Nobuya Yamada

HER2 overexpression correlates with survival after curative resection of pancreatic cancer.

HER2 overexpression has been linked to clinical outcomes in several solid tumors, such as breast cancer. However, the correlation between HER2 overexpression and survival in pancreatic carcinoma remains unclear. The impact of HER2 overexpression on survival in pancreatic ductal cancer was examined. Immunohistochemical staining of 129 pancreatic cancers without hematogenous metastases or peritoneal dissemination treated by macroscopically curative resection were analyzed in association with survival data. To determine HER2 overexpression in this pancreatic cancer series, the polyclonal antibody included in HercepTest, which is used worldwide for clinical examination of HER2 overexpression in breast cancer, was used. Immunoreactivity was classified according to the scale presented in the HercepTest Scoring Guidelines. Twenty‐two cases (17.1%) had a score of 0, 28 cases (21.7%) had of a score of 1+, 41 cases (31.8%) had a score of 2+, and 38 cases (29.4%) had a score of 3+. Therefore, HER2 overexpression (score 2+ or 3+) was observed in 79 cases (61.2%). Patients with HER2 overexpression tumors had significantly shorter survival times than those with HER2 normal expression (score 0 or 1+) tumors (median survival time, 14.7 vs 20.7 months, respectively; P = 0.0078 on the log‐rank test). On multivariate survival analysis, HER2 overexpression remained an independent prognostic factor (hazard ratio, 1.806; P = 0.0258). A significant percentage of pancreatic cancers were demonstrated to have HER2 overexpression, and overexpression of this tyrosine kinase receptor proved to be an independent factor for a worse prognosis. These results should encourage further investigation of treatments using new molecular targeting agents against HER2 protein to improve the survival of pancreatic cancer patients. (Cancer Sci 2009; 100: 1243–1247)

Increased sialyl Lewis A expression and fucosyltransferase activity with acquisition of a high metastatic capacity in a colon cancer cell line

A human colon cancer cell line, OCUC-LM1(LM), was established from a liver metastasis in our laboratory. Intrasplenic injection of LM into nude mice was repeated three and five times, and the daughter cell lines were designated as LM-H3 and LM-H5 respectively. The level of sialyl Lewis A (SLA) in the supernatant of LM-H3 and LM-H5 was 3 and 4.5 times higher than that of LM respectively. Flow cytometric analysis of SLA expression showed that the peak channel for LM was 113; for LM-H3, 126; and for LM-H5, 146. The mean fluorescence intensity of LM was 102.3 +/- 43.5; for LM-H3, 126.2 +/- 28.4; and for LM-H5, 144.8 +/- 23.4. In endothelial cell adhesion assays, the percentages of adherent LM-H3 and LM-H5 cells were significantly higher than for LM. The activity of alpha1-->4 fucosyltransferase was higher in LM-H3 and LM-H5 than in LM, but there was no difference in alpha2-->3 sialyltransferase activities for type 1 chain among the cell lines. Our results suggest that SLA expression is associated with acquisition of a high capacity for liver metastasis of colon cancer; increased SLA expression is due mainly to increased fucosyltransferase activity.

Inhibitory effect of a selective cyclooxygenase-2 inhibitor on liver metastasis of colon cancer.

COX‐2 overexpression is recognized in various cancers, but the role of COX‐2 in the progression of cancer, including the liver metastasis of colon cancer, is not clearly understood. We examined the role of COX‐2 in the mechanism of liver metastasis of colon cancer, using a highly metastasizable colon carcinoma cell line, LM‐H3. A COX‐2 inhibitor, JTE‐522, inhibited cell proliferation and invasion of LM‐H3 in vitro and clearly reduced the number of metastatic nodules on the surface of nude mouse livers in vivo. We also examined the effects of JTE‐522 on the production of growth factors and MMPs through the use of ELISA and gelatin zymography, respectively. JTE‐522 downregulated PDGF production by LM‐H3 but had no influence on VEGF production. JTE‐522 also inhibited MMP‐2 secretion by LM‐H3. JTE‐522 downregulated PGE2 production, but the associated changes in PGE2 did not affect PDGF and VEGF production by LM‐H3. We conclude that JTE‐522 downregulated the cell proliferation and invasive potential of LM‐H3 by reducing the production of PDGF and MMP‐2 and hypothesize that these inhibitory effects on the production of PDGF and MMP‐2 can lead to inhibition of liver metastasis of colon cancer. These data indicate that the COX‐2 inhibitor JTE‐522 has a high potential for use as a clinical agent for the treatment of liver metastasis of colon cancer.

