenografts and lung metastases.

BACKGROUND: Melanoma represents one of the most aggressive and therapeutically challenging malignancies as it often gives rise to metastases and develops resistance to classical chemotherapeutic agents. Although diverse therapies have been generated, no major improvement of the patient prognosis has been noticed. One promising alternative to the conventional therapeutic approaches currently available is the inactivation of proteins essential for survival and/or progression of melanomas by means of RNA interference. Survivin and cyclin B1, both involved in cell survival and proliferation and frequently deregulated in human cancers, are good candidate target genes for siRNA mediated therapeutics. METHODS: We used our newly developed sticky siRNA-based technology delivered with linear polyethyleneimine (PEI) to inhibit the expression of survivin and cyclin B1 both in vitro and in vivo, and addressed the effect of this inhibition on B16-F10 murine melanoma tumor development. RESULTS: We confirm that survivin and cyclin B1 downregulation through a RNA interference mechanism induces a blockage of the cell cycle as well as impaired proliferation of B16-F10 cells in vitro. Most importantly, PEI-mediated systemic delivery of sticky siRNAs against survivin and cyclin B1 efficiently blocks growth of established subcutaneaous B16-F10 tumors as well as formation and dissemination of melanoma lung metastases. In addition, we highlight that inhibition of survivin expression increases the effect of doxorubicin on lung B16-F10 metastasis growth inhibition. CONCLUSION: PEI-mediated delivery of sticky siRNAs targeting genes involved in tumor progression such as survivin and cyclin B1, either alone or in combination with chemotherapeutic drugs, represents a promising strategy for melanoma treatment.

Systemic delivery of sticky siRNAs targeting the cell cycle for lung tumor metastasis inhibition.

RNA interference allows the design of new inhibitors that target deregulated pathways in cancer. However systemic delivery of siRNA for the treatment of solid tumors still remains an issue. In our study, in order to suppress the progression of lung cancer metastasis in mice, we developed sticky siRNA (ssiRNA) to inhibit survivin and cyclin B1, two candidates involved in cell survival and proliferation. We exploited the linear polyethylenimine (PEI) as potent non-viral carrier to efficiently deliver our inhibitors. As a proof of concept, we have chosen a very aggressive mammary adenocarcinoma model (TSA-Luc cells), which forms lung metastases upon systemic cell injection. We confirmed in vitro, that the ssiRNAs delivered with PEI are not only able to inhibit our target genes at the mRNA and protein levels, but are also able to block the cell cycle and cell proliferation through a mechanism of RNA interference. More importantly, we showed in vivo by luciferase dosage, bioimaging and tissue section, an inhibition of lung tumor metastases after systemic delivery of cyclin B1 and survivin ssiRNA complexed with PEI. Alternating treatment with cisplatin and ssiRNA/PEI showed an additive effect between the two anticancer drugs on lung tumor inhibition leading to a significant increase in animal survival. Moreover a promising window between activity (IC50) and toxicity (LD50), essential for therapeutic application, was observed. Our data show that systemic delivery of ssiRNA/PEI complexes targeting the cell cycle is a valuable strategy for the treatment of lung tumor metastasis and that it can be combined with chemotherapy.

Conjugating phosphospermines to siRNAs for improved stability in serum, intracellular delivery and RNAi-mediated gene silencing.

siRNAs are usually formulated with cationic polymers or lipids to form supramolecular particles capable of binding and crossing the negatively charged cell membrane. However, particles hardly diffuse through tissues when administered in vivo. We therefore are developing cationic siRNAs, composed of an antisense sequence annealed to an oligophosphospermine-conjugated sense strand. Cationic siRNAs have been previously shown to display gene silencing activity in human cell line (Nothisen et al. J. Am. Chem. Soc.2009). We have improved the synthesis, purification and characterization of oligospermine-oligoribonucleotide conjugates which provide cationic siRNAs with enhanced biological activity. We show data supporting their carrier-free intracellular delivery in a molecular, soluble state. Additional results on the relationship between global charge, uptake and silencing activity confirm the requirement for an overall positive charge of the conjugated siRNA in order to enter cells. Importantly, conjugated siRNAs made of natural phosphodiester nucleotides are protected from nuclease degradation by the oligophosphospermine moiety, operate through the RNAi mechanism and mediate specific gene silencing at submicromolar concentration in the presence of serum.

