Kazunori Aoki

Downregulation and growth inhibitory effect of epithelial-type Krüppel-like transcription factor KLF4, but not KLF5, in bladder cancer.

Krüppel-like factors (KLFs) are key transcriptional regulators of cell differentiation and proliferation. Among the KLF family, the expression of KLF4 (GKLF) and KLF5 (IKLF) is highly restricted in the epithelial cells of several organs such as the gut and skin, and it has been reported that these epithelial-type KLF genes may be involved in colon carcinogenesis. Recently we found that Klf4 and Klf5 genes were significantly expressed in the developmental bladder epithelium of mice as well. Therefore, in this report we studied the involvement of the KLF4 and KLF5 genes in bladder carcinogenesis. First, we analyzed the expression of KLF4 and KLF5 in a variety of human bladder cancer cell lines and surgical specimens by RNA blot and in situ hybridization analyses. Both genes were highly expressed in the normal bladder epithelium, whereas KLF4, but not KLF5, was frequently downregulated in bladder cancer cell lines and cancer tissues. We then transduced the KLF4 and KLF5 genes into the bladder cancer cell lines using adenoviral vectors to examine the biological activities of the genes on those cells. The transduction of KLF4, but not KLF5, suppressed cell growth and induced apoptosis. Our study suggests that inactivation of KLF4 is one of the frequent steps towards bladder carcinogenesis.

Polyethylenimine-mediated gene transfer into pancreatic tumor dissemination in the murine peritoneal cavity

Although peritoneal dissemination of cancer cells often occurs at the advanced stages of pancreatic, gastric or ovarian cancers, no effective therapy has been established. Cationic lipid-mediated gene transfer into peritoneal dissemination may offer a prospect of safe therapies, but vector improvements are needed with regard to the efficiency and specificity of the gene transfer. In this study, the intraperitoneal injection of plasmid DNA:polyethylenimine (PEI) complexes into mice was evaluated as a gene delivery system for the peritoneal disseminations. The luciferase and β-galactosidase genes were used as marker genes. PEI was more efficient than the cationic lipids examined in this study in vivo, and the transgene was preferentially expressed in the tumors. Although PCR analysis showed that the injected DNA was delivered to various organs, the distributed DNA became undetectable by 6 months after the gene transfer. Blood chemistry and histological analysis showed no significant toxicity in the injected mice. This study demonstrated that the intraperitoneal injection of DNA:PEI is a promising delivery method to transduce a gene into disseminated cancer nodules in the peritoneal cavity.

Extracellular matrix interacts with soluble CD95L: Retention and enhancement of cytotoxicity

Fas ligand (CD95L) is synthesized both on the cell surface membrane and in a soluble form. Although CD95L contributes to immune privilege in the cornea and testis, the functions of these alternatively processed proteins are not well understood. Some reports suggest that the cytotoxicity of soluble CD95L is insignificant, whereas others show potent responses in vivo, including hepatocyte apoptosis that causes liver failure. We show here that extracellular matrix proteins interact with soluble CD95L and potentiate its pro-apoptotic activity. The cytotoxicity of supernatants from CD95L-expressing cells was increased by incubation on tissue culture plates coated with these matrix proteins; this effect was mediated by trimeric soluble CD95L. With the use of immunoprecipitation, it was found that CD95L binds directly to fibronectin. In addition, immunohistochemical analysis of the cornea revealed that soluble CD95L binds primarily to extracellular matrix. The retention of soluble CD95L on extracellular matrices is likely to play an important role in the development of peripheral tolerance in immune-privileged sites.

Suppression of Ki-ras p21 levels leading to growth inhibition of pancreatic cancer cell lines with Ki-ras mutation but not those without Ki-ras mutation.

