Nozomu Yanaihara

MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma.

CONTEXT MicroRNAs have potential as diagnostic biomarkers and therapeutic targets in cancer. No study has evaluated the association between microRNA expression patterns and colon cancer prognosis or therapeutic outcome. OBJECTIVE To identify microRNA expression patterns associated with colon adenocarcinomas, prognosis, or therapeutic outcome. DESIGN, SETTING, AND PATIENTS MicroRNA microarray expression profiling of tumors and paired nontumorous tissues was performed on a US test cohort of 84 patients with incident colon adenocarcinoma, recruited between 1993 and 2002. We evaluated associations with tumor status, TNM staging, survival prognosis, and response to adjuvant chemotherapy. Associations were validated in a second, independent Chinese cohort of 113 patients recruited between 1991 and 2000, using quantitative reverse transcription polymerase chain reaction assays. The final date of follow-up was December 31, 2005, for the Maryland cohort and August 16, 2004, for the Hong Kong cohort. MAIN OUTCOME MEASURES MicroRNAs that were differentially expressed in tumors and microRNA expression patterns associated with survival using cancer-specific death as the end point. RESULTS Thirty-seven microRNAs were differentially expressed in tumors from the test cohort. Selected for validation were miR-20a, miR-21, miR-106a, miR-181b, and miR-203, and all 5 were enriched in tumors from the validation cohort (P < .001). Higher miR-21 expression was present in adenomas (P = .006) and in tumors with more advanced TNM staging (P < .001). In situ hybridization demonstrated miR-21 to be expressed at high levels in colonic carcinoma cells. The 5-year cancer-specific survival rate was 57.5% for the Maryland cohort and was 49.5% for the Hong Kong cohort. High miR-21 expression was associated with poor survival in both the training (hazard ratio, 2.5; 95% confidence interval, 1.2-5.2) and validation cohorts (hazard ratio, 2.4; 95% confidence interval, 1.4-3.9), independent of clinical covariates, including TNM staging, and was associated with a poor therapeutic outcome. CONCLUSIONS Expression patterns of microRNAs are systematically altered in colon adenocarcinomas. High miR-21 expression is associated with poor survival and poor therapeutic outcome.

MicroRNA expression in squamous cell carcinoma and adenocarcinoma of the esophagus: Associations with survival

Purpose: The dismal outcome of esophageal cancer patients highlights the need for novel prognostic biomarkers, such as microRNAs (miRNA). Although recent studies have established the role of miRNAs in esophageal carcinoma, a comprehensive multicenter study investigating different histologic types, including squamous cell carcinoma (SCC) and adenocarcinoma with or without Barretts, is still lacking. Experimental Design: miRNA expression was measured in cancerous and adjacent noncancerous tissue pairs collected from 100 adenocarcinoma and 70 SCC patients enrolled at four clinical centers from the United States, Canada, and Japan. Microarray-based expression was measured in a subset of samples in two cohorts and was validated in all available samples. Results: In adenocarcinoma patients, miR-21, miR-223, miR-192, and miR-194 expression was elevated, whereas miR-203 expression was reduced in cancerous compared with noncancerous tissue. In SCC patients, we found elevated miR-21 and reduced miR-375 expression levels in cancerous compared with noncancerous tissue. When comparing cancerous tissue expression between adenocarcinoma and SCC patients, miR-194 and miR-375 were elevated in adenocarcinoma patients. Significantly, elevated miR-21 expression in noncancerous tissue of SCC patients and reduced levels of miR-375 in cancerous tissue of adenocarcinoma patients with Barretts were strongly associated with worse prognosis. Associations with prognosis were independent of tumor stage or nodal status, cohort type, and chemoradiation therapy. Conclusions: Our multicenter-based results highlight miRNAs involved in major histologic types of esophageal carcinoma and uncover significant associations with prognosis. Elucidating miRNAs relevant to esophageal carcinogenesis is potentially clinically useful for developing prognostic biomarkers and identifying novel drug targets and therapies. (Clin Cancer Res 2009;15(19):6192–200)

