Shinji Ohki

Circulating myeloid-derived suppressor cells are increased and correlate to immune suppression, inflammation and hypoproteinemia in patients with cancer

Recent studies have identified myeloid-derived suppressor cells (MDSCs) that are potent suppressors of tumor immunity and therefore a significant impediment to cancer immunotherapy. It has been reported that MDSCs are generated by malignant diseases or inflammation. However, no systematic studies in patients have been described. In order to clinically characterize MDSCs, we tested PBMCs from patients with various types of cancer including cholangiocellular, hepatocellular and pancreatic carcinoma, esophageal, gastric and colorectal cancer, breast cancer and thyroid cancer, and GIST, and those from normal volunteers using flow cytometry analysis. A significant increase was seen in the percentages of MDSCs in PBMCs from patients compared with normal volunteers. Among these patients, MDSC level was higher in patients with cancer of the digestive system and patients with breast cancer compared with normal volunteers. MDSC level was significantly and inversely correlated to stimulation indices (SI) of PHA-blastogenesis of lymphocytes and serum concentration of total protein, and positively correlated to neutrophil count. MDSC percentage in patients with gastric and colorectal cancer was also significantly correlated to neutrophil count and inversely correlated with lymphocyte count, and showed highly significant correlation to neutrophil/lymphocyte rate (NLR). In patients with breast cancer, MDSC levels in preoperative patients was significantly increased compared to normal volunteers and significantly decreased in postoperative patients. Thus, it is clear that MDSCs are increased in patients with cancer and closely related to suppression of cell-mediated immune responses. These data also suggest that they are related to chronic inflammation and that their levels are increased further in the terminal stages of patients whose nutritional status is impaired as observed in hypoproteinemia. MDSC levels have also been shown to decrease after removal of tumors in patients with breast cancer.

INHIBITORY EFFECTS OF PROSTAGLANDIN D2 AGAINST THE PROLIFERATION OF HUMAN COLON CANCER CELL LINES AND HEPATIC METASTASIS FROM COLORECTAL CANCER

The inhibitory action of prostaglandin D2 (PGD2) and its effect on the cell cycle were examined in cell lines SW480 and LS174T of human colon cancer. The growth of the cell lines were assessed 24h and 48h after the addition of 1.0μg/ml and 10.0μg/ml PGD2. The growth of SW480 cells and 24h and 48h after the addition of 10.0μ/ml, while that of LS174T was inhibited by both doses after 24h and 48h. S-Phase DNA synthesis in the SW480 cells was significantly blocked 24h after the addition of 10.0μg/ml PGD2. The cell cycle of LS174T cells was arrested at the G0+G1 phase 24h after the addition of 1.0μg/ml and 10.0μg/ml PGD2. The correlation between hepatic metastasis and PGD2 concentration in human cancer tissue was examined. The mean value of PGD2 concentrations in the primary cancer tissue was significantly lower in the hepatic metastasis group than that in the group without hepatic metastasis. These findings suggest that measuring the PGD2 in cancer tissue may be useful for detecting and predicting the hepatic metastasis from human colorectal cancer.

Cathepsin L is highly expressed in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract that are diagnosed by c-kit staining in most cases. A lysosomal cysteine proteinase termed cathepsin L has been commonly associated with malignancy in several cancer types, but this finding has not been reported for GISTs. We analyzed the cathepsin L mRNA and protein expression in GISTs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that cathepsin L levels were higher in GISTs than those in gastric or colorectal tumors; this finding was supported by results of the Western blot analysis. Immunohistochemistry revealed that cathepsin L was localized to the cytoplasm of GIST cells as an intense granular signal, which was not observed in the cells of leiomyoma, a mesenchymal tumor that was analyzed as a control specimen. Double immunofluorescence microscopy revealed that a portion of the granular signal colocalized with lysosome-associated membrane protein-1 (LAMP-1), which is a lysosomal marker. Moreover, immunohistochemical analysis of 43 tumor specimens revealed that 86.0% (n=37) were cathepsin-L positive, and this positivity was significantly correlated with c-kit positivity but not with other clinicopathological factors, including gender, age, region, size, mitosis and risk of recurrence. From these results, we conclude that cathepsin L is highly expressed in GISTs compared to its expression in other cancerous lesions; this identifies cathepsin-L as a new diagnostic marker for GISTs.

