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Background: Noninvasive detection of early stage cancers with accurate prediction of tumor tissue-of-origin could improve patient prognosis. Because miRNA profiles differ between organs, circulating miRNomics represent a promising method for early detection of cancers, but this has not been shown conclusively., Methods: A serum miRNA profile (miRNomes)-based classifier was evaluated for its ability to discriminate cancer types using advanced machine learning. The training set comprised 7931 serum samples from patients with 13 types of solid cancers and 5013 noncancer samples. The validation set consisted of 1990 cancer and 1256 noncancer samples. The contribution of each miRNA to the cancer-type classification was evaluated, and those with a high contribution were identified., Results: Cancer type was predicted with an accuracy of 0.88 (95% confidence interval [CI] = 0.87 to 0.90) in all stages and an accuracy of 0.90 (95% CI = 0.88 to 0.91) in resectable stages (stages 0-II). The F1 score for the discrimination of the 13 cancer types was 0.93. Optimal classification performance was achieved with at least 100 miRNAs that contributed the strongest to accurate prediction of cancer type. Assessment of tissue expression patterns of these miRNAs suggested that miRNAs secreted from the tumor environment could be used to establish cancer type-specific serum miRNomes., Conclusions: This study demonstrates that large-scale serum miRNomics in combination with machine learning could lead to the development of a blood-based cancer classification system. Further investigations of the regulating mechanisms of the miRNAs that contributed strongly to accurate prediction of cancer type could pave the way for the clinical use of circulating miRNA diagnostics., (© The Author(s) 2022. Published by Oxford University Press.)

The SWI/SNF chromatin remodeling complex regulates transcription through the control of chromatin structure and is increasingly thought to play an important role in human cancer. Lung adenocarcinoma (LADC) patients frequently harbor mutations in SMARCA4, a core component of this multisubunit complex. Most of these mutations are loss-of-function mutations, which disrupt critical functions in the regulation of chromatin architecture and can cause DNA replication stress. This study reports that LADC cells deficient in SMARCA4 showed increased DNA replication stress and greater sensitivity to the ATR inhibitor (ATRi) in vitro and in vivo . Mechanistically, loss of SMARCA4 increased heterochromatin formation, resulting in stalled forks, a typical DNA replication stress. In the absence of SMARCA4, severe ATRi-induced single-stranded DNA, which caused replication catastrophe, was generated on nascent DNA near the reversed forks around heterochromatin in an Mre11-dependent manner. Thus, loss of SMARCA4 confers susceptibility to ATRi, both by increasing heterochromatin-associated replication stress and by allowing Mre11 to destabilize reversed forks. These two mechanisms synergistically increase susceptibility of SMARCA4-deficient LADC cells to ATRi. These results provide a preclinical basis for assessing SMARCA4 defects as a biomarker of ATRi efficacy., (© The Author(s) 2020. Published by Oxford University Press on behalf of NAR Cancer.)

