PRDM14 promotes malignant phenotype and correlates with poor prognosis in colorectal cancer.

BACKGROUND: Emerging evidence suggests that stemness in cancer cells is a cause of drug resistance or metastasis and is an important therapeutic target. PR [positive regulatory domain I-binding factor 1 (PRDI-BF1) and retinoblastoma protein-interacting zinc finger gene (RIZ1)] domain containing 14 (PRDM14), that regulates pluripotency in primordial germ cell, has reported the overexpression and function of stemness in various malignancies, suggesting it as the possible therapeutic target. However, to our knowledge, there have been no reports on the expression and function of PRDM14 in colorectal cancer (CRC). Therefore, we investigated the expression and the role of PRDM14 in CRC. METHODS: We performed immunohistochemistry evaluations and assessed PRDM14 expression on 414 primary CRC specimens. Colon cancer cell lines were subjected to functional and stemness assays in vitro and in vivo. RESULTS: We found that PRDM14 positive staining exhibited heterogeneity in the CRC primary tumor, especially at the tumor invasion front. The aberrant expression of PRDM14 at the invasion front was associated with lymph node metastasis and disease stage in patients with CRC. Furthermore, the multivariate analysis revealed high PRDM14 expression as an independent prognostic factor in the patients with Stage III CRC. Overexpression of PRDM14 enhanced the invasive, drug-resistant and stem-like properties in colon cancer cells in vitro and tumorigenicity in vivo. CONCLUSION: Our findings suggest that PRDM14 is involved in progression and chemoresistance of CRC, and is a potential prognostic biomarker and therapeutic target in the CRC patients.

Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer.

BACKGROUND: LINE-1 methylation level is a surrogate marker of global DNA methylation. LINE-1 methylation in primary colorectal cancers (CRCs) is highly variable and strongly associated with a poor prognosis. However, no study has examined LINE-1 methylation levels of metastatic CRCs in relation to prognosis or assessed the heterogeneity of LINE-1 methylation level within the primary CRCs. METHODS: Pyrosequencing was used to quantify LINE-1 methylation level in 42 liver metastases, 26 matched primary tumours, and 6 matched lymph node (LN) metastases. KRAS, BRAF, and PIK3CA mutation status and microsatellite instability (MSI) status were also examined. RESULTS: The distribution of LINE-1 methylation level in liver metastases was as follows: mean, 67.3; range, 37.1-90.1. Primary tumours showed LINE-1 methylation levels similar to those of matched liver and LN metastases. The difference in LINE-1 methylation level between superficial areas and invasive front areas was within 7.0 in all six cases evaluated. Prognostic impact of LINE-1 hypomethylation in liver metastases on overall survival was not observed. The concordance rate was 94% for KRAS, 100% for BRAF, 88% for PIK3CA, and 97% for MSI. CONCLUSION: Alteration of LINE-1 methylation level may occur in early CRC tumorigenesis, and the LINE-1 methylation level is relatively stable during CRC progression.

Prognostic significance of AMP-activated protein kinase expression and modifying effect of MAPK3/1 in colorectal cancer.

BACKGROUND: AMP-activated protein kinase (AMPK, PRKA) has central roles in cellular metabolic sensing and energy balance homeostasis, and interacts with various pathways (e.g., TP53 (p53), FASN, MTOR and MAPK3/1 (ERK)). AMP-activated protein kinase activation is cytotoxic to cancer cells, supporting AMPK as a tumour suppressor and a potential therapeutic target. However, no study has examined its prognostic role in colorectal cancers. METHODS: Among 718 colon and rectal cancers, phosphorylated AMPK (p-AMPK) and p-MAPK3/1 expression was detected in 409 and 202 tumours, respectively, by immunohistochemistry. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for clinical and tumoral features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. RESULTS: Phosphorylated AMPK expression was not associated with survival among all patients. Notably, prognostic effect of p-AMPK significantly differed by p-MAPK3/1 status (P(interaction)=0.0017). Phosphorylated AMPK expression was associated with superior colorectal cancer-specific survival (adjusted HR 0.42; 95% confidence interval (CI), 0.24-0.74) among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases (adjusted HR 1.22; 95% CI: 0.85-1.75). CONCLUSION: Phosphorylated AMPK expression in colorectal cancer is associated with superior prognosis among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases, suggesting a possible interaction between the AMPK and MAPK pathways influencing tumour behaviour.

Cyclin D1 is frequently overexpressed in microsatellite unstable colorectal cancer, independent of CpG island methylator phenotype.

