The Necroptotic Process-Related Signature Predicts Immune Infiltration and Drug Sensitivity in Kidney Renal Papillary Cell Carcinoma.

BACKGROUND: It remains controversial whether the current subtypes of kidney renal papillary cell carcinoma (KIRP) can be used to predict the prognosis independently. OBJECTIVE: This observational study aimed to identify a risk signature based on necroptotic pro-cess-related genes (NPRGs) in KIRP. METHODS: In the training cohort, LASSO regression was applied to construct the risk signature from 158 NPRGs, followed by the analysis of Overall Survival (OS) using the Kaplan-Meier method. The signature accuracy was evaluated by the Receiver Operating Characteristic (ROC) curve, which was further validated by the test cohort. Wilcoxon test was used to compare the expressions of immune-related genes, neoantigen genes, and immune infiltration between differ-ent risk groups, while the correlation test was performed between NPRGs expressions and drug sensitivity. Gene set enrichment analysis was used to investigate the NPRGs' signature's biologi-cal functions. RESULTS: We finally screened out 4-NPRGs (BIRC3, CAMK2B, PYGM, and TRADD) for con-structing the risk signature with the area under the ROC curve (AUC) reaching about 0.8. The risk score could be used as an independent OS predictor. Consistent with the enriched signaling, the NPRGs signature was found to be closely associated with neoantigen, immune cell infiltration, and immune-related functions. Based on NPRGs expressions, we also predicted multiple drugs potentially sensitive or resistant to treatment. CONCLUSION: The novel 4-NPRGs risk signature can predict the prognosis, immune infiltration, and therapeutic sensitivity of KIRP.

Significance of UGT1A6, UGT1A9, and UGT2B7 genetic variants and their mRNA expression in the clinical outcome of renal cell carcinoma.

UDP-glucuronosyltransferase (UGT) metabolizes a number of endogenous and exogenous substrates. Renal cells express high amounts of UGT; however, the significance of UGT in patients with renal cell carcinoma (RCC) remains unknown. In this study, we profile the mRNA expression of UGT subtypes (UGT1A6, UGT1A9, and UGT2B7) and their genetic variants in the kidney tissue of 125 Japanese patients with RCC (Okayama University Hospital, Japan). In addition, we elucidate the association between the UGT variants and UGT mRNA expression levels and clinical outcomes in these patients. The three representative genetic variants, namely, UGT1A6 541A > G, UGT1A9 i399C > T, and UGT2B7-161C > T, were genotyped, and their mRNA expression levels in each tissue were determined. We found that the mRNA expression of the three UGTs (UGT1A6, UGT1A9, and UGT2B7) are significantly downregulated in RCC tissues. Moreover, in patients with RCC, the UGT2B7-161C > T variant and high UGT2B7 mRNA expression are significantly correlated with preferable cancer-specific survival (CSS) and overall survival (OS), respectively. As such, the UGT2B7-161C > T variant and UGT2B7 mRNA expression level were identified as significant independent prognostic factors of CSS and CSS/OS, respectively. Taken together, these findings indicate that UGT2B7 has a role in RCC progression and may, therefore, represent a potential prognostic biomarker for patients with RCC.

Identification of MICALL2 as a Novel Prognostic Biomarker Correlating with Inflammation and T Cell Exhaustion of Kidney Renal Clear Cell Carcinoma.

Purpose: The interplay of inflammation and immunity affects all stages from tumorigenesis to progression, and even tumor response to therapy. A growing interest has been attracted from the biological function of MICALL2 to its effects on tumor progression. This study was designed to verify whether MICALL2 could be a prognostic biomarker to predict kidney renal clear cell carcinoma (KIRC) progression, inflammation, and immune infiltration within tumor microenvironment (TME). Methods: We firstly analyzed MICALL2 expressions across 33 cancer types from the UCSC Xena database and verified its expression in KIRC through GEPIA platform and GEO datasets. The clinicopathological characteristics were further analyzed based on the median expression. Kaplan-Meier method, univariate and multivariate analyses were applied to compare survival outcomes. ESTIMATE and CIBERSORT algorithms were performed to assess immune infiltration, and a co-expression analysis was conducted to evaluate the correlation between MICALL2 and immunoregulatory genes. Enrichment analysis was finally performed to explore the biological significance of MICALL2. Results: MICALL2 was highly expressed in 16 types of cancers compared with normal tissues. MICALL2 expression increased with advanced clinicopathological parameters and was an independent predictor for poor prognosis in KIRC. Moreover, MICALL2 closely correlated with inflammation-promoting signatures and immune infiltration including T cell exhaustion markers. Consistently, MICALL2 involved in the regulation of signaling pathways associated with tumor immunity, tumor progression, and impaired metabolic activities. Conclusion: MICALL2 can function as a prognostic biomarker mediating inflammation, immune infiltration, and T cell exhaustion within the microenvironment of KIRC.

