CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKC␦ to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein. CUB-domain-containing protein 1 (CDCP1)3 is an 836 amino acid integral membrane glycoprotein with a type I orientation at the cell surface (1-4), that is up-regulated in a number of malignancies including breast (1, 5, 6), colon (1, 2, 7), and lung (1) cancers. Of potential clinical significance, CDCP1 expression correlates with recurrence and patient survival rate in renal cell carcinomas (8) and lung adenocarcinomas (9), indicating that it may be suitable as a prognostic marker. Consistent with a role in cancer progression, silencing of CDCP1 reduced the metastatic ability of lung cancer A549 cells (10) and the peritoneal dissemination of gastric cancer 44As3 cells (11) in mice. Although its biological function is not known, the potential of CDCP1 as a therapeutic target for cancer treatment has been highlighted by studies showing that antibody-mediated inhibition of CDCP1 reduced metastasis of prostate cancer PC3 cells in mice (12, 13) and chicken embryos (13). Currently the mechanisms regulating CDCP1 in cancer and normal physiology are not well defined (14).During cellular processing, the 29-residue CDCP1 N-terminal signal peptide is removed generating a protein with molecular mass identified as either 135 kDa (2, 10, 15) or 140 kDa (3, 4) that contains 30 -40 kDa of N-linked glycans (2). In addition to this full-length form, there is evidence that a shorter CDCP1 species is expressed endogenously by a range of cell lines or is generated through the action of exogenous serine proteases. For example, lung cancer A549, PC14, H520, H322, and H157 cells (10) and gastric cancer 44As3 and 58As9 cells (11) resuspended non-enzymatically ...
The E2F family of transcription factors plays a crucial role in the regulation of genes involved in cell proliferation, differentiation, and apoptosis. In keratinocytes, the inhibition of E2F is a key step in the control and initiation of squamous differentiation. Because the product of the recently identified E2F7a/E2F7b gene has been shown to repress E2F-regulated promoters, and to be abundant in skin, we examined its role in the epidermis. Our results indicate that E2F7b mRNA expression is selectively associated with proliferation-competent keratinocytes. Moreover, E2F7 was able to antagonize E2F1-induced proliferation and apoptosis. In contrast, although E2F7 was able to inhibit proliferation and initiate differentiation, it was unable to antagonize the differentiation suppression induced by E2F1. These data indicate that E2F7-mediated suppression of proliferation and apoptosis acts through E2F1-dependent pathways, whereas E2F7-induced differentiation acts through an E2F1-independent pathway. These data also suggest that proliferation, differentiation, and survival of primary human keratinocytes can be controlled by the relative ratio of E2F1 to E2F7. Because deregulated proliferation, differentiation, and apoptosis are hallmarks of cancer, we examined the expression levels of E2F1 and E2F7 in cutaneous squamous cell carcinomas (CSCC). We found that both genes were overexpressed in CSCCs compared with normal epidermis. Furthermore, inhibition of E2F7 in a SCC cell line sensitized the cells to UV-induced apoptosis and doxorubicin-induced apoptosis. Combined, these data suggest that the selected disruption of E2F1 and E2F7 in keratinocytes is likely to contribute to CSCC formation and may prove to be a viable therapeutic target.
It has been shown previously that recombinant virus-like particles (VLPs) of papillomavirus can induce VLP-specific humoral and cellular immune responses following parenteral administration. To test whether mucosal administration of bovine papillomavirus type 1 (BPV1) VLPs could produce mucosal as well as systemic immune responses to VLPs, 50 micrograms chimeric BPV1 VLPs containing an HPV16 E7 CTL epitope (BPVL1/E7 VLP) was administered intranasally to mice. After two immunisations, L1-specific serum IgG and IgA were observed. L1-specific IgG and IgA were also found in respiratory and vaginal secretions. Both serum and mucosal antibody inhibited papillomavirus VLP-induced agglutination of RBC, indicating that the antibody induced by mucosal immunisation may recognize conformational determinants associated with virus neutralisation. For comparison, VLPs were given intramuscularly, and systemic and mucosal immune responses were generally comparable following systemic or mucosal delivery. However, intranasal administration of VLP induced significantly higher local IgA response in lung, suggesting that mucosally delivered HPV VLP may be more effective for mediating local mucosal immune responses. Intranasal immunisation with HPV6b L1 VLP produced VLP-specific T proliferative responses in splenocytes, and immunisation with BPVL1 VLP containing an HPV16 E7 CTL epitope induced E7-specific CTL responses. We conclude that immunisation with papillomavirus VLPs via mucosal and intramuscular routes, without adjuvant, can elicit specific antibody at mucosal surfaces and also systemic VLP epitope specific T cell responses. These findings suggest that mucosally delivered VLPs may offer an alternative HPV VLP vaccine strategy for inducing protective humoral immunity to anogenital HPV infection, together with cell-mediated immune responses to eliminate any cells which become infected.
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