MYO18B, a candidate tumor suppressor gene at chromosome 22q12.1, deleted, mutated, and methylated in human lung cancer

Loss of heterozygosity on chromosome 22q has been detected in approximately 60% of advanced nonsmall cell lung carcinoma (NSCLC) as well as small cell lung carcinoma (SCLC), suggesting the presence of a tumor suppressor gene on 22q that is involved in lung cancer progression. Here, we isolated a myosin family gene, MYO18B, located at chromosome 22q12.1 and found that it is frequently deleted, mutated, and hypermethylated in lung cancers. Somatic MYO18B mutations were detected in 19% (14/75) of lung cancer cell lines and 13% (6/46) of primary lung cancers of both SCLC and NSCLC types. MYO18B expression was reduced in 88% (30/34) of NSCLC and 47% (8/17) of SCLC cell lines. Its expression was restored by treatment with 5-aza-2′-deoxycytidine in 11 of 14 cell lines with reduced MYO18B expression, and the promoter CpG island of the MYO18B gene was methylated in 17% (8/47) of lung cancer cell lines and 35% (14/40) of primary lung cancers. Furthermore, restoration of MYO18B expression in lung carcinoma cells suppressed anchorage-independent growth. These results indicate that the MYO18B gene is a strong candidate for a novel tumor suppressor gene whose inactivation is involved in lung cancer progression.

Copy Number Analysis Identifies Novel Interactions Between Genomic Loci in Ovarian Cancer

Ovarian cancer is a heterogeneous disease displaying complex genomic alterations, and consequently, it has been difficult to determine the most relevant copy number alterations with the scale of studies to date. We obtained genome-wide copy number alteration (CNA) data from four different SNP array platforms, with a final data set of 398 ovarian tumours, mostly of the serous histological subtype. Frequent CNA aberrations targeted many thousands of genes. However, high-level amplicons and homozygous deletions enabled filtering of this list to the most relevant. The large data set enabled refinement of minimal regions and identification of rare amplicons such as at 1p34 and 20q11. We performed a novel co-occurrence analysis to assess cooperation and exclusivity of CNAs and analysed their relationship to patient outcome. Positive associations were identified between gains on 19 and 20q, gain of 20q and loss of X, and between several regions of loss, particularly 17q. We found weak correlations of CNA at genomic loci such as 19q12 with clinical outcome. We also assessed genomic instability measures and found a correlation of the number of higher amplitude gains with poorer overall survival. By assembling the largest collection of ovarian copy number data to date, we have been able to identify the most frequent aberrations and their interactions.

Reduced expression of MYO18B, a candidate tumor-suppressor gene on chromosome arm 22q, in ovarian cancer.

Allelic imbalance on chromosome arm 22q has been detected in 50–70% of ovarian cancers, suggesting the presence of a tumor‐suppressor gene on this chromosome arm that is involved in ovarian carcinogenesis. Recently, we isolated a candidate tumor‐suppressor gene, MYO18B, at 22q12.1, which is deleted, mutated and hypermethylated in approximately 50% of lung cancers. In our study, we analyzed genetic and epigenetic alterations of the MYO18B gene in ovarian cancers. Missense MYO18B mutations were detected in 1 of 4 (25%) ovarian cancer cell lines and in 1 of 17 (5.9%) primary ovarian cancers. MYO18B expression was reduced in all 4 ovarian cancer cell lines and in 12 of 17 (71%) of primary ovarian cancers. MYO18B expression was restored by treatment with 5‐aza‐2′‐deoxycytidine and/or trichostatin A in 3 of 4 cell lines with reduced MYO18B expression, and hypermethylation of the promoter CpG island for MYO18B was observed in 2 of these 3 cell lines. Its hypermethylation was also observed in 2 of 15 (13%) primary ovarian cancers. Thus, it was indicated that MYO18B expression is reduced in a considerable fraction of ovarian cancers by several mechanisms, including hypermethylation, while the MYO18B gene is mutated in a small subset of ovarian cancers. The present results suggest that MYO18B alterations, including both epigenetic and genetic alterations, play an important role in ovarian carcinogenesis.

