SummaryKeratin filaments form cytoskeletal networks in epithelial cells. Dynamic rearrangement of keratin filament networks is required for epithelial cells to perform cellular processes such as cell migration and polarization; however, the mechanism governing keratin filament rearrangement remains unclear. Here, we describe a novel mechanism of keratin cytoskeleton organization mediated by casein kinase Ia (CK-1a) and a newly identified keratin-associated protein, FAM83H. Knockdown of FAM83H induces keratin filament bundling, whereas overexpression of FAM83H disassembles keratin filaments, suggesting that FAM83H regulates the filamentous state of keratins. Intriguingly, keratin filament bundling is concomitant with the dissociation of CK-1a from keratin filaments, whereas aberrant speckle-like localization of CK-1a is observed concomitantly with keratin filament disassembly. Furthermore, CK-1a inhibition, similar to FAM83H knockdown, causes keratin filament bundling and reverses keratin filament disassembly induced by FAM83H overexpression, suggesting that CK-1a mediates FAM83H-dependent reorganization of keratin filaments. Because the N-terminal region of FAM83H interacts with CK-1a and the C-terminal region interacts with keratins, FAM83H might tether CK-1a to keratins. Colorectal cancer tissue also shows keratin filament disassembly accompanied with FAM83H overexpression and aberrant CK-1a localization, and FAM83H-overexpressing cancer cells exhibit loss or alteration of epithelial cell polarity. Importantly, knockdown of FAM83H inhibits cell migration accompanied by keratin cytoskeleton rearrangement in colorectal cancer cells. These results suggest that keratin cytoskeleton organization is regulated by FAM83H-mediated recruitment of CK-1a to keratins, and that keratin filament disassembly caused by overexpression of FAM83H and aberrant localization of CK-1a could contribute to the progression of colorectal cancer.

Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized pH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.

BackgroundDiagnosis of esophageal squamous cell carcinoma (SCC) may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available.ResultsFifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning) using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of these antigens were similar in amino acid sequences, and they were designated as ECSA (esophageal carcinoma SEREX antigen)-1, -2 and -3. The ECSA family was also similar to an EST clone, hepatocellular carcinoma-associated antigen 25a (HCA25a). Serum antibody levels to ECSA-1, -2 and -3 were significantly higher in patients with esophageal SCC than in healthy donors. Based on the conserved amino acid sequences, three peptides were synthesized and used for enzyme-linked immunosorbent assays (ELISA). The serum antibody levels against one of these peptides were significantly higher in patients with esophageal SCC. This peptide sequence was also conserved in FAM119A, GOSR1 and BBS5, suggesting that these are also ECSA family members. Reverse transcription followed by quantitative PCR analysis showed that the mRNA expression levels of ECSA-1, -2 and -3 and FAM119A but not of HCA25a, GOSR1 and BBS5 were frequently elevated in esophageal SCC tissues.ConclusionsWe have identified a new gene family designated ECSA. Serum antibodies against the conserved domain of the ECSA family may be a promising tumor marker for esophageal SCC.

Purpose: The histone deacetylase inhibitor FK228 shows strong activity as a potent antitumor drug but its precise mechanism is still obscure. The purpose of this study is to reveal the effect of FK228 on gene expression in the cell and to determine the mechanism of the antitumor activity of FK228 for further clinical applications. Experimental Design and Results: Microarray analysis was applied to verify the gene expression profiles of 4,608 genes after FK228 treatment using human esophageal squamous cell cancer cell lines T.Tn and TE2. Among them, peroxiredoxin 1 (Prdx1), a member of the peroxiredoxin family of antioxidant enzymes having cell growth suppression activity, as well as p21 WAF1 , were significantly activated by FK288. In addition, FK228 strongly inhibited the cell growth of T.Tn and TE2 by the induction of apoptosis. Further, chromatin immunoprecipitation analysis revealed that FK228 induced the accumulation of acetylated histones H3 and H4 in Prdx1 promoter, including the Sp1-binding site. In mouse xenograft models of T.Tn and TE2 cells, FK228 injection resulted in significant tumor regression as well as activated Prdx1 expression in tumor tissues. Prdx1 suppression by RNA interference hindered the antitumor effect of FK228. Conclusion: Our results indicate that the antitumor effect of FK228 in esophageal cancer cells is shown at least in part through Prdx1 activation by modulating acetylation of histones in the promoter, resulting in tumor growth inhibition with apoptosis induction.

BACKGROUND:The expression of Fra-1 (Fos related antigen 1) involves tumor progression and invasion, and its gene ablation could suppress the invasive phenotypes of human tumor cells. The authors investigated the significance of Fra-1 expression in esophageal squamous cell carcinoma (ESCC) and studied the effect of its down-regulation on cell proliferation, motility, and invasion. METHODS: Surgical specimens from 164 patients with ESCC were evaluated. Fra-1 expression in the primary tumor along with metastatic lymph nodes was compared among various clinicopathological characteristics, and overall survival was analyzed. The rate and intensity of Fra-1 immunoreactivity were also investigated. The molecular role of Fra-1 was assessed by its down-regulation in human ESCC cell lines. RESULTS: Fra-1 expression was positive in 127 (77.4%) ESCC patients. Immunoreactivity was localized to the marginal areas of the ESCC tumors. Positive Fra-1 expression correlated with depth of tumor, lymph node metastasis, stage, and infiltrative growth pattern. A significant difference was seen in the survival between tumors with and without Fra-1, and positive Fra-1 expression was revealed to be an independent factor related to poor prognosis. Patients with metastatic lymph nodes with positive Fra-1 expression presented decreased survival compared with negative Fra-1 expression. After the down-regulation of Fra-1 expression, a significant decrease in cell proliferation, motility, and invasion was observed. CONCLUSIONS: This study demonstrated ESCC patients positive for Fra-1 to be associated with poor prognosis. The findings also suggest that Fra-1 regulation may play an important role in the progression of ESCC.