Metastasis is the main cause of cancer mortality for many types of cancer; however, difficulties remain in effectively preventing metastasis. It has been recently and widely reported that cancer-derived extracellular vesicles (EVs) contribute to cancer metastasis. Thus, therapeutic strategies targeting cancer-derived EVs hold great promise because of the possibility of EVs driving the cancer microenvironment toward metastasis. Here, we provide a novel strategy for therapeutic antibody treatment to target cancer-derived EVs and inhibit the metastasis of breast cancer in a mouse model, establishing a rationale for further clinical investigation. Treatment with human-specific anti-CD9 or anti-CD63 antibodies significantly decreased metastasis to the lungs, lymph nodes, and thoracic cavity, although no obvious effects on primary xenograft tumor growths were observed. In in vitro and in vivo experiments, the EVs incubated with the targeted antibodies were preferentially internalized by macrophages, suggesting that antibody-tagged cancer-derived EVs would be eliminated by macrophages. Our results suggested that therapeutic antibody administration effectively suppresses EV-triggered metastasis in cancer and that the removal of EVs could be a novel strategy for cancer therapy., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Long-term outcomes of patients with peritoneal dissemination of gastric cancer remain unsatisfactory despite advances in treatment modalities. Internal luminescence conditionally replicative adenovirus (CRAd) presents a novel approach for cancer treatment and imaging., Materials and Methods: 3CL is a modified cyclooxygenase-2 (COX2) promoter-driven CRAd which contains the luciferase expression gene for bioluminescence imaging. The visualizing and therapeutic effect of 3CL was evaluated in a mouse model of peritoneal dissemination., Results: Intraperitoneal injection of 3CL achieved the shrinkage and reduction of lesions of peritoneal dissemination. Six model mice treated with 3CL had a significantly longer mean survival time than 6 mock-treated mice (85.7 versus 34.3 days, p=0.0005). By whole-body bioluminescent imaging, the sensitivity and specificity of peritoneal dissemination detection through macroscopic inspection were 58.1% and 83.2%, respectively, whereas 3CL viral imaging modality yielded corresponding values of 78.8% and 99.3%. Peritoneal lesions detected by imaging histologically contained cancer cells and necrotic tissue, which originated from viral oncolytic effects., Conclusion: Cox2 CRAds with 5/3 chimeric-fiber modification, therefore, appear to be a promising imaging and therapeutic tools for peritoneal dissemination of gastric cancer., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Ribophorin II (RPN2), part of the N-oligosaccharyltransferase complex, is highly expressed in breast cancer stem cells and is associated with tumor metastasis through interaction with mutant p53. The clinicopathological implication of RPN2 expression is undetermined. We examined immunohistochemically the expression levels of RPN2 and p53 in primary breast cancer tissues surgically resected from 218 patients. The correlations of RPN2 expression with the intrinsic subtype defined by hormone receptors (HRs) and HER2, clinicopathological parameters, p53 expression, and patients' clinical outcomes were examined. RPN2 was positive in 139 (64%), and the incidence of RPN2 expression was higher in the triple-negative breast cancer (TNBC) (HR-/HER2-) (65%) and HER2-enriched (HR-/HER2+) subtype (95%) than in the luminal A-like (HR+/HER2-) subtype (58%) (P = 0.0009). RPN2 expression was also correlated with p53 nuclear accumulation (P = 0.04). The RPN2-positive/p53-positive patient group showed significantly poorer prognosis than the RPN2-negative group for disease-free survival (P = 0.05) and for overall survival (P = 0.02). By multivariate analyses, the combination of RPN2 and p53 was not an independent prognostic factor. RPN2 expression was correlated with clinically aggressive features of breast cancer. These data support the further clinical application of anti-RPN2 therapy and the development of personalized medicine., (© 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Programmed cell death ligand-1 (PD-L1) has recently gained considerable attention for its role in tumor immune escape. Here, we identify a miR-197/CKS1B/STAT3-mediated PD-L1 network in chemoresistant non-small-cell lung cancer (NSCLC), independent of immunoinhibitory signals. miR-197 is downregulated in platinum-resistant NSCLC specimens, resulting in the promotion of chemoresistance, tumorigenicity, and pulmonary metastasis in vitro and in vivo. Mechanistic investigations reveal that a miR-197-mediated CKS1B/STAT3 axis exerts tumor progression regulated by various oncogenic genes (Bcl-2, c-Myc, and cyclin D1), and PD-L1 is a putative biomarker of this axis. Furthermore, we demonstrate that a miR-197 mimic sensitizes PD-L1(high) drug-resistant cells to chemotherapy. These results indicate that the biological interaction between PD-L1 and chemoresistance occurs through the microRNA regulatory cascade. More importantly, expression levels of miR-197 are inversely correlated with PD-L1 expression (n = 177; P = 0.026) and are associated with worse overall survival (P = 0.015). Our discoveries suggest that the miR-197/CKS1B/STAT3-mediated network can drive tumor PD-L1 expression as a biomarker of this cascade, and miR-197 replacement therapy may be a potential treatment strategy for chemoresistant NSCLC.

In lung cancer progression, p53 mutations are more often observed in invasive tumors than in noninvasive tumors, suggesting that p53 is involved in tumor invasion and metastasis. To understand the nature of p53 function as a tumor suppressor, it is crucial to elucidate the detailed mechanism of the alteration in epithelial cells that follow oncogenic KRAS activation and p53 inactivation. Here, we report that KRAS activation induces epithelial-mesenchymal transition and that p53 inactivation is required for cell motility and invasiveness. Furthermore, TSPAN2, a transmembrane protein, is responsible for cell motility and invasiveness elicited by p53 inactivation. TSPAN2 is highly expressed in p53-mutated lung cancer cells, and high expression of TSPAN2 is associated with the poor prognosis of lung adenocarinomas. TSPAN2 knockdown suppresses metastasis to the lungs and liver, enabling prolonged survival. TSPAN2 enhances cell motility and invasiveness by assisting CD44 in scavenging intracellular reactive oxygen species., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA interference-based therapies are proving to be powerful tools for combating various diseases, including cancer. Scientists are researching the development of safe and efficient systems for the delivery of small RNA molecules, which are extremely fragile in serum, to target organs and cells in the human body. A dozen pre-clinical and clinical trials have been under way over the past few years involving biodegradable nanoparticles, lipids, chemical modification and conjugation. On the other hand, microRNAs, which control the balance of cellular biological processes, have been studied as attractive therapeutic targets in cancer treatment. In this review, we provide an overview of RNA interference-based therapeutics in clinical trials and discuss the latest technology for the systemic delivery of nucleic acid drugs. Furthermore, we focus on dysregulated microRNAs in human cancer, which have progressed in pre-clinical trials as therapeutic targets, and describe a wide range of strategies to control the expression levels of endogenous microRNAs. Further development of RNA interference technologies and progression of clinical trials will contribute to the achievement of practical applications of nucleic acid drugs.

