Takuya Egami

S100P Is an Early Developmental Marker of Pancreatic Carcinogenesis

Purpose: Our goal was to clarify the involvement and clinical significance of S100P in pancreatic carcinogenesis. Experimental Design: We examined S100P expression in 45 bulk pancreatic tissues; in microdissected cells, including invasive ductal carcinoma (IDC) cells (20 sections), pancreatic intraepithelial neoplasia (PanIN) cells (12 sections), intraductal papillary mucinous neoplasm (IPMN) cells (19 sections), and normal epithelial cells (11 sections); and in pancreatic juice samples from 99 patients with pancreatic diseases (32 cancer, 35 IPMN, and 32 chronic pancreatitis samples). We used quantitative real-time reverse transcription-PCR with gene-specific priming to measure S100P in these various types of samples. Results: In bulk tissue analyses, pancreatic cancer and IPMN expressed significantly higher levels of S100P than did nonneoplastic pancreas (P < 0.017 and P = 0.0013, respectively). Microdissection analyses revealed that IPMN expressed significantly higher levels of S100P than did IDC (P < 0.0001) and PanIN (P = 0.0031), although S100P expression did not differ between IDC and PanIN (P = 0.077). In pancreatic juice analyses, cancer and IPMN juice expressed significantly higher levels of S100P than did pancreatitis juice (both P < 0.0001). Receiver operating characteristic curve analyses revealed that measurement of S100P in pancreatic juice was useful for discriminating neoplastic disease from chronic pancreatitis (area under the curve = 0.837; 95% confidence interval, 0.749-0.903). Conclusion:S100P may be an early developmental marker of pancreatic carcinogenesis, and measurement of S100P in pancreatic juice may be useful for early detection of pancreatic cancer or screening of early pancreatic carcinogenesis.

Tumor–stromal interactions with direct cell contacts enhance proliferation of human pancreatic carcinoma cells

Pancreatic ductal adenocarcinoma is often characterized by an abundant desmoplastic stroma that is partially induced by activated pancreatic stellate cells (PSCs). Indirect co‐culture has often been used to investigate the effects of cancer–stromal interactions on the proliferation of cancer cells, but the effects of cell–cell adhesion and juxtacrine signaling between cancer and stromal cells cannot be evaluated using this method. This study aimed to establish a simplified direct co‐culture system that could be used to quantify populations of cancer cells in co‐culture with PSCs, and to evaluate the effects of direct cell contact on the proliferation of cancer cells. We established three green fluorescent protein (GFP)‐expressing pancreatic cancer cell lines and were able to quantify them with high reliability and reproducibility, even when co‐cultured directly with PSCs, using a color plate reader. We assessed the differential effects of direct and indirect co‐culture with PSCs on the proliferation of cancer cells, and found that the proliferation of GFP‐expressing pancreatic cancer cell lines was dramatically enhanced by direct co‐culture with PSCs, compared with the indirect co‐culture system. We also found that direct co‐culture of cancer cells and PSCs activated the Notch signaling pathway in both cell types. Direct cell contact between cancer cells and PSCs plays an important role in the control of cancer cell proliferation, and is essential to the understanding of tumor–stromal interactions. (Cancer Sci 2009; 100: 2309–2317)

Radiation Enhances Adenoviral Gene Therapy in Pancreatic Cancer via Activation of Cytomegalovirus Promoter and Increased Adenovirus Uptake

