Identification of hepatic microvascular adhesion-related genes of human colon cancer cells using random homozygous gene perturbation.

Random homozygous gene perturbation (RHGP), in combination with liver sinusoidal endothelial cell (LSEC) adhesion screening of clonal colon cancer cells with perturbed genes, was used to identify genes contributing to the hepatic microvascular adhesion of colon cancer cells. Plasmid vector encoding transactivator and gene search vector were transfected into HT-29 human colorectal cancer cells to create a HT-29 RHGP cell library; the adhesion of these library cells to primary cultured mouse LSEC significantly decreased in the presence of RSL1 ligand (inducer), indicating that most of the genes contributing to HT-29 adhesion to LSEC were altered. Next, HT-29 RHGP cell library fractions with upregulated or silenced LSEC adhesion-related genes were isolated. Around 160 clones having altered expression in LSEC adhesion-related genes were obtained, and nine relevant protein-coding genes were identified. Some were proadhesive genes detected because of their overexpression in adherent HT-29 cells (DGCR8 and EFEMP1 genes) and their silenced status in nonadherent HT-29 cells (DGKE, DPY19L1, KIAA0753, PVR and USP11 genes). Others were antiadhesive genes detected because of their overexpression in nonadherent HT-29 cells (ITPKC gene) and their silenced status in adherent HT-29 cells (PPP6R2 gene). Silencing of PVR, DGCR8 and EFEMP1 genes decreased adhesion to LSEC and hepatic microvascular retention of HT-29 cells. The results conclude that RHGP was a valuable strategy for the discovery of mechanisms regulating microvascular adhesion of circulating colon cancer cells before hepatic metastasis formation. Identified genes may contribute to understand the metastatic process of colon cancer and to discovering molecular targets for hepatic metastasis therapeutics.

Identification of annexin A13 as a regulator of chemotherapy resistance using random homozygous gene perturbation.

OBJECTIVE: To utilize a powerful new technology for target discovery, Random Homozygous Gene Perturbation (RHGP), and to identify novel targets that cause tumor cells to become chemoresistant. STUDY DESIGN: RHGP was used to identify and validate genetic changes that cause chemoresistance of tumor cells to Rapamycin. RESULTS: A series of targets was identified that allowed tumor cells to survive treatment with Rapamycin. We validated these targets and focused on Annexin A13, a target where decreased expression caused tumor cell insensitivity to Rapamycin. Ectopic overexpression of Annexin A13 was likewise sufficient to sensitize malignant breast cancer cells to treatment with Rapamycin. CONCLUSION: These findings expand our knowledge of mechanisms that allow tumor cell drug resistance and demonstrate the power of RHGP to identify novel targets and mechanisms.

The Rps23rg gene family originated through retroposition of the ribosomal protein s23 mRNA and encodes proteins that decrease Alzheimer's beta-amyloid level and tau phosphorylation.

Retroposition is an important mechanism for gene origination. However, studies to elucidate the functions of new genes originated through retroposition, especially the functions related to diseases, are limited. We recently identified a mouse gene, Rps23 retroposed gene 1 (Rps23rg1), that regulates beta-amyloid (Abeta) level and tau phosphorylation, two major pathological hallmarks of Alzheimer's disease (AD), and found that Rps23rg1 originated through retroposition of the mouse ribosomal protein S23 (Rps23) mRNA. Here we show that retroposition of Rps23 mRNA occurred multiple times in different species but only generated another functionally expressed Rps23rg1-homologous gene, Rps23rg2, in mice, whereas humans may not possess functional Rps23rg homologs. Both Rps23rg1 and Rps23rg2 are reversely transcribed relative to the parental Rps23 gene, expressed in various tissues and encode proteins that interact with adenylate cyclases. Similar to the RPS23RG1 protein, RPS23RG2 can upregulate protein kinase A activity to reduce the activity of glycogen synthase kinase-3, Abeta level and tau phosphorylation. However, the effects of RPS23RG2 are weaker than those of RPS23RG1 and such a difference could be attributed to the extra carboxyl-terminal region of RPS23RG2, which may have an inhibitory effect. In addition, we show that the transmembrane domain of RPS23RG1 is important for its function. Together, our results present a new gene family, whose products and associated signaling pathways might prevent mice from developing AD-like pathologies.

Identification of PTCH1 requirement for influenza virus using random homozygous gene perturbation.