The release of humoral factors between cancer cells and the microenvironmental cells is critical for metastasis; however, the roles of secreted miRNAs in non-cell autonomous cancer progression against microenvironmental cells remain largely unknown. Here, we demonstrate that the neutral sphyngomyelinase 2 (nSMase2) regulates exosomal microRNA (miRNA) secretion and promotes angiogenesis within the tumor microenvironment as well as metastasis. We demonstrate a requirement for nSMase2-mediated cancer cell exosomal miRNAs in the regulation of metastasis through the induction of angiogenesis in inoculated tumors. In addition, miR-210, released by metastatic cancer cells, was shown to transport to endothelial cells and suppress the expression of specific target genes, which resulted in enhanced angiogenesis. These findings suggest that the horizontal transfer of exosomal miRNAs from cancer cells can dictate the microenviromental niche for the benefit of the cancer cell, like "on demand system" for cancer cells.

Many reports have indicated that the abnormal expression of microRNAs (miRNAs) is associated with the progression of disease and have identified miRNAs as attractive targets for therapeutic intervention. However, the bifunctional mechanisms of miRNA guide and passenger strands in RNA interference (RNAi) therapy have not yet been clarified. Here, we show that miRNA (miR)-582-5p and -3p, which are strongly decreased in high-grade bladder cancer clinical samples, regulate tumor progression in vitro and in vivo. Significantly, the overexpression of miR-582-5p or -3p reduced the proliferation and invasion of UM-UC-3 human bladder cancer cells. Furthermore, transurethral injections of synthetic miR-582 molecule suppressed tumor growth and metastasis in an animal model of bladder cancer. Most interestingly, our study revealed that both strands of miR-582-5p and -3p suppressed the expression of the same set of target genes such as protein geranylgeranyltransferase type I beta subunit (PGGT1B), leucine-rich repeat kinase 2 (LRRK2) and DIX domain containing 1 (DIXDC1). Knockdown of these genes using small interfering RNA (siRNA) resulted in the inhibition of cell growth and invasiveness of UM-UC-3. These findings uncover the unique regulatory pathway involving tumor suppression by both strands of a single miRNA that is a potential therapeutic target in the treatment of invasive bladder cancer.

MicroRNAs (miRNAs) act to fine-tune cellular responses in a variety of biological circumstances such as development, organogenesis, and homeostasis. The dysregulation of miRNA expression accelerates disease progression, including metabolic disease, immunological disease and cancer, through the gene network disorder. Therefore, understanding the miRNA maturation process may unravel the mechanisms of cancer malignancy; however, the life of miRNA has not been clarified. In this article, we summarize the recent findings regarding the novel forms of miRNA, especially secretory miRNAs, focusing on exosomal miRNAs. Recent research has revealed that exosomal miRNAs affect many aspects of physiological and pathological conditions, and may be useful as novel therapy. Here, we propose a method for the delivery of tumor-suppressive miRNAs to desired sites using exosomes, and we named this method "exocure"., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The prognosis of patients with advanced diffuse-type gastric cancer (GC), especially scirrhous gastric cancer (SGC) remains extremely poor. Peritoneal carcinomatosis is a frequent form of metastasis of SGC. With survival rates of patients with peritoneal metastasis at 3 and 5 years being only 9.8% and 0%, respectively, development of a new treatment is urgently crucial. For such development, the establishment of a therapeutic mouse model is required. Among the 11 GC cell lines we examined, HSC-60 showed the most well-preserved expression profiles of the Hedgehog and epithelial-mesenchymal transition pathways found in primary SGCs. After six cycles of harvest of ascitic tumor cells and their orthotopic inoculation in scid mice, a highly metastatic subclone of HSC-60, 60As6 was obtained, by means of which we successfully developed peritoneal metastasis model mice. The mice treated with small interfering (si) RNA targeting NEDD1, which encodes a gamma-tubulin ring complex-binding protein, by the atelocollagen-mediated delivery system showed a significantly prolonged survival. Our mouse model could thus be useful for the development of a new therapeutic modality. Intraperitoneal administration of siRNAs of targeted genes such as NEDD1 could provide a new opportunity in the treatment of the peritoneal metastasis of SGC., (© 2012 Japanese Cancer Association.)

