Naoki Hijiya

Overexpression of miR‐210, a downstream target of HIF1α, causes centrosome amplification in renal carcinoma cells

MiR‐210 is significantly up‐regulated in clear cell renal cell carcinoma (CCC), but the mechanism and biological consequences of miR‐210 up‐regulation are poorly understood. Here, we show that miR‐210 is highly expressed in renal carcinoma cell lines and that its expression is clearly correlated with accumulation of hypoxia‐inducible factor 1α (HIF1α) under normoxia as well as hypoxia, suggesting that miR‐210 up‐regulation in renal carcinoma cells is most likely due to accumulation of HIF1α. To reveal the effects of miR‐210 up‐regulation, the miR‐210 precursor was transfected into renal carcinoma cells. After transfection, the cells accumulated at the G2/M phase of the cell cycle and their viability was decreased, suggesting that miR‐210 overexpression may trigger an event that hinders normal cell division. Immunocytochemistry demonstrated a multipolar spindle accompanied by centrosome amplification in cells overexpressing miR‐210. It has been reported that centrosome amplification induces chromosome mis‐segregation, finally leading to chromosome instability and aneuploidy. Indeed, the proportion of aneuploid cells (>4n) was increased in miR‐210 overexpressed cells. By using the TargetScan and PicTar algorithms, E2F3 was identified as one of the possible targets of miR‐210 and was suppressed at the protein level by miR‐210. Moreover, the proportion of aneuploid cells was increased in E2F3 siRNA transfected cells. On the basis of these results, we propose that miR‐210 up‐regulation due to HIF1α accumulation may induce aneuploidy via E2F3 down‐regulation at least in part, and may play a role in tumourigenesis and/or progression of CCC. Copyright

MiR-29c is downregulated in gastric carcinomas and regulates cell proliferation by targeting RCC2

BackgroundPreviously, using miRNA microarray, we have found that miR-29c is significantly downregulated in advanced gastric carcinoma. In the present study, we investigated whether miR-29c functions as a tumor-suppressor miRNA in gastric carcinoma cells. For this purpose, we verified the downregulation of miR-29c in gastric carcinoma tissues, and assessed the biological effect of miR-29c on gastric carcinoma cells.ResultsIn miR-29c-transfected cells, both proliferation and colony formation ability on soft agar were significantly decreased. Although apoptosis was not induced, BrdU incorporation and the proportion of cells positive for phospho-histone H3 (S10) were significantly decreased in miR-29c-transfected cells, indicating that miR-29c may be involved in the regulation of cell proliferation. To explain the mechanism of growth suppression by miR-29c, we explored differentially expressed genes (>2-fold) in miR-29c-transfected cells in comparison with negative control transfected cells using microarray. RCC2, PPIC and CDK6 were commonly downregulated in miR-29c-transfected MKN45, MKN7 and MKN74 cells, and all of the genes harbored miR-29c target sequences in the 3’-UTR of their mRNA. RCC2 and PPIC were actually upregulated in gastric carcinoma tissues, and therefore both were identified as possible targets of miR-29c in gastric carcinoma. To ascertain whether downregulation of RCC2 and/or PPIC is involved in the growth suppression by miR-29c, we transfected siRNAs against RCC2 and PPIC into MKN45 and determined cell viability, the rate of BrdU incorporation, and caspase activity. We found that RCC2-knockdown decreased both cell viability and BrdU incorporation without any increase of caspase activity, while PPIC-knockdown did not, indicating that downregulation of RCC2 may be at least partly responsible for the growth suppression by miR-29c.ConclusionsOur findings indicate that miR-29c may have tumor-suppressive functions in gastric carcinoma cells, and that its decreased expression may confer a growth advantage on tumor cells via aberrant expression of RCC2.

Analysis of p53 Mutations and the Expression of p53 and p21WAF1/CIP1 Protein in 15 Cases of Sebaceous Carcinoma of the Eyelid

PURPOSEnThe purpose of this study was to detect mutation of the p53 gene, to assess its relationship with p53 or p21(WAF1/CIP1) expression, and to evaluate the correlation between p53 mutation or p21(WAF1/CIP1) expression and clinicopathologic findings in sebaceous carcinoma of the eyelid.nnnMETHODSnFifteen conventional paraffin-embedded samples of sebaceous carcinoma of the eyelid were analyzed. Using the single-strand conformation polymorphism technique, the authors sequenced coding exons 5-8 of the p53 gene. The expression of p53 and p21(WAF1/CIP1) protein was analyzed by immunohistochemistry.nnnRESULTSnIn 10 of the 15 cases (66.7%), point mutations were detected in the p53 gene. CC to TT double-base changes (tandem mutations), which are known to be induced only by UV, were not detected in any of the mutations. Correlations between p53 mutation and expression were found to be statistically significant (P = 0.007). There was no significant correlation between p53 mutation and clinicopathologic findings or p21(WAF1/CIP1) expression. However, there was a significant inverse correlation between p21(WAF1/CIP1) expression and presence of lymph node metastasis (P = 0.007).nnnCONCLUSIONSnAmong human cancers, sebaceous carcinoma of the eyelid may be one of those showing most frequent mutation of the p53 gene, which may not be caused by exposure to UV. p21(WAF1/CIP1) downregulation may be associated with lymph node metastasis.

Downregulation of SAV1 plays a role in pathogenesis of high-grade clear cell renal cell carcinoma

BackgroundClinical outcome of patients with high-grade ccRCC (clear cell renal cell carcinoma) remains still poor despite recent advances in treatment strategies. Molecular mechanism of pathogenesis in developing high-grade ccRCC must be clarified. In the present study, we found that SAV1 was significantly downregulated with copy number loss in high-grade ccRCCs. Therefore, we investigated the SAV1 function on cell proliferation and apoptosis in vitro. Furthermore, we attempted to clarify the downstream signaling which is regulated by SAV1.MethodsWe performed array CGH and gene expression analysis of 8 RCC cell lines (786-O, 769-P, KMRC-1, KMRC-2, KMRC-3, KMRC-20, TUHR4TKB, and Caki-2), and expression level of mRNA was confirmed by quantitative RT-PCR (qRT-PCR) analysis. We next re-expressed SAV1 in 786-O cells, and analyzed its colony-forming activity. Then, we transfected siRNAs of SAV1 into the kidney epithelial cell line HK2 and renal proximal tubule epithelial cells (RPTECs), and analyzed their proliferation and apoptosis. Furthermore, the activity of YAP1, which is a downstream molecule of SAV1, was evaluated by western blot analysis, reporter assay and immunohistochemical analysis.ResultsWe found that SAV1, a component of the Hippo pathway, is frequently downregulated in high-grade ccRCC. SAV1 is located on chromosome 14q22.1, where copy number loss had been observed in 7 of 12 high-grade ccRCCs in our previous study, suggesting that gene copy number loss is responsible for the downregulation of SAV1. Colony-forming activity by 786-O cells, which show homozygous loss of SAV1, was significantly reduced when SAV1 was re-introduced exogenously. Knockdown of SAV1 promoted proliferation of HK2 and RPTEC. Although the phosphorylation level of YAP1 was low in 786-O cells, it was elevated in SAV1-transduced 786-O cells. Furthermore, the transcriptional activity of the YAP1 and TEAD3 complex was inhibited in SAV1-transduced 786-O cells. Immunohistochemistry frequently demonstrated nuclear localization of YAP1 in ccRCC cases with SAV1 downregulation, and it was preferentially detected in high-grade ccRCC.ConclusionsTaken together, downregulation of SAV1 and the consequent YAP1 activation are involved in the pathogenesis of high-grade ccRCC. It is an attractive hypothesis that Hippo signaling could be candidates for new therapeutic target.

Genomic profiling of gastric carcinoma in situ and adenomas by array-based comparative genomic hybridization†