Repurposing of posaconazole as a hedgehog/SMO signaling inhibitor for embryonal rhabdomyosarcoma therapy.

Posaconazole (POS) is a novel antifungal agent, which has been repurposed as an anti-tumor drug for its potential inhibition of Hedgehog signaling pathway. Hedgehog pathway is reported to be abnormally activated in embryonal rhabdomyosarcoma (ERMS), this study aimed to reveal whether POS could inhibit Hedgehog signaling pathway in ERMS. Following POS treatment, XTT viability assay was used to determine the cell proliferation of ERMS cell lines. Protein changes related to Hedgehog signaling, cell cycle and autophagy were detected by Western blot. The cell cycle distribution was analyzed by flow cytometry. Moreover, a subcutaneous tumor mouse model of ERMS was established to assess the anti-tumor effect of POS. POS was found to inhibit tumor progression by inducing G0/G1 arrest and autophagy of RD, RMS-YM, and KYM-1 cells dose-dependently. Western blot demonstrated that POS downregulated the expressions of SMO, Gli1, c-Myc, CDK4, and CDK6, while upregulated the expressions of autophagy-related proteins. Immunofluorescence microscopy revealed a significant increase of LC3B puncta in POS-treated ERMS cells. Furthermore, POS treatment led to a significant inhibition of tumor growth in mice bearing ERMS. Our findings could provide a theoretical basis and have important clinical implications in developing POS as a promising agent against ERMS by targeting Hedgehog pathway.

Relevance of CYP3A5 Expression on the Clinical Outcome of Patients With Renal Cell Carcinoma.

BACKGROUND/AIM: This study aimed to elucidate the detailed characteristics of CYP3A5 expression and the association between CYP3A5 expression and clinical outcomes in patients with renal cell carcinoma (RCC). PATIENTS AND METHODS: This study retrospectively enrolled 124 Japanese patients with RCC treated at the Okayama University Hospital. The commonest CYP3A5 gene polymorphism, CYP3A5*3, and expression levels of CYP3A5 mRNA and protein in each tissue were examined. RESULTS: Expression of CYP3A5 mRNA and protein in RCC tissues was significantly down-regulated compared to that in adjacent normal tissues. High level of CYP3A5 mRNA expression significantly extended cancer-specific survival (p=0.004) and overall survival (p=0.002). The CYP3A5 mRNA expression level was identified as a significant independent prognostic factor for both cancer-specific survival and overall survival. CONCLUSION: CYP3A5 could serve as a potential marker for prognostication and treatment planning for patients with RCC.

Tumor suppressor REIC/Dkk-3 and its interacting protein SGTA inhibit glucocorticoid receptor to nuclear transport.

REIC/Dkk-3 is a tumor suppressor, and its expression is significantly downregulated in a variety of human cancer types. A previous study performed yeast two-hybrid screening and identified the small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, which is a novel interacting partner of REIC/Dkk-3. The previous study also indicated that the REIC/Dkk-3 protein interferes with the dimerization of SGTA and then upregulates the AR transport and signaling in human prostate cancer PC3 cells. Since the transport of some steroid receptors to nucleus is conducted similarly by dynein motor-dependent way, the current study aimed to investigate the role of SGTA and REIC/Dkk-3 in the transport of other glucocorticoid receptors (GR). In vitro reporter assays for the cytoplasmic GR transport were performed in human prostate cancer PC3 cells and 293T cells. As for the SGTA protein, a suppressive effect on the GR transport to the nucleus was observed in the cells. As for the REIC/Dkk-3 protein, an inhibitory effect was observed for the GR transport in PC3 cells. Under the depleted condition of SGTA by short-hairpin (sh)RNA, the downregulation of GR transport by REIC/Dkk-3 was significantly enhanced compared with the non-depleted condition in PC3 cells, suggesting a compensatory role of REIC/Dkk-3 in the SGTA mediated inhibition of GR transport. The current study therefore demonstrated that SGTA inhibited the cytoplasmic transport of GR in 293T and PC3 cells, and REIC/Dkk-3 also inhibited the cytoplasmic transport of GR in PC3 cells. These results may be used to gain novel insight into the GR transport and signaling in normal and cancer cells.