Purpose: Adenovirus-mediated gene therapy combined with radiation is expected to be a new approach to treat pancreatic cancer. However, there are no reports of definitive effects of radiation on adenovirus-mediated gene therapies. In the present study, we investigated the effect of radiation on the transduction efficiency of an adenovirus-based gene therapy. Experimental Design: We used adenovirus expressing NK4 (Ad-NK4), an antagonist for hepatocyte growth factor, as a representative gene therapy. Pancreatic cancer cells preinfected with Ad-NK4 were irradiated, and NK4 levels in culture media of these cells were measured. We investigated cytomegalovirus (CMV) promoter activity and uptake of adenovirus in these cells. To examine the effect of radiation in vivo, Ad-NK4 was given to irradiated subcutaneous tumors in nude mice, and NK4 levels in tumors were measured. Results: NK4 levels in culture media of irradiated cells were 4.5-fold (P < 0.01) higher than those of nonirradiated cells. Radiation enhanced activation of the CMV promoter and adenovirus uptake (P < 0.01), leading to increased levels of NK4. We found that activation of p38 mitogen-activated protein kinase and up-regulation of dynamin 2 may be involved in the radiation-induced activation of the CMV promoter and adenovirus uptake, respectively. NK4 levels in irradiated tumors were 5.8-fold (P = 0.017) higher than those in nonirradiated tumors. Conclusions: The present findings suggest that radiation significantly improves the efficiency of adenovirus-mediated gene transfer in pancreatic cancer and probably contributes to decreasing the dose of adenovirus required for gene transfer and controlling side effects of adenovirus infection in nonirradiated normal tissue.

LIM only 4 is overexpressed in late stage pancreas cancer

BackgroundLIM-only 4 (LMO4), a member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors, was initially reported to have an oncogenic role in breast cancer. We hypothesized that LMO4 may be related to pancreatic carcinogenesis as it is in breast carcinogenesis. If so, this could result in a better understanding of tumorigenesis in pancreatic cancer.MethodsWe measured LMO4 mRNA levels in cultured cells, pancreatic bulk tissues and microdissected target cells (normal ductal cells; pancreatic intraepithelial neoplasia-1B [PanIN-1B] cells; PanIN-2 cells; invasive ductal carcinoma [IDC] cells; intraductal papillary-mucinous adenoma [IPMA] cells; IPM borderline [IPMB] cells; and invasive and non-invasive IPM carcinoma [IPMC]) by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR).Results9 of 14 pancreatic cancer cell lines expressed higher levels of LMO4 mRNA than did the human pancreatic ductal epithelial cell line (HPDE). In bulk tissue samples, expression of LMO4 was higher in pancreatic carcinoma than in intraductal papillary-mucinous neoplasm (IPMN) or non-neoplastic pancreas (p < 0.0001 for both). We carried out microdissection-based analyses. IDC cells expressed significantly higher levels of LMO4 than did normal ductal epithelia or PanIN-1B cells (p < 0.001 for both) or PanIN-2 cells (p = 0.014). IPMC cells expressed significantly higher levels of LMO4 than did normal ductal epithelia (p < 0.001), IPMA (p < 0.001) and IPMB cells (p = 0.003).ConclusionPancreatic carcinomas (both IDC and IPMC) expressed significantly higher levels of LMO4 mRNA than did normal ductal epithelia, PanIN-1B, PanIN-2, IPMA and IPMB. These results suggested that LMO4 is overexpressed at late stages in carcinogenesis of pancreatic cancer.

Combination with low-dose gemcitabine and hTERT-promoter-dependent conditionally replicative adenovirus enhances cytotoxicity through their crosstalk mechanisms in pancreatic cancer

To overcome the limited clinical efficacy of conditionally replicative adenoviruses (CRAds), we investigated the effects of combination therapy with gemcitabine (GEM) and the hTERT-promoter-dependent CRAd (hTERT-CRAd), Ad5/3hTERTE1. This combination therapy exhibited enhanced cytotoxic effects on pancreatic cancer both in vitro and in vivo. Furthermore, we revealed that this enhancement effect was due to the multiple bidirectional interactions between hTERT-CRAd and GEM. The GEM-sensitizing effect of E1 expression derived from hTERT-CRAd, and the enhancement effect by GEM on hTERT promoter activity which led to the increase of adenovirus E1 and viral infectivity. This combination therapy may be a promising therapeutic approach for pancreatic cancer.

