RPN2-mediated glycosylation of tetraspanin CD63 regulates breast cancer cell malignancy.

BACKGROUND: The tetraspanin CD63 is a highly N-glycosylated protein that is known to regulate cancer malignancy. However, the contribution of glycosylation of CD63 to cancer malignancy remains unclear. Previously, we reported that ribophorin II (RPN2), which is part of an N-oligosaccharyle transferase complex, is responsible for drug resistance in breast cancer cells. In this study, we demonstrate that cancer malignancy associated with the glycosylation of CD63 is regulated by RPN2. RESULTS: Inhibition of RPN2 expression led to a reduction in CD63 glycosylation. In addition, the localization of CD63 was deregulated by knockdown of RPN2. Interestingly, multidrug resistance protein 1 (MDR1) localization was displaced from the cell surface in CD63-silenced cells. CD63 silencing reduced the chemoresistance and invasion ability of malignant breast cancer cells. Furthermore, the enrichment of CD63/MDR1-double positive cells was associated with lymph node metastasis. Taken together, these results indicated that high glycosylation of CD63 by RPN2 is implicated in clinical outcomes in breast cancer patients. CONCLUSIONS: These findings describe a novel and important function of RPN2-mediated CD63 glycosylation, which regulates MDR1 localization and cancer malignancy, including drug resistance and invasion.

Local and systemic delivery of siRNAs for oligonucleotide therapy.

RNA interference (RNAi) is a relatively new found phenomenon of posttranscriptional gene silencing to regulate the expression of multiple genes involved in a wide range of biological processes. The gene-silencing technology via RNAi has also been developed into a commonly anti-gene method. Furthermore, in vivo data indicate that small interfering RNAs (siRNAs) may be used to treat human diseases. However, the most challenging issue to a successful in vivo application is the development of a delivery system that can transport siRNA molecules into the tissues and/or the cells of interest. Also, the evaluation of siRNA potency in vivo is central for the selection of therapeutic siRNAs. In this chapter, the effects of atelocollagen-delivered siRNAs in live animals were monitored using bioluminescence imaging.

Type I collagen gene suppresses tumor growth and invasion of malignant human glioma cells.

BACKGROUND: Invasion is a hallmark of a malignant tumor, such as a glioma, and the progression is followed by the interaction of tumor cells with an extracellular matrix (ECM). This study examined the role of type I collagen in the invasion of the malignant human glioma cell line T98G by the introduction of the human collagen type I alpha1 (HCOL1A1) gene. RESULTS: The cells overexpressing HCOL1A1 were in a cluster, whereas the control cells were scattered. Overexpression of HCOL1A1 significantly suppressed the motility and invasion of the tumor cells. The glioma cell growth was markedly inhibited in vitro and in vivo by the overexpression of HCOL1A1; in particular, tumorigenicity completely regressed in nude mice. Furthermore, the HCOL1A1 gene induced apoptosis in glioma cells. CONCLUSION: These results indicate that HCOL1A1 have a suppressive biological function in glioma progression and that the introduction of HCOL1A1 provides the basis of a novel therapeutic approach for the treatment of malignant human glioma.

[Application of atelocollagen-mediated siRNA delivery for RNAi therapies].

RNAi has rapidly become a powerful tool for drug target discovery and validation in an in vitro culture system and, consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Novel treatments and drug discovery in pre-clinical studies based on RNAi are currently targeting a wide range of diseases, including viral infections and cancers by the local administration of synthetic small interfering RNA (siRNA) that target local lesions. Recently, specific methods for the systemic administration of siRNAs have been reported to treat non-human primates or a cancer metastasis model. In vivo siRNA-delivery technology is a key hurdle to the successful therapeutic application of RNAi. This article reviews the non-viral delivery system of atelocollagen for siRNA, which could be useful for functional screening of the genes in vitro and in vivo, and will provide a foundation for further development of RNAi therapeutics.

Central genetic alterations common to all HCV-positive, HBV-positive and non-B, non-C hepatocellular carcinoma: a new approach to identify novel tumor markers.

