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DEPDC1 (DEP domain containing 1) is overexpressed in multiple cancers and is associated with cell cycle progression. In this report, we have investigated the expression, localization, phosphorylation and function of DEPDC1 during mitosis. DEPDC1 has two isoforms (isoform a and isoform b), and both of them are increased in mitosis and degraded once cells exit mitosis. DEPDC1a is localized to the centrosome in metaphase, whereas DEPDC1b is localized to the entire cell cortex during mitosis. DEPDC1a, but not DEPDC1b, was required for the integrity of centrosome and organization of the bipolar spindle. Mass spectrometry and biochemical analyses revealed phosphorylation of DEPDC1 at Ser110. The phosphorylation of Ser110 is essential for localization of DEPDC1a to the centrosome. Consistently, non-phosphorylation mutants of DEPDC1a did not rescue disruption of centrosome organization by depletion of endogenous DEPDC1. Our results show a novel role for DEPDC1 in maintaining centrosome integrity during mitosis for the accurate distribution of chromosomes.

Homeobox (HOX) genes are conserved transcription factors which determine the anterior-posterior body axis patterning. HOXD8 is a member of HOX genes deregulated in several tumors such as lung carcinoma, neuroblastoma, glioma and colorectal cancer (CRC) in a context-dependent manner. In CRC, HOXD8 is downregulated in cancer tissues and metastatic foci as compared to normal tissues. Whether HOXD8 acts as a tumor suppressor of malignant progression and metastasis is still unclear. Also, the underlying mechanism of its function including the downstream targets is totally unknown. Here, we clarified the lower expression of HOXD8 in clinical colorectal cancer vs. normal colon tissues. Also, we showed that stable expression of HOXD8 in colorectal cancer cells significantly reduced the cell proliferation, anchorage-independent growth and invasion. Further, using The Cancer Genome Atlas (TCGA), we identified the genes associated with HOXD8 in order to demonstrate its function as a suppressor or a promoter of colorectal carcinoma. Among inversely related genes, apoptotic inhibitors like STK38 kinase and MYC were shown to be negatively associated with HOXD8. We demonstrated the ability of HOXD8 to upregulate executioner caspases 6 & 7 and cleaved PARP, thus inducing the apoptotic events in colorectal cancer cells.

Ovarian cancer cells shed from primary tumors can spread easily to the peritoneum via the peritoneal fluid. To allow further metastasis, the cancer cells must interact with the mesothelial cell layer, which covers the entire surface of the peritoneal organs. Although the clinical importance of this interaction between cancer and mesothelial cells has been increasingly recognized, the molecular mechanisms utilized by cancer cells to adhere to and migrate through the mesothelial cell layer are poorly understood. To investigate the molecular mechanisms of cancer cell trans-mesothelial migration, we set up an in vitro trans-mesothelial migration assay using primary peritoneal mesothelial cells. Using this method, we found that downregulation of filopodial protein fascin-1 or myosin X expression in ES-2 cells significantly inhibited the rate of trans-mesothelial migration of cancer cells, whereas upregulation of fascin-1 in SK-OV-3 cells enhanced this rate. Furthermore, downregulation of N-cadherin or integrin β1 inhibited the rate of cancer cell trans-mesothelial migration. Conversely, downregulation of cortactin or TKS5 or treatment with the MMP inhibitor GM6001 or the N-WASP inhibitor wiskostatin did not have any effect on cancer cell trans-mesothelial migration. These results suggest that filopodia, but not lamellipodia or invadopodia, play an important role in the trans-mesothelial migration of ovarian cancer cells.

Invadopodia are specialized actin-based microdomains of the plasma membrane that combine adhesive properties with matrix degrading activities. Proper functioning of the bone, immune, and vascular systems depend on these organelles, and their relevance in cancer cells is linked to tumor metastasis. The elucidation of the mechanisms driving invadopodia formation is a prerequisite to understanding their role and ultimately to controlling their functions. Special AT-rich sequence-binding protein 2 (SATB2) was reported to suppress tumor cell migration and metastasis. However, the mechanism of action of SATB2 is unknown. Here, we show that SATB2 inhibits invadopodia formation in HCT116 cells and that the molecular scaffold palladin is inhibited by exogenous expression of SATB2. To confirm this association, we elucidated the function of palladin in HCT116 using a knock down strategy. Palladin knock down reduced cell migration and invasion and inhibited invadopodia formation. This phenotype was confirmed by a rescue experiment. We then demonstrated that palladin expression in SATB2-expressing cells restored invasion and invadopodia formation. Our results showed that SATB2 action is mediated by palladin inhibition and the SATB2/palladin pathway is associated with invadopodia formation in colorectal cancer cells.