Chemotherapeutic agents potentiate adenoviral gene therapy for pancreatic cancer

Adenovirus‐mediated gene therapy combined with chemotherapeutic agents is expected to represent a new approach for treating pancreatic cancer. However, there have been no reports of definitive effects of chemotherapeutic agents on adenovirus‐mediated gene therapies. In the present study, we investigated the effects of chemotherapeutic agents on the transduction efficiency of an adenovirus‐based gene therapy. Adenovirus (Ad‐NK4) expressing NK4, which acts as a hepatocyte growth factor antagonist, was used as a representative gene therapy. Pancreatic cancer cells infected with Ad‐NK4 were treated with chemotherapeutic agents (5‐fluorouracil [5FU], cisplatin or etoposide), and the NK4 levels in their culture media were measured. To examine the effects of chemotherapeutic agents in vivo, Ad‐NK4 was administered to subcutaneous tumors in mice after treatment with the agents, and the tumor NK4 levels were measured. The NK4 levels in culture media from cells treated with 5FU, cisplatin and etoposide were 5.2‐fold (P = 0.026), 6‐fold (P < 0.001) and 4.3‐fold (P < 0.001) higher than those of untreated cells, respectively. The chemotherapeutic agents also increased Ad‐NK4 uptake. The NK4 levels in tumors treated with 5FU, cisplatin and etoposide were 5.4‐fold (P = 0.006), 11.8‐fold (P < 0.001) and 4.9‐fold (P = 0.017) higher than those in untreated tumors, respectively. The present findings suggest that chemotherapeutic agents significantly improve the efficiency of adenovirus‐mediated gene transfer in pancreatic cancer. Furthermore, they will contribute to decreases in the adenovirus doses required for gene transfer, thereby controlling the side‐effects of adenovirus infection in normal tissues. (Cancer Sci 2009; 100: 722–729)

Quantitative analysis of hTERT mRNA levels in cells microdissected from cytological specimens

Clinicians frequently require cytopathological assessment of tumor samples for preoperative diagnosis, but in some specimens, diagnosis remains inconclusive after cytological examination. To date, several molecular markers, including human telomerase reverse transcriptase (hTERT), have been assessed for the ability to detect malignancy. However, analyses using whole cytological specimens are generally affected by contamination of untargeted cells. The present study investigated the feasibility of more sensitive examination by quantitative mRNA analysis of target cells microdissected from cytological specimens. Laser capture microdissection (LCM) was used to obtain target cells from cytological specimens. hTERT mRNA levels were then measured in target cells by quantitative real‐time RT‐PCR (qRT‐PCR). The effect of RNA fragmentation on qRT‐PCR was also assessed. Total RNA from cytological specimens was sometimes fragmented to a large degree. To avoid the effect of RNA fragmentation, gene specific priming and PCR primers generating short PCR products were used and no difference in delta Ct values between fragmented and non‐fragmented RNA were found. hTERT mRNA levels were measured in cells microdissected from 33 cytological specimens. The levels of hTERT mRNA were significantly higher in malignant cases compared to those in non‐malignant cases (P = 0.0003). The sensitivity was 96.2%, even when the specificities were 100%. High levels of hTERT mRNA were also found in three cases that were not diagnosed as malignant by cytological examination. Quantitative assessment of hTERT mRNA levels in cells microdissected from cytological specimens is a potential diagnostic tool to potentiate cytological examination in diagnosing malignancy. (Cancer Sci 2008; 99: 2244–2251)

Gemcitabine synergistically enhances the effect of adenovirus gene therapy through activation of the CMV promoter in pancreatic cancer cells