Hepatocellular carcinoma (HCC) is a common malignancy, but the prognosis remains poor due to the lack of sensitive diagnostic markers. To gain insight into the central molecular features common to all types of HCC, and to identify novel diagnostic markers or therapeutic targets for HCC, we performed a gene expression profiling analysis using a high throughput RT-PCR system. After examining the mRNA expression of 3,072 genes in 204 (119 tumor and 85 non-tumor) liver samples, we identified differential gene expression between the HCV group (n=80), HBV group (n=19) and non-B, non-C group (n=20) with a principal component analysis and a correlation spectrum analysis. After selection of genes differentially expressed between tumor and non-tumor tissues (p<0.01) within each HCC group, a total of 51 differentially expressed genes (23 upregulated and 28 downregulated genes) were found to be common to the three HCC groups. Gene Ontology grouping analysis revealed that genes with functions related to cell proliferation or differentiation and genes encoding extracellular proteins were found to be significantly enriched in these 51 common genes. Using an atelocollagen-based cell transfection array for functional analysis of eight upregulated genes, five (CANX, FAM34A, PVRL2, LAMR1, and GBA) significantly inhibited cellular apoptosis by two independent assays. In conclusion, we identified 51 differentially expressed genes, common to all HCC types. Among these genes, there was a high incidence of anti-apoptotic activity. This combination approach with the advanced statistical methods and the bioinformatical analysis may be useful for finding novel molecular targets for diagnosis and therapy.

Atelocollagen-mediated systemic DDS for nucleic acid medicines.

The goal of our research is to provide a practical platform for drug delivery in oligonucleotide therapy. We report here the efficacy of an atelocollagen-mediated oligonucleotide delivery system applied to systemic siRNA and antisense oligonucleotide treatments in animal disease models. Atelocollagen and oligonucleotides formed a complex of nanosized particles, which was highly stable against nucleases. The complex allowed oligonucleotides to be delivered efficiently into several organs and tissues via intravenous administration. In a tumor metastasis model, the complex successfully delivered siRNA to metastasized tumors in bone tissue and inhibited their growth. We also demonstrated that a single intravenous treatment of the antisense oligodeoxynucleotide complex suppressed ear dermatitis in a contact hypersensitivity model. These results indicate the strong potential of the atelocollagen-mediated drug delivery system for practical therapeutic technology.

Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo.

Silencing gene expression by siRNAs is rapidly becoming a powerful tool for the genetic analysis of mammalian cells. However, the rapid degradation of siRNA and the limited duration of its action call for an efficient delivery technology. Accordingly, we describe here that Atelocollagen complexed with siRNA is resistant to nucleases and is efficiently transduced into cells, thereby allowing long-term gene silencing. Site-specific in vivo administration of an anti-luciferase siRNA/Atelocollagen complex reduced luciferase expression in a xenografted tumor. Furthermore, Atelocollagen-mediated transfer of siRNA in vivo showed efficient inhibition of tumor growth in an orthotopic xenograft model of a human non-seminomatous germ cell tumor. Thus, for clinical applications of siRNA, an Atelocollagen-based non-viral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo.

Gene expression profiling of cerebellar development with high-throughput functional analysis.

We measured the expression levels of 450 genes during mouse postnatal cerebellar development by quantitative PCR using RNA purified from layers of the cerebellar cortex. Principal component analysis of the data matrix demonstrated that the first and second components corresponded to general levels of gene expression and gene expression patterns, respectively. We introduced 288 of the 450 genes into PC12 cells using a high-throughput transfection assay based on atelocollagen and determined the ability of each gene to promote neurite outgrowth or cell proliferation. Five genes induced neurite outgrowth, and seven genes enhanced proliferation. Evaluation of the functional data and gene expression patterns showed that none of these genes exhibited elevated expression at maturation, suggesting that genes characteristic of mature neurons are not likely to participate in neuronal development. These results demonstrate that functional data can facilitate interpretation of expression profiles and identification of new molecules that participate in biological processes.

Atelocollagen for protein and gene delivery.

