The human tumor/chick embryo model involving grafting of human HT-1080 fibrosarcoma cells on the chorioallantoic membrane was used in conjunction with quantitative real-time Alu PCR to select in vivo a pair of isogenic cell lines (HT-hi/diss and HT-lo/diss), dramatically differing in their ability to disseminate from the primary tumor (i.e., intravasate into the chorioallantoic membrane vasculature and metastasize to the lungs). During an immunohistochemical time course study, HT-hi/diss cells were sequentially visualized having escaped from the primary tumors, engaged with the blood vessels, and eventually observed inside the chorioallantoic membrane capillaries, thus reflecting early intravasating events. In contrast, HT-lo/diss cells seemed restricted to their primary tumor. Importantly, after i.v. inoculation, both variants arrested, extravasated, and proliferated in host tissues with similar efficiencies, highlighting that the observed earlier events at the periphery of the primary tumor could account for their differential dissemination. In a mechanistic probing of these events, we determined that HT-hi/diss intravasation was sensitive to a broad-range matrix metalloproteinase (MMP) inhibitor. To analyze the possible role of individual MMPs, membrane-bound MMP-14 and secreted MMP-9 were individually down-regulated in HT-hi/diss cells with their corresponding small interfering RNAs. Despite efficient down-regulation of MMP-14, neither intravasation nor metastasis of HT-hi/diss cells was affected significantly. However, a substantial down-regulation of MMP-9 was accompanied by a surprising 3-fold increase in intravasation and metastasis. The results emphasize a rising awareness that targeting certain MMPs might result in an enhanced malignancy, exemplified herein at the intravasation level as this step of the metastatic cascade is dissected and quantified.

The function of CUB domain-containing protein 1 (CDCP1), a recently described transmembrane protein expressed on the surface of hematopoietic stem cells and normal and malignant cells of different tissue origin, is not well defined. The contribution of CDCP1 to tumor metastasis was analyzed by using HeLa carcinoma cells overexpressing CDCP1 (HeLa-CDCP1) and a high-disseminating variant of prostate carcinoma PC-3 naturally expressing high levels of CDCP1 (PC3-hi/diss). CDCP1 expression rendered HeLa cells more aggressive in experimental metastasis in immunodeficient mice. Metastatic colonization by HeLa-CDCP1 was effectively inhibited with subtractive immunization-generated, CDCP1-specific monoclonal antibody (mAb) 41-2, suggesting that CDCP1 facilitates relatively late stages of the metastatic cascade. In the chick embryo model, time-and dose-dependent inhibition of HeLa-CDCP1 colonization by mAb 41-2 was analyzed quantitatively to determine when and where CDCP1 functions during metastasis. Quantitative PCR and immunohistochemical analyses indicated that CDCP1 facilitated tumor cell survival soon after vascular arrest. Live cell imaging showed that the function-blocking mechanism of mAb 41-2 involved enhancement of tumor cell apoptosis, confirmed by attenuation of mAb 41-2-mediated effects with the caspase inhibitor z-VAD-fmk. Under proapoptotic conditions in vitro, CDCP1 expression conferred HeLa-CDCP1 cells with resistance to doxorubicin-induced apoptosis, whereas ligation of CDCP1 with mAb 41-2 caused additional enhancement of the apoptotic response. The functional role of naturally expressed CDCP1 was shown by mAb 41-2-mediated inhibition of both experimental and spontaneous metastasis of PC3-hi/diss. These findings confirm that CDCP1 functions as an antiapoptotic molecule and indicate that during metastasis CDCP1 facilitates tumor cell survival likely during or soon after extravasation.

We report a novel nucleolar interaction between the AAA ATPase p97/VCP and the Werner protein (WRNp), a member of the RecQ helicase family. p97/VCP mediates several important cellular functions in eucaryotic cells, including membrane fusion of the endoplasmic reticulum and Golgi and ubiquitin-dependent protein degradation. Mutations in the WRN gene cause Werner syndrome, a genetic disorder characterized by premature onset of aging symptoms, a higher incidence of cancer, and a high susceptibility to DNA damage caused by topoisomerase inhibitors. We observed that both WRNp and valosin-containing protein (VCP) were present in the nucleoplasm and in nucleolar foci in mammalian cells and that WRNp and p97/VCP physically interacted in the nucleoli. Importantly, the nucleolar WRNp/VCP complex was dissociated by treatment with camptothecin, an inhibitor of topoisomerase I, whereas other WRNp-associated protein complexes, such as WRNp/Ku 80, were not dissociated by this drug. Because WRN syndrome cells are sensitive to topoisomerase inhibitors, these observations suggest that the VCP/WRNp interaction plays an important role in WRN biology. We propose a novel role for VCP in the DNA damage response pathway through modulation of WRNp availability. INTRODUCTIONThe ATPases associated with diverse cellular activities (AAA) proteins are a common family of Mg 2ϩ -dependent ATPases that contain one or two conserved ATP-binding domains (Ogura and Wilkinson, 2001). These domains, or AAA cassettes , consist of a conserved sequence of 230-amino acid residues that include the Walker A and B motifs. Another sequence-conserved domain, the second region of homology, is found in part of the AAA cassette (Confalonieri and Duguet, 1995) but is not present in the closely related AAAϩ family (Neuwald et al., 1999) of proteins. Cdc48p, p97, and valosin-containing protein (VCP) are 92-to 97-kDa orthologous members of a subfamily of AAA ATPases originally defined in yeast, Xenopus, and mammals, respectively (Moir et al., 1982;Peters et al., 1990;Frohlich et al., 1991). This subfamily has a known propensity to oligomerize and form homohexamers (Peters et al., 1992) that are a part of multiprotein complexes (Ogura and Wilkinson, 2001).Cdc48p/p97/VCP is involved in two major and distinct cellular pathways: homotypic membrane fusion of endoplasmic reticulum (ER) and Golgi fragments and ubiquitindependent protein degradation. This versatility is achieved through different sets of adaptor proteins . In ER membrane fusion, Cdc48p/p97/VCP is complexed with Ufe1p and Shp1p (Latterich et al., 1995;Lin et al., 2001) or with their vertebrate orthologs p47 and syntaxin 5 in Golgi membrane fusion (Acharya et al., 1995;Rabouille et al., 1995). It is likely that the role of Cdc48p in ER membrane fusion is to remove a fusion inhibitor, thus permitting membrane fusion to occur (Lin et al., 2001). Cdc48p/p97/VCP also participates in the degradation of ubiquitinated proteins in yeast via Ufd1p and Npl4p and in mammals (Ghislain et al., 1996;Dai et al., 1998;Meyer ...