Induction of cells with prostate cancer stem-like properties from mouse induced pluripotent stem cells via conditioned medium.

Cancer stem cells (CSCs) that closely correlated with tumor growth, metastasis, provide a plausible explanation for chemoresistance and cancer relapse. CSCs are usually isolated and enriched from carcinoma cells, which is inconvenient, low-efficient, and even unreliable. Here, we converted mouse induced pluripotent stem cells (miPSCs) into prostate cancer stem-like cells with carcinoma microenvironment following exposure to conditioned medium (CM) derived from RM9, a mouse prostate cancer cell line. These transformed cells, termed as miPS-RM9CM, displayed CSCs properties, including spheroids morphology and expression of both stemness genes and cancer stem cells surface markers, such as Oct3/4, Sox2, Nanog, Klf-4, c-Myc, CD44, and CD133. In addition, in vivo transplantation experiment was performed to confirm the tumorigenicity. Furthermore, we used the model to assess conventional chemotherapeutic agent, docetaxel. The results showed that miPS-RM9CM cells exhibited increased resistance to docetaxel, however, high susceptibility to the cancer cell stemness inhibitor I (BBI-608). Our current study demonstrates that CM from cultured RM9 cells play a crucial role in the determination of cell fate from miPSCs to cancer stem-like cells and provide a potentially valuable system for the study of CSCs.

Role of Egr1 on Pancreatic Endoderm Differentiation.

The low efficiency of in vitro differentiation of human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (iPSCs) into insulin-producing cells is a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). Our previous investigation into the key factors for the differentiation of PSCs into insulin-producing cells suggested that the expression of GATA binding protein 6 (GATA6) and Gremlin 1 (GREM1) and inhibition of early growth response protein 1 (Egr1) may be important factors. In this study, we investigated the role of Egr1 in pancreas development. The transfection of small interfering RNA (siRNA) of Egr1 in the early phase induced the differentiation of iPSCs derived from fibroblasts (FiPSCs) into pancreatic endoderm and insulin-producing cells. In contrast, the downregulation of Egr1 in the late phase suppressed the differentiation of FiPSCs into pancreatic endoderm and insulin-producing cells. In addition, the overexpression of Egr1 suppressed the differentiation of iPSCs derived from pancreatic cells into pancreatic endoderm and insulin-producing cells. These data suggest that the downregulation of Egr1 in the early phase can efficiently induce the differentiation of iPSCs into insulin-producing cells.

Robust cancer-specific gene expression by a novel cassette with hTERT and CMV promoter elements.

We developed and validated a novel hTERT/CMV promoter element-driven gene expression cassette that can robustly enhance cancer-specific gene expression. The following gene expressional elements were located in tandem within the plasmid construct: [hTERT core promoter, cytomegalovirus (CMV) minimized promoter, RU5' sequence, an inserted gene, BGH polyA, hTERT enhancer]; this is hereafter referred to as the hT/Cm-R-hT construct. Using various human cancer cell lines and normal cells, the cancer-specific transcription of the green fluorescent protein (GFP) gene was examined by western blotting and fluorescence microscopy. Cancer-specific gene expression was robustly achieved in the hT/Cm-R-hT plasmid in comparison to the other control hT/Cm-driven construct. Notably, the expression level of GFP observed in the hT/Cm-R-hT-driven construct was superior to that of the control plasmid with the conventional CMV promoter in HEK293 cells, which are known to possess higher hTERT activity than normal cells. We next examined the availability of hT/Cm-R-hT in detecting the target GFP expressing cancer cells from human peripheral blood mononuclear cells (PBMCs). The hT/Cm-R-hT plasmid successfully induced cancer-specific gene expression; the robust expression of GFP was observed in target HeLa cancer cells, whereas GFP was not visibly expressed in normal PBMCs. The plasmid allowed for the selective visualization of viable HeLa cancer cells in mixed cell cultures containing up to 10000-fold more PBMCs. These findings indicate that the hT/Cm-R-hT expressional system is a valuable tool for detecting viable cancer cells mixed with normal cells. The current system can therefore be applied to the in vitro detection of cancer cells that are disseminated in the blood and other types of body fluid in vivo. Since the current system can also be applied to other types of vectors, including virus vectors, this approach using the hTERT promoter-based construct is expected to become a valuable tool for enhancing cancer-specific gene expression.