Although genomic copy number aberrations (CNAs) of gastric carcinoma at the advanced stage have already been extensively characterized by array comparative genomic hybridization (array CGH) analysis, those of gastric carcinoma in situ (CIS) are still poorly understood. Furthermore, no reports have demonstrated correlations between CNAs and histopathological features of gastric adenoma. In this study, we investigated CNAs of 20 gastric CISs (Vienna category 4.2) and 20 adenomas including seven low‐grade adenomas (LGA; Vienna category 3) and 13 high‐grade adenomas (HGA; Vienna category 4.1), using oligonucleotide‐based array CGH. The most frequent aberrations in CIS were gains at 8q (85%) and 20q (50%), and losses at 5q (50%) and 17p (50%), suggesting that these CNAs are involved in the development of CIS. We found that the pattern of CNAs in HGA was quite different from that in LGA. The most frequent CNAs in HGA were gains at 8q (62%) and 7pq (54%), whereas those in LGA were gain at 7q21.3‐q22.1 (57%) and loss at 5q (43%). Interestingly, gains at 8q and 7pq, both of which occurred most frequently in HGA, were not detected in any cases of LGA. Of note, 8q gain was detected most frequently in both HGA and CIS but was undetected in LGA. Since HGA is believed to have a higher risk of progression to invasive carcinoma than LGA, these data suggest that 8q gain is important for the malignant transformation of gastric adenoma. Copyright

Genomic profiling of renal cell carcinoma in patients with end‐stage renal disease

The purpose of the present study was to determine the genomic profile of renal cell carcinoma (RCC) in end‐stage renal disease (ESRD) by analyzing genomic copy number aberrations. Seventy‐nine tumor samples from 63 patients with RCC‐ESRD were analyzed by array comparative genomic hybridization using the Agilent Whole Human Genome 4u2003×u200344K Oligo Micro Array (Agilent Technologies Inc., Palo Alto, CA, USA). Unsupervised hierarchical clustering analysis revealed that the 63 cases could be divided into two groups, Clusters A and B. Cluster A was comprised mainly of clear cell RCC (CCRCC), whereas Cluster B was comprised mainly of papillary RCC (PRCC), acquired cystic disease (ACD)‐associated RCC, and clear cell papillary RCC. Analysis of the averaged frequencies revealed that the genomic profiles of Clusters A and B resembled those of sporadic CCRCC and sporadic PRCC, respectively. Although it has been proposed on the basis of histopathology that ACD‐associated RCC, clear cell papillary RCC and PRCC‐ESRD are distinct subtypes, the present data reveal that the genomic profiles of these types, categorized as Cluster B, resemble one another. Furthermore, the genomic profiles of PRCC, ACD‐associated RCC and clear cell papillary RCC admixed in one tissue tended to resemble one another. On the basis of genomic profiling of RCC‐ESRD, we conclude that the molecular pathogenesis of CCRCC‐ESRD resembles that of sporadic CCRCC. Although various histologic subtypes of non‐clear cell RCC‐ESRD have been proposed, their genomic profiles resemble those of sporadic PRCC, suggesting that the molecular pathogenesis of non‐CCRCC‐ESRD may be related to that of sporadic PRCC. (Cancer Sci 2012; 103: 569–576)

Genomic Profiling of Oral Squamous Cell Carcinoma by Array-Based Comparative Genomic Hybridization

We designed a study to investigate genetic relationships between primary tumors of oral squamous cell carcinoma (OSCC) and their lymph node metastases, and to identify genomic copy number aberrations (CNAs) related to lymph node metastasis. For this purpose, we collected a total of 42 tumor samples from 25 patients and analyzed their genomic profiles by array-based comparative genomic hybridization. We then compared the genetic profiles of metastatic primary tumors (MPTs) with their paired lymph node metastases (LNMs), and also those of LNMs with non-metastatic primary tumors (NMPTs). Firstly, we found that although there were some distinctive differences in the patterns of genomic profiles between MPTs and their paired LNMs, the paired samples shared similar genomic aberration patterns in each case. Unsupervised hierarchical clustering analysis grouped together 12 of the 15 MPT-LNM pairs. Furthermore, similarity scores between paired samples were significantly higher than those between non-paired samples. These results suggested that MPTs and their paired LNMs are composed predominantly of genetically clonal tumor cells, while minor populations with different CNAs may also exist in metastatic OSCCs. Secondly, to identify CNAs related to lymph node metastasis, we compared CNAs between grouped samples of MPTs and LNMs, but were unable to find any CNAs that were more common in LNMs. Finally, we hypothesized that subpopulations carrying metastasis-related CNAs might be present in both the MPT and LNM. Accordingly, we compared CNAs between NMPTs and LNMs, and found that gains of 7p, 8q and 17q were more common in the latter than in the former, suggesting that these CNAs may be involved in lymph node metastasis of OSCC. In conclusion, our data suggest that in OSCCs showing metastasis, the primary and metastatic tumors share similar genomic profiles, and that cells in the primary tumor may tend to metastasize after acquiring metastasis-associated CNAs.