Recent progress in recombinant gene technology and cell culture technology has made it possible to use protein and polynucleotides as effective drugs. However, because of their short half-lives in the body and the necessity of delivering to target site, those substances do not always exhibit good potency as expected. Therefore, delivery systems of such drugs are important research subjects in the field of pharmacology, and to prolong the effect of these drugs, many studies are being conducted to control the release of proteins and polynucleotides from various carrier materials. Collagen is one of the most useful carrier materials for this purpose. In this article, we report on the controlled release of protein drugs using collagen, focusing on a new drug delivery system (DDS), the Minipellet, as our basic technology. Then we introduce our recent work about gene therapy using collagen-based DDS. Basic formulation study showed that collagen DDS protects DNA degradation from both chemical cleavage and enzymatic digestion. A single injection of collagen DDS containing plasmid DNA produced physiologically significant levels of gene-encoding proteins in the local site and systemic circulation of animals and resulted in prolonged biological effects. These results suggest that collagen DDS containing plasmid DNA may enhance the clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Also, variety kinds of application of collagen DDS for gene therapy using adenovirus vector, antisense DNA and DNA vaccine, will be discussed.

The role of atelocollagen-based cell transfection array in high-throughput screening of gene functions and in drug discovery.

The human genome project has been completed, but the function of many genes is unknown. It is, therefore, necessary to elucidate the function of a large number of genes within a short time. To achieve this goal, materials are needed that condense or package DNA into nano-particles that can easily be taken up by cells and would allow DNA to be retained without degradation. Atelocollagen is a reliable carrier for gene delivery because it is considered safe and appropriate for practical use. We developed a basic technique for high-throughput gene transfer and expression screening by pre-coating a multi-well plate with an Atelocollagen/DNA complex in which cells are then seeded. Complexes with a nano-particle form were efficiently transduced into cells without the use of additional transfection reagents, and they allowed for long-term gene expression. The complex spotted onto the well of a plate was stable for a long period and allowed the cells to transduce and express reporter genes. We also showed that the present method with Atelocollagen-based gene transfer is applicable to gene medicines, such as antisense ODNs, siRNA, and adenovirus vectors. These results suggest that an Atelocollagen-based cell transfection array may be appropriate for general use in the high-throughput screening of large sets of gene medicines with functions in mammalian cells.

Ribophorin II regulates breast tumor initiation and metastasis through the functional suppression of GSK3ß.

Mutant p53 (mtp53) gain of function (GOF) contributes to various aspects of tumor progression including cancer stem cell (CSC) property acquisition. A key factor of GOF is stabilization and accumulation of mtp53. However, the precise molecular mechanism of the mtp53 oncogenic activity remains unclear. Here, we show that ribophorin II (RPN2) regulates CSC properties through the stabilization of mtp53 (R280K and del126-133) in breast cancer. RPN2 stabilized mtp53 by inactivation of glycogen synthase kinase-3ß (GSK3ß) which suppresses Snail, a master regulator of epithelial to mesenchymal transition. RPN2 knockdown promoted GSK3ß-mediated suppression of heat shock proteins that are essential for mtp53 stabilization. Furthermore, our study reveals that high expression of RPN2 and concomitant accumulation of mtp53 were associated with cancer tissues in a small cohort of metastatic breast cancer patients. These findings elucidate a molecular mechanism for mtp53 stabilization and suggest that RPN2 could be a promising target for anti-CSC therapy.

Imaging exosome transfer from breast cancer cells to stroma at metastatic sites in orthotopic nude-mouse models.

Exosomes play an important role in cell-to-cell communication to promote tumor metastasis. In order to image the fate of cancer-cell-derived exosomes in orthotopic nude mouse models of breast cancer, we used green fluorescent protein (GFP)-tagged CD63, which is a general marker of exosomes. Breast cancer cells transferred their own exosomes to other cancer cells and normal lung tissue cells in culture. In orthotopic nude-mouse models, breast cancer cells secreted exosomes into the tumor microenvironment. Tumor-derived exosomes were incorporated into tumor-associated cells as well as circulating in the blood of mice with breast cancer metastases. These results suggest that tumor-derived exosomes may contribute to forming a niche to promote tumor growth and metastasis. Our results demonstrate the usefulness of GFP imaging to investigate the role of exosomes in cancer metastasis.