Adenovirus-mediated gene therapy shows remarkable promise as a new strategy for advanced pancreatic cancer, but satisfactory clinical results have not yet been obtained. To improve this gene therapy, we investigated the effects of gemcitabine (GEM) on transgene expression by adenoviral vectors and their biological effects. We used Ad-lacZ and adenoviral vector-expressing NK4 (Ad-NK4) as representative adenoviral vectors. These vectors express β-galactosidase (β-gal) and NK4 (which inhibits the invasion of cancer cells), respectively, under the control of the CMV promoter. Cells were infected with the individual adenoviruses and then treated with GEM. GEM increased β-gal mRNA expression and β-gal activity, and increased NK4 expression in both culture media and within infected cells, in dose-dependent manners. The increased expression of NK4 delivered by Ad-NK4 had biological effects by inhibiting the invasion of cancer cells. GEM also enhanced NK4 expression in SUIT-2 cells transfected with an NK4-expressing plasmid, suggesting that GEM enhanced CMV promoter activity. In in vivo experiments, NK4 expression within subcutaneously implanted tumors was increased in GEM-treated mice compared with control mice. These results suggest that adenovirus-mediated gene therapy with GEM may be a promising approach for treating pancreatic cancer, and that this combination therapy may decrease the risks of side effects.

Tumor–stroma interactions reduce the efficacy of adenoviral therapy through the HGF-MET pathway

Many preclinical studies have shown the potential of adenovirus‐based cancer gene therapy. However, successful translation of these promising results into the clinic has not yet been achieved. Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant desmoplastic stroma, and tumor–stromal cell interactions play a critical role in tumor progression. Therefore, we hypothesized that tumor–stroma interactions reduce the efficacy of adenoviral therapy. We investigated the effect of fibroblasts on adenovirus‐based gene therapy using SUIT‐2 and PANC‐1 pancreatic cancer cells cultured with or without fibroblast‐conditioned culture supernatant then infected with Ad‐LacZ. After 48 h, the cells were stained for β‐galactosidase. The results showed that the number of β‐galactosidase‐positive cells was significantly reduced after culture with fibroblast‐conditioned supernatant (P < 0.05). Because the hepatocyte growth factor (HGF)/MET pathway plays an important role in tumor–stroma interactions we next investigated the involvement of this pathway in tumor–stroma interactions leading to the decreased efficacy of adenoviral therapy. SUIT‐2 cells were cultured with or without SU11274 (a MET inhibitor) and/or fibroblast‐conditioned culture supernatant, then infected with Ad‐GFP. After 48 h, GFP‐positive cells were counted. The number of GFP‐positive cells in cultures containing fibroblast‐conditioned supernatant plus SU11274 was significantly greater than in cultures without SU11274. In conclusion, our results suggest that stromal cells in PDAC reduce the efficacy of adenoviral therapy through a mechanism involving the HGF/MET pathway. Control of such tumor–stroma interactions may lead to improvements in adenoviral gene therapy for PDAC. (Cancer Sci 2011; 102: 484–491)

Up‐regulation of integrin β3 in radioresistant pancreatic cancer impairs adenovirus‐mediated gene therapy

Adenovirus‐mediated gene therapy is a promising approach for the treatment of pancreatic cancer. We previously reported that radiation enhanced adenovirus‐mediated gene expression in pancreatic cancer, suggesting that adenoviral gene therapy might be more effective in radioresistant pancreatic cancer cells. In the present study, we compared the transduction efficiency of adenovirus‐delivered genes in radiosensitive and radioresistant cells, and investigated the underlying mechanisms. We used an adenovirus expressing the hepatocyte growth factor antagonist, NK4 (Ad‐NK4), as a representative gene therapy. We established two radioresistant human pancreatic cancer cell lines using fractionated irradiation. Radiosensitive and radioresistant pancreatic cancer cells were infected with Ad‐NK4, and NK4 levels in the cells were measured. In order to investigate the mechanisms responsible for the differences in the transduction efficiency between these cells, we measured expression of the genes mediating adenovirus infection and endocytosis. The results revealed that NK4 levels in radioresistant cells were significantly lower (P < 0.01) than those in radiosensitive cells, although there were no significant differences in adenovirus uptake between radiosensitive cells and radioresistant cells. Integrin β3 was up‐regulated and the Coxsackie virus and adenovirus receptor was down‐regulated in radioresistant cells, and inhibition of integrin β3 promoted adenovirus gene transfer. These results suggest that inhibition of integrin β3 in radioresistant pancreatic cancer cells could enhance adenovirus‐mediated gene therapy. (Cancer Sci 2009; 100: 1902–1907)