23136

EPB41L3

erythrocyte membrane protein band 4.1-like 3;EPB41L3;erythrocyte membrane protein band 4.1-like 3

band 4.1-like protein 3|differentially expressed in adenocarcinoma of the lung protein 1

18p11.32

protein-coding

BACKGROUND: The tumor suppressor genes CADM1/TSLC1 and DAL-1/4.1B are frequently inactivated by promoter methylation in non-small cell lung cancer. The proteins they encode, CADM1 and 4.1B, form a complex in human epithelial cells and are involved in cell-cell adhesion. METHODS: expression of CADM1 and 4.1B proteins was examined by immunohistochemistry in 67 primary breast cancer and adjacent noncancerous tissues. CADM1 and 4.1B messenger RNA (mRNA) was detected by reverse-transcription polymerase chain reaction (RT-PCR). The methylation status of the CADM1 and 4.1B promoters was determined quantitatively by bisulfite treatment followed by pyrosequencing. RESULTS: CADM1 and 4.1B protein signals were detected along the cell membrane in normal mammary epithelia. By contrast, 47 (70%) and 49 (73%) of 67 primary breast cancers showed aberrant CADM1 and 4.1B staining, respectively. Aberrant CADM1 staining was more frequently observed in pT2 and pT3 tumors and for stages II and III (P = 0.045 and P = 0.020, respectively), while aberrant 4.1B staining was more often observed in tumors with lymph node metastasis, for pT2 and pT3 tumors, and for stages II and III (P = 0.0058, P = 0.0098, and P = 0.0007, respectively). Furthermore, aberrant CADM1 and 4.1B expression was preferentially observed in invasive relative to noninvasive lesions from the same specimen (P = 0.036 and P = 0.0009, respectively). Finally, hypermethylation of CADM1 and 4.1B genes was detected in 46% and 42% of primary breast cancers, respectively. CONCLUSIONS: Our findings suggest that aberrant CADM1 and 4.1B expression is involved in progression of breast cancer, especially in invasion into the stroma and metastasis.

Renal clear cell carcinoma (RCCC) is a malignant tumor with poor prognosis caused by the high incidence of metastasis to distal organs. Although metastatic RCCC cells frequently show aberrant cytoskeletal organization, the underlying mechanism has not been elucidated. DAL-1/4.1B is an actin-binding protein implicated in the cytoskeleton-associated processes, while its inactivation is frequently observed in lung and breast cancers and meningiomas, suggesting that 4.1B is a potential tumor suppressor. We studied a possible involvement of 4.1B in RCCCs and evaluated it as a clinical indicator. 4.1B protein was detected in the proximal convoluted tubules of human kidney, the presumed cell of origin of RCCC. On the other hand, loss or marked reduction of its expression was observed in 10 of 19 (53%) renal cell carcinoma (RCC) cells and 12 of 19 (63%) surgically resected RCCC by reverse transcription-PCR. Bisulfite sequencing or bisulfite SSCP analyses revealed that the 4.1B promoter was methylated in 9 of 19 (47%) RCC cells and 25 of 55 (45%) surgically resected RCCC, and inversely correlated with 4.1B expression (p < 0.0001). Aberrant methylation appeared to be a relatively early event because more than 40% of the tumors with pT1a showed hypermethylation. Furthermore, 4.1B methylation correlated with a nuclear grade (p = 0.017) and a recurrence-free survival (p = 0.0036) and provided an independent prognostic factor (p = 0.038, relative risk 10.5). These results indicate that the promoter methylation of the 4.1B is one of the most frequent epigenetic alterations in RCCC and could predict the metastatic recurrence of the surgically resected RCCC.

The differentially expressed in adenocarcinoma of the lung-1 (DAL-1) protein is a member of the membrane-associated cytoskeleton protein 4.1 family. This protein was previously found to be downregulated or lost in more than half of primary non-small cell lung cancers (NSCLC). In this study, the relationship between DAL-1 expression and NSCLC metastasis was examined. DAL-1 mRNA and protein levels were measured in NSCLC cell lines and in tumor cells isolated from the pleural fluid of NSCLC patients clinically diagnosed with distant metastases to the bone or brain. The results revealed that DAL-1 expression was observed in two (GLC-82 and NCI-H460) out of seven metastatic NSCLC cell lines examined. DAL-1 expression was not observed in the cells isolated from the pleural fluid in nine out of ten patients. Overexpression of DAL-1 in A549 cells, a cell line lacking endogenous DAL-1, inhibited cell migration and invasion by approximately 38 and 48 %, respectively. In contrast, DAL-1 knockdown in NCI-H460 cells enhanced the migration and invasion potential of this cell line 4.6- and 3-fold, respectively. Furthermore, DAL-1 promoter methylation was observed in six of nine pleural fluid NSCLC cell isolates and in two cell lines (A549 and H1299), as evidenced by a lack of endogenous DAL-1. Demethylation in A549 cells successfully restored DAL-1 mRNA and protein expression levels, resulting in a parallel remarkable inhibition of migration and invasion. These results indicated that DAL-1 was pivotal in triggering NSCLC migration and invasion and that loss of DAL-1 expression was due to the epigenetic methylation.

Protein 4.1B is a 4.1/ezrin/radixin/moesin domain-containing protein whose expression is frequently lost in a variety of human tumors, including meningiomas, non-small-cell lung cancers, and breast carcinomas. However, its potential tumor-suppressive function under in vivo conditions remains to be validated. In a screen for genes involved with prostate cancer metastasis, we found that 4.1B expression is reduced in highly metastatic tumors. Down-regulation of 4.1B increased the metastatic propensity of poorly metastatic cells in an orthotopic model of prostate cancer. Furthermore, 4.1B-deficient mice displayed increased susceptibility for developing aggressive, spontaneous prostate carcinomas. In both cases, enhanced tumor malignancy was associated with reduced apoptosis. Because expression of Protein 4.1B is frequently down-regulated in human clinical prostate cancer, as well as in a spectrum of other tumor types, these results suggest a more general role for Protein 4.1B as a negative regulator of cancer progression to metastatic disease.