Ectopic expression of MECA‐79 as a novel prognostic indicator in gastric cancer

The aim of this study was to clarify the clinical implications of a unique carbohydrate determinant, MECA‐79, in gastric cancer specimens and cells. Immunohistochemical analysis showed that 62 of 225 (27.6%) cases were defined as positive for MECA‐79. MECA‐79 expression was correlated with depth of invasion, venous invasion, TNM stage, and distant metastasis. In survival analyses, patients with MECA‐79 expression had worse prognosis by the log–rank test. Multivariate analysis of the Cox proportional hazard model showed that MECA‐79 expression was an independent factor of a worse cancer‐specific survival. Among 11 gastric cancer cells, MECA‐79 was observed in only MKN7 cells, which also expressed GlcNAc6ST‐2 transcript. A knockdown of GlcNAc6ST‐2 in MKN7 cells showed a markedly reduced expression of MECA‐79, suggesting that GlcNAc‐sulfation of MECA‐79 is mainly synthesized by GlcNAc6ST‐2. Furthermore, real‐time RT‐PCR analysis revealed that GlcNAc6ST‐2 was significantly increased in cancer tissues compared with paired normal mucosa. In conclusion, the expression of MECA‐79 could be a useful marker for the prognosis of gastric cancer. Our results might also provide novel perspectives on the biology of MECA‐79 and GlcNAc6ST‐2 in cancer progression and metastasis. (Cancer Sci 2011; 102: 1088–1094)

Expression of circadian clock genes in human colorectal adenoma and carcinoma

Circadian rhythms are fundamental biological systems in most organisms. Epidemiological and animal studies have demonstrated that disruption of circadian rhythms is linked to tumor progression and mammalian tumorigenesis. However, the clinical significance of in situ clock gene expression in precancerous and cancerous colorectal lesions remains unknown. The present study aimed to investigate mRNA transcript levels of circadian clock genes within human colorectal cancer and adenoma tissue sections. Using in situ hybridization, the expression of key clock genes, including period circadian protein homolog (Per) 1 and 2, cryptochrome 1 (Cry1), circadian locomoter output cycles protein kaput (Clock), brain and muscle ARNT-like protein 1 (Bmal1) and casein kinase 1ε (CK1ε) were retrospectively examined in 51 cases of colorectal carcinoma and 10 cases of adenoma. The expression of clock genes was almost undetectable in the majority of adenomas, whereas positive expression of clock genes was observed in 27–47% of carcinomas. Notably, positive Per1, Per2 and Clock staining in colorectal carcinomas were each significantly associated with a larger tumor size (P=0.012, P=0.011 and P=0.009, respectively). Tumors with positive Per2 and Clock expression tended to exhibit deeper depth of invasion and were generally more advanced than tumors that did not express these genes (P=0.052 and P=0.064, respectively). However, no statistically significant association was observed between clock gene expression and clinicopathological variables, including histopathological differentiation, lymph node metastasis, depth of invasion or disease stage, although Per2-positive tumors tended to be associated with poorer overall survival (P=0.060). The results of the current study suggest that dysregulated expression of clock genes may be important in human colorectal tumorigenesis.

Stromal VCAN expression as a potential prognostic biomarker for disease recurrence in stage II-III colon cancer.

To develop prognostic biomarkers that can discriminate stage II-III colorectal cancer patients with high risk of postoperative recurrence, we conducted a genome-wide screening of relapse-related genes utilizing multiple microarray cohorts. Among differentially expressed genes between tumor and nontumor, we identified eight candidate genes associated with relapse in two datasets of stage II-III patients (n = 94 and 145, respectively, P < 0.05). Using datasets of laser-microdissected samples and FACS-purified cell populations, the localization of candidate genes, including COL4A2, COL4A1, VCAN and SERPINE1, were found predominantly in cancer stroma rather than epithelial components. Among those relapse-related stromal genes, VCAN mRNA, specifically expressed in cancer-associated fibroblasts, was further validated to be a prognostic factor in two additional independent datasets, consisting of 453 (P = 0.0334) and 89 (P = 0.0041) stage II-III patients. Furthermore, in our large set of formalin-fixed paraffin-embedded cohort (n = 338), VCAN protein was detected exclusively in cancer stroma by immunohistochemistry, demonstrating a stepwise increase of stromal VCAN from normal tissues through stage 0 to stage IV tumors. Stromal VCAN protein was associated with shorter relapse-free survival (RFS) in stage II-III colon cancer, independent of other clinical factors by multivariate analysis (P = 0.004). Stratified analyses revealed that stromal VCAN was a strong prognostic indicator particularly in stage II colon cancer (P = 0.0029). In all five analyzed cohorts, the expression of VCAN, in transcript or protein levels, was associated with poor RFS in stage II-III patients. We conclude that VCAN is a promising biomarker to identify stage II-III patients at high risk of relapse who may benefit from intensive postoperative management.

