Wataru Yasui

Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis

BACKGROUND Analyses of microRNA expression profiles have shown that many microRNAs are expressed aberrantly and correlate with tumorigenesis, progression, and prognosis of various haematological and solid tumours. We aimed to assess the relation between microRNA expression and progression and prognosis of gastric cancer. METHODS 353 gastric samples from two independent subsets of patients from Japan were analysed by microRNA microarray. MicroRNA expression patterns were compared between non-tumour mucosa and cancer samples, graded by diffuse and intestinal histological types and by progression-related factors (eg, depth of invasion, metastasis, and stage). Disease outcome was calculated by multivariable regression analysis to establish whether microRNAs are independent prognostic factors. FINDINGS In 160 paired samples of non-tumour mucosa and cancer, 22 microRNAs were upregulated and 13 were downregulated in gastric cancer; 292 (83%) samples were distinguished correctly by this signature. The two histological subtypes of gastric cancer showed different microRNA signatures: eight microRNAs were upregulated in diffuse-type and four in intestinal-type cancer. In the progression-related signature, miR-125b, miR-199a, and miR-100 were the most important microRNAs involved. Low expression of let-7g (hazard ratio 2.6 [95% CI 1.3-4.9]) and miR-433 (2.1 [1.1-3.9]) and high expression of miR-214 (2.4 [1.2-4.5]) were associated with unfavourable outcome in overall survival independent of clinical covariates, including depth of invasion, lymph-node metastasis, and stage. INTERPRETATION MicroRNAs are expressed differentially in gastric cancers, and histological subtypes are characterised by specific microRNA signatures. Unique microRNAs are associated with progression and prognosis of gastric cancer. FUNDING National Cancer Institute.

Expression of Wnt-5a Is Correlated with Aggressiveness of Gastric Cancer by Stimulating Cell Migration and Invasion

Wnt-5a is a representative ligand that activates a beta-catenin-independent pathway in the Wnt signaling. Although abnormal activation of beta-catenin-dependent pathway is often observed in human cancer, the relationship between beta-catenin-independent pathway and tumorigenesis is not clear. We sought to clarify how Wnt-5a is involved in aggressiveness of gastric cancer. Abnormal expression of Wnt-5a was observed in 71 of 237 gastric cancer cases by means of immunohistochemistry. The positivity of Wnt-5a expression was correlated with advanced stages and poor prognosis of gastric cancer. Wnt-5a had the abilities to stimulate cell migration and invasion in gastric cancer cells. Wnt-5a activated focal adhesion kinase and small GTP-binding protein Rac, both of which are known to play a role in cell migration. Cell migration, membrane ruffling, and turnover of paxillin were suppressed in Wnt-5a knockdown cells. Furthermore, anti-Wnt-5a antibody suppressed gastric cancer cell migration. These results suggest that Wnt-5a stimulates cell migration by regulating focal adhesion complexes and that Wnt-5a is not only a prognostic factor but also a good therapeutic target for gastric cancer.

Frequent amplification of the c-met gene in scirrhous type stomach cancer.

Amplification of the c-met gene, that encodes hepatocyte growth factor receptor, was examined on human esophageal, gastric and colorectal carcinomas. Six (55%) of the 11 gastric carcinoma cell lines and 15 (23%) of the 64 advanced gastric carcinomas showed the c-met gene amplification. Among them, c-met amplification was detected in 5 gastric cancer cell lines, derived from scirrhous gastric carcinoma and in 5 (38%) of 13 scirrhous gastric carcinoma tissues. Furthermore, patients of gastric carcinoma with c-met amplification showed significantly advanced tumor stage and poorer prognosis than those without the amplification. Conversely, no amplification was detected in any of the esophageal and colorectal carcinoma cell lines as well as carcinoma tissues except one colonic carcinoma. These results overall suggest that amplification of the c-met gene might participate in carcinogenesis and progression of stomach cancer, especially scirrhous type stomach carcinoma.

Genetic variation in PSCA is associated with susceptibility to diffuse-type gastric cancer

Gastric cancer is classified into intestinal and diffuse types, the latter including a highly malignant form, linitis plastica. A two-stage genome-wide association study (stage 1: 85,576 SNPs on 188 cases and 752 references; stage 2: 2,753 SNPs on 749 cases and 750 controls) in Japan identified a significant association between an intronic SNP (rs2976392) in PSCA (prostate stem cell antigen) and diffuse-type gastric cancer (allele-specific odds ratio (OR) = 1.62, 95% CI = 1.38–1.89, P = 1.11 × 10−9). The association was far less significant in intestinal-type gastric cancer. We found that PSCA is expressed in differentiating gastric epithelial cells, has a cell-proliferation inhibition activity in vitro and is frequently silenced in gastric cancer. Substitution of the C allele with the risk allele T at a SNP in the first exon (rs2294008, which has r2 = 0.995, D′ = 0.999 with rs2976392) reduces transcriptional activity of an upstream fragment of the gene. The same risk allele was also significantly associated with diffuse-type gastric cancer in 457 cases and 390 controls in Korea (allele-specific OR = 1.90, 95% CI = 1.56–2.33, P = 8.01 × 10−11). The polymorphism of the PSCA gene, which is possibly involved in regulating gastric epithelial-cell proliferation, influences susceptibility to diffuse-type gastric cancer.