Antitumor Effect of Trastuzumab for Pancreatic Cancer with High HER-2 Expression and Enhancement of Effect by Combined Therapy with Gemcitabine

Purpose: The purpose of the present study was to evaluate whether trastuzumab has antitumor effect against pancreatic cancer and whether this effect is concordant with levels of HER-2, which is reportedly overexpressed in pancreatic cancer. We also investigated whether the effect is potentiated in combined therapy with gemcitabine. Experimental Design: Using immunohistochemistry and FACScan, we analyzed HER-2 expression in 16 pancreatic cancer cell lines. The in vitro antiproliferative effect of trastuzumab, alone and in combination with gemcitabine, was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The in vitro antibody-dependent cell-mediated cytotoxicity of trastuzumab was investigated by 51Cr release assay. The in vivo antitumor effect of trastuzumab, alone and in combination with gemcitabine, was evaluated in nude mouse xenograft growth. The survival benefit was evaluated in a Capan-1 orthotopic implanted nude mouse model. Results: HER-2 expression of 2+ or more was observed in 10 and of 3+ in 2 of the 16 cell lines. No in vitro growth-inhibitory effect of trastuzumab was found in any cell line, but trastuzumab induced antibody-dependent cell-mediated cytotoxicity in proportion to HER-2 expression level. Trastuzumab inhibited tumor growth in Capan-1 (HER-2: 3+) xenografts and prolonged survival in the orthotopic model. These effects were increased by combined therapy with gemcitabine. In contrast, trastuzumab exhibited no antitumor effect against PANC-1 (HER-2: 1+) or SW1990 (HER-2: 2+) xenografts. Conclusions: The antitumor effect of trastuzumab in pancreatic cancer with high HER-2 expression was shown in vitro and in vivo. Clinical application of trastuzumab is expected in pancreatic cancer with 3+ HER-2 expression.

Hypoxia Stimulates the EMT of Gastric Cancer Cells through Autocrine TGFβ Signaling

Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor β1 (TGFβ1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFβ1 and TGFβR was evaluated by real time RT-PCR. The TGFβ1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFβ1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFβR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFβR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFβ1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFβ/TGFβR signaling.

Chemokine Receptor CCR7 Expression Correlates with Lymph Node Metastasis in Pancreatic Cancer

Aims: The clinical significance of chemokine receptor CCR7 expression in pancreatic ductal cancer was investigated. Methods: Immunohistochemical staining of 89 pancreatic cancers treated macroscopically with curative resection without hematogenous metastases or peritoneal dissemination were analyzed in association with clinicopathological data. Results: The positivity of CCR7 in pancreatic cancer was 32.6% (29/89). A significant correlation was detected between CCR7-positive expression and lymph node metastasis. Patients with CCR7-positive tumors had significantly shorter survival times than those with CCR7-negative tumors (median, 12.8 vs. 21.9 months, respectively; p = 0.0039). CCR7 expression was an independent prognostic factor (hazard ratio, 1.949; p = 0.0364) by multivariate survival analysis; however, it was not an indicator for any particular site of recurrence. Conclusion: The survival impact of CCR7 expression on resectable pancreatic cancer may be associated with lymphatic spread. The results from the present study should foster further investigation of treatments using an inhibitor for the CCR7 protein to improve the survival of pancreatic cancer.

In vivo and in vitro interactions between human colon carcinoma cells and hepatic stellate cells.

Stromal reaction is important for the growth of cancer both in primary and metastatic sites. To demonstrate this reaction during the hepatic metastasis of human colon carcinoma, we histologically investigated alterations to the distribution and phenotype of hepatic stellate cells (HSCs), the only mesenchymal cells in the liver parenchyma, using a nude mouse model. Intrasplenically injected colon carcinoma LM‐H3 cells migrated into the space of Disse and underwent proliferation, in close association with hepatocytes and HSCs, at 2 days. At 14 days, HSCs were accumulated around the tumor mass and expressed α‐smooth muscle actin, a marker for HSC activation. We next investigated in vitro the growth factors involved in the interactions between LM‐H3 cells and HSCs. Conditioned medium of rat HSCs which underwent culture‐induced activation contained platelet‐derived growth factor (PDGF)‐AB, hepatocyte growth factor (HGF) and transforming growth factor (TGF)β, and could augment LM‐H3‐cell proliferation and migration. Neutralizing antibodies against PDGF‐AA and PDGF‐BB and those against PDGF‐BB and HGF inhibited proliferation and migration, respectively, of LM‐H3 cells, whereas antibody against TGF‐β had no effect. LM‐H3 cells expressed PDGF receptors‐α and ‐β and c‐met. Conditioned medium of LM‐H3 cells contained PDGF‐AB, and could enhance HSC proliferation and migration. This augmenting effect was suppressed by treatment with anti‐PDGF‐AB antibody. The present study has demonstrated that bidirectional interactions involving PDGF and HGF take place in vitro between colon carcinoma cells and HSCs, raising the possibility that similar interactions might be involved in the stromal reaction during hepatic metastasis.