AIMS: Cyclin D1 and cyclin-dependent kinases are commonly activated in colorectal cancer. Microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) are important molecular classifiers in colorectal cancer. The aim was to clarify the relationship between cyclin D1, MSI and CIMP. METHODS AND RESULTS: Among 865 colorectal cancers with MSI and CIMP data, 246 tumours (28.4%) showed cyclin D1 overexpression by immunohistochemistry. DNA methylation in p14 and eight CIMP-specific promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) was quantified by real-time polymerase chain reaction (MethyLight). Both MSI-high and CIMP-high were associated with cyclin D1 overexpression (P < 0.0001). After tumours were stratified by MSI and CIMP status, the relationship between MSI-high and cyclin D1 persisted (P < or = 0.02), whereas the relationship between CIMP-high and cyclin D1 did not. Cyclin D1 overexpression was correlated with BRAF mutation (P = 0.0001), p27 loss (P = 0.0007) and p16 loss (P = 0.02), and inversely with p53 expression (P = 0.0002) and p21 loss (P < 0.0001). After stratification by MSI status, the inverse relationship between cyclin D1 and p21 loss still persisted (P < 0.008). CONCLUSIONS: Cyclin D1 activation is associated with MSI and inversely with p21 loss in colorectal cancers. Cyclin D1 may play an important role in the development of MSI-high tumours, independent of CIMP status.

Transcriptional silencing of hedgehog-interacting protein by CpG hypermethylation and chromatic structure in human gastrointestinal cancer.

Hedgehog-interacting protein (HHIP) was identified as a putative antagonist of the Hh pathway and as a target of Hh signalling. Our aim was to clarify the expression profiles and epigenetic alterations of the HHIP gene in gastrointestinal cancer. The expression and promoter epigenetic status of HHIP in cancer cell lines and freshly resected gastrointestinal cancer tissues were examined using RT-PCR, tissue microarray analysis, methylation-specific PCR, and chromatin immunoprecipitation assay. Cells were treated with the demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. WST-8 assays and in vitro invasion assays after treatment with HHIP-specific siRNA were performed. HHIP expression levels were reduced in most of the gastrointestinal cancer cell lines and in a certain subset of cancer tissues, and these were correlated with promoter hypermethylation. A heterochromatic structure characterized by neither acetylated H3 nor acetylated H4, and histone H3 lysine 9 hypermethylation and histone H3 lysine 4 hypomethylation was observed in cancer cells in which the HHIP gene was aberrantly silenced. On the other hand, overexpression of the HHIP gene was also found in some cancer tissues and there were significant correlations between protein expression levels of HHIP and those of Sonic hedgehog (Shh), Indian hedgehog, Patched, and glioma-associated oncogene homologue-1. An association was found between lymph node metastasis and HHIP silencing in colorectal cancer tissues with strong Shh expression and between advanced TNM stage and HHIP silencing in diffuse-type gastric cancer tissues with strong Shh expression. Down-regulation of HHIP expression by siRNA resulted in a significant increase in colon cancer cell growth and invasion in vitro. Silencing of the HHIP gene due to hypermethylation and chromatin remodelling appears to be frequently involved in gastrointestinal tumourigenesis.

Gene expression profiling of colorectal adenomas and early invasive carcinomas by cDNA array analysis.

It is generally accepted that most colorectal carcinomas arise in pre-existing adenomas. Morphologically, colorectal adenomas can be divided into two groups, protruded type and flat type. The aim of this study was to clarify relevant alterations of gene expression associated with the early stage of colorectal carcinogenesis. Using cDNA array, we analysed the expression profiles of 550 cancer-related genes in 36 colorectal adenomas (18 flat-type and 18 protruded-type adenomas) and 14 early invasive carcinomas. Among the 550 genes, we chose 32 genes the average expression levels of which were at least three-fold up- or downregulated in tumour tissues compared with levels in matched normal tissues. A total of 13 and 19 genes were identified as up- and downregulated genes in tumour tissues, respectively. Among the upregulated genes, the average expression levels of E1AF, bone morphogenic protein (BMP)-4, insulin-like growth factor (IGF)-2, inducible nitric oxide synthase (iNOS), tissue inhibitors of metalloproteinase (TIMP)-1, Smad4, and nm23 in tumour tissues were over five times higher than those in matched normal tissues. Colorectal adenomas and early invasive carcinomas were divided into two major clusters by clustering analysis. Moreover, flat- and protruded-type adenomas were divided into two major clusters by clustering analysis. The expression profiles obtained by the cDNA array clearly indicate that colorectal adenomas and early invasive carcinomas have specific expression profiles. Likewise, the gene expression profiles of flat- and protruded-type adenomas are different. These results indicate that molecular classification of early colorectal tumours by a cDNA array is feasible.