The Downregulation of the Expression of CD147 by Tumor Suppressor REIC/Dkk-3, and Its Implication in Human Prostate Cancer Cell Growth Inhibition.

The cluster of differentiation 147 (CD147), also known as EMMPRIN, is a key molecule that promotes cancer progression. We previously developed an adenoviral vector encoding a tumor suppressor REIC/Dkk-3 gene (Ad-REIC) for cancer gene therapy. The therapeutic effects are based on suppressing the growth of cancer cells, but, the underlying molecular mechanism has not been fully clarified. To elucidate this mechanism, we investigated the effects of Ad-REIC on the expression of CD147 in LNCaP prostate cancer cells. Western blotting revealed that the expression of CD147 was significantly suppressed by Ad-REIC. Ad-REIC also suppressed the cell growth of LNCaP cells. Since other researchers have demonstrated that phosphorylated mitogen-activated protein kinases (MAPKs) and c-Myc protein positively regulate the expression of CD147, we investigated the correlation between the CD147 level and the activation of MAPK and c-Myc expression. Unexpectedly, no positive correlation was observed between CD147 and its possible regulators, suggesting that another signaling pathway was involved in the downregulation of CD147. This is the first study to show the downregulation of CD147 by Ad-REIC in prostate cancer cells. At least some of the therapeutic effects of Ad-REIC may be due to the downregulation of the cancer-progression factor, CD147.

Tumor suppressor REIC/DKK-3 and co-chaperone SGTA: Their interaction and roles in the androgen sensitivity.

REIC/DKK-3 is a tumor suppressor, however, its intracellular physiological functions and interacting molecules have not been fully clarified. Using yeast two-hybrid screening, we found that small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, is a novel interacting partner of REIC/DKK-3. Mammalian two-hybrid and pull-down assay results indicated that the SGTA-REIC/DKK-3 interaction involved the N-terminal regions of both REIC/DKK-3 and SGTA and that REIC/DKK-3 interfered with the dimerization of SGTA, which is a component of the AR complex and a suppressor of dynein motor-dependent AR transport and signaling. A reporter assay in human prostate cancer cells that displayed suppressed AR signaling by SGTA showed recovery of AR signaling by REIC/DKK-3 expression. Considering these results and our previous data that REIC/DKK-3 interacts with the dynein light chain TCTEX-1, we propose that the REIC/DKK-3 protein interferes with SGTA dimerization, promotes dynein-dependent AR transport and then upregulates AR signaling.

A vaccine strategy with multiple prostatic acid phosphatase-fused cytokines for prostate cancer treatment.