Proliferating Cell Nuclear Antigen Labeling Index of Preoperative Biopsy Specimens in Gastric Carcinoma with Special Reference to Prognosis

Background. Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase δ and is considered to correlate with the cells proliferative state. Immunohistochemical methods with a anti‐PCNA antibody (PC10) have particular advantages compared with other techniques because of the maintenance of the tissue architecture and simplicity of the methodology.

RNA interference suppression of mucin 5AC (MUC5AC) reduces the adhesive and invasive capacity of human pancreatic cancer cells

BackgroundMUC5AC is a secretory mucin normally expressed in the surface muconous cells of stomach and bronchial tract. It has been known that MUC5AC de novo expression occurred in the invasive ductal carcinoma and pancreatic intraepithelial neoplasm with no detectable expression in normal pancreas, however, its function remains uncertain. Here, we report the impact of MUC5AC on the adhesive and invasive ability of pancreatic cancer cells.MethodsWe used two MUC5AC expressing cell lines derived from human pancreatic cancer, SW1990 and BxPC3. Small-interfering (si) RNA directed against MUC5AC were used to assess the effects of MUC5AC on invasion and adhesion of pancreas cancer cells in vitro and in vivo. We compared parental cells (SW1990 and BxPC3) with MUC5AC suppressed cells by si RNA (si-SW1990 and si-BxPC3).ResultsMUC5AC was found to express in more than 80% of pancreatic ductal carcinoma specimens. Next we observed that both of si-SW1990 and si-BxPC3 showed significantly lower adhesion and invasion to extracellular matrix components compared with parental cell lines. Expression of genes associated with adhesion and invasion including several integerins, matrix metalloproteinase (MMP) -3 and vascular endothelial growth factor (VEGF) were down-regulated in both MUC5AC suppressed cells. Furthermore, production of VEGF and phosphorylation of VEGFR-1 were significantly reduced by MUC5AC down regulation. Both of si-SW1990 and si-BxPC3 attenuated activation of Erk1/2. In vivo, si-SW1990 did not establish subcutaneous tumor in nude mice.ConclusionsKnockdown of MUC5AC reduced the ability of pancreatic cancer cells to adhesion and invasion, suggesting that MUC5AC might contribute to the invasive motility of pancreatic cancer cells by enhancing the expression of integrins, MMP-3, VEGF and activating Erk pathway.

IGF-1 receptor and IGF binding protein-3 might predict prognosis of patients with resectable pancreatic cancer

BackgroundThe present study aimed to elucidate the clinicopathologic role of insulin-like growth factor-1 receptor (IGF1R) and IGF binding protein-3 (IGFBP3) in patients with pancreatic cancer. The function of IGFBP3 is controversial, because both inhibition and facilitation of the action of IGF as well as IGF-independent effects have been reported. In this study, IGF1R and IGFBP3 expression was examined, and their potential roles as prognostic markers in patients with pancreatic cancer were evaluated.MethodsClinicopathological features of 122 patients with curatively resected pancreatic cancer were retrospectively reviewed, and expression of IGF1R and IGFBP3 was immunohistochemically analyzed.ResultsExpression of IGF1R and IGFBP3 was observed in 50 (41.0%) and 37 (30.3%) patients, respectively. IGF1R expression was significantly associated with histological grade (p = 0.037). IGFBP3 expression had a significant association with tumor location (p = 0.023), and a significant inverse association with venous invasion (p = 0.037). Tumors with IGF1R-positive and IGFBP3-negative expression (n = 32) were significantly frequently Stage II and III (p = 0.011). The prognosis for IGF1R positive patients was significantly poorer than that for IGF1R negative patients (p = 0.0181). IGFBP3 protein expression did not correlate significantly with patient survival. The subset of patients with both positive IGF1R and negative IGFBP3 had worse overall survival (8.8 months versus 12.6 months, respectively, p < 0.001).ConclusionIGF1R signaling might be associated with tumor aggressiveness, and IGFBP3 might show antiproliferative effects in pancreatic cancer. Both high IGF1R expression and low IGFBP3 expression represent useful prognostic markers for patients with curatively resected pancreatic cancer.