Proteolysis-induced N-terminal ectodomain shedding of the integral membrane glycoprotein CUB domain-containing protein 1 (CDCP1) is accompanied by tyrosine phosphorylation of its C-terminal domain and recruitment of Src and PKCdelta.

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 PKCdelta 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.

E2F7 can regulate proliferation, differentiation, and apoptotic responses in human keratinocytes: implications for cutaneous squamous cell carcinoma formation.

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.

Nasopharyngeal carcinoma-associated Epstein-Barr virus-encoded oncogene latent membrane protein 1 potentiates regulatory T-cell function.

Sequence variation in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) oncogene structure may affect antigen-presenting cell (APC) function of infected B cells and immune escape by EBV-specific T cells and thus contribute to the development of malignancy. Normal B cell-associated LMP1 (B-LMP1) upregulates B cell APC function through activation of the necrosis factor (NF)-kappaB subunit, RelB. We examined the ability of B-LMP1 and a nasopharyngeal carcinoma-associated LMP1 (NPC-LMP1) to modulate B cell APC function and T-cell responses. B lymphoma cells transfected with NPC-LMP1 stimulated resting T cells in mixed lymphocyte reaction less efficiently than B-LMP1 transfectants. Unexpectedly, antigen presentation to CD4(+) T helper cells was reduced owing to potentiation of regulatory T-cell function by NPC-LMP1 transfectants, which produce increased levels of interleukin-10, rendering CD4(+) T cells hyporesponsive. Thus, after primary EBV infection, T cells may escape activation by NPC-LMP1. These observations have important implications for the establishment of EBV-associated malignancy in the context of infection with tumour-associated EBV LMP1 variants.

E2F modulates keratinocyte squamous differentiation: implications for E2F inhibition in squamous cell carcinoma.

E2F regulation is essential for normal cell cycle progression. Therefore, it is not surprising that squamous cell carcinoma cell lines (SCC) overexpress E2F1 and exhibit deregulated E2F activity when compared with normal keratinocytes. Indeed, deliberate E2F1 deregulation has been shown to induce hyperplasia and skin tumor formation. In this study, we report on a dual role for E2F as a mediator of keratinocyte proliferation and modulator of squamous differentiation. Overexpression of E2F isoforms in confluent primary keratinocyte cultures resulted in suppression of differentiation-associated markers. Moreover, we found that the DNA binding domain and the trans-activation domain of E2F1 are important in mediating suppression of differentiation. Use of a dominant/negative form of E2F1 (E2F d/n) found that E2F inhibition alone is sufficient to suppress the activity of proliferation-associated markers but is not capable of inducing differentiation markers. However, if the E2F d/n is expressed in differentiated keratinocytes, differentiation marker activity is further induced, suggesