Hirofumi Sonoda

Aberrant expression of lysophosphatidic acid (LPA) receptors in human colorectal cancer

Lysophosphatidic acid (LPA) is a simple bioactive phospholipid with diverse effects on various cells, that interacts with three G protein-coupled transmembrane receptors, LPA1, LPA2, and LPA3. The expression pattern and functions of these LPA receptors in various tumors have not been fully examined, except in ovarian cancer. To evaluate the LPA receptor expression profile in human colorectal cancer and in normal mucosa, we used real-time reverse transcription-polymerase chain reaction (RT-PCR) and measured the expression levels of LPA1, LPA2, and LPA3 messenger RNA (mRNA) in 26 colorectal cancers and 16 corresponding normal tissue samples. Normal epithelium expressed both LPA1 and LPA2 mRNA at similar levels. In comparison, colorectal cancers expressed LPA1 mRNA at a significantly lower level (0.3-fold; P<0.05), and LPA2 mRNA at a significantly higher level (three-fold; P<0.05), as compared with normal tissues. Thus, the ratio of LPA2/LPA1 increased markedly during malignant transformation (18-fold increase). LPA3 mRNA was expressed at only a low level in both normal and cancer tissues. We also assessed LPA2 expression immunohistochemically using a rat anti-LPA2 monoclonal antibody, and confirmed high expression of LPA2 in colorectal cancer at the protein level. As for LPA1, we examined Western blot analysis for 16 matched normal and cancer tissues. It revealed a significant decrease in the expression of LPA1 protein in cancer tissues compared to normal mucosa in nine of 16 cases, and in the remaining seven cases the expression levels was much the same. These results suggested that alteration of LPA receptor expression might be an important event in the development of colorectal cancer, and therefore, LPA and its receptors could be a chemopreventive target against colorectal cancer.

Protein Kinase D-mediated Phosphorylation and Nuclear Export of Sphingosine Kinase 2

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling.

A Novel Phospholipase A1 with Sequence Homology to a Mammalian Sec23p-interacting Protein, p125

p125, a mammalian Sec23p-interacting protein, exhibits sequence homology with bovine testis phosphatidic acid-preferring phospholipase A1. In this study, we identified and characterized a new homologue of p125, KIAA0725p. KIAA0725p exhibited remarkable sequence similarity with p125 throughout the entire sequence determined but lacked an N-terminal proline-rich, Sec23p-interacting region. In vitro binding analysis showed that KIAA0725p does not bind to Sec23p. KIAA0725p possessed phospholipase A1 activity preferentially for phosphatidic acid. We examined the effects of overexpression of KIAA0725p on the morphology of organelles. Overexpression of KIAA0725p, like that of p125, caused dispersion of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus. Different from the case of p125, overexpression of KIAA0725p resulted in dispersion of tethering proteins located in the Golgi region and caused aggregation of the endoplasmic reticulum. Our results indicate that KIAA0725p is a new member of the phosphatidic acid-preferring phospholipase A1protein family and suggest that the cellular function of KIAA0725p is different from that of p125.

Requirement of phospholipase D for ilimaquinone-induced Golgi membrane fragmentation

Although organelles such as the endoplasmic reticulum and Golgi apparatus are highly compartmentalized, these organelles are interconnected through a network of vesicular trafficking. The marine sponge metabolite ilimaquinone (IQ) is known to induce Golgi membrane fragmentation and is widely used to study the mechanism of vesicular trafficking. Although IQ treatment causes protein kinase D (PKD) activation, the detailed mechanism of IQ-induced Golgi membrane fragmentation remains unclear. In this work, we found that IQ treatment of cells caused a robust activation of phospholipase D (PLD). In the presence of 1-butanol but not 2-butanol, IQ-induced Golgi membrane fragmentation was completely blocked. In addition, IQ failed to induce Golgi membrane fragmentation in PLD knock-out DT40 cells. Furthermore, IQ-induced PKD activation was completely blocked by treatment with either 1-butanol or propranolol. Notably, IQ-induced Golgi membrane fragmentation was also blocked by propranolol treatment. These results indicate that PLD-catalyzed formation of phosphatidic acid is a prerequisite for IQ-induced Golgi membrane fragmentation and that enzymatic conversion of phosphatidic acid to diacylglycerol is necessary for subsequent activation of PKD and IQ-induced Golgi membrane fragmentation.

CD133 expression predicts post-operative recurrence in patients with colon cancer with peritoneal metastasis

Despite extensive research on cancer stem cells in colorectal cancer, the impact of stem cell markers on patient survival remains unclear, particularly in those with distant metastasis. In this study, we focused on colon cancer with peritoneal metastasis and investigated the association between the expression of CD133, aldehyde dehydrogenase-1 (ALDH1) and leucine-rich repeating G-protein coupled receptor-5 (Lgr5), and disease prognosis. Putative stem cell marker expression was immunohistochemically evaluated in samples from 142 primary tumours and 75 peritoneal nodules. The associations between the expression of these markers and clinicopathological characteristics, overall survival and disease-free survival were analysed. The expression of CD133, ALDH1 and Lgr5 was found to be positive in 55.6, 47.2 and 78.9% of the primary tumour samples, respectively. While their expression was not associated with overall survival, disease-free survival was significantly worse in the CD133-negative group (36.1 vs. 13.7%, P=0.041). Multivariable analysis confirmed that a negative CD133 expression was an independent risk factor for a reduced disease-free survival (P=0.005). Furthermore, the benefit of systemic chemotherapy was significantly greater in the CD133-negative group (P=0.039). On the whole, our data indicated that patients with colon cancer with CD133-negative expression had a reduced disease-free survival. Thus, we propose that CD133 expression may be a useful clinical biomarker in the treatment of colon cancer with peritoneal metastasis.

Prognostic Significance and Clinicopathological Features of Synchronous Colorectal Cancer

Aim: This study aimed to clarify the difference in the clinicopathological and prognostic features between synchronous colorectal cancer (CRC) and solitary CRC. Materials and Methods: A retrospective analysis was conducted in patients with synchronous and solitary CRC. Results: A total of 92 (7.1%) out of 1,295 consecutive patients had synchronous CRC. Mucinous adenocarcinoma was more frequent in patients with synchronous CRC than in those with solitary CRC (13.0% vs. 3.7%; p<0.001). The 5-year relapse-free survival (RFS) rate was poorer in patients with synchronous CRC than in those with solitary CRC (65.3% vs. 75.1%; p=0.035), which was contrived by the multivariate analysis (hazard ratio=1.52(HR); p=0.039). Conclusion: Patients with synchronous CRC had a poorer RFS than those with solitary CRC; thus, patients with synchronous CRC might require more intensive care than those with solitary CRC in follow-up.

Lymphogenous metastasis to the transverse colon that originated from signet-ring cell gastric cancer: A case report and review of the literature.

Metastases to the colon are rare and a high-frequency primary region is the stomach. In cases of metastases to the colon, the morphological type of the metastatic region is mostly the infiltrating type of poorly differentiated or undifferentiated adenocarcinoma with lymph and blood vessel invasion. A case of cancer metastasis to the transverse colon that originated from advanced gastric cancer, which shows the difficulties in the precise diagnosis of metastases to the colon, is presented. In the present case, the gastric carcinoma was determined to be an advanced infiltrative ulcerative adenocarcinoma and the colon carcinoma was determined to be a superficial depressed adenocarcinoma. After surgery, the colon carcinoma was diagnosed as a metastatic adenocarcinoma from gastric adenocarcinoma with high invasion of vessels, by immunohistopathological analysis of CK7, CK20, p53 and HER-2. In this report, previously reported cases of metastases to the colon from gastric cancer were reviewed and their morphological characteristics were analyzed.