Molecular-pathological prognostic factors of gastric cancer: a review

Invasion and metastasis are critical determinants of cancer morbidity. Genes and molecules participating in these steps must be regarded as potential prognostic factors. Growth factors and their receptors, cell-cycle regulators, cell-adhesion molecules and matrix-degrading enzymes are those to be used as prognostic factors, including epidermal growth factor (EGF), EGF receptor, K-sam, HER-2, interleukin (IL)-8, vascular endothelial growth factor (VEGF), cyclin E, p27, E-cadherin, CD44v6, matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Alterations in epigenetics, such as aberrant DNA methylation and histone modification that are, in part, associated with the tumor progression of gastric cancer, can be candidate prognostic factors. The number of methylated genes may serve as a marker of tumor progression. Genetic polymorphism not only affects cancer susceptibility but also influences malignant phenotype; examples include single-nucleotide polymorphism in the HER-2 and MMP-9 genes. Comprehensive gene expression analyses are useful to search for novel genes related to invasion and metastasis and potential prognostic factors. Serial analysis of gene expression (SAGE) has identified several these genes, such as CDH17, APOE, FUS, COL1A1, COL1A2, GW112, and MIA. Overexpression of MIA is found to be associated with poor prognosis. Microarray analysis has great potential for identifying the characteristics of individual cancers, from the view point of gene expression profiles. A combination of these examinations can not only foretell a patient’s prognosis but can also give information directly connected with personalized cancer medicine and prevention.

Expression of receptors for advanced glycation end‐products (RAGE) is closely associated with the invasive and metastatic activity of gastric cancer

The receptor for advanced glycation end‐products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. This study investigated the expression of RAGE in gastric carcinomas and its association with invasion and metastasis. Of eight gastric cancer cell lines examined, seven constitutively expressed RAGE messenger ribonucleic acid (mRNA), MKN45 being the exception. RAGE protein expression of MKN28 cells treated with RAGE antisense S‐oligodeoxynucleotide was nine times less than that of sense S‐oligodeoxynucleotide‐treated cells. Growth of cells under RAGE antisense S‐oligodeoxynucleotide treatment was not different from that seen under sense S‐oligodeoxynucleotide treatment in MKN28 (a cell line producing high levels of RAGE) and MKN45 (a non‐RAGE‐expressing cell line). RAGE antisense S‐oligodeoxynucleotide treatment suppressed the invasive activity of RAGE‐positive MKN28 cells, as estimated by in vitro invasion assay. The number of MKN28 cells invading the type IV collagen‐coated membrane under RAGE antisense S‐oligodeoxynucleotide treatment was significantly lower than under RAGE sense S‐oligodeoxynucleotide treatment (p<0.0001). In contrast, antisense and sense S‐oligodeoxynucleotide‐treated RAGE‐negative MKN45 cells showed no difference. A wound‐healing assay showed that no RAGE antisense S‐oligodeoxynucleotide‐treated MKN28 cells migrated into the scraped area, whereas sense S‐oligodeoxynucleotide‐treated cells showed many budding nests in the scraped area. Immunohistochemistry of gastric carcinoma tissue showed that 62 (65%) of the 96 cases examined were RAGE‐positive and that poorly differentiated adenocarcinomas preferentially expressed RAGE protein (38/42, 90%) (p<0.0001). Strong RAGE immunoreactivity was also correlated with depth of invasion and lymph node metastasis (p<0.0001). RAGE‐positive cancer cells tended to be distributed at the invasive front of primary tumours and were detected in all metastatic foci in lymph nodes. In contrast, a major RAGE ligand, amphoterin, was expressed in 82 (85%) of the 96 cases, regardless of histological type and disease progression. RAGE expression appears to be closely associated with invasion and metastasis in gastric cancer. Copyright

Mesenchymal stem cells enhance growth and metastasis of colon cancer

Recently, mesenchymal stem cells (MSCs) were reported to migrate to tumor stroma as well as injured tissue. We examined the role of human MSCs in tumor stroma using an orthotopic nude mice model of KM12SM colon cancer. In in vivo experiments, systemically injected MSCs migrated to the stroma of orthotopic colon tumors and metastatic liver tumors. Orthotopic transplantation of KM12SM cells mixed with MSCs resulted in greater tumor weight than did transplantation of KM12SM cells alone. The survival rate was significantly lower in the mixed‐cell group, and liver metastasis was seen only in this group. Moreover, tumors resulting from transplantation of mixed cells had a significantly higher proliferating cell nuclear antigen labeling index, significantly greater microvessel area and significantly lower apoptotic index. Splenic injection of KM12SM cells mixed with MSCs, in comparison to splenic injection of KM12SM cells alone, resulted in a significantly greater number of liver metastases. MSCs incorporated into the stroma of primary and metastatic tumors expressed α‐smooth muscle actin and platelet‐derived growth factor receptor‐β as carcinoma‐associated fibroblast (CAF) markers. In in vitro experiments, KM12SM cells recruited MSCs, and MSCs stimulated migration and invasion of tumor cells through the release of soluble factors. Collectively, MSCs migrate and differentiate into CAFs in tumor stroma, and they promote growth and metastasis of colon cancer by enhancing angiogenesis, migration and invasion and by inhibiting apoptosis of tumor cells.

