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Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.

TSLC1 is a tumor suppressor gene encoding a member of the immunoglobulin (Ig) superfamily. The significant homology of its extracellular domain with those of otherIg superfamily cell adhesionmolecules (IgCAMs) has raised the possibility that TSLC1 participates in cell-cell interactions. In this study, the physiological properties of TSLC1 were investigated in Madin-Darby canine kidney (MDCK) cells expressing TSLC1 tagged with green fluorescent protein (GFP) as well as in the cells that express endogenous TSLC1. Biochemical analysis has revealed that TSLC1 is anN-linked glycoprotein with a molecular mass of 75 kDa and that it forms homodimers through cis interaction within the plane of the cell membranes. Confocal laser scanning microcopy of the cells expressing TSLC1 showed the localization patterns characteristic to adhesion molecules. At the beginning of cell attachment, TSLC1 accumulated in interdigitated structures at cell-cell boundaries, but, when cells reached a confluence, TSLC1 was distributed all along the cell membranes. In polarized cells, TSLC1 was recruited to the lateral membrane, implying trans interaction of TSLC1 between neighboring cells. In support of this notion, MDCK cells expressing TSLC1-GFP showed a significant level of cell aggregation in the absence or presence of Ca²⁺ and Mg²⁺. Taken together, these results indicate that TSLC1 mediates intracellular adhesion through homophilic interactions in a Ca²⁺/Mg²⁺-independent manner.

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.

Purpose: DAL-1/4.1B is an actin-binding protein originally identified as a molecule whose expression is down-regulated in lung adenocarcinoma. We have previously shown that a lung tumor suppressor, TSLC1, associates with DAL-1, suggesting that both proteins act in the same cascade. The purpose of this study is to understand the molecular mechanisms and clinical significance of DAL-1 inactivation in lung cancer. Experimental Design: We studied aberration of the DAL-1 in 103 primary non–small cell lung cancers (NSCLC) and 18 lung cancer cells. Expression and allelic and methylation status of DAL-1 was examined by reverse transcription-PCR, microsatellite analysis, and bisulfite sequencing or bisulfite single-strand conformational polymorphism, respectively. Results: Loss of DAL-1 expression was strongly correlated with promoter methylation in lung cancer cells, whereas DAL-1 expression was restored by a demethylating agent, 5-aza-2′-deoxycytidine. The DAL-1 promoter was methylated in 59 (57%) primary NSCLC tumors, 37% of which were associated with loss of heterozygosity around the DAL-1 on chromosomal region 18p11.3. In squamous cell carcinomas, DAL-1 methylation was observed in 9 of 10 tumors at stage I, whereas the incidence of methylation gradually increased in adenocarcinomas as they progressed [13 of 36 (36%), 4 of 12 (33%), 14 of 17 (82%), and 3 of 3 (100%) tumors at stages I, II, III, and IV, respectively; P = 0.0026]. Furthermore, in adenocarcinomas, disease-free survival and overall survival were significantly shorter in patients with tumors harboring the methylated DAL-1 (P = 0.0011 and P = 0.045, respectively). Conclusions:DAL-1 methylation is involved in the development and progression of NSCLC and provides an indicator for poor prognosis.