Death receptor pathway activation and increase of ROS production by the triple epigenetic inhibitor UVI5008.

Deregulation of the epigenome is recognized as cause of cancer and epigenetic factors are receiving major attention as therapeutic targets; yet, the molecular mode of action of existing epi-drugs is largely elusive. Here, we report on the decryption of the mechanism of action of UVI5008, a novel epigenetic modifier, that inhibits histone deacetylases, sirtuins, and DNA methyltransferases. UVI5008 highly efficiently induces cancer cell-selective death in a variety of models and exerts its activities in several human tumor xenografts and genetic mouse models of human breast cancer in vivo. Its anticancer activity involves independent activation of death receptors and reactive oxygen species production. Importantly, UVI5008 action is not critically dependent on p53, Bcl-2 modifying factor, and/or TNF-related apoptosis-inducing ligand as cell death is efficiently induced in cells mutated or deficient for these factors limiting the risk of drug resistance development and maximizing its application spectrum. The simultaneous modulation of multiple (epigenetic) targets promises to open new avenues with unanticipated potential against cancer.

Transformation-dependent silencing of tumor-selective apoptosis-inducing TRAIL by DNA hypermethylation is antagonized by decitabine.

TNF-related apoptosis-inducing ligand (TRAIL) kills tumor cells selectively. We asked how emerging tumor cells escape elimination by TRAIL and how tumor-specific killing by TRAIL could then be restored. We found that TRAIL expression is consistently downregulated in HRAS(G12V)-transformed cells in stepwise tumorigenesis models derived from four different tissues due to DNA hypermethylation of CpG clusters within the TRAIL promoter. Decitabine de-silenced TRAIL, which remained inducible by interferon, while induction of TRAIL by blocking the HRAS(G12V)-activated mitogen-activated protein kinase pathway was subordinated to epigenetic silencing. Decitabine induced apoptosis through upregulation of endogenous TRAIL in cooperation with favorable regulation of key players acting in TRAIL-mediated apoptosis. Apoptosis induction by exogenously added TRAIL was largely increased by decitabine. In vivo treatment of xenografted human HRAS(G12V)-transformed human epithelial kidney or syngenic mice tumors by decitabine blocked tumor growth induced TRAIL expression and apoptosis. Our results emphasize the potential of decitabine to enhance TRAIL-induced apoptosis in tumors and thus provide a rationale for combination therapies with decitabine to increase tumor-selective apoptosis.

Targeted expression of tumor necrosis factor-related apoptosis-inducing ligand TRAIL in skin protects mice against chemical carcinogenesis.

BACKGROUND: Gene ablation studies have revealed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L, TNFSF10) plays a crucial role in tumor surveillance, as TRAIL-deficient mice exhibit an increased sensitivity to different types of tumorigenesis. In contrast, possible tumor-protective effect of increased levels of endogenous TRAIL expression in vivo has not been assessed yet. Such models will provide important information about the efficacy of TRAIL-based therapies and potential toxicity in specific tissues. METHODS: To this aim, we engineered transgenic mice selectively expressing TRAIL in the skin and subjected these mice to a two-step chemical carcinogenesis protocol that generated benign and preneoplastic lesions. We were therefore able to study the effect of increased TRAIL expression at the early steps of skin tumorigenesis. RESULTS: Our results showed a delay of tumor appearance in TRAIL expressing mice compared to their wild-type littermates. More importantly, the number of tumors observed in transgenic animals was significantly lower than in the control animals, and the lesions observed were mostly benign. Interestingly, Wnt/ß-catenin signaling differed between tumors of wild-type and TRAIL transgenics. CONCLUSION: Altogether, these data reveal that, at least in this model, TRAIL is able on its own to act on pre-transformed cells, and reduce their tumorigenic potential.

The roles of CUX1 homeodomain proteins in the establishment of a transcriptional program required for cell migration and invasion.