Alzheimer's disease (AD) is characterized by the accumulation of β-amyloid peptide (Aβ) in the brain because of an imbalance between Aβ production and clearance. Neprilysin (NEP) is the most important Aβ-degrading enzyme in the brain. Thus, researchers have explored virus-mediated NEP gene delivery. However, such strategies may entail unexpected risks, and thus exploration of a new possibility for NEP delivery is also required. Here, we show that human adipose tissue-derived mesenchymal stem cells (ADSCs) secrete exosomes carrying enzymatically active NEP. The NEP-specific activity level of 1 μg protein from ADSC-derived exosomes was equivalent to that of ~ 0.3 ng of recombinant human NEP. Of note, ADSC-derived exosomes were transferred into N2a cells, and were suggested to decrease both secreted and intracellular Aβ levels in the N2a cells. Importantly, these characteristics were more pronounced in ADSCs than bone marrow-derived mesenchymal stem cells, suggesting the therapeutic relevance of ADSC-derived exosomes for AD.

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-3β (GSK3β) which suppresses Snail, a master regulator of epithelial to mesenchymal transition. RPN2 knockdown promoted GSK3β-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.

Hyaluronic acid (HA) has been implicated in the proliferation and metastasis of tumor cells. However, most previous studies were conducted on extracellular matrix or pericellular HA, and the role of circulating HA in vivo has not been studied. HA is rapidly cleared from the bloodstream. The scavenger receptor Stabilin-2 (Stab2) is considered a major clearance receptor for HA. Here we report a dramatic elevation in circulating HA levels in Stab2-deficient mice without any overt phenotype. Surprisingly, the metastasis of B16F10 melanoma cells to the lungs was markedly suppressed in the Stab2-deficient mice, whereas cell proliferation was not affected. Furthermore, administration of an anti-Stab2 antibody in Stab2(+) mice elevated serum HA levels and prevented the metastasis of melanoma to the lung, and also suppressed spontaneous metastasis of mammary tumor and human breast tumor cells inoculated in the mammary gland. Administration of the antibody or high-dose HA in mice blocked the lodging of melanoma cells to the lungs. Furthermore, HA at high concentrations inhibited the rolling/tethering of B16 cells to lung endothelial cells. These results suggest that blocking Stab2 function prevents tumor metastasis by elevating circulating HA levels. Stab2 may be a potential target in antitumor therapy.

Normal epithelial cells regulate the secretion of autocrine and paracrine factors that prevent aberrant growth of neighboring cells, leading to healthy development and normal metabolism. One reason for tumor initiation is considered to be a failure of this homeostatic cell competitive system. Here we identify tumor-suppressive microRNAs (miRNAs) secreted by normal cells as anti-proliferative signal entities. Culture supernatant of normal epithelial prostate PNT-2 cells attenuated proliferation of PC-3M-luc cells, prostate cancer cells. Global analysis of miRNA expression signature revealed that a variety of tumor-suppressive miRNAs are released from PNT-2 cells. Of these miRNAs, secretory miR-143 could induce growth inhibition exclusively in cancer cells in vitro and in vivo. These results suggest that secretory tumor-suppressive miRNAs can act as a death signal in a cell competitive process. This study provides a novel insight into a tumor initiation mechanism.

RNA interference (RNAi) has rapidly become a powerful tool for drug-target discovery and therapeutics. Cancer is an important application for RNAi therapeutics, since abnormal gene regulation is thought to contribute to the pathogenesis and maintenance of the metastatic phenotype of cancer. Many oncogenic genes present enticing therapeutic target possibilities for RNAi. Small interfering RNA (siRNA) and microRNA (miRNA) are potent and specific examples of RNAi are able to silence tumor-related genes and multiple oncogenic pathways and appear to be a rational approach to inhibit tumor growth. In subsequent in vivo studies, an appropriate animal model must be developed for a better evaluation of gene-silencing effects on tumors. How to evaluate the effect of siRNA and miRNA in an in vivo therapeutic model is also important. Bioluminescence imaging is an optical imaging method that can evaluate RNAi in vivo.

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.