Screening of potential molecular targets for colorectal cancer therapy.

Colorectal cancer is a leading cause of cancer death worldwide. To identify molecular targets for colorectal cancer therapy, we tested small interfering RNAs (siRNAs) against 97 genes whose expression was elevated in human colorectal cancer tissues for the ability to promote apoptosis of human colorectal cancer cells (HT-29 cells). The results indicate that the downregulation of PSMA7 (proteasome subunit, alpha-type, 7) and RAN (ras-related nuclear protein) most efficiently induced apoptosis of HT-29 cells. PSMA7 and RAN were highly expressed in colorectal cancer cell lines compared with normal colon tissues. Furthermore, PSMA7 and RAN were overexpressed in not only colon tumor tissues but also the other tumor tissues. Moreover, in vivo delivery of PSMA7 siRNA and RAN siRNA markedly induced apoptosis in HT-29 xenograft tumors in mice. Thus, silencing of PSMA7 and RAN induces cancer cells to undergo apoptosis, and PSMA7 and RAN might be promising new molecular targets for drug and RNA interference-based therapeutics against colorectal cancer.

RPN2 gene confers docetaxel resistance in breast cancer.

Drug resistance acquired by cancer cells has led to treatment failure. To understand the regulatory network underlying docetaxel resistance in breast cancer cells and to identify molecular targets for therapy, we tested small interfering RNAs (siRNAs) against 36 genes whose expression was elevated in human nonresponders to docetaxel for the ability to promote apoptosis of docetaxel-resistant human breast cancer cells (MCF7-ADR cells). The results indicate that the downregulation of the gene encoding ribophorin [corrected] II (RPN2), which is part of an N-oligosaccharyl transferase complex, most efficiently induces apoptosis of MCF7-ADR cells in the presence of docetaxel. RPN2 silencing induced reduced glycosylation of the P-glycoprotein, as well as decreased membrane localization, thereby sensitizing MCF7-ADR cells to docetaxel. Moreover, in vivo delivery of siRNA specific for RPN2 markedly reduced tumor growth in two types of models for drug resistance. Thus, RPN2 silencing makes cancer cells hypersensitive response to docetaxel, and RPN2 might be a new target for RNA interference-based therapeutics against drug resistance.

Atelocollagen-mediated drug discovery technology.

RNA interference (RNAi) was first reported in nematodes in 1998. Since that time, RNAi has been discovered in fish, insects and mammals. Novel treatments and drug discovery in preclinical studies based on RNAi are targeting a wide range of diseases at present, including viral infections and cancers. In addition to the local administration of synthetic small interfering RNA (siRNA) targeting local lesions, specific methods for the systemic administration of these molecules to treat infectious diseases or metastatic cancers have also been reported. In vivo delivery technology is a key hurdle that must be addressed for the successful clinical application of synthetic siRNA. In this review, the authors evaluate the recent findings on atelocollagen-siRNA complexes for the treatment for metastatic cancers and outline the cancer therapies and drug discovery studies that are based on RNAi technology.

Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery for siRNAs toward treatment of bone-metastatic cancer. Accordingly, we report here that i.v. injection of GL3 luciferase siRNA complexed with atelocollagen showed effective reduction of luciferase expression from bone-metastatic prostate tumor cells developed in mouse thorax, jaws, and/or legs. We also show that the siRNA/atelocollagen complex can be efficiently delivered to tumors 24 h after injection and can exist intact at least for 3 days. Furthermore, atelocollagen-mediated systemic administration of siRNAs such as enhancer of zeste homolog 2 and phosphoinositide 3'-hydroxykinase p110-alpha-subunit, which were selected as candidate targets for inhibition of bone metastasis, resulted in an efficient inhibition of metastatic tumor growth in bone tissues. In addition, upregulation of serum IL-12 and IFN-alpha levels was not associated with the in vivo administration of the siRNA/atelocollagen complex. Thus, for treatment of bone metastasis of prostate cancer, an atelocollagen-mediated systemic delivery method could be a reliable and safe approach to the achievement of maximal function of siRNA.