In cancer cells, loss of E-cadherin gene expression caused dysfunction of the cell-cell junction system, triggering cancer invasion and metastasis. Therefore, E-cadherin is an important tumor-suppressor gene. To understand how E-cadherin gene expression is regulated in cancer cells, we have used E-cadherin-positive and -negative expressing cells to find out the possible up-or downregulating transcription factors in human E-cadherin regulatory sequences. Functional analysis of human E-cadherin regulatory sequences constructs indicated that AML1, Sp1, and p300 may play important roles in promoting E-cadherin expression. In addition, we found there are four HNF3-binding sites in human E-cadherin regulatory sequences. The exogenous HNF3 can enhance the E-cadherin promoter activity in metastatic breast cancer cells and the metastatic breast cancer cells stably transfected with HNF3 showed re-expression of E-cadherin. The HNF3 stable transfectants changed from mesenchymal-like into epithelial morphology. The transwell assays showed the re-expressed E-cadherin reduced cell motility of metastatic breast cancer cells. These results suggested HNF3 may play important roles in the upregulation of the E-cadherin promoter, with the consequent re-expression of E-cadherin, thus reducing the metastatic potential of breast cancer cells. These findings suggested HNF3 plays important roles in the upregulation of the E-cadherin gene and may be able to reduce the motility of metastatic breast cancer cells.

PARK2, an ubiquitin ligase closely correlated with Parkinson's disease and cancer, has been shown to accumulate at centrosomes to ubiquitinate misfolded proteins accumulated during interphase. In the present study, we demonstrated that PARK2 can also localize to centrosomes in mitosis and that the protein does not fluctuate through the S- to M-phase. A C-terminal truncation of PARK2 resulted in a spindle assembly checkpoint defect, characterized by HeLa cells able to bypass mitotic arrest induced by nocodazole and form multinucleated cells when overexpressing the C-terminal truncated PARK2 protein. The spindle assembly checkpoint defect may be due to a change in a biochemical or structural property of PARK2 caused by the C-terminal truncation, resulting in a loss of self-interaction between PARK2 proteins.

No abstract

Abstract. Aneuploidy is a common characteristic of human solid tumors. It has been proposed that a defect of the spindle assembly checkpoint (SAC) generates aneuploidy and might facilitate tumorigenesis. However, a direct link between the SAC proteins and tumorigenesis has not yet been elucidated. Here, we demonstrate the association of the SAC protein MAD1 with the RNA polymerase II complex and its role in gene expression. Furthermore, MAD1 binds to the E-cadherin promoter region. Knockdown of endogenous MAD1 by siRNA reduces E-cadherin expression and enhances the migration ability of non-metastatic breast cancer cells, indicating that reduced MAD1 expression is a new potential diagnostic symptom of tumor metastasis. IntroductionOver 90% of cancer death is attributed to metastasis (1). Remodeling of the extracellular matrix and an enhanced migratory ability are critical steps in the metastatic dissemination of cancer cells (2). E-cadherin is a component of cell-cell adhesion junctions (3), and its linkage to the cytoskeleton requires peripheral membrane proteins, including α-catenin, β-catenin, vinculin, and α-actinin. In addition, β-catenin directly binds to E-cadherin (4,5). The actin cytoskeleton is also required for the association of E-cadherin with another cell adhesion protein, nectin (6). E-cadherin is thought of as a hallmark symptom of metastatic cells because the loss of E-cadherin may lead to an invasiveness of human carcinoma cells (3). Several transcriptional repressors of E-cadherin, such as the zinc-finger-containing proteins SIP-1, δEF-1, Snail, and Slug and the helix-loop-helix proteins E12/E47 and Twist, have been reported to be highly associated with tumor progression (7-10).Abnormal chromosome number (i.e., aneuploidy) is also a common phenomenon in cancer cells. The spindle assembly checkpoint (SAC) monitors the fidelity of chromosomal segregation in mitosis to prevent aneuploidy in daughter cells. Mutations and/or reduced levels of mitotic checkpoint proteins can cause checkpoint malfunction and chromosomal instability (CIN) and thereby contribute to tumor formation (11). The heterozygous deletion of mitotic arrest-deficient protein 1 (MAD1), a component of the SAC, increases the incidence of tumors in mice (12). MAD1 has also been reported to have lower expression level in gastric tumors, hepatocellular carcinoma and renal cell carcinomas (13-16). Although it has been shown that MAD1 interacts with HDAC1 (17), the transcriptional activity of MAD1 and the role of MAD1 in tumor progression are not known. Furthermore, the direct role of SAC proteins in tumorigenesis remains to be elucidated.Here, we demonstrate that MAD1 associates with the RNA polymerase II complex and may regulate MAD1 and E-cadherin expression. Depletion of MAD1 causes lower E-cadherin expression and enhances cell migration ability, which is a critical sign of metastatic cancer cells. Materials and methodsPlasmid constructions and antibody production. Full-length MAD1 was cloned into phrGFP-N1 (Stratagene) and pCMVFlag2 ...