Ki‐ras point mutation characteristically occurs frequently in human pancreatic cancer. To clarify the effect of antisense Ki‐ras RNA on various pancreatic cancer cell lines, a plasmid expressing an antisense Ki‐ras gene fragment (AS‐K‐ras‐LNSX) was transduced into seven human pancreatic cancer cell lines (AsPC‐1, MIA PaCa‐2, PANC‐1, PSN‐1, BxPC‐3, Hs 700T, and Hs 766T) by liposome‐mediated transfection. Western blot analysis showed that transfection of AS‐K‐ras‐LNSX led to a significant reduction in the amounts of Ki‐ras p21 protein in all the pancreatic cancer cell lines except BxPC‐3. The growth of pancreatic cancer cell lines having Ki‐ras point mutations (AsPC‐1, MIA PaCa‐2, PANC‐1, and PSN‐1) was suppressed after transduction of AS‐K‐ras‐LNSX, whereas the effect of the antisense construct on the growth was not significant in cell lines with a wild‐type Ki‐ras gene (BxPC‐3, Hs 700T, and Hs 766T). These results suggest that the pancreatic cancer cells with activated Ki‐ras depend heavily on a Ki‐ras p21–mediated growth signal pathway for their growth because they were far more susceptible to the suppression of the Ki‐ras p21 protein than the cells with wild‐type Ki‐ras. The remarkably increased dependence of the cancer‐cell growth circuitry on one or a few crucial regulatory molecules may thus be a common feature of the cancer cells and implies a novel rationale for the targeting of cancer therapy. Mol. Carcinog. 20:251–258, 1997.

A Phase 2 Study of Cisplatin in Patients with Hepatocellular Carcinoma

A phase 2 study of cisplatin was performed in 28 previously untreated patients with unresectable hepatocellular carcinoma. The drug was given intravenously at a dose of 80 mg/m2/day every 4 weeks. Of 26 patients evaluated, 4 (15.4%) showed partial responses lasting for > 3 months, while no patient achieved a complete response. Of 22 patients whose serum level of alpha-fetoprotein (AFP) was high (> 400 ng/ml) before treatment, 6 (27.3%) showed a > 50% reduction in serum AFP levels after treatment. The current study indicates that cisplatin is an anticancer agent worthy of further testing in patients with this disease.

Cloning of human bone morphogenetic protein type IB receptor (BMPR- IB) and its expression in prostate cancer in comparison with other BMPRs

Bone metastasis is a common event in prostate cancer, and it is known that some of the bone morphogenetic proteins (BMPs) are expressed in prostate cancer cells, while no study on the expression of their receptors, BMPRs, has been reported. Here we report cloning and sequence analysis of the human BMPR-IB cDNA. We also analysed the expression of transcripts of three types of the BMPR genes in human tissues and prostate cancer cell lines. The BMPR-IB mRNA was present in various organs, but the highest level was found in the prostate. Moreover, the amount of BMPR-IB mRNA was significantly low in prostate cancer tissues after androgen withdrawal and was also low in prostate cancer cell lines. RT - PCR analysis showed that the BMPR-IB message was upregulated by androgen stimulation in the LNCaP cell line which expresses the androgen receptor. By contrast, the mRNA levels of BMPR-IA and BMPR-II were not significantly different among non-cancerous and cancerous prostate tissues. It was also suggested that human BMPR-IA and BMPR-IB might have different biological functions in the prostate, although their sequences were 85.3% identical in the serine-threonine kinase domain.

Prognostic factors in patients with advanced pancreatic cancer treated with systemic chemotherapy

The clinical features of 65 patients with advanced pancreatic cancer treated between 1984 and 1993 were analyzed retrospectively to identify the significant prognostic factors. All the patients had presented unresectable or metastatic disease on imaging diagnostic evaluation and had received systemic chemotherapy. The overall median survival time and 1-year survival rate were 3.9 months and 9.8%, respectively. The independent favorable prognostic factors identified by multivariate analysis using the Cox proportional hazards model were a performance status of 0–1, a serum carcinoernbryonic antigen level of < 10 ng/ml, and an absence of distant metastasis. A prognostic index calculated from the regression coefficients for these three factors was used to classify the patients into three groups, with good, intermediate, and poor prognoses. The median survival time for these three groups was 7.4, 3.5, and 2.0 months, respectively (p < 0.001). The results of this study may be useful in the design and analysis of future clinical trials of systemic chemotherapy for advanced pancreatic cancer.

