Yoshinobu Toda

Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer

The mainstay in the management of invasive bladder cancer continues to be radical cystectomy. With regard to improvement of quality of life, however, therapies that preserve the bladder are desirable. We investigated the use of intravesical PLK-1 small interfering RNA (siRNA) against bladder cancer. Patients with bladder cancers expressing high levels of PLK-1 have a poor prognosis compared with patients with low expression. Using siRNA/cationic liposomes, the expression of endogenous PLK-1 could be suppressed in bladder cancer cells in a time- and dose-dependent manner. As a consequence, PLK-1 functions were disrupted. Inhibition of bipolar spindle formation, accumulation of cyclin B1, reduced cell proliferation, and induction of apoptosis were observed. In order to determine the efficacy of the siRNA/liposomes in vivo, we established an orthotopic mouse model using a LUC-labeled bladder cancer cell line, UM-UC-3(LUC). PLK-1 siRNA was successfully transfected into the cells, reduced PLK-1 expression, and prevented the growth of bladder cancer in this mouse model. This is the first demonstration, to our knowledge, of inhibition of cancer growth in the murine bladder by intravesical siRNA/cationic liposomes. We believe intravesical siRNA instillation against bladder cancer will be useful as a therapeutic tool.

GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion

Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.

Cyclin-D1-gene amplification is a more potent prognostic factor than its protein over-expression in human head-and-neck squamous-cell carcinoma.

To evaluate the prognostic significance of cyclin D1 protein/gene expressions in human head‐and‐neck squamous‐cell carcinoma (HNSCC), we examined amplification of the cyclin‐D1 gene (CCND1) by the differential PCR method and over‐expression of cyclin‐D1 protein by immunohistochemistry in 45 paraffin‐embedded sections from HNSCC. Amplification of CCND1 was found in 10 (22%) cases and over‐expression of cyclin D1 was found in 24 (53%) cases. CCND1 amplification was also found in 3 (25%) of 12 cases of dysplastic lesions adjacent to HNSCC. The overall 5‐year survival of patients with CCND1 amplification or with protein over‐production was significantly lower than that of patients without (p < 0.0001 and p < 0.05, respectively). However, with multivariate analysis, only amplification of CCND1 retained an independent prognostic value (p = 0.0018). These suggest that CCND1 amplification occurs at early stages of HNSCC tumorigenesis and is a more useful prognostic factor than over‐expression of cyclin D1 in HNSCC. Int. J. Cancer 74:576–581, 1997.© 1997 Wiley‐Liss, Inc.

Application of tyramide signal amplification system to immunohistochemistry : A potent method to localize antigens that are not detectable by ordinary method

Tyramide signal amplification–avidin–biotin complex (TSA‐ABC) method is a powerful technique used to detect antigens that are not detectable by ordinary immunohistochemistry. It is worth trying in cases where localization of antigens by the conventional method has failed and antibodies are precious.

Identification of EP4 as a Potential Target for the Treatment of Castration-Resistant Prostate Cancer Using a Novel Xenograft Model

More effective therapeutic approaches for castration-resistant prostate cancer (CRPC) are urgently needed, thus reinforcing the need to understand how prostate tumors progress to castration resistance. We have established a novel mouse xenograft model of prostate cancer, KUCaP-2, which expresses the wild-type androgen receptor (AR) and which produces the prostate-specific antigen (PSA). In this model, tumors regress soon after castration, but then reproducibly restore their ability to proliferate after 1 to 2 months without AR mutation, mimicking the clinical behavior of CRPC. In the present study, we used this model to identify novel therapeutic targets for CRPC. Evaluating tumor tissues at various stages by gene expression profiling, we discovered that the prostaglandin E receptor EP4 subtype (EP4) was significantly upregulated during progression to castration resistance. Immunohistochemical results of human prostate cancer tissues confirmed that EP4 expression was higher in CRPC compared with hormone-naïve prostate cancer. Ectopic overexpression of EP4 in LNCaP cells (LNCaP-EP4 cells) drove proliferation and PSA production in the absence of androgen supplementation in vitro and in vivo. Androgen-independent proliferation of LNCaP-EP4 cells was suppressed when AR expression was attenuated by RNA interference. Treatment of LNCaP-EP4 cells with a specific EP4 antagonist, ONO-AE3-208, decreased intracellular cyclic AMP levels, suppressed PSA production in vitro, and inhibited castration-resistant growth of LNCaP-EP4 or KUCaP-2 tumors in vivo. Our findings reveal that EP4 overexpression, via AR activation, supports an important mechanism for castration-resistant progression of prostate cancer. Furthermore, they prompt further evaluation of EP4 antagonists as a novel therapeutic modality to treat CRPC.