Cholangiocarcinoma is a particularly devastating carcinoma with limited treatment options. Tousled-like kinase 1 (TLK1) is a serine/threonine protein kinase that regulates DNA replication and DNA repair pathways. Here, we show that TLK1 is abundantly expressed in cholangiocarcinoma as well as in cell lines derived from cholangiocarcinoma. Although siRNA knockdown of TLK1 did not affect the viability of cholangiocarcinoma cells, it sensitized cholangiocarcinoma cells to cisplatin-induced apoptosis. Immunofluorescence analysis of gammaH2AX foci showed that silencing of TLK1 enhanced DNA damage induced by cisplatin treatment. Our results suggest that TLK1 plays a pivotal role for the repair of cisplatin-induced DNA damage.

Nitric oxide (NO) plays a pivotal role in tumorigenesis, particularly with relation to cancer cell invasion and metastasis. NO can reversibly couple to cysteine thiols to form an S-nitrosothiol, which regulates the enzymatic activities of target proteins. c-Src is a tyrosine kinase that promotes cancer cell invasion and metastasis. Interestingly, c-Src can be activated by NO stimulation. However, mechanisms by which NO stimulates Src kinase activity have not been elucidated. We report here that NO causes S-nitrosylation of c-Src at cysteine 498 (Cys(498)) to stimulate its kinase activity. Cys(498) is conserved among Src family kinases, and Cys(506) of c-Yes, which corresponds to Cys(498) of c-Src, was also important for the NO-mediated activation of c-Yes. Estrogens may work synergistically with NO to induce the proliferation and migration of many kinds of breast cancer cells. For example, beta-estradiol induces the expression of endothelial nitric synthase and production of NO in MCF7 cells. We found that activation of c-Src in MCF7 cells by beta-estradiol stimulation was mediated by the S-nitrosylation of Cys(498). In addition, we report that disruption of E-cadherin junctions and enhancement of cell invasion by beta-estradiol stimulation was mediated by NO-dependent activation of c-Src. These results identify a novel signaling pathway that links NO and Src family kinases to cancer cell invasion and metastasis.

Both MMP-2 and MMP-9 play critical roles in tumor invasion, but their productions are differentially controlled. While the promoter region of MMP-9 has the conserved proximal AP-1 binding site, that of the MMP-2 has a noncanonical AP-1 site. To assess the role of AP-1 function, we examined the effects of dominant-negative Fos (DeltaFos), BATF and siRNA against c-Jun on MMP production in v-Crk-transformed cells which have augmented production of MMP-2 and MMP-9. Suppression of AP-1 dependent transcription by conditional expression of dominant-negative Fos (DeltaFos) and BATF substantially inhibited not only MMP-9 production but also MMP-2 production. The ChIP analysis showed the direct association of AP-1 and MMP-2 promoter region. In addition, silencing of c-Jun expression by siRNA transfection suppressed MMP-2 and MMP-9 production and in vitro invasiveness. Furthermore, the invadopodia formation of v-Crk-transformed cells could be suppressed by BATF expression or c-Jun siRNA treatment. Taken together, AP-1 appears to play a critical role in the production of MMP-2 and MMP-9 and invadopodia formation of v-Crk-transformed cells.

A chemical derivatization technique in TOF-SIMS along with ultra-low angle sample cutting technique were used to perform a quantitative study of the surface and in-depth double bond profile of the photo-initiated polymerized thin film. We found out that the characteristic peaks at m / z 185 and 199 were obtained from the thin film composed of acrylate monomer and methacrylate monomer, respectively, after reaction with bromine gas. The detection sensitivity of certain chemical indicators is affected by changing the primary ion species. The Bi 3 + primary ion results in the best chemical sensitivity. The surface double-bond density obtained by TOF-SIMS and the Br 3d signal intensity of XPS showed a good linear relationship in the limited region due to the effect of matrix hardness. The thin film was cut with microtome about 1° angle and was left to react with bromine and was measured using TOF-SIMS. It was clearly observed from this technique that double bond of acrylate and methacrylate monomer remained much more at the surface of the photo-initiated polymerized thin film, due to the inhibition of polymerization by oxygen. From the surface to 1 μm depth, both monomers show the same behavior, but the rate of polymerization of methacrylate monomer was lower than that of acrylate in deeper layers.