Recent reports have linked the expression of specific microRNAs (miRNAs) with tumorigenesis and metastasis. Here, we show that microRNA (miR)-16, which is expressed at lower levels in prostate cancer cells, affects the proliferation of human prostate cancer cell lines both in vitro and in vivo. Transient transfection with synthetic miR-16 significantly reduced cell proliferation of 22Rv1, Du145, PPC-1, and PC-3M-luc cells. A prostate cancer xenograft model revealed that atelocollagen could efficiently deliver synthetic miR-16 to tumor cells on bone tissues in mice when injected into tail veins. In the therapeutic bone metastasis model, injection of miR-16 with atelocollagen via tail vein significantly inhibited the growth of prostate tumors in bone. Cell model studies indicate that miR-16 likely suppresses prostate tumor growth by regulating the expression of genes such as CDK1 and CDK2 associated with cell-cycle control and cellular proliferation. There is a trend toward lower miR-16 expression in human prostate tumors versus normal prostate tissues. Thus, this study indicates the therapeutic potential of miRNA in an animal model of cancer metastasis with systemic miRNA injection and suggest that systemic delivery of miR-16 could be used to treat patients with advanced prostate cancer.

Background and Aim: Multipotential mesenchymal stem cells (MSC), present in many organs and tissues, represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of regeneration medicine. Adipose tissue mesenchymal stem cells (AT-MSC), known as adipose-derived stem cells (ASC) are especially attractive in the context of future clinical applications because of their high accessibility and minimal invasiveness during the procedure to obtain them. The goal of the present study was to induce human ASC into functional hepatocytes in vitro within a very short period of time and to check their therapeutic potential in vivo., Methods: In vitro generated ASC-derived hepatocytes were checked for hepatocyte-specific markers and functions. Afterwards, they were transplanted into nude mice with liver injury. Twenty-four hours after transplantation, biochemical parameters were evaluated in blood serum., Results: We have shown here that ASC can be differentiated into hepatocytes within 13 days and can reach the functional properties of primary human hepatocytes. After transplantation into mice with acute liver failure, ASC-derived hepatocytes can restore such liver functions as ammonia and purine metabolism. Markers of liver injury, alanine aminotransferase, aspartate aminotransferase, as well as ammonia, were decreased after ASC-derived hepatocyte transplantation., Conclusions: Our data highlight the properties of ASC as having a special affinity for hepatocyte differentiation in vitro and liver regeneration in vivo. Thus, ASC may be a superior choice for the establishment of a therapy for injured liver.

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.

Myo18B is an unconventional myosin family protein expressed predominantly in muscle cells. Although conventional myosins are known to be localized on the A-bands and function as a molecular motor for muscle contraction, Myo18B protein was localized on the Z-lines of myofibrils in striated muscles. Like Myo18A, another 18th class of myosin, the N-terminal unique domain of the protein and not the motor domain and the coiled-coil tail is critical for its localization to F-actin in myocytes. Myo18B expression was induced by myogenic differentiation through the binding of myocyte-specific enhancer factor-2 to its promoter. Deficiency of Myo18B caused an embryonic lethality in mice accompanied by disruption of myofibrillar structures in cardiac myocytes at embryonic day 10.5. Thus, Myo18B is a unique unconventional myosin that is predominantly expressed in myocytes and whose expression is essential for the development and/or maintenance of myofibrillar structure.

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.

Viral capsids of simian virus 40 (SV40) are highly efficient gene delivery vehicles that infect a broad range of cells and tissues. To develop a controlled, cell type-specific delivery system, we sought to display foreign peptides on the capsid surface by genetically manipulating the major capsid protein Vp1. Here we report the identification of two sites within the surface loops of Vp1 that can accommodate foreign peptides in such a way that the foreign peptides are displayed on the surface of the virus-like particles (VLPs) without interfering with VLP assembly or the packaging of viral DNA. Insertion of Flag-tags but not RGD integrin-binding motifs at these sites strongly inhibited cell attachment of VLPs, which normally associate with host cells through cell surface molecules such as major histocompatibility complex (MHC) class I and ganglioside GM1. Instead, VLPs carrying the RGD motifs bound to integrin in vitro and to the cell surface in an RGD-dependent manner. Thus, insertion of foreign sequences into the surface loops of Vp1 can reduce natural virus-cell interactions and even confer an ability to bind to a new target receptor. This study demonstrates the potential usefulness of this strategy for the development of novel delivery vehicles with different cell tropisms.