Molecular Genetic Alteration and DNA Ploidy in Hereditary Nonpolyposis Colorectal Cancer

BackgroundTo clarify the biologic characteristics of hereditary nonpolyposis colorectal cancer (HNPCC), an investigation was made of the association of this cancer with mutations of theTP53 (aliasp53) and K-ras genes, and with DNA ploidy.MethodsSamples from 19 patients with HNPCC and from 56 patients with sporadic colorectal cancer were analyzed by polymerase chain reaction-single strand conformation polymorphism and flow cytometric analyses.ResultsMutations of thep53 gene were found in 7 (32%) of 22 samples from HNPCC patients, and K-ras mutations were found in 8 (44%) of 18 samples from HNPCC patients. Aneuploidy was noted in 8 (44%) of 18 samples. Each incidence in HNPCC was similar to the corresponding incidence in the sporadic colorectal cancers.ConclusionThe results of this investigation indicate that HNPCC shows no difference from sporadic colorectal cancer in terms ofp53 and K-ras alterations, or DNA ploidy.

CDX2 is involved in microRNA‑associated inflammatory carcinogenesis in gastric cancer

The development of gastric cancer is significantly associated with chronic inflammation, such as caused by Helicobacter pylori (H. pylori) infection. Caudal-type homeobox 2 (CDX2) is a homeobox protein involved in intestinal differentiation in normal and in aberrant locations, and is associated with inflammation. The authors of the present study have previously reported that CDX2 may have a suppressive role in the progression and carcinogenesis of gastric carcinoma. In the present study, the authors initially confirmed that a decreased expression of CDX2, as detected by immunohistochemistry, is associated with poor cancer-specific survival in 210 gastric cancer cases, which is consistent with several previously published studies. To elucidate the potential mechanisms underlying this association, the authors investigated the mechanism of CDX2 suppression, which included interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) and p53 signaling pathways. The present study confirmed that CDX2 was suppressed by activation of the IL-6/STAT3 signaling pathway via miR-181b in vitro. It was further revealed that gastric cancer with negative CDX2 expression is associated with negative p53 staining, and this association was particularly significant in undifferentiated gastric cancer. The activation of the IL-6/STAT3 signaling pathway suppressed miR-34a, which is induced by p53. This suggests that the activation of the IL-6/STAT3 signaling pathway inflammation signaling pathway suppresses the p53 signaling pathway in tumors without TP53 mutation, which results in poor prognostic outcomes. In conclusion, CDX2 may be a useful prognostic biomarker for gastric cancer and is associated with p53 inactivation.

Enhanced expression of KIF4A in colorectal cancer is associated with lymph node metastasis

Kinesin family member 4A (KIF4A) is a member of the kinesin 4 subfamily of kinesin-related proteins and serves an important role in cell division. The expression levels of KIF4A have been investigated in numerous types of cancer, including cervical, lung, oral, and breast cancer, and are established to be associated with poor patient prognosis. However, the role of KIF4A, as well as its expression in colorectal cancer (CRC), remains to be elucidated. Therefore, the current study investigated KIF4A expression levels in patients with CRC and demonstrated that its levels were increased in tumor tissues compared with non-tumor tissues. To investigate the functional role of KIF4A, KIF4A was knocked down in CRC cells and cell viability was evaluated. CRC cells with KIF4A knockdown exhibited lower cell proliferation compared with control cells. In addition, KIF4A expression levels, as determined by immunohistochemistry, were compared with the expression of Ki-67, but no significant associations were observed in the patients with CRC. Therefore, KIF4A was found to be upregulated in patients with CRC and downregulation of KIF4A reduced cell proliferation in CRC cells. These results suggest that KIF4A may be a potential therapeutic target, which may improve the outcomes of patients with CRC.

Immunogenic tumor cell death induced by chemotherapyin patients with breast cancer and esophageal squamous cell carcinoma

It has been reported that chemo-radiotherapy can induce immunogenic tumor cell death (ICD), which triggers T-cell immunity mainly mediated by high-mobility group box 1 protein (HMGB1) and calreticulin. However, there is still limited information to support this theory relating to chemotherapy alone. In the present study, the expression of HMGB1 and calreticulin was evaluated by immunohistochemistry in pre-treatment biopsy specimens and surgically resected specimens, which were obtained from patients with breast cancer (n=52) and esophageal squamous cell carcinoma (ESCC) (n=8) who had been treated with neoadjuvant chemotherapy (NAC). We also analyzed HMGB1 and calreticulin expression in breast cancer cell lines treated with chemotherapeutic drugs. As a result, both HMGB1 and calreticulin expression levels were significantly upregulated after NAC in both breast cancer and ESCC tissues. However, no significant correlation was observed between HMGB1 expression and pathological response after NAC or between HMGB1 expression and patient survival. Furthermore, although overall survival in the high infiltration group of CD8-positive T cells was significantly superior to that in the low infiltration group in breast cancer patients, there were no correlations between the number of CD8-positive T cells and HMGB1 or calreticulin expression levels. In addition, chemotherapeutic drugs induced upregulation of HMGB1 and calreticulin in all tested cell lines. Our findings indicate that chemotherapy alone can significantly induce ICD regardless of the degree of pathological response after chemotherapy.