Immunotherapy is one of the attractive treatment strategies for advanced prostate cancer. The US Food and Drug Administration (FDA) previously approved the therapeutic vaccine, sipuleucel-T, which is composed of autologous antigen-presenting cells cultured with a fusion protein [prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GMCSF)]. Although sipuleucel-T has been shown to prolong the median survival of patients for 4.1 months, more robust therapeutic effects may be expected by modifying the vaccination protocol. In the present study, we aimed to develop and validate a novel vaccination strategy using multiple PAP-fused cytokines for prostate cancer treatment. Using a super gene expression (SGE) system that we previously established to amplify the production of a recombinant protein, significant amounts of PAP-fused cytokines [human GMCSF, interleukin-2 (IL2), IL4, IL7 and mouse GMCSF and IL4] were obtained. We examined the activity of the fusion proteins in vitro to validate their cytokine functions. A significant upregulation of dendritic cell differentiation from monocytes was achieved by PAP-GMCSF when used with the other PAP-fused cytokines. The PAP-fused human IL2 significantly increased the proliferation of lymphocytes, as determined by flow cytometry. We also investigated the in vivo therapeutic effects of multiple PAP-fused cytokines in a mouse prostate cancer model bearing prostate-specific antigen (PSA)- and PAP-expressing tumors. The simultaneous intraperitoneal administration of PAP-GMCSF, -IL2, -IL4 and -IL7 significantly prevented tumor induction and inhibited the tumor growth in the PAP-expressing tumors, yet not in the PSA-expressing tumors. The in vivo therapeutic effects with the multiple PAP-fused cytokines were superior to the effects of PAP-GMCSF alone. We thus demonstrated the advantages of the combined use of multiple PAP-fused cytokines including PAP-GMCSF, and propose a promising prostatic antigen-vaccination strategy to enhance the therapeutic effects.

Potential Factors for the Differentiation of ESCs/iPSCs Into Insulin-Producing Cells.

The low efficiency of in vitro differentiation of human embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs) into insulin-producing cells thus creates a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). In this study, we investigated the key factors for the differentiation of PSCs into insulin-producing cells. We obtained microarray data of HUES8 and HUES6 from two GeneChips (GPL3921: Affymetrix HT Human Genome U133A Array, GPL570: Affymetrix Human Genome U133 Plus 2.0 Array) in a database of GEO (NCBI), since HUES8 can differentiate into pancreatic cells, while HUES6 hardly demonstrates any differentiation at all. The genes with more than fourfold higher expressions in HUES8 compared to HUES6 included RPS4Y1, DDX3Y, EIF1AY, GREM1, GATA6, and NLGN4Y. Since there were four genes, RPS4Y1, DDX3Y, EIF1AY, and NLGN4Y, on the Y chromosome and HUES8 was a male cell line and HUES6 was a female cell line, we excluded these genes in this study. On the other hand, genes with more than fourfold higher expressions in HUES6 compared to HUES8 included NLRP2, EGR1, and SMC3. We next compared iPSCs derived from pancreatic cells (PiPSCs) and iPSCs derived from fibroblasts (FiPSCs). PiPSCs differentiated into insulin-producing cells more easily than FiPSCs because of their epigenetic memory. The gene expressions of GREM1, GATA6, NLRP2, EGR1, and SMC3 in PiPSCs and FiPSCs were also investigated. The expression level of GREM1 and GATA6 in PiPSCs were higher than in FiPSCs. On the other hand, EGR1, which was lower in HUES8 than in HUES6, was predictably lower in PiPSCs than FiPSCs, while NLRP2 and SMC3 were higher in PiPSCs than FiPSCs. These data suggest that the expression of GATA6 and GREM1 and the inhibition of EGR1 may be important factors for the differentiation of PSCs into insulin-producing cells.

Significant association between the Axin2 rs2240308 single nucleotide polymorphism and the incidence of prostate cancer.

The Wnt signaling pathway plays a crucial role in human cancer development, and axis inhibition protein 2 (Axin2) is a master scaffold protein involved in Wnt signaling. Axin2 negatively regulates Wnt signaling and acts as a tumor suppressor protein. The present study evaluated the association between the Axin2 single nucleotide polymorphism (SNP) rs2240308 [guanine (G)/adenine (A)] and the incidence of prostate cancer. In total, 103 patients with prostate cancer and 100 cancer-free control males were included in this case-control study, and were genotyped using the genomic DNA extracted from peripheral blood samples. The results revealed a higher incidence of prostate cancer in the subjects with the homozygous GG genotype and a reduced cancer incidence in the patients with the GA genotype of the rs2240308 SNP (G/A) in the Axin2 gene. The adjusted odds ratio for carriers with the GA genotype was 0.377 (95% CI, 0.206-0.688; P=0.001) and that for the AA genotype was 0.830 (95% CI, 0.309-2.232; P=0.712) compared with the GG genotype. Therefore, the GA genotype was found to exhibit a protective effect that decreased the risk of prostate cancer. To the best of our knowledge, this is the first study to demonstrate the significant association between this SNP (rs2240308, G/A) and the risk of prostate cancer. This association indicates the possibility that the variations in the Axin2 gene in this position may play a significant role in promoting the development of cancer in the prostate. We believe that the Axin2 SNP (rs2240308) could be a useful biomarker for the predisposition and early diagnosis of the disease.