We have previously reported that expression of SIRPalpha1/SHPS-1 was strongly suppressed in v-Src-transformed cells and its forced expression resulted in the suppression of anchorage-independent growth of the cells [K. Machida, S. Matsuda, K. Yamaki, T. Senga, A.A. Thant, H. Kurata, K. Miyazaki, K. Hayashi, T. Okuda, T. Kitamura, T. Hayakawa, M. Hamaguchi, v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells, Oncogene 19 (2000) 1710-1718]. We examined the effect of human SIRPalpha1 expression in breast cancer cell lines, Hs578T and MCF7, and compared with the effect of SIRPalpha2 expression in Hs578T. Forced expression of either SIRPalpha1 or SIRPalpha2 did not perturb the growth of Hs578T in a conventional attached condition. Their expression, however, enforced the actin stress fiber formation and induced activation of Rho, but not Rac, in Hs578T cells. Moreover, forced expression of either SIRPalpha1 or SIRPalpha2 displayed distinct suppressive effect on the anchorage-independent growth of Hs578T cells. Similarly, forced expression of SIRPalpha1 in MCF7 specifically suppressed the anchorage-independent growth of the cells. Taken together, our results strongly suggest the function of SIRPalpha1 and 2 as type II tumor suppressors for human breast carcinoma.

To search for the signaling critical for the production of tissue inhibitor of metalloproteinase-2 (TIMP-2), we investigated the role of SHP-2 in TIMP-2 production with Concanavalin A (Con A)-treated cells. In wild-type fibroblasts, Con A-treatment dramatically activated TIMP-2 production. In contrast, production of TIMP-2 in response to Con A-treatment was severely impaired in cells expressing mutant SHP-2 whose 65 amino acids in the SH2-N domain were deleted. Con A-treatment activated dual signaling pathways, Erk and p38, in a SHP-2-dependent manner. Pretreatment of wild-type cells with U0126, a potent inhibitor of MEK1, significantly inhibited the production of TIMP-2, whereas SB203580, a specific inhibitor for p38, could not. Finally, expression of exogenous wild-type SHP-2 in SHP-2 mutant cells clearly rescued Erk activation and TIMP-2 production in response to Con A-treatment. Taken together, our results strongly suggest that SHP-2 plays a critical role as a positive modulator for the production of TIMP-2 via MEK1-Erk signaling in fibroblasts.

Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. Here we show that the N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage.

Geminin is a cellular protein that associates with Cdt1 and inhibits Mcm2-7 loading during S phase. It prevents multiple cycles of replication per cell cycle and prevents episome replication. It also directly inhibits the HoxA11 transcription factor. Here we report that geminin forms a parallel coiled-coil homodimer with atypical residues in the dimer interface. Point mutations that disrupt the dimerization abolish interaction with Cdt1 and inhibition of replication. An array of glutamic acid residues on the coiled-coil domain surface interacts with positive charges in the middle of Cdt1. An adjoining region interacts independently with the N-terminal 100 residues of Cdt1. Both interactions are essential for replication inhibition. The negative residues on the coiled-coil domain and a different part of geminin are also required for interaction with HoxA11. Therefore a rigid cylinder with negative surface charges is a critical component of a bipartite interaction interface between geminin and its cellular targets.

We investigated the role of MEK1 signaling in MMP-2 activation by use of constitutive active/dominant negative forms of MEK1 and MEK1-specific inhibitor. We found that cell transformation with active forms of MEK1 dramatically increased secretion and proteolytic activation of MMP-2 and subsequently stimulated invasiveness of cells. Contrary, expression of dominant negative form of MEK1 in v-src-transformed cells or in Con A-activated cells resulted in the suppression of the augmented secretion and proteolytic activation of MMP-2. In addition, treatment of v-src-transformed cells with PD98059, a MEK1-specific inhibitor, strongly suppressed the secretion and activation of MMP-2, whereas treatment with wortmannin, a PI3 kinase inhibitor, showed no clear effect on MMP-2 secretion. Taken together, these results strongly suggest that MEK-MAP kinase signaling, but not PI3 kinase signaling, plays a critical role in the activation of MMP-2 secretion and, subsequently, in the invasiveness of v-src-transformed cells.

The Src homology 3 (SH3) domain, originally identified in v-Crk, plays an important role in signal transduction. The comparative study with c-src has revealed that v-src oncogene of Schmidt-Ruppin strain of Rous sarcoma virus has three point mutations in its SH3 domain and one in the upstream of SH3. To assess the role of these mutations, each of the single mutations was introduced into c-Src by oligonucleotide-directed mutagenesis and its effect on cell transformation was examined. While variant Src proteins that carry each one of single mutations could not transform cells, double mutation at positions 95 and 117 converted c-Src to be oncogenic and active in kinase. An additional mutation at position 124 together with one at 95 and 117 further activated Src kinase. By use of GST-fusion forms of v-Src SH3 and c-Src SH3, we found that these mutations in SH3 suppressed the binding of SH3 with c-Src protein, possibly with a linker region, while v-SrcSH3 retained the ability to bind a subset of cellular protein to the level similar to those of c-SrcSH3. Taken together, our results suggest that point mutations accumulated in SH3 region can activate, in concert, Src kinase by relaxing the interaction between SH3 and the linker region and subsequently convert Src to be oncogenic.