Clinicopathological significance of vascular endothelial growth factor (VEGF)‐C in human esophageal squamous cell carcinomas

The purpose of this study was to investigate the expression of vascular endothelial growth factor (VEGF) ‐C in human esophageal squamous cell carcinomas to elucidate its role in lymph node metastasis and tumor progression. The expression of VEGF‐C and flt‐4 genes was examined in 5 esophageal carcinoma cell lines, 12 fresh biopsy specimens and 48 archival surgical specimens of human esophageal carcinoma tissues by RT‐PCR and immunohistochemistry. Immunohistochemistry using antibodies against CD34 (endothelial cell specific) was also carried out and microvessels were quantified by counting vessels in a 200× field in the most vascular area of the tumor. Of the 5 human esophageal carcinoma cell lines, 4 constitutively expressed VEGF‐C mRNA. In 8 (66.7%) of 12 cases, VEGF‐C mRNA was detected in only tumor tissues but not in normal mucosa by RT‐PCR. There was a significant relationship between VEGF‐C and flt‐4 mRNA expression. Out of the 48 surgical specimens of esophageal carcinomas, 19 (39.6%) and 10 (20.8%) exhibited intense VEGF‐C immunoreactivity in the cytoplasm of many cancer cells and the stromal cells, respectively. In contrast, Flt‐4 was mainly expressed on the lymphatic endothelial cells. Normal and dysplastic esophageal squamous epithelium exhibited no or faint cytoplasmic staining of VEGF‐C. VEGF‐C expression correlated with depth of tumor invasion, tumor stage, venous invasion, lymphatic invasion and lymph node metastasis. Vessel count was significantly higher in the VEGF‐C positive tumors than in the negative tumors. These results overall suggest that VEGF‐C may play a role in tumor progression via lymphangiogenesis and angiogenesis in human esophageal carcinoma.

Co‐expression of osteopontin and CD44v9 in gastric cancer

We have examined the expression of osteopontin (OPN) in 40 human primary gastric carcinoma tissues, 5 metastatic foci (lymph nodes) and corresponding normal mucosas. Twenty‐nine of 40 primary tumors (72.5%) and 3 of 5 lymph node metastases (60%) overexpressed OPN mRNA in comparison with those of the corresponding normal mucosa. The incidence as well as relative expression level of OPN mRNA was higher in well differentiated gastric cancers than poorly differentiated ones. Moreover, increased OPN mRNA expression in primary tumor specimens was observed along with the advancement of the clinico‐pathological stage. Using in situ hybridization (ISH) analysis, not only inflammatory cells in tumor stroma but also tumor cells showed positive signals for OPN mRNA. By immunohistochemistry, co‐immunoreaction of OPN and CD44v9 in tumor cells obviously correlated with the degree of lymphatic vessel invasion or long distant lymph node metastases in poorly differentiated gastric cancer. Interestingly, strong co‐immunoreaction of OPN and CD44v9 of tumor cells was concommitant with cluster formation in the lymphatic vessels. Our results suggest that overexpression of OPN correlated with the progression of human gastric carcinoma. Especially in CD44‐bearing poorly differentiated gastric cancer, interaction between OPN and CD44 may parallel lymphogenous metastasis. Int. J. Cancer (Pred. Oncol.) 79:127–132, 1998.© 1998 Wiley‐Liss, Inc.

Wnt5a signaling is involved in the aggressiveness of prostate cancer and expression of metalloproteinase

Wnt5a is a representative ligand that activates the β-catenin-independent pathway in Wnt signaling. Although it has been reported that abnormal activation of the Wnt/β-catenin-dependent pathway is often observed in human prostate cancer, the involvement of the β-catenin-independent pathway in this cancer is unclear. Abnormal expression of Wnt5a and β-catenin was observed in 27 (28%) and 49 (50%) of 98 prostate cancer cases, respectively, by immunohistochemical analyses. Simultaneous expression of Wnt5a and β-catenin was observed in only five cases, suggesting their exclusive expression. The positive detection of Wnt5a was correlated with high Gleason scores and biochemical relapse of prostate cancer, but that of β-catenin was not. Knockdown and overexpression of Wnt5a in human prostate cancer cell lines reduced and stimulated, respectively, their invasion activities, and the invasion activity required Frizzled2 and Ror2 as Wnt receptors. Wnt5a activated Jun-N-terminal kinase through protein kinase D (PKD) and the inhibition of PKD suppressed Wnt5a-dependent cell migration and invasion. In addition, Wnt5a induced the expression of metalloproteinase-1 through the recruitment of JunD to its promoter region. These results suggest that Wnt5a promotes the aggressiveness of prostate cancer and that its expression is involved in relapse after prostatectomy.