ING2 is upregulated in colon cancer and increases invasion by enhanced MMP13 expression

Inhibitor of growth 2 (ING2) is associated with chromatin remodeling and regulation of gene expression by binding to a methylated histone H3K4 residue and recruiting HDAC complexes to the region. The aim of our study is to investigate the regulation of ING2 expression and the clinical significance of upregulated ING2 in colon cancer. Here, we show that the ING2 mRNA level in colon cancer tissue increased to more than twice than that in normal mucosa in the 45% of colorectal cancer cases that we examined. A putative NF‐κB binding site was found in the ING2 promoter region. We confirmed that NF‐κB could bind to the ING2 promoter by EMSA and luciferase assays. Subsequent microarray analyses revealed that ING2 upregulates expression of matrix metalloproteinase 13 (MMP13), which enhances cancer invasion and metastasis. ING2 regulation of MMP13 expression was confirmed in both ING2 overexpression and knock down experiments. MMP13 expression was further induced by coexpression of ING2 with HDAC1 or with mSin3A, suggesting that the ING2‐HDAC1‐mSin3A complex members regulates expression of MMP13. In vitro invasion assay was performed to determine functional significance of ING2 upregulation. ING2 overexpressed cells exhibited greater invasive potential. Taken together, upregulation of ING2 was associated with colon cancer and MMP13‐dependent cellular invasion, indicating that ING2 expression might be involved with cancer invasion and metastasis. Published 2009 UICC.

Genomic consequences of aberrant DNA repair mechanisms stratify ovarian cancer histotypes

We studied the whole-genome point mutation and structural variation patterns of 133 tumors (59 high-grade serous (HGSC), 35 clear cell (CCOC), 29 endometrioid (ENOC), and 10 adult granulosa cell (GCT)) as a substrate for class discovery in ovarian cancer. Ab initio clustering of integrated point mutation and structural variation signatures identified seven subgroups both between and within histotypes. Prevalence of foldback inversions identified a prognostically significant HGSC group associated with inferior survival. This finding was recapitulated in two independent cohorts (n = 576 cases), transcending BRCA1 and BRCA2 mutation and gene expression features of HGSC. CCOC cancers grouped according to APOBEC deamination (26%) and age-related mutational signatures (40%). ENOCs were divided by cases with microsatellite instability (28%), with a distinct mismatch-repair mutation signature. Taken together, our work establishes the potency of the somatic genome, reflective of diverse DNA repair deficiencies, to stratify ovarian cancers into distinct biological strata within the major histotypes.

Cytokine gene expression signature in ovarian clear cell carcinoma

Cytokine expression in a tumor microenvironment can impact both host defense against the tumor and tumor cell survival. In this study, we sought to clarify whether the cytokine gene expression profile could have clinical associations with ovarian cancer. We analyzed the expression of 16 cytokine genes (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-γ, TNF-α, IL-6, HLA-DRA, HLA-DPA1 and CSF1) in 50 ovarian carcinomas. Hierarchical clustering analysis of these tumors was carried out using Cluster software and differentially expressed genes were examined between clear cell carcinoma (CCC) and other subtypes. Following this examination we evaluated the biological significance of IL-6 knockdown in CCC. Unsupervised hierarchical clustering analysis of cytokine gene expression revealed two distinct clusters. The relationship between the two clusters and clinical parameters showed statistically significant differences in CCC compared to other histologies. CCC showed a dominant Th-2 cytokine expression pattern driven largely by IL-6 expression. Inhibition of IL-6 in CCC cells suppressed Stat3 signaling and rendered cells sensitive to cytotoxic agents. The unique cytokine expression pattern found in CCC may be involved in the pathogenesis of this subtype. In particular, high IL-6 expression appears likely to be driven by the tumor cells, fueling an autocrine pathway involving IL-6 expression and Stat3 activation and may influence survival when exposed to cytotoxic chemotherapy. Modulation of IL-6 expression or its related signaling pathway may be a promising strategy of treatment for CCC.