Intraductal papillary mucinous neoplasms of the pancreas complicated with intraductal hemorrhage, perforation, and fistula formation: CT and MR imaging findings with pathologic correlation.

ObjectiveTo correlate the CT and MR images with pathologic findings on intraductal papillary mucinous neoplasms (IPMNs) complicated with intraductal hemorrhage, perforation, and fistula.Materials and methodsWe retrospectively evaluated the CT (nxa0=xa05), MR imaging (nxa0=xa04), and pathological features of five IPMN patients complicated with intraductal hemorrhage (nxa0=xa05), perforation (nxa0=xa01), and fistula into the duodenum and jejunum (nxa0=xa01).ResultsIntraductal hemorrhage could be detected as high attenuation on non-contrast CT in two of the five cases, and as high signal intensity on fat-suppressed T1-weighted MR images in all four of the cases. Perforation and fistula could be recognized on CT images. In all IPMNs, denuded epitheliums were observed pathologically. Dissolution of the duct wall and extension of mucinous materials were seen at the area of denuded epithelium. Perforations and fistula, without evidence of cancer invasion, were recognized in the dissolved duct wall. Pathogenesis of the perforations and fistula formations appeared to be related to excessive pressure in the dilated ducts and autodigestion of enzyme-rich fluids.ConclusionComplications with IPMN could be recognized on CT and fat-suppressed T1-weighted MR images. Intraductal hemorrhage might be predictive sign of perforation and fistula formation.

Induction of GNE in myofibers after muscle injury.

Objective: The aim of the present study was to clarify the expression of uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) protein and mRNA in damaged or regenerating myofibers. Methods: We investigated the muscle expression pattern of GNE protein by immunohistochemistry using a murine model involving intramuscular injection of cardiotoxin (CTX), and the expression level of GNE mRNA by quantitative real-time polymerase chain reaction analysis of damaged or regenerating myofibers that had been collected directly from tissue sections using laser-capture microdissection. Results: The expression of GNE protein was increased in severely damaged myofibers as well as in regenerating myofibers with central nuclei, both of which also showed an increase in the expression of GNE mRNA. In regenerating myofibers, immunoreactivity for GNE protein in nuclei relative to that in the cytoplasm was higher at 7 days than at 4 days after CTX injection. Conclusion: Our findings suggest that GNE expression is induced when myofibers are damaged or regenerating, and that GNE plays a role in muscle regeneration.

Visinin-like protein-1 overexpression is an indicator of lymph node metastasis and poor prognosis in colorectal cancer patients

Lymph node metastasis is an important factor determining outcome from colorectal cancer (CRC). Identification of molecular markers useful to predict lymph node metastasis is urgently needed. Our objective was to identify genes useful for characterization and prediction of lymph node metastasis in CRC. Gene expression profiles of cancer were determined by human U133 Plus 2.0 GeneChip® in 24 CRC patients, and patients with and without lymph node metastasis were compared. We focused on the visinin‐like protein‐1 (VSNL‐1) gene and evaluated VSNL‐1 mRNA expression levels with reverse transcriptase‐polymerase chain reaction and immunohistochemical methods. Immunohistochemical evaluation of VSNL‐1 mRNA expression was also performed in 143 other CRC patients to determine clinicopathological significance of VSNL‐1. Twenty‐four novel discriminating genes showed expression significantly different between patients with and without lymph node metastasis. Mean level of VSNL‐1 mRNA expression in tumor tissue with lymph node metastasis was significantly higher than that in tissue without lymph node metastasis. Immunohistochemical examination demonstrated immunoreactivity for VSNL‐1 in cytoplasm of the cancer cells with lymph node metastasis. High VSNL‐1 expression was significantly associated with lymphatic invasion in stage II disease (p = 0.0061) and number of lymph node metastases in stage III disease (p = 0.0461). Patients with high VSNL‐1 expression had significantly poorer prognosis than those with low expression in stage III disease (p = 0.045). This study is the first to demonstrate a prognostic role for VSNL‐1 at the mRNA level, suggesting the possible usefulness of VSNL‐1 as a predictor of lymph node metastasis and poor prognosis in CRC.