Akira Sekikawa

STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells

Signal transduction and activator of transcription 3(STAT3) signaling is constitutively activated in various tumors, and is involved in cell survival and proliferation during oncogenesis. There are few reports, however, on the role of STAT3 signaling in gastric cancer. The aim of the present study was to clarify the role of STAT3 signaling in apoptosis and cellular proliferation in gastric cancer. Here we reported that STAT3 was constitutively activated in various human gastric cancer cells and its inhibition by ectopic dominant-negative STAT3 or Janus kinase inhibitor, tyrphostin AG490, induced apoptosis. Furthermore, STAT3 inhibition markedly decreased survivin expression, and forced expression of survivin rescued AGS cells from apoptosis induced by STAT3 inhibition. Although some reports demonstrated that the PI3K/Akt pathway regulates survivin expression, inhibition of the PI3K/Akt pathway did not affect survivin expression in AGS and MKN1 cells. Finally, activated form of STAT3, Tyr-705 phospho-stat3, was found in the nucleus of cancer cells in 11 of 40 (27.5%) human gastric cancer specimens. These findings suggest that constitutively activated STAT3 signaling supports gastric cancer cell survival in association with survivin expression.

Expression of SDF-1α and nuclear CXCR4 predicts lymph node metastasis in colorectal cancer

Although stromal cell-derived factor (SDF)-1α and its receptor CXCR4 are experimentally suggested to be involved in tumorigenicity, the clinicopathological significance of their expression in human disease is not fully understood. We examined SDF-1α and CXCR4 expression in colorectal cancers (CRCs) and their related lymph nodes (LNs), and investigated its relationship to clinicopathological features. Specimens of 60 primary CRCs and 27 related LNs were examined immunohistochemically for not only positivity but also immunostaining patterns for SDF-1α and CXCR4. The relationships between clinicopathological features and SDF-1α or CXCR4 expression were then analysed. Stromal cell-derived factor-1α and CXCR4 expression were significantly associated with LN metastasis, tumour stage, and survival of CRC patients. Twenty-nine of 47 CXCR4-positive CRCs (61.7%) showed clear CXCR4 immunoreactivity in the nucleus and a weak signal in the cytoplasm (nuclear type), whereas others showed no nuclear immunoreactivity but a diffuse signal in the cytoplasm and at the plasma membrane (cytomembrane type). Colorectal cancer patients with nuclear CXCR4 expression showed significantly more frequent LN metastasis than did those with cytomembrane expression. Colorectal cancer patients with nuclear CXCR4 expression in the primary lesion frequently had cytomembrane CXCR4-positive tumours in their LNs. In conclusion, expression of SDF-1α and nuclear CXCR4 predicts LN metastasis in CRCs.

Growth arrest‐specific gene 6 and Axl signaling enhances gastric cancer cell survival via Akt pathway

Activation of tyrosine kinases is an important factor during cancer development. Axl, one of the receptor tyrosine kinases, binds to the specific ligand growth arrest‐specific gene 6 (Gas6), which encodes a vitamin K‐dependent γ‐carboxyglutamyl protein. Although many receptor tyrosine kinases and their ligands are involved in gastric carcinogenesis, whether Gas6‐Axl signaling is involved in gastric carcinogenesis has not been elucidated. The aim of this study was to investigate the expression of Gas6 and Axl in gastric cancer and also their roles during gastric carcinogenesis. mRNA and protein of Gas6 and Axl were highly expressed in a substantial proportion of human gastric cancer tissue and cell lines, and Gas6 expression was significantly associated with lymph node metastasis. With recombinant Gas6 and a decoy‐receptor of Axl in vitro, we demonstrated that Gas6‐Axl signaling pathway enhanced cellular survival and invasion and suppressed apoptosis via Akt pathway. Our results suggests that Gas6‐Axl signaling plays a role during gastric carcinogenesis, and that targeting Gas6‐Axl signaling could be a novel therapeutic for gastric cancer.

Nuclear expression of phosphorylated EGFR is associated with poor prognosis of patients with esophageal squamous cell carcinoma.

Objectives: Although it has been reported that epidermal growth factor receptor (EGFR) is able to translocate from the plasma membrane to the nucleus, the pathophysiological role of this translocation in tumorigenicity is still unclear. In the present study, to elucidate the pathophysiological significance of EGFR translocation, we investigated the expression not only of conventional EGFR but also its phosphorylated form (pEGFR), focusing on its cellular localization in esophageal cancer tissues. Methods: Fifty-two specimens of esophageal squamous cell carcinoma (SCC) obtained by surgery were examined immunohistochemically for their EGFR and pEGFR immunostaining patterns. The relationships between clinicopathological parameters and EGFR or pEGFR immunostaining patterns were then analyzed. Results: In 37 (71.2%) of the 52 esophageal SCCs, EGFR immunoreactivity was clearly localized at the plasma membrane of the cancer cells, whereas pEGFR immunoreactivity was clearly localized in the nucleus in 19 (36.5%) cases. Nuclear expression of pEGFR significantly correlated with TNM stage and lymph node metastasis, and moreover was associated with a poor outcome of esophageal SCC. Conclusions: Nuclear translocalization of pEGFR is associated with an increase in the malignant potential of esophageal SCC and may affect prognosis in patients with esophageal SCC.

Possible role of REG Iα protein in ulcerative colitis and colitic cancer

Background and aims: Although regenerating gene (REG) Iα protein may be involved in the inflammation and carcinogenesis in the gastrointestinal tract, its pathophysiological role in ulcerative colitis (UC) and the resulting colitic cancer remains unclear. We investigated expression of the REG Iα gene and its protein in UC and colitic cancer tissues. We examined whether cytokines are responsible for REG Iα gene expression and whether REG Iα protein has a trophic and/or an antiapoptotic effect on colon cancer cells. Methods: Expression of REG Iα mRNA and its gene product in UC tissues was analysed by real time reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. The effects of cytokines on REG Iα promoter activity were examined in LoVo cells by luciferase reporter assay. The effects of REG Iα protein on growth and H2O2 induced apoptosis were examined in LoVo cells by MTT and TUNEL assays, respectively. Results: REG Iα protein was strongly expressed in inflamed epithelium and in dysplasias and cancerous lesions in UC tissues. The level of REG Iα mRNA expression in UC tissues correlated significantly with severity of inflammation and disease duration. REG Iα promoter activity was enhanced by stimulation with interferon γ or interleukin 6. REG Iα protein promoted cell growth and conferred resistance to H2O2 induced apoptosis in LoVo cells. REG Iα protein promoted Akt phosphorylation and enhanced Bcl-xL and Bcl-2 expression in LoVo cells. Conclusions: The REG Iα gene is inducible by cytokines and its gene product may function as a mitogenic and/or an antiapoptotic factor in the UC-colitic cancer sequence.

Involvement of the IL-22/REG Iα axis in ulcerative colitis

The Regenerating gene (REG) Iα protein, a trophic and/or anti-apoptotic factor, is important in the pathophysiology of gastrointestinal inflammation. Interleukin (IL)-22 is a recently identified cytokine that is suggested to have pivotal roles in inflammatory bowel diseases. We therefore investigated the involvement of the IL-22/REG Iα axis and examined the mechanism of regulation of REG Iα expression by IL-22 stimulation in ulcerative colitis (UC) mucosa. Expression of IL-22, IL-22 receptor 1 (IL-22R1), and REG Iα in UC mucosa was analyzed by real-time RT-PCR and immunohistochemistry. The effects of IL-22 on REG Iα protein expression were examined using a small-interfering RNA for STAT3, an MAPK inhibitor or a PI3K inhibitor. The element responsible for IL-22-induced REG Iα promoter activation was determined by a promoter deletion and electrophoretic mobility shift assay. The expression of IL-22 was enhanced in infiltrating inflammatory cells, and that of IL-22R1 and REG Iα was concurrently enhanced in the inflamed epithelium in UC mucosa. The levels of REG Iα and IL-22 mRNA expression were strongly correlated, and the distributions of REG Iα- and IL-22R1-positive epithelial cells were very similar. IL-22 simulation enhanced the expression of REG Iα protein through STAT3 tyrosine phosphorylation in colon cancer cells. The IL-22-responsive element was located between −142 and −134 in the REG Iα promoter region. REG Iα protein may have a pathophysiological role as a biological mediator for immune cell-derived IL-22 in the UC mucosa.

Colonic expression of heme oxygenase-1 is associated with a better long-term survival in patients with colorectal cancer

Objective. Heme oxygenase-1 (HO-1) has emerged as a crucial mediator of mucosal defense in the gastrointestinal tract. Its metabolic pathway products, biliverdin/bilirubin and carbon monoxide, can reduce oxidative stress and inflammation, and promote resistance to apoptosis. The role of HO-1 in gastrointestinal malignancies, however, remains to be elucidated. The purpose of this study was to analyze HO-1 expression in human colon adenoma and cancer samples. Material and methods. Fifty-five paraffin-embedded surgical specimens of colorectal cancer and 19 colonic adenoma samples were stained immunhistochemically for HO-1 expression using an anti-HO-1 monoclonal antibody. HO-1 expression was evaluated independently by two different investigators and subsequently correlated to clinical data and patients’ life expectancy. Results. Focal HO-1 expression could be documented in 41.8% (23/55) of patients with colorectal cancer. HO-1 expression in colonic adenoma was detectable in 36.8% (7/19) of cases. The rate of lymphatic tumor invasion was significantly lower in colorectal cancer samples expressing HO-1 (p=0.048). Additionally, fewer lymph node metastases were found in colorectal cancer samples with HO-1 expression, but these differences did not reach statistical significance. Mean observation period was 65.87±3.96 months. Kaplan-Meier analysis showed a significantly better survival for colorectal cancer patients with colonic HO-1 expression (p=0.018). Conclusions. This study demonstrates that colonic HO-1 may be a prognostic marker of colorectal-cancer outcome.