Prostate cancers generally acquire an androgen-independent growth capacity with progression, resulting in resistance to antiandrogen therapy. Therefore, identification of the genes regulated through this process may be important for understanding the mechanisms of prostate carcinogenesis. We here utilized androgen-dependent/independent transplantable tumors, newly established with the 'transgenic rat adenocarcinoma in prostate' (TRAP) model, to analyze their gene expression using microarrays. Among the overexpressed genes in androgen-independent prostate cancers compared with the androgen-dependent tumors, glutathione S-transferase pi (GST-pi) was included. In line with this, human prostate cancer cell lines PC3 and DU145 (androgen independent) had higher expression of GST-pi compared with LNCaP (androgen dependent) as determined by semiquantitative reverse transcription-polymerase chain reaction analysis. To investigate the roles of GST-pi expression in androgen-independent human prostate cancers, GST-pi was knocked down by a small interfering RNA (siRNA), resulting in significant decrease of the proliferation rate in the androgen-independent PC3 cell line. In vivo, administration of GST-pi siRNA-atelocollagen complex decreased GST-pi protein expression, resulting in enhanced numbers of TdT mediated dUTP-biotin nick-end labering (TUNEL)-positive apoptotic cells. These findings suggest that GST-pi might play important roles in proliferation of androgen-independent human prostate cancer cells.

Deregulated proliferation is one of the fundamental characteristics of carcinogenesis. p27 is one of the most well characterized negative cell cycle regulator. In our previous study, expression of p27 protein was found to be dramatically suppressed on stimulation of cell proliferation by calculi in the rodent urinary bladder, withdrawal of the insult resulting in re-expression of p27 and regression of urothelial hyperplastic lesions. In the present study, to evaluate how loss of function impacts on urinary bladder carcinogenesis, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), a bladder carcinogen was given to p27 knockout mice. Males and females with either null, hetero or wild-type p27 alleles were divided into 2 groups, one given drinking water containing 0.05% BBN for 10 weeks and the other receiving distilled water, then, killed at week 20. The experiment was repeated for confirmation of the outcome. In the second experiment, performed with a larger number of animals, the incidence of urinary bladder carcinomas was significantly higher in female p27-null mice than in their wild-type counterparts. p27 deficiency also resulted in their increase of relative weights of urinary bladders and section areas of carcinomas in BBN-treated mice. Interestingly, while BrdU labeling indices generally increased with progression of mucosal proliferative lesions, from normal epithelium, through hyperplasia to carcinoma, there was no significant variation with the p27 genotype, in tumors as well as normal urothelium. These findings suggest that p27 deficient mice have elevated susceptibility to BBN-induction of urinary bladder carcinogenesis through a mechanism which might be independent of acceleration of cell cycling., ((c) 2007 Wiley-Liss, Inc.)

In this study, we describe pancreatic cell ontogeny in renal capsule-transplanted embryonic stem cells (ES) after injury by streptozocin (STZ), showing pancreatogenesis in situ. Seven-week-old female BALB/c nude mice were treated with either a single 175- or 200-mg/kg STZ dose, a regimen that induces substantial beta-cell damage without overt hyperglycemia, and transplanted 24 hr later with 1 x 10(5) ES. Immunohistochemistry was performed on ES tissue at 15, 21, and 28 days after transplantation using antibodies against stage- and lineage-specific pancreatic markers. After 21 days, PDX-1+ pancreatic foci first appeared in the renal capsule and expressed both amylase and endocrine hormones (insulin, glucagon, and somatostatin). These foci increased in size by day 28 because of acinar and duct cell proliferation, whereas endocrine cells remained non-dividing, and made up 2-4% of ES tumor volume. PDX-1, Nkx6.1, Ngn3, and ISL-1 protein localization patterns in pancreatic foci were comparable with embryonic pancreatogenesis. A prevalence of multihormonal endocrine cells, a characteristic of adult beta-cell regeneration, indicated a possible divergence from embryonic islet cell development. The results indicate that beta-cell damage, without overt hyperglycemia, induces a process of fetal-like pancreatogenesis in renal capsule-transplanted ES, leading to beta-cell neogenesis.

Unlabelled: Recent observations indicate that several stem cells can differentiate into hepatocytes; thus, cell-based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vitro hepatic differentiation potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs). We used AT-MSCs from different age patients and found that, after incubation with specific growth factors (hepatocyte growth factor [HGF], fibroblast growth factor [FGF1], FGF4) the CD105(+) fraction of AT-MSCs exhibited high hepatic differentiation ability in an adherent monoculture condition. CD105(+) AT-MSC-derived hepatocyte-like cells revealed several liver-specific markers and functions, such as albumin production, low-density lipoprotein uptake, and ammonia detoxification. More importantly, CD105(+) AT-MSC-derived hepatocyte-like cells, after transplantation into mice incorporated into the parenchyma of the liver., Conclusion: Adipose tissue is a source of multipotent stem cells that can be easily isolated, selected, and induced into mature, transplantable hepatocytes. The fact that they are easy to procure ex vivo in large numbers makes them an attractive tool for clinical studies in the context of establishing an alternative therapy for liver dysfunction.