A Phase II Study of Cisplatin in Patients with Biliary Tract Carcinoma

A phase II study of cisplatin was performed in 13 previously untreated patients with unresectable biliary tract carcinoma. The drug was given intravenously at a dose of 80 mg/m2/day once every 4 weeks. Of 13 patients evaluated, 1 showed partial response lasting 3 months, while no patients showed complete response. Of 9 patients, whose serum level of carcinoembryonic antigen (CEA) was high (> or = 10 ng/ml) before treatment, 4 showed > or = 50% reduction in serum CEA level after treatment. The current study indicates that cisplatin does not have significant antitumor activity against biliary tract carcinoma.

Development of gene therapy to target pancreatic cancer

Pancreatic cancer remains one of the most difficult cancers to treat. Its high propensity to infiltrate and metastasize early from a small primary focus necessitates development of a new therapy which can track down the disseminated cancer cells in vivo. Gene therapy may offer new opportunities for a variety of targeting strategies, and we review here some of our work related to the development of targeted gene therapy: 1) Targeting by specific molecular abnormality: Many pancreatic cancer cells show “addiction” to K‐as mutation, while normal cells appear resistant to suppression of K‐ras‐mediated signaling by antisense K‐ras RNA expression adenoviral vector. 2) Targeting by in vivo tumor characteristics: In a peritoneal dissemination model, intraperitoneal lipofection/polyfection can deliver and express transgenes highly preferentially in tumor nodules. 3) Targeting by vector: An efficient protocol for construction of an adenovirus expression vector library has been developed, which will enable a direct functional selection of fiber knob‐modified targeting vector species for given cells. 4) Targeting by tumor immunity: Several cytokines not only induce direct cytotoxicity, but are also expected to activate specific immunity to achieve targeted suppression of cancer cells in vivo. Unlike parenteral administration of short‐lived recombinant interferon protein, local interferon gene transfer can provide a target tissue‐restricted distribution and sustained expression, which may improve the efficacy/safety balance of cytokine therapy. Cancer gene therapy development is, in general, at the stage of proof of principles and safety. However, it is an art of integrated science. The recent rapid progress of related sciences and technologies will expand the potential and consolidate the clinical reality of gene therapy.

Proinflammatory Proteins S100A8/S100A9 Activate NK Cells via Interaction with RAGE

S100A8/A9, a proinflammatory protein, is upregulated in inflammatory diseases, and also has a tumor-promoting activity by the recruitment of myeloid cells and tumor cell invasion. However, whether the expression of S100A8/A9 in tumors predicts a good or poor prognosis is controversial in the clinical setting. In this study, to clarify the in vivo role of S100A8/A9 in the tumor microenvironment, we s.c. inoculated Pan02 cells stably expressing S100A8 and S100A9 proteins (Pan02-S100A8/A9) in syngeneic C57BL/6 mice. Unexpectedly, after small tumor nodules were once established, they rapidly disappeared. Flow cytometry showed that the number of NK cells in the tumors was increased, and an administration of anti-asialoGM1 Ab for NK cell depletion promoted the growth of Pan02-S100A8/A9 s.c. tumors. Although the S100A8/A9 proteins alone did not change the IFN-γ expression of NK cells in vitro, a coculture with Pan02 cells, which express Rae-1, induced IFN-γ production, and Pan02-S100A8/A9 cells further increased the number of IFN-γ+ NK cells, suggesting that S100A8/A9 enhanced the NK group 2D ligand-mediated intracellular activation pathway in NK cells. We then examined whether NK cell activation by S100A8/A9 was via their binding to receptor of advanced glycation end product (RAGE) by using the inhibitors. RAGE antagonistic peptide and anti-RAGE Ab inhibited the IFN-γ production of NK cells induced by S100A8/A9 proteins, and an administration of FPS-ZM1, a RAGE inhibitor, significantly enhanced the in vivo growth of Pan02-S100A8/A9 tumors. We thus found a novel activation mechanism of NK cells via S100A8/A9–RAGE signaling, which may open a novel perspective on the in vivo interaction between inflammation and innate immunity.