Hypoxia promotes glycogen synthesis and accumulation in human ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) has several significant characteristics based on molecular features that are distinct from those of ovarian high-grade serous carcinoma. Cellular glycogen accumulation is the most conspicuous feature of OCCC and in the present study its metabolic mechanism was investigated. The amount of glycogen in cells cultured under hypoxia increased significantly and approximately doubled after 48 h (P<0.01) compared to that under normoxic conditions. Periodic acid-Schiff positive staining also demonstrated intracellular glycogen storage. Western blot analysis revealed that HIF1α, which was overexpressed and stabilized under hypoxic conditions, led to an increase in the levels of cellular glycogen synthase 1, muscle type (GYS1), and conversely to a decrease in inactive phosphorylated GYS1 at serine (Ser) 641. Additional increases were observed in both protein phosphatase 1, which dephosphorylates and thereby induces GYS1 enzyme activity, and glycogen synthase kinase 3 beta (GSK3β) phosphorylated at Ser9, which is inactive on phosphorylation of GYS1 and subsequently induces its enzyme activity. By contrast, the level of PYGM-b decreased. These results indicated that the glycogen accumulation under a hypoxic environment resulted in the promotion of glycogen synthesis, but did not lead to inhibition of glycogen degradation and/or consumption. Under hypoxic conditions, HAC2 cells showed activation of the PI3K/AKT pathway caused by a mutation in exon 20 of PIK3CA, encoding the catalytic subunit p110α of PI3K. The resulting activation of AKT (phosphoSer473) also plays a role as a central enhancer in glycogen synthesis through suppression of GSK3β via phosphorylation at Ser9. Hypoxia decreased the cytocidal activity of cisplatin and doxorubicin to various degrees. In conclusion, the hypoxic conditions together with HIF1 expression and stabilization increased the intracellular glycogen contents and resistance to the anticancer drugs.

DYRK2 regulates epithelial-mesenchymal-transition and chemosensitivity through Snail degradation in ovarian serous adenocarcinoma

Epithelial-mesenchymal-transition (EMT) plays essential roles in ovarian cancer invasion, metastasis, and drug resistance. A hallmark of EMT is the loss of E-cadherin, which is regulated by Snail. Recently, it was shown that dual-specificity tyrosine-regulated kinase 2 (DYRK2) controls Snail degradation in breast cancer. The aim of this study is to clarify whether DYRK2 regulates EMT through Snail degradation in ovarian serous adenocarcinoma (SA). Expression of DYRK2 and Snail in two pairs of cisplatin-resistant and the original cisplatin-sensitive ovarian cancer cell line were analyzed by immunoblotting and real-time RT-PCR analysis. Morphological change, invasion ability, and chemosensitivity were evaluated by using DYRK2 stable knockdown cell line in 2008 (2008 shDYRK2). Immunohistochemical analyses for DYRK2 and Snail were performed with surgical specimens. The correlations between the expression of these proteins and the clinicopathological parameters, including prognosis, were determined. Moreover, we conducted a hypodermic administration test in nude mice and examined reproductive and cisplatin response activities. DYRK2 protein expression was posttranslationally reduced in cisplatin-resistant SA cell lines. 2008 shDYRK2 showed mesenchymal phenotype and resistant to cisplatin. Immunohistochemistry demonstrated that DYRK2 expression inversely correlated with Snail expression, and reduced expression of DYRK2 was associated with shorter overall survival in SA. DYRK2 may regulate EMT through Snail degradation in ovarian SA and might be a predictive marker for a favorable prognosis in the treatment of this cancer.