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.

Pancreatic ductal adenocarcinoma is one of the most debilitating malignancies in humans. Currently, radiation and chemotherapy are ineffective, with median survival times after treatment of <12 months. Animal models that reflect the human condition and can be used to explore screening and therapeutic approaches are clearly desirable. One feature of human pancreatic adenocarcinoma is an exceedingly high frequency of K-ras mutation. The present study was conducted to determine if targeted activation of a human oncogenic-ras transgene in rat pancreas would induce carcinomas correspondent to human pancreatic ductal adenocarcinomas. We established transgenic (Hras250) rats in which expression of a human Ha-rasG12V oncogene is regulated by the Cre/lox system. Targeted pancreatic activation of the transgene was accomplished by injection of Cre-carrying adenovirus into the pancreatic ducts and acini through the common bile duct. Adenoviral infection of injected animals was exclusive to the pancreas; infected cells could be identified in duct, intercalated duct, centroacinar and, less frequently, acinar cells, but not in endocrine islet cells. Four weeks after injection, proliferative lesions in the duct epithelium, intercalated ducts and centroacinar cells, but not acinar cells, were widespread. Tumorigenesis in other tissues was not observed. Most lesions, including atypical duct proliferative lesions, PanIN-like lesions and carcinomas, were positive for cytokeratins 19 and 7, cyclooxygenase 2 and MMP-7 but negative for amylase and chymotrypsin. Many adenocarcinoma lesions were positive for EGF and EGFR. Duct epithelial and atypical duct proliferative lesions and carcinoma lesions were all positive for transduced Ha-rasG12V oncogene expression. The cytogenesis of pancreatic ductal type carcinoma was depicted. This model exhibits important similarities to the human disease and promises to advance our understanding of the behavior of pancreas adenocarcinomas and expedite screening and therapy.

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.

One of the most dramatic events of the past 5 years in the field of molecular biology has been the discovery of RNA interference (RNAi). Although RNAi is an evolutionarily conserved phenomenon for sequence-specific gene silencing in mammalian cells, exogenous small interfering RNA (siRNA) and vector-based short hairpin RNA (shRNA) can also invoke RNAi responses. Both are now not only experimental tools for analyzing gene function but are expected to be excellent avenues for drug target discovery and the emerging class of gene medicine for targeting incurable diseases such as cancer. The success of cancer therapeutic use of RNAi relies on the development of safe and efficacious delivery systems that introduce siRNA and shRNA expression vectors into target tumor cells. For their delivery, a variety of strategies have been used, most of them based on traditional gene therapy delivery systems. In this review, we present siRNA delivery method strategies and discuss the potential of RNAi-based gene therapy in cancer treatment.

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.

Previously, we established the in vivo lung metastasis model of rat HCC induced by two hepatocarcinogens, diethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR) at a dose of 120 ppm. This model allows us to investigate modifying factors leading to the inhibition of metastasis formation. However, low survival rates made the evaluation of metastasis formation difficult. The current experiments were conducted to modify the experimental protocol to improve survival and to establish a better animal metastasis model. Lower doses of NMOR (80 or 40 ppm in drinking water) were given to F344 rats for 14 weeks after DEN treatment. Survival rates in the 80 ppm group and in the 40 ppm group were 57% and 81%, respectively and these values were significantly higher than that in 120 ppm. Incidences of lung metastasis in the 40 ppm group steadily increased up to 67% by week 36 while that in the 80 ppm increased sharply up to 86% by week 24. Severity of lung metastases in the 40 ppm group at week 36 was mild compared with the 80 ppm group at week 24. In the second experiment, in order to characterize HCC development and lung metastasis in the 40 ppm group, rats given DEN and then followed with 40 ppm NMOR were killed sequentially. Development of HCC was observed at week 14 and reached 100% incidence at week 20. First lung metastatic lesions were evident at week 22, and incidence of lung metastasis reached 100%. Tumor cells were identified in the blood at week 20 by RT-PCR. The current study revealed that 40 ppm NMOR for 14 weeks after DEN treatment developed HCC without lung metastases at week 22, then HCC with a frequent lung metastasis at week 40. Thus, it can be said that this system is a more appropriate model for elucidation of mechanisms of metastasis and also for analysis of factors to inhibit natural metastasis.