Expression of Reg Iα Protein in Human Gastric Cancers

Background/Aims: Although regeneratinggene(Reg) Iα protein has a trophic effect on gastric epithelial cells, it is unclear whether Reg Iα protein and its receptor are involved in gastric carcinogenesis. Therefore, we investigated the Reg Iα protein expression in human gastric cancers and assessed its relationship to clinicopathological factors. Methods: Sixty-one gastric cancer specimens were examined, using immunohistochemistry, for Reg Iα protein, p53, and proliferating cell nuclear antigen. The expression of both Reg Iα and Reg receptor mRNA was examined in seven human gastric cancer cell lines (MKN1, MKN28, MKN45, MKN74, KATOIII, GCIY, and AGS) by reverse transcription-polymerase chain reaction and Northern blot analysis. Results: Twenty-three (37.7%) of the 61 gastric cancer tissues samples were positive for Reg Iα protein. The Reg Iα expression was significantly related to the presence of lymphatic invasion but not to tumor size, tumor stage, Lauren’s classification, presence of venous invasion, lymph node metastases, or p53 overexpression. Gastric cancers positive for Reg Iα protein showed a significantly higher proliferating cell nuclear antigen labeling index than negative ones. The expression of both Reg Iα and Reg receptor mRNA was detected in all seven gastric cancer cell lines. Conclusion: Reg Iα protein may play a role in the development of gastric cancers.

Expression of the REG IV gene in ulcerative colitis

The regenerating gene (REG) IV gene was isolated from a cDNA library of ulcerative colitis (UC) tissues. However, its role in the pathophysiology of UC and subsequent development of colitic cancer is still unclear. We investigated the expression of the REG IV gene in UC and colitic cancer tissues and examined whether cytokines or growth factors are responsible for REG IV gene expression and whether REG IV gene induction affects cell growth and apoptosis in colon cancer cells. The expressions of REG IV and growth factor genes in UC tissues were analyzed by real time reverse transcription-polymerase chain reaction. The effects of cytokines and growth factors on REG IV gene expression were examined in SW403 cells by Northern blot analysis. The effects of REG IV gene induction on cell growth and H2O2-induced apoptosis were examined in DLD-1 cells by MTT and TUNEL assays, respectively. REG IV mRNA was strongly expressed in inflamed epithelium and in dysplasias and cancerous lesions in UC tissues. The level of REG IV mRNA expression was correlated with that of basic fibroblast growth factor (bFGF) as well as hepatocyte growth factor (HGF) mRNA expression in UC tissues. The REG IV gene expression in SW403 colon cancer cells was enhanced by stimulation with transforming growth factor-α, epidermal growth factor, bFGF, and HGF. REG IV gene induction promoted cell growth and conferred resistance to H2O2-induced apoptosis in DLD-1 cells. The REG IV gene is inducible by growth factors and may function as a growth promoting and/or an antiapoptotic factor in the pathophysiology of UC.

DMBT1 is a novel gene induced by IL‐22 in ulcerative colitis

Background: Interleukin (IL)‐22 is a recently identified cytokine that is suggested to play pivotal roles in various inflammatory diseases. Although the IL‐22 receptor 1 (IL‐22R1) is restrictively expressed in epithelial cells in the colon, the role of IL‐22 in colonic diseases still remains unclear. In this study microarray analyses revealed that deleted in malignant brain tumors 1 (DMBT1) is a novel upregulated gene in IL‐22‐stimulated colon cancer cells. Therefore, we investigated the involvement of DMBT1 and IL‐22 in ulcerative colitis (UC) tissues and examined the mechanism regulating the expression of DMBT1 in response to IL‐22 stimulation. Methods: Changes of gene expression in IL‐22‐stimulated SW403 cells were investigated by microarray analyses. The effects of IL‐22 on DMBT1 expression were examined in SW403 cells using a small interfering RNA (si)RNA for STAT3 or inhibitors for MEK, PI3K, and nuclear factor kappa B (NF‐&kgr;B). The element responsible for IL‐22‐induced DMBT1 promoter activation was determined by a promoter deletion and electrophoretic mobility shift assay (EMSA). Expression of IL‐22, IL‐22R1, and DMBT1 in UC tissues was analyzed by real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) and immunohistochemistry. Results: IL‐22 treatment enhanced the expression of DMBT1 through STAT3 tyrosine phosphorylation and NF‐&kgr;B activation in colon cancer cells. The IL‐22‐responsive element was located between −187 and −179 in the DMBT1 promoter region. In the UC mucosa the levels of DMBT1 and IL‐22 mRNA expression were significantly enhanced and positively correlated, the numbers of IL‐22‐positive lymphocytes were increased, and the expression of IL‐22R1 and DMBT1 was enhanced in the inflamed epithelium. Conclusions: The IL‐22/DMBT1 axis may play a pivotal role in the pathophysiology of UC. (Inflamm Bowel Dis 2010;)