Impairment of bone formation with aluminum and ferric nitrilotriacetate complexes

SummaryThe deleterious effects of aluminum(Al) and iron(Fe) on bone formation were studied in the presence of nitrilotriacetate (NTA) as a chelator. Both Al-NTA (1.0–1.5 mg Al/kg/day, n=12)- and ferric nitrilotriacetate (Fe-NTA) (2.0 mg/kg/day, n=4)—treated Wistar rats showed renal insufficiency blood urea nitrogen [BUN] levels of 25±8.8−20±0.7 compared to 12±0.7–11±0.4 mg/dl), osteomalacia with a relative osteoid volume of 31.5±5.6−13.2±2.4 compared to 4.6±1.8−0.83±0.12%, and bone growth retardation (3.1±0−3.0±0.2 compared to 3.4±0−3.3±0.1 cm) in 24 control rats. Dietary vitamin E(VE) supplementation prevented the Fe-NTA-induced impairment, but not the Al-NTA toxicity. Aluminum was deposited at the interface between osteoid and mineralized bone, while Fe was deposited in the osteoblasts and osteoclasts. There seems to be a positive correlation between hypophosphatemia and osteomalacia but carboxy-terminal parathyroid hormone (C-PTH) and calcium (Ca) levels in the serum were not related to the degree of osteomalacia. Administration of Al-NTA results in more bone Al deposition than that of aluminum chloride (AlCl3) (450±40 compared to 211±18 mg/kg fat-free dry weight). The Fe-NTA bone change is related to VE-preventable cellular injury, being consistent with the notion that Fe-NTA toxicity is caused by lipid peroxidation. Al-NTA can be used as an animal model of renal osteodystrophy. Osteodystrophy by Al in chronic renal failure may be mediated by the intrinsic chelator or chelating substance(s) retained in the body fluid due to renal insufficiency.

Microsatellite instability and alternative genetic pathway in intrahepatic cholangiocarcinoma

BACKGROUND/AIMS Intrahepatic cholangiocarcinoma (ICC) arises from intrahepatic bile duct epithelium and is the second most prevalent among primary liver cancers. The aim of this study was to clarify the mechanism of cholangiocarcinogenesis. METHODS We studied the incidence of microsatellite instability (MSI) involving eight highly polymorphic microsatellite markers and alternations of the K-ras, p53 and mdm-2 genes in human ICC tissues. Overexpression of mdm-2 oncoprotein was also immunohistochemically studied. RESULTS Of all 65 cases examined, K-ras gene mutation was found in three cases (4.6%) at codon 12. Analysis of p53 alterations was performed in 28 cases including 22 frozen samples and mutations were found in three cases (10.7%). Overexpression of mdm-2 protein was observed in 25 (41.7%) out of 60 cases analyzed. In 22 frozen samples, seven (31.8%) cases showed mdm-2 amplification and four (18.2%) cases revealed MSI-positive phenotype. Among the cases analyzed, all the tumors with mdm-2 amplification/overexpression harbored the wild-type p53 gene and all the microsatellite instability-positive cases were from mass-forming (MF) + periductal-infiltrating (PI) subtype. CONCLUSIONS These results suggest that mdm-2 plays a role, which might be partially through inhibiting p53 activity, in cholangiocarcinogenesis and that M

Longitudinal and Simultaneous Imaging of Retinal Ganglion Cells and Inner Retinal Layers in a Mouse Model of Glaucoma Induced by N-Methyl-D-Aspartate

