Regulation of the actin cytoskeleton may play a crucial role in cell motility and cancer invasion. We have produced a monoclonal antibody (NCC- Lu-632, IgM, k) reactive with an antigenic protein that is upregulated upon enhanced cell movement. The cDNA for the antigen molecule was found to encode a novel isoform of nonmuscle α-actinin. This isoform (designated actinin-4) was concentrated in the cytoplasm where cells were sharply extended and in cells migrating and located at the edge of cell clusters, but was absent from focal adhesion plaques or adherens junctions, where the classic isoform (actinin-1) was concentrated. Actinin-4 shifted steadily from the cytoplasm to the nucleus upon inhibition of phosphatidylinositol 3 kinase or actin depolymerization. The cytoplasmic localization of actinin-4 was closely associated with an infiltrative histological phenotype and correlated significantly with a poorer prognosis in 61 cases of breast cancer. These findings suggest that cytoplasmic actinin-4 regulates the actin cytoskeleton and increases cellular motility and that its inactivation by transfer to the nucleus abolishes the metastatic potential of human cancers.

JapicCTI-101021.

No abstract

We report the cloning and characterization of a cancer-associated cell membrane glycoprotein recognized by mAb NCC-3G10. The antibody showed strong reactivity to a wide variety of cancer cells, but only to a limited number of normal cells including lymphocytes, endothelial cells, and basal cells of stratified squamous epithelium. The cDNA for the antigen encodes 178 aa, which includes a putative signal sequence, a potential O-glycosylated extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. Transfection of the cDNA into PLC͞PRF͞5 liver cancer cells resulted in reduced cell-cell adhesiveness, based on both morphology and results of Ca 2؉ -dependent cell aggregation assay. In transfected cells, E-cadherin was markedly decreased at the protein level in inverse proportion to the expression level of the antigen recognized by NCC-3G10, but not at the mRNA level. Aggregation of the antigen by NCC-3G10-coated beads triggered accumulation of actin, suggesting some interplay between this antigen and Ecadherin through actin. When metastatic ability was examined in severe combined immunodeficient mice by injecting PLC͞PRF͞5 cells into the spleen, the transfectants formed a markedly higher number of metastatic nodules in comparison with controls. We have named this cell membrane glycoprotein, which downregulates E-cadherin and promotes metastasis, dysadherin. T he cadherins are members of a large family of transmembrane glycoproteins that mediate calcium-dependent, homophilic cell-cell adhesion and play an important role in the maintenance of normal tissue architecture (1). Cadherins are connected indirectly to the actin cytoskeleton by means of a group of proteins known as the catenins. The transmembrane cadherin is associated with either ␤-catenin or plakoglobin, which in turn associates with ␣-catenin, and then ␣-catenin mediates the interaction between the cadherin-catenin complex and the actin cytoskeleton (2-4). Numerous studies have demonstrated the importance of the E-cadherin͞catenin complex in normal development, maintenance, and repair of tissue and tumor development. Early studies showed that inhibition of E-cadherin activity with function-perturbing antibodies altered the morphology of Madin-Darby canine kidney cells and conferred on them the ability to invade collagen gels and embryonic chicken heart tissue (5, 6). Invasive fibroblast-like carcinoma cells could be converted to a noninvasive phenotype by transfection with a cDNA encoding E-cadherin (7). Several studies have reported a correlation between decreased function of the E-cadherin͞catenin complex and the initiation and progression of human tumors. Several mechanisms for the irreversible and reversible inactivation of the E-cadherin͞catenin complex in human tumors have been reported (8). Mutations have been reported in the genes for E-cadherin and ␣-and ␤-catenins (9-12). Transcriptional inactivation of E-cadherin expression was shown to occur by deoxycytidylate-phosphate-deoxyguanylate methylation around the promoter regio...

To clarify the significance of DNA methylation alterations during renal carcinogenesis, methylome analysis using single-CpG-resolution Infinium array was performed on 29 normal renal cortex tissue (C) samples, 107 non-cancerous renal cortex tissue (N) samples obtained from patients with clear cell renal cell carcinomas (RCCs) and 109 tumorous tissue (T) samples. DNA methylation levels at 4830 CpG sites were already altered in N samples compared with C samples. Unsupervised hierarchical clustering analysis based on DNA methylation levels at the 801 CpG sites, where DNA methylation alterations had occurred in N samples and were inherited by and strengthened in T samples, clustered clear cell RCCs into Cluster A (n = 90) and Cluster B (n = 14). Clinicopathologically aggressive tumors were accumulated in Cluster B, and the cancer-free and overall survival rates of patients in this cluster were significantly lower than those of patients in Cluster A. Clear cell RCCs in Cluster B were characterized by accumulation of DNA hypermethylation on CpG islands and considered to be CpG island methylator phenotype (CIMP)-positive cancers. DNA hypermethylation of the CpG sites on the FAM150A, GRM6, ZNF540, ZFP42, ZNF154, RIMS4, PCDHAC1, KHDRBS2, ASCL2, KCNQ1, PRAC, WNT3A, TRH, FAM78A, ZNF671, SLC13A5 and NKX6-2 genes became hallmarks of CIMP in RCCs. On the other hand, Cluster A was characterized by genome-wide DNA hypomethylation. These data indicated that DNA methylation alterations at precancerous stages may determine tumor aggressiveness and patient outcome. Accumulation of DNA hypermethylation on CpG islands and genome-wide DNA hypomethylation may each underlie distinct pathways of renal carcinogenesis. Abbreviations:BAMCAbacterial artificial chromosome array-based methylated CpG island amplificationCnormal renal cortex tissue obtained from patients without any primary renal tumorCIMPCpG island methylator phenotypeHCChepatocellular carcinomaNnon-cancerous renal cortex tissue obtained from patients with clear cell renal cell carcinomasNCBINational Center for Biotechnology InformationRCCrenal cell carcinomaTtumorous tissueTNMTumor-Node-Metastasis