Dramatic increase in expression of a transgene by insertion of promoters downstream of the cargo gene.

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5' located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

A novel gene expression system strongly enhances the anticancer effects of a REIC/Dkk-3-encoding adenoviral vector.

Gene expression systems with various promoters, including the cytomegalovirus (CMV) promoter, have been developed to increase the gene expression in a variety of normal and cancer cells. In particular, in the clinical trials of cancer gene therapy, a more efficient and robust gene expression system is required to achieve sufficient therapeutic outcomes. By inserting the triple translational enhancer sequences of human telomerase reverse transcriptase (hTERT), Simian virus 40 (SV40) and CMV downstream of the sequence of the BGH polyA, we were able to develop a novel gene expression system that significantly enhances the expression of the genes of interest. We termed this novel gene expression cassette the super gene expression (SGE) system, and herein verify the utility of the SGE cassette for a replication-deficient adenoviral vector. We newly developed an adenoviral vector expressing the tumor suppressor, reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), based on the CMV promoter-driven SGE system (Ad-SGE-REIC) and compared the therapeutic utility of Ad-SGE-REIC with that of the conventional adenoviral vectors (Ad-CMV-REIC or Ad-CAG-REIC). The results demonstrated that the CMV promoter-SGE system allows for more potent gene expression, and that the Ad-SGE-REIC is superior to conventional adenoviral systems in terms of the REIC protein expression and therapeutic effects. Since the SGE cassette can be applied for the expression of various therapeutic genes using various vector systems, we believe that this novel system will become an innovative tool in the field of gene expression and gene therapy.

Cancer stem cell-like characteristics of a CD133+ subpopulation in the J82 human bladder cancer cell line.

Cancer stem cells (CSCs) are thought to be crucial for understanding the biological roots of cancer, and are of increasing importance as a target for new anticancer agents. According to an expression analysis of the cell surface antigens of various types of cancer, CD133 is considered to be a potential marker of cancer stemness. In this study, a human urinary bladder cancer cell line (J82) was used to analyze the cancer stem cell-like characteristics of CD133+ bladder cancer cells in vitro and in vivo. The CD133 expression in the J82 cells was examined and the cells were immunomagnetically categorized into positive and negative subsets. The CD133- and CD133+ subsets were phenotypically divergent with regard to the cell growth pattern, while CD133+ cells tended to colonize during their growth. In CD133+ cells, the pluripotent stem cell factors Oct-4 and Sox-2 were upregulated, and a statistically significant proliferation increase was observed when compared to CD133- cells. The CD133+ subpopulation was more tolerant to the chemotherapeutic agent cisplatin, and Bacillus Calmette-Guérin (BCG), an agent instilled intravesically to treat bladder cancer. In addition, CD133+ J82 cells were more resistant to radiation treatment when compared to CD133- cells. The in vivo tumorigenesis of the CD133- and CD133+ subsets of J82 cancer cells was also examined by subcutaneously injecting them into nude mice. The tumor growth was more aggressive in the CD133+ subpopulation, showing a significant difference in the tumorigenic potential in these subsets. In conclusion, J82 human bladder cancer cells include CD133- and CD133+ subpopulations, while the CD133 molecule is a potential marker of the potential malignancy of human bladder cancer. In the present study, the CD133+ subpopulation was herein demonstrated to have certain characteristics consistent with those of cancer stem cells.

Preclinical biodistribution and safety study of reduced expression in immortalized cells/Dickkopf-3-encoding adenoviral vector for prostate cancer gene therapy.