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.

Phenylethyl isothiocyanate (PEITC), generally thought to be a chemopreventive agent for various kinds of genotoxic carcinogens, has been found to induce rat urinary bladder carcinomas in our laboratory. To cast light on the underlying mechanisms, the reversibility of urothelial proliferative lesions and the frequencies of H-ras and p53 mutations in the induced rat urinary bladder tumors were investigated. F344 male rats were given diet containing 0.1% PEITC for 48 weeks and then killed, or for 32 weeks and then returned to normal diet without supplement for 1, 3, 7 days or 16 weeks before death. At 7 days after withdrawal of PEITC treatment, carcinomas were observed in only two of 24 rats but a high incidence of dysplasias was evident. Furthermore, 16 weeks after withdrawal, seven of 12 (58.3%) rats had carcinomas. In addition, carcinomas were induced in 11 of 12 (91.7%) rats continuously receiving PEITC for 48 weeks. Most of the carcinomas were characterized as of non-papillary transitional cell type with occasional squamous cell differentiation and/or glandular components. Bromodeoxyuridine labeling indices (LIs) were increased by PEITC administration even in normal-looking epithelium. After withdrawal of treatment, LIs in simple and papillary or nodular (PN) hyperplasias were markedly decreased and these lesions gradually disappeared, while values for dysplasias and carcinomas, which persisted, were only slightly decreased. A silent point mutation was found in H-ras in one of 12 tumor samples (8.3%), whereas seven (58.3%) had mutations in p53. These results indicate that PEITC itself is a carcinogen for the rat urinary bladder, and that while the simple and PN hyperplasia induced by PEITC are reversible, dysplasia is irreversible with the potential to give rise to non-papillary carcinomas with frequent p53 mutations.

To examine the effect of non-steroidal anti-inflammatory drugs on metastasis formation, aspirin (ASP, 0.5% in diet) and indomethacin (IM, 0.005% in drinking water) were applied to an in vivo highly metastatic rat hepatocellular carcinoma (HCC) model in F344 male rats. Administration for 8 weeks after induction of highly metastatic HCC by sequential treatment with diethylnitrosamine and N-nitrosomorpholine did not cause any significant change in survival rate or body weight. Multiplicity of HCC in the liver increased during ASP or IM treatment without any significant histological alteration. Although absent in the rats killed at the end of the period of carcinogen exposure, lung metastasis at the end of the experiment was found in 100%, 89% and 100% of rats in the control, ASP and IM groups, respectively. Degree of metastasis was classified into three groups according to the number of metastatic nodules, i.e., slight (1 - 5 nodules), moderate (6 - 50) and severe (more than 51), which amounted to 0%, 43% and 57% in the control group. ASP significantly reduced the degree of metastasis, the incidences being 33%, 44%, and 11%, respectively, whereas IM was without significant influence. Both agents suppressed cell proliferation in HCCs, without any alteration of pan-cadherin expression. However, expression in HCC of mRNAs for intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, both of which are considered to play key roles in attachment of cancer cells to the endothelium, was significantly suppressed by ASP. Thus, the present study demonstrated that ASP, but not IM, has the potential to inhibit lung metastasis of rat HCC in vivo, possibly via reduced attachment of tumor cells to the vascular endothelium. Moreover, these data indicate this in vivo model for induction of rat highly metastatic HCC to be a useful tool for the assessment of the efficacy of therapeutic treatments to block metastasis formation.

Cyclin D1 is a critical gene involved in the regulation of progression through the G(1) phase of the cell cycle, thereby contributing to cell proliferation. Gene amplification and abnormal expression of Cyclin D1 have been described in several human cancers. To understand their biological significance in skin carcinogenesis, we established Cyclin D1-conditional transgenic mice with C57BL/6J background, in which skin-specific overexpression of Cyclin D1 transgene was observed. The mice were subjected to dimethylbenz[a]anthracene complete skin carcinogenesis studies. After 40 weeks of repeated administration of dimethylbenz[a]anthracene on the skin (once a week), all of the mice with high Cyclin D1 expression had papillomas, whereas only 9.5% of the control mice without the transgene developed papillomas. Primary cultured keratinocytes with induced Cyclin D1 transgene expression showed resistance to calcium-induced terminal differentiation and continued to replicate in vitro. These results clearly provide us with direct experimental evidence that overexpression of CyclinD1 induces excessive dermal cell proliferation via the altered differentiation state of keratinocytes. The conditional transgenic mice described here provide excellent in vivo and in vitro model systems to understand the role of cyclin D1 and deregulation of the cell cycle in carcinogenesis.