Influenza infection remains a leading cause of infectious disease-mediated morbidity and mortality. Accumulating evidence indicates that most variants of seasonal and pandemic influenza have developed resistance to conventional therapies. Such information has spawned new interest in identifying novel approaches to target influenza. Our laboratories have been developing a new strategy of Host-Oriented Therapeutics, which seeks to target host molecules in a safe and effective manner that prevents the virus from causing disease. Using an improved discovery technology, Random Homozygous Gene Perturbation (RHGP), we identified the PTCH1 protein as an essential host target that critically controls influenza virus infection. We further demonstrated that targeted intervention against PTCH1 using antibodies or siRNA decreases influenza infection. Finally, we demonstrated the involvement of PTCH1 in influenza infection outside of the laboratory by showing that genetic variations of PTCH1 relate to decreased disease morbidity in the field. Altogether, these findings have important implications for the development of novel, host-directed therapeutics to improve influenza disease management.

A functional mouse retroposed gene Rps23r1 reduces Alzheimer's beta-amyloid levels and tau phosphorylation.

Senile plaques consisting of beta-amyloid (Abeta) and neurofibrillary tangles composed of hyperphosphorylated tau are major pathological hallmarks of Alzheimer's disease (AD). Elucidation of factors that modulate Abeta generation and tau hyperphosphorylation is crucial for AD intervention. Here, we identify a mouse gene Rps23r1 that originated through retroposition of ribosomal protein S23. We demonstrate that RPS23R1 protein reduces the levels of Abeta and tau phosphorylation by interacting with adenylate cyclases to activate cAMP/PKA and thus inhibit GSK-3 activity. The function of Rps23r1 is demonstrated in cells of various species including human, and in transgenic mice overexpressing RPS23R1. Furthermore, the AD-like pathologies of triple transgenic AD mice were improved and levels of synaptic maker proteins increased after crossing them with Rps23r1 transgenic mice. Our studies reveal a new target/pathway for regulating AD pathologies and uncover a retrogene and its role in regulating protein kinase pathways.

Identification of novel host-oriented targets for Human Immunodeficiency Virus type 1 using Random Homozygous Gene Perturbation.

BACKGROUND: Human Immunodeficiency Virus (HIV) is a global threat to public health. Current therapies that directly target the virus often are rendered ineffective due to the emergence of drug-resistant viral variants. An emerging concept to combat drug resistance is the idea of targeting host mechanisms that are essential for the propagation of the virus, but have a minimal cellular effect. RESULTS: Herein, using Random Homozygous Gene Perturbation (RHGP), we have identified cellular targets that allow human MT4 cells to survive otherwise lethal infection by a wild type HIV-1NL4-3. These gene targets were validated by the reversibility of the RHGP technology, which confirmed that the RHGP itself was responsible for the resistance to HIV-1 infection. We further confirmed by siRNA knockdowns that the RHGP-identified cellular pathways are responsible for resistance to infection by either CXCR4 or CCR5 tropic HIV-1 variants. We also demonstrated that cell clones with these gene targets disrupted by RHGP were not permissible to the replication of a drug resistant HIV-1 mutant. CONCLUSION: These studies demonstrate the power of RHGP to identify novel host targets that are essential for the viral life cycle but which can be safely perturbed without overt cytotoxicity. These findings suggest opportunities for the future development of host-oriented therapeutics with the broad spectrum potential for safe and effective inhibition of HIV infection.

Function-first approaches to improve target identification in cancer.

Target discovery for cancer is undergoing a sort of revival with an increasing need for improved therapeutics. Likewise, the strategies to discover new and better therapeutic targets have come full circle, with greater emphasis placed upon targets that are functionally relevant to the disease process. In this article, we review the evolution of cancer target discovery and discuss random homozygous gene perturbation, an emerging technology that combines the practicality of screening for new targets by emphasizing function as the primary criterion, with cutting-edge advances in gene-based screening of all potential targets in a cell.

The use of Random Homozygous Gene Perturbation to identify novel host-oriented targets for influenza.

Conventional approaches for therapeutic targeting of viral pathogens have consistently faced obstacles arising from the development of resistant strains and a lack of broad-spectrum application. Influenza represents a particularly problematic therapeutic challenge since high viral mutation rates have often confounded many conventional antivirals. Newly emerging or engineered strains of influenza represent an even greater threat as typified by recent interest in avian subtypes of influenza. Based on the limitations associated with targeting virally-encoded molecules, we have taken an orthogonal approach of targeting host pathways in a manner that prevents viral propagation or spares the host from virus-mediated pathogenicity. To this end, we report herein the application of an improved technology for target discovery, Random Homozygous Gene Perturbation (RHGP), to identify host-oriented targets that are well-tolerated in normal cells but that prevent influenza-mediated killing of host cells. Improvements in RHGP facilitated a thorough screening of the entire genome, both for overexpression or loss of expression, to identify targets that render host cells resistant to influenza infection. We identify a set of host-oriented targets that prevent influenza killing of host cells and validate these targets using multiple approaches. These studies provide further support for a new paradigm to combat viral disease and demonstrate the power of RHGP to identify novel targets and mechanisms.