The biodistribution and safety of adenoviral vectors encoding the human REIC/Dkk-3 tumor suppressor gene (Ad-REIC) were examined in this preclinical study for in situ prostate cancer gene therapy. First, the in vitro apoptotic effects of Ad-REIC in normal and cancer cells derived from the prostate and liver were examined. Significant apoptotic effects were observed at 100 MOI (multiplicity of infection) in prostate cancer cells (LNCaP, PC3) and hepatoma cells (HEP3B and HEPG2); however, no effects were seen in normal cells. To analyze the safety of intraprostatic Ad-REIC administration, the biodistribution and histology after Ad-REIC injection were evaluated in various organs of normal male C57BL6 mice. In a supporting study, vector dissemination following intravenous injection of Ad-REIC into tail veins was determined. To evaluate whether Ad-REIC was present in the collected tissue specimens, human REIC gene detection was performed using DNA-PCR. Intraprostatic treatment administered at lower doses showed vector biodistribution into the colon, urinary bladder and prostate. At higher doses, vector dissemination was observed in tissues more distant from the prostate, including the lung, thymus, heart, liver and adrenal gland. After intravenous injection of Ad-REIC, dissemination was observed in the liver and spleen. These results indicate that the biodistribution of Ad-REIC is determined by the dose and route of administration. Although acute inflammatory effects were observed in the prostate after intraprostatic administration at higher doses, no abnormal histological findings were noted in the other tissues, including those of intravenously treated mice. Regarding the safety of Ad-REIC administration, no deaths and no signs of toxicity or unusual behavior were observed in the mice in any treatment group. Based on these preclinical experiments, adenovirus-mediated in situ REIC/Dkk-3 gene therapy is considered to be safe for use as a treatment for human prostate cancer.

A novel gene expression system for detecting viable bladder cancer cells.

A novel transcriptional system was developed that can robustly enhance cancer-specific gene expression. In the system, hTERT promoter-driven gene expression was enhanced by an advanced two-step transcriptional amplification (TSTA). This construct was used to develop a novel system for detection of bladder cancer cells. The current study evaluated the advanced TSTA system by examining the cancer-specific gene transcription in various bladder cancer cell lines. The system significantly enhanced cancer-specific luciferase gene expression in the bladder cancer cell lines in comparison to the previous expression system of one-step or conventional TSTA. The fold gain of the enhancement was significantly correlated to the telomerase activity of the cell lines. A green fluorescent protein (GFP) gene encoding plasmid vector was constructed where hTERT promoter-driving transcription is enhanced by the advanced TSTA to utilize the system for the imaging and detection of viable bladder cancer cells. The advanced TSTA-hTERT-GFP plasmid successfully induced cancer-specific gene expression, showing robust GFP expression in human bladder cancer cell lines, but no visible GFP expression in normal bladder urothelial cells. The control GFP plasmid with a CMV promoter yielded GFP expression in both normal bladder cells and cancer cells. The advanced TSTA-hTERT-GFP plasmid allowed selective visualization of viable human bladder cancer cells in mixed cell culture containing 10- and 100-fold more normal bladder urothelial cells. These findings indicate that the advanced TSTA-hTERT expressional system is a valuable tool for detecting viable bladder cancer cells. The current system can be applied for in vitro detection of bladder cancer cells in urine and other types of cancer cells disseminated in vivo.

Implications of transcriptional factor, OCT-4, in human bladder malignancy and tumor recurrence.

OCT-4, which is also known as POU5f1, is a key regulator of self-renewal in embryonic stem cells. The new cancer stem cell concept proposes that the expression of such genes is potentially correlated with tumorigenesis and can affect some aspects of the cancer behavior, such as recurrence or metastasis. This study investigated the association between OCT-4 expression in cancer tissues obtained by transurethral surgery and the clinical data to clarify the involvement of OCT-4 in human bladder malignancy. Immunohistochemical analysis demonstrated that a positive rate of OCT-4 expression was significantly associated with the higher-grade cancer (G2 and G3) in comparison with that of the lower grade (G1). In addition, positive OCT-4 expression was significantly associated with the intra-bladder tumor recurrence after the operation. The staining intensity of OCT-4 expression was also correlated with tumor recurrence. These data indicate that positive OCT-4 expression may be involved in the development of high-grade bladder cancer and with the bladder cancer recurrence. This is the first study showing a correlation between the expression of OCT-4 and bladder cancer recurrence. OCT-4 may be a valuable clinical marker for the progression of bladder cancer and may be an attractive therapeutic target for the development of new medicines for the treatment of malignancy.