Based on the multidomain structure of the bacterial Pseudomonas exotoxin A, a recombinant fusion protein was constructed which serves as a target cell-specific carrier for the transfer of DNA via receptor-mediated endocytosis. The protein consists of three functional domains: 1) an ErbB-2 -specific single chain antibody confers target cell specificity, 2) the exotoxin A translocation domain facilitates endosome escape, and 3) a DNA binding domain derived from the yeast GAL4 protein enable sequence-specific high affinity binding to DNA. Carrier protein purified from bacterial lysates displayed both ErbB-2-specific and DNA sequence-specific binding in vitro. Complexes which formed spontaneously by the interaction of the fusion protein with a luciferase reporter gene construct carrying a GAL4-specific recognition sequence, after condensation of the DNA and compensation of excess negative charge with poly-L-lysine were able to transfect ErbB-2-expressing cells in vitro in a cell-specific manner. Transient expression of the luciferase gene driven by the SV40 early promoter was observed and correlates with the amount of carrier protein in the complex. Truncated forms of the carrier protein lacking either the cell recognition domain or the translocation domain failed to facilitate efficient DNA transfer.

We undertook a large-scale genetic screen to identify genes able to alter the cellular response to physiological signals and provide selective advantage once tumorigenesis has begun. We identified MAP17, a small 17 kDa non-glycosylated membrane protein previously identified, being overexpressed in carcinomas. We found that MAP17 is overexpressed in a great variety of human carcinomas. Immunohistochemical analysis of MAP17 during cancer progression shows, at least in prostate and ovarian carcinomas, that overexpression of the protein strongly correlates with tumoral progression (P < 0.0001). Many tumor cells also express MAP17 and its expression does not correlate with expression of SCL, a neighbor gene reported to be co-expressed in some hematopoietic cell lines. SCL neither is expressed in most MAP17-positive tumors, indicating the independent transcription of MAP17, at least in carcinomas. We cloned 5' genomic region to MAP17 and described the minimal promoter necessary to produce independent activation of MAP17. Moreover, we have found that MAP17 promoter is activated by oncogenes. Taken together, our data show an independent activation of MAP17 promoter that can be driven by oncogenes and that might explain the common overexpression of MAP17 in human carcinomas.

Tumorigenesis occurs when the mechanisms involved in the control of tissue homeostasis are disrupted and cells stop responding to physiological signals. Therefore, genes capable of desensitizing tumoral cells from physiological signals may provide a selective advantage within the tumoral mass and influence the outcome of the disease. We undertook a large-scale genetic screen to identify genes able to alter the cellular response to physiological signals and provide selective advantage once tumorigenesis has begun. We identified MAP17, a small 17 kDa non-glycosylated membrane protein previously identified by differential display being over-expressed in carcinomas. Tumor cells that over-express MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in presence or absence of contact inhibition, decreased apoptotic sensitivity and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 are associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. Treatment of melanoma cells with inhibitors of Na+-coupled co-transporters lead to an inhibition of ROS increase and a decrease in the malignant cell behavior in MAP17-expressing clones. Finally, we show that MAP17-dependent ROS increase and tumorigenesis are dependent on its PDZ-binding domain, since disruption of its sequence by point mutations abolishes its ability to enhance ROS production and tumorigenesis. Our work shows the tumorigenic capability of MAP17 through a connection between Na+-coupled co-transporters and ROS.

Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is one the most frequent genetic events in human cancer. A cell-based imaging assay that monitored the translocation of the Akt effector protein, Forkhead box O (FOXO), from the cytoplasm to the nucleus was employed to screen a collection of 33,992 small molecules. The positive compounds were used to screen kinases known to be involved in FOXO translocation. Pyrazolopyrimidine derivatives were found to be potent FOXO relocators as well as biochemical inhibitors of PI3K␣. A combination of virtual screening and molecular modeling led to the development of a structure-activity relationship, which indicated the preferred substituents on the pyrazolopyrimidine scaffold. This leads to the synthesis of ETP-45658, which is a potent and selective inhibitor of phosphoinositide 3-kinases and demonstrates mechanism of action in tumor cell lines and in vivo in treated mice.The phosphoinositide 3-kinase (PI3K) 4 /Akt pathway is activated in a variety of solid and non-solid tumors (1) and therefore is considered as a potential intervention point for anticancer therapeutics. Activation of the pathway is frequently caused by mutations in PI3K␣ that enhance its catalytic activity, leading to the generation of phosphatidyl 3,4,5-trisphosphate (PIP3) (2) or by mutations or deletions in the tumor suppressor PTEN (phosphatase and tensin homolog) that result in its loss of function. PTEN antagonizes the activity of PI3K␣ through the dephosphorylation PIP3 (3). In addition, PI3K␣ can be activated by mutations in certain receptor-tyrosine kinases as well as by mutations in the oncogene KRAS (4, 5).The PIP3 generated by activation of PI3K␣ or sustained by the inactivation of PTEN binds to a subset of lipid-binding domains in downstream targets such as the pleckstrin homology (PH) domain of the oncogene Akt (6, 7); thereby, recruiting it to the plasma membrane. Once at the plasma membrane, Akt can be activated (8, 9). When active, Akt phosphorylates several effector molecules including the Forkhead box O (FOXO) transcription factors (10, 11). FOXO proteins are a family of conserved polypeptides that bind to DNA as a monomer and activate the transcription of genes that are involved in numerous biologically relevant processes such as metabolism, differentiation, proliferation, longevity, and apoptosis (12, 13). Akt phosphorylates FOXO proteins at three conserved consensus sites, which leads to conformational changes that facilitate CRM-1-mediated nuclear export (14, 15). Nuclear FOXO proteins function as regulators of transcription, whereas cytoplasmic FOXO proteins are considered inactive. It is well established that FOXO is negatively regulated by various proliferative and antiapoptotic signaling pathways that activate the PI3K/Akt signaling cascade (11). Therefore, we chose to employ a high content imaging approach to monitor the nucleocytoplasmic translocation of a GFP-FOXO3a fusion protein in U2OS cells (U2foxRELOC) (16,17) as the readout for biological inhibition...

Modular fusion proteins that combine distinct functions required for cell type-specific uptake and intracellular delivery of DNA present an attractive approach for the development of self-assembling vectors for targeted gene delivery. Here, we describe a novel DNA carrier protein termed GD5 that mimics the structure of the bacterial diphtheria toxin (DT) and facilitates target cell-specific gene transfer via receptor-mediated endocytosis. GD5 carries at the N terminus the DNA-binding domain of the yeast transcription factor Gal4, which is connected to a C-terminal antibody fragment specific for the tumor-associated ErbB2 antigen via an internal DT translocation domain as an endosome escape activity. Bacterially expressed GD5 protein specifically bound to ErbB2-expressing cells and formed protein-DNA complexes with a luciferase reporter gene construct. These complexes, after compensation of excess negative charge with poly-L-lysine, served as a specific transfection vector for ErbB2-expressing cells. Inhibitors of endosomal acidification drastically reduced GD5-mediated transfection, indicating that the DT translocation domain of GD5, similar to the parental toxin, is strictly dependent on the transit through an acidic environment. Our results suggest that fusion proteins that employ the natural endosome escape mechanism of bacterial toxins might aid in the development of efficient nonviral vectors for applications in gene therapy.

Senescence is a mechanism that limits cellular lifespan and constitutes a barrier against cellular immortalization. To identify new senescence regulatory genes that might play a role in tumorigenesis, we have designed and performed a large-scale antisense-based genetic screen in primary mouse embryo fibroblasts (MEFs). Out of this screen, we have identified five different genes through which loss of function partially bypasses senescence. These genes belong to very different biochemical families: csn2 (component of the Cop9 signalosome), aldose reductase (a metabolic enzyme) and brf1 (subunit of the RNA polymerase II complex), S-adenosyl homocysteine hydrolase and Bub1. Inactivation, at least partial, of these genes confers resistance to both p53-and p16INK4a-induced proliferation arrest. Furthermore, such inactivation inhibits p53 but not E2F1 transcriptional activity and impairs DNA-damage-induced transcription of p21. Since the aim of the screen was to identify new regulators of tumorigenesis, we have tested their inactivation in human tumors. We have found, either by northern blot or quantitative reverse transcriptase-PCR analysis, that the expression of three genes, Csn2, Aldose reductase and Brf1, is lost at different ratios in tumors of different origins. These genes are located at common positions of loss of heterogeneity (15q21.2, 7q35 and 14q32.33); therefore,we have measured genomic losses of these specific genes in different tumors. We have found that Csn2 and Brf1 also show genomic losses of one allele in different tumors. Our data suggest that the three genes identified in the genome-wide loss-of-function genetic screen are putative tumor suppressors located at 15q21.2; 7q35 and 14q32.33.

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