[Chemotherapy-resistant CD7-positive stem cell lymphoma presenting with intra-abdominal mass].

We report a case of CD7+ stem cell lymphoma. A 47-year-old man presented with general malaise and lumbago in April 1997. The patient exhibited swollen left cervical lymph-nodes and an intra-abdominal bulky mass. He was referred to us because lymph-node biopsy specimens indicated a diagnosis of diffuse type malignant lymphoma. An abdominal CT scan disclosed large retroperitoneal, para-aortic, and mesenteric root masses. Bone marrow involvement was shown by bone marrow biopsy specimens, though no circulating blasts were detected at presentation. The patient was treated with high-dose CHOP therapy without any benefit. Though ESHAP therapy was performed as salvage chemotherapy, the abdominal masses did not shrink at all. The patient died of tumor progression in November 1997. In the terminal stage, the lymphoma cells emerged in the peripheral blood and thus became available for analysis. The cells expressed CD5, 7, 34, 38, 71, but were negative for CD1, 2, 3, 4, 8, 10, 13, 14, 16, 19, 20, 21, 25, HLA-DR, and EMA. An immunoglobulin heavy chain gene rearrangement band was detected by Southern blot analysis. However, no T cell receptor lambda or beta chain gene rearrangement bands were detected.

[A case of systemic lupus erythematosus overlapping with progressive systemic sclerosis accompanied by deposition of AA amyloid in the stomach].

A 52-year-old woman was admitted to our department because of fever and dysphasia in November 1994. She had noticed Raynaud's phenomenon, arthralgia, and stiffness in the skin for the past 20 years without receiving specific treatment. A diagnosis of progressive systemic sclerosis (PSS) was made based on the presence of sclerosis of the skin, sclerodactyly, pulmonary fibrosis, and the dilatation of the lower esophagus. It was also confirmed that systemic lupus erythematosus (SLE) overlapped based on the presence of an oral ulcer, polyarthralgia, leukocytopenia, renal dysfunction, positive tests for antinuclear antibodies and anti-DNA antibodies. A gastric biopsy specimen revealed amyloid deposits which showed green birefrigence by Congo red staining under polarized light microscopy. The disappearance of the green birefrigence after potassium permanganate treatment indicated that the deposits consisted of AA amyloid. No amyloid deposits were detected in the rectum or the kidney. The average of the SAA level in active SLE patients is 174 micrograms/ml in our department and the SAA level in the present case was 280 micrograms/ml. Although AA amyloidosis is rarely associated with SLE and PSS, a long-term elevation of SAA level could cause the development of amyloidosis even in the cases of SLE and PSS.

[A case of protein-losing gastroenteropathy in association with mixed connective tissue disease which was successfully treated with cyclophosphamide pulse therapy].

A 47-year-old man had been given a diagnosis of mixed connective tissue disease (MCTD) on 1987 when he had presented with Raynaud's phenomenon, polyarthralgia, sclerodactyly, and a high titre of anti-RNP antibody. Once his symptoms had improved following the administration of prednisolone orally and the treatment was discontinued since 1995. He noticed dyspnea and chest pain in February 1997. The bilateral pleural effusion was pointed out in the local hospital and he was admitted to our hospital in March 1997 for further examination. In addition to pleural effusion and ascites, laboratory studies revealed hypoalbuminemia and low serum levels of complements. Renal and liver function tests were normal and the urine gave a trace test for protein. The presence of protein loss in the gut was confirmed by an elevated alpha 1-antitrypsin clearance and 99mTc-albumin scintigraphy showing abnormal radioactivity in the gastrointestinal tract. Although endoscopic examination showed no abnormal findings macroscopically and gastrointestinal biopsies revealed nonspecific inflammation only, immunofluorescent studies demonstrated deposits of C 3, C 4 and IgG in the stomach, colon, and pleura. These findings supported the pathogenesis that immune deposits in tissues caused protein-losing gastroenteropathy (PLGE) in MCTD. Intravenous administration of cyclophosphamide started since July 1997, while the high-dose corticosteroid therapy including methylprednisolone pulse therapy were not effective. Hypoalbuminemia and low serum levels of complements improved remarkably and the pleural effusion and ascites disappeared after cyclophosphamide pulse therapy four times monthly. Cyclophosphamide pulse therapy should be considered as a possibly effective treatment for PLGE in association with collagen disease resistant to corticosteroid therapy.