PURPOSE To investigate the longitudinal profile of N-methyl-D-aspartate (NMDA) injection-induced damage in retinal ganglion cells (RGCs) by imaging retinal Thy 1-cyan fluorescent protein (CFP) expression and inner retinal layers using a custom-made imaging device containing short-wavelength confocal scanning laser ophthalmoscope (scSLO) and speckle noise-reduced spectral-domain optical coherence tomography (SD-OCT). METHODS Simultaneous scSLO and SD-OCT examinations were performed in Thy 1-CFP mice injected with NMDA (1-20 nanomoles). CFP-expressing RGCs were counted using scSLO images. Ganglion cell complex (GCC: retinal nerve fiber layer, ganglion cell layer, and inner plexiform layer) thickness around the optic disc was measured in SD-OCT images. RESULTS The RGCs rapidly decreased 1 day after NMDA injection in a dose-dependent manner (65.3%, 71.7%, 49.5%, and 27.1% of the preinjection level, 2, 5, 10, and 20 nanomoles, respectively) and continued to decrease slightly (to 53.7%, 44.1%, 28.3%, and 20.2% of the preinjection level on days 14, 2, 5, 10, and 20 nanomoles, respectively). In contrast, dose-dependent reduction of GCC thickness was first detected 4 days after injection. The thickness further decreased to 84.6%, 75.7%, 76.5%, and 71.4% of the preinjection level on day 14 (2, 5, 10, and 20 nanomoles, respectively). CONCLUSIONS NMDA-induced RGC damage is characterized by rapid RGCs loss followed by gradual reduction in GCC thickness. Simultaneous imaging of CFP expression in the RGCs and inner retinal layers provides a sensitive, reliable, and new method for longitudinal evaluation of progressive RGC damage in experimental models of glaucoma.

GEP100-Arf6-AMAP1-Cortactin Pathway Frequently Used in Cancer Invasion Is Activated by VEGFR2 to Promote Angiogenesis

Angiogenesis and cancer invasiveness greatly contribute to cancer malignancy. Arf6 and its effector, AMAP1, are frequently overexpressed in breast cancer, and constitute a central pathway to induce the invasion and metastasis. In this pathway, Arf6 is activated by EGFR via GEP100. Arf6 is highly expressed also in human umbilical vein endothelial cells (HUVECs) and is implicated in angiogenesis. Here, we found that HUVECs also highly express AMAP1, and that vascular endothelial growth factor receptor-2 (VEGFR2) recruits GEP100 to activate Arf6. AMAP1 functions by binding to cortactin in cancer invasion and metastasis. We demonstrate that the same GEP100-Arf6-AMAP1-cortactin pathway is essential for angiogenesis activities, including cell migration and tubular formation, as well as for the enhancement of cell permeability and VE-cadherin endocytosis of VEGF-stimulated HUVECs. Components of this pathway are highly expressed in pathologic angiogenesis, and blocking of this pathway effectively inhibits VEGF- or tumor-induced angiogenesis and choroidal neovascularization. The GEP100-Arf6-AMAP1-cortactin pathway, activated by receptor tyrosine kinases, appears to be common in angiogenesis and cancer invasion and metastasis, and provides their new therapeutic targets.

Novel Roles of Hakai in Cell Proliferation and Oncogenesis

During tumor development, cells acquire multiple phenotypic changes upon misregulation of oncoproteins and tumor suppressor proteins. Hakai was originally identified as an E3 ubiquitin-ligase for the E-cadherin complex that regulates cell-cell contacts. Here, we present evidence that Hakai plays a crucial role in various cellular processes and tumorigenesis. Overexpression of Hakai affects not only cell-cell contacts but also proliferation in both epithelial and fibroblast cells. Furthermore, the knockdown of Hakai significantly suppresses proliferation of transformed epithelial cells. Expression of Hakai is correlated to the proliferation rate in human tissues and is highly up-regulated in human colon and gastric adenocarcinomas. Moreover, we identify PTB-associated splicing factor (PSF), an RNA-binding protein, as a novel Hakai-interacting protein. By using cDNA arrays, we have determined various specific PSF-associated mRNAs encoding proteins that are involved in several cancer-related processes. Hakai affects the ability of PSF to bind these mRNAs, and expression of PSF short hairpin RNA or a dominant-negative PSF mutant significantly suppresses proliferation of Hakai-overexpressing cells. Collectively, these results suggest that Hakai is an important regulator of cell proliferation and that Hakai may be an oncoprotein and a potential molecular target for cancer treatment.