The Cut homeobox gene 1 (CUX1) codes for several homeodomain proteins that display distinct DNA binding and transcriptional properties. Some CUX1 isoforms were previously shown to stimulate entry into S phase. More recently, siRNA-mediated knockdown of CUX1 was shown to cause a decrease in cell migration and invasion. In contrast, ectopic expression of p110 or p75 CUX1 stimulated cell migration and invasion in tissue culture. Moreover, metastasis to the lung was observed in a few cases following development of mammary tumors in p75 CUX1 transgenic mice. Chromatin immunoprecipitation (ChIP) assays followed by hybridization on promoter arrays (ChIP-chip) led to the identification of more than 20 genes that are directly regulated by CUX1 and code for proteins involved in cytoskeleton remodeling, cell-cell and cell-matrix adhesion, epithelial to mesenchymal transition and transcriptional regulation. Many targets of CUX1 are regulators of Rho GTPases that play a role both in cell cycle progression and cell motility. Interestingly, some genes that promote cell motility are activated by CUX1, while some genes that inhibit cell motility are repressed by CUX1. The dual function of CUX1 as an activator and repressor is best exemplified by the regulatory cascade whereby CUX1 activates expression of the Snail and Slug transcription factors and then cooperates with them to repress the E-cadherin and occludin genes, thereby causing a severe disorganization of cell-cell junctions. Together, these studies indicate that CUX1 stimulates cell motility by regulating a large number of genes involved in various molecular functions.

Mouse mammary tumor virus p75 and p110 CUX1 transgenic mice develop mammary tumors of various histologic types.

The p75 and p110 isoforms of the CUX1 homeodomain protein are overexpressed in breast tumors and cancer cell lines. To assess and compare the ability of these short CUX1 isoforms in driving mammary tumor development, we used site-specific transgenesis into the Hprt locus to generate transgenic mice expressing p75 or p110 CUX1 under the control of the mouse mammary tumor virus-long terminal repeat. We report that mammary tumors developed after a long latency period, and although various histopathologies were observed, the proportion of adenosquamous carcinomas was significantly higher in p75 CUX1 than in p110 CUX1 transgenic mice. Metastasis to the lung was observed in three p75 CUX1 transgenic mice. Comparisons between tumors and adjacent normal mammary glands revealed that transgenes were overexpressed in most but not all tumors, yet in all cases tested, CUX1 DNA binding was increased, suggesting that both higher expression and changes in post-translational modifications can contribute to stimulate transgene activity. Interestingly, higher expression of erbB2 mRNA was seen in most tumors, not only solid carcinomas but also adenosquamous carcinomas, whereas higher expression of various Wnt genes and activation of the beta-catenin pathway was observed primarily in adenosquamous carcinomas. Activation of erbB2 expression appeared to represent a cooperating event that occurred independently of CUX1. In contrast, chromatin immunoprecipitation, short hairpin RNA-mediated knockdown, and reporter assays established that CUX1 is involved in the transcriptional regulation of several Wnt genes. Together, these results support the notion that oncogenic activity of CUX1 can facilitate the establishment of a Wnt/beta-catenin autocrine loop.

p110 CUX1 homeodomain protein stimulates cell migration and invasion in part through a regulatory cascade culminating in the repression of E-cadherin and occludin.

In this study, we investigated the mechanism by which the CUX1 transcription factor can stimulate cell migration and invasion. The full-length p200 CUX1 had a weaker effect than the proteolytically processed p110 isoform; moreover, treatments that affect processing similarly impacted cell migration. We conclude that the stimulatory effect of p200 CUX1 is mediated in part, if not entirely, through the generation of p110 CUX1. We established a list of putative transcriptional targets with functions related to cell motility, and we then identified those targets whose expression was directly regulated by CUX1 in a cell line whose migratory potential was strongly stimulated by CUX1. We identified 18 genes whose expression was directly modulated by p110 CUX1, and its binding to all target promoters was validated in independent chromatin immunoprecipitation assays. These genes code for regulators of Rho-GTPases, cell-cell and cell-matrix adhesion proteins, cytoskeleton-associated proteins, and markers of epithelial-to-mesenchymal transition. Interestingly, p110 CUX1 activated the expression of genes that promote cell motility and at the same time repressed genes that inhibit this process. Therefore, the role of p110 CUX1 in cell motility involves its functions in both activation and repression of transcription. This was best exemplified in the regulation of the E-cadherin gene. Indeed, we uncovered a regulatory cascade whereby p110 CUX1 binds to the snail and slug gene promoters, activates their expression, and then cooperates with these transcription factors in the repression of the E-cadherin gene, thereby causing disorganization of cell-cell junctions.

Tumor necrosis factor receptor-associated factor 4 is a dynamic tight junction-related shuttle protein involved in epithelium homeostasis.

BACKGROUND: Despite numerous in vivo evidences that Tumor Necrosis Factor Receptor-Associated Factor 4 (TRAF4) plays a key biological function, how it works at the cellular and molecular level remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we show using immunofluorescence and immuohistochemistry that TRAF4 is a novel player at the tight junctions (TJs). TRAF4 is connected to assembled TJs in confluent epithelial cells, but accumulates in the cytoplasm and/or nucleus when TJs are open in isolated cells or EGTA-treated confluent cells. In vivo, TRAF4 is consistently found at TJs in normal human mammary epithelia as well as in well-differentiated in situ carcinomas. In contrast, TRAF4 is never localized at the plasma membrane of poorly-differentiated invasive carcinomas devoid of correct TJs, but is observed in the cytoplasm and/or nucleus of the cancer cells. Moreover, TRAF4 TJ subcellular localization is remarkably dynamic. Fluorescence recovery after photobleaching (FRAP) experiments show that TRAF4 is highly mobile and shuttles between TJs and the cytoplasm. Finally, we show that intracellular TRAF4 potentiates ERK1/2 phosphorylation in proliferating HeLa cells, an epithelial cell line known to be devoid of TJs. CONCLUSIONS/SIGNIFICANCE: Collectively, our data strongly support the new concept of TJs as a dynamic structure. Moreover, our results implicate TRAF4 in one of the emerging TJ-dependent signaling pathways that responds to cell polarity by regulating the cell proliferation/differentiation balance, and subsequently epithelium homeostasis. Drastic phenotypes or lethality in TRAF4-deficient mice and drosophila strongly argue in favor of such a function.

TRAF4, the unique family member.

The fourth member of the TRAF protein family (TRAF4) presents several characteristics that distinguish it from the other members of the family. These characteristics concern the primary sequence of the protein, a strong evolutionary conservation, and a tightly regulated physiological expression during development. The subcellular localization of TRAF4 is controversial as it has been detected at the cell membrane, in the cytoplasm and in the nucleus. Using mouse and fly models, it has been established that TRAF4 is a key molecule in diverse ontogenic processes, particularly in the nervous system. However, the molecular mechanisms of action of TRAF4 remain evasive as it was found to interact with diverse types of proteins, leading either to pro-apoptotic or anti-apoptotic functions. Finally, few studies implicated TRAF4 in human diseases.

Spatial and temporal distribution of the traf4 genes during zebrafish development.

The tumor necrosis factor-associated factor 4 (TRAF4) is a particular member of the TRAF protein family since it is not involved in the Tumor Necrosis Factor (TNF) and Interleukin-1 (IL-1) signaling pathways. In the present study, we cloned two zebrafish orthologs of the human traf4, traf4a and traf4b, which are the first TRAFs described in zebrafish. During embryogenesis, traf4b expression is present in a weak ubiquitous manner. In contrast, traf4a exhibits a highly specific expression pattern in the sensorial and neural cells, and the somites of embryos. This gene is tightly regulated during embryogenesis. Together, our data show that traf4 is conserved during evolution, and traf4a is the zebrafish ortholog of traf4.

Functional characterization of the MENTAL domain.

Human metastatic lymph node (MLN) 64 is composed of two conserved regions. The amino terminus contains a conserved membrane-spanning MENTAL (MLN64 NH(2)-terminal) domain shared with an unique protein called MENTHO (MLN64 NH(2)-terminal domain homologue) and targets the protein to late endosome. The carboxyl-terminal domain is composed of a cholesterol binding steroidogenic acute regulatory-related lipid transfer domain exposed to the cytoplasm. MENTHO overexpression leads to the accumulation of enlarged endosomes. In this study, we show that MLN64 overexpression also induces the formation of enlarged endosomes, an effect that is probably mediated by the MENTAL domain. Using an in vivo photocholesterol binding assay, we find that the MENTAL domain of MLN64 is a cholesterol binding domain. Moreover, glutathione S-transferase pull-down or co-immunoprecipitation experiments demonstrate that this domain mediates homo- and hetero-interaction of MLN64 and MENTHO. In living cells, the expression of paired yellow fluorescent and cyan fluorescent fusion proteins show MENTHO homo-interaction and its interaction with MLN64. These data indicate that within late-endosomal membranes, MLN64 and MENTHO define discrete cholesterol-containing subdomains. The MENTAL domain might serve to maintain cholesterol at the membrane of late endosomes prior to its shuttle to cytoplasmic acceptor(s).

Association of the breast cancer protein MLN51 with the exon junction complex via its speckle localizer and RNA binding module.

MLN51 is a nucleocytoplasmic shuttling protein that is overexpressed in breast cancer. The function of MLN51 in mammals remains elusive. Its fly homolog, named barentsz, as well as the proteins mago nashi and tsunagi have been shown to be required for proper oskar mRNA localization to the posterior pole of the oocyte. Magoh and Y14, the human homologs of mago nashi and tsunagi, are core components of the exon junction complex (EJC). The EJC is assembled on spliced mRNAs and plays important roles in post-splicing events including mRNA export, nonsense-mediated mRNA decay, and translation. In the present study, we show that human MLN51 is an RNA-binding protein present in ribonucleo-protein complexes. By co-immunoprecipitation assays, endogenous MLN51 protein is found to be associated with EJC components, including Magoh, Y14, and NFX1/TAP, and subcellular localization studies indicate that MLN51 transiently co-localizes with Magoh in nuclear speckles. Moreover, we demonstrate that MLN51 specifically associates with spliced mRNAs in co-precipitation experiments, both in the nucleus and in the cytoplasm, at the position where the EJC is deposited. Most interesting, we have identified a region within MLN51 sufficient to bind RNA, to interact with Magoh and spliced mRNA, and to address the protein to nuclear speckles. This conserved region of MLN51 was therefore named SELOR for speckle localizer and RNA binding module. Altogether our data demonstrate that MLN51 associates with EJC in the nucleus and remains stably associated with mRNA in the cytoplasm, suggesting that its overexpression might alter mRNA metabolism in cancer.

Impaired neural tube closure, axial skeleton malformations, and tracheal ring disruption in TRAF4-deficient mice.

TRAF4 belongs to the tumor necrosis factor receptor-associated factor (TRAF) family of proteins but, unlike other family members, has not yet been clearly associated to any specific receptor or signaling pathway. To investigate the biological function of TRAF4, we have generated traf4-deficient mice by gene disruption. The traf4 gene mutation is embryonic lethal but with great individual variation, as approximately one third of the homozygous mutant embryos died in utero around embryonic day 14, whereas the others reach adulthood. Surviving mutant mice manifest numerous developmental abnormalities; notably, 100% of homozygous mutant mice suffer respiratory disorder and wheezing caused by tracheal ring disruption. Additional malformations concern mainly the axial skeleton, as the ribs, sternum, tail, and vertebral arches are affected, with various degrees of penetrance. Traf4-deficient mice also exhibit a high incidence of spina bifida, a defect likened to neural tube defects (NTD) that are common congenital malformations in humans. Altogether, our results demonstrate that TRAF4 is required during embryogenesis in key biological processes including the formation of the trachea, the development of the axial skeleton, and the closure of the neural tube. Considering the normal expression pattern of TRAF4 in neural tissues, we can conclude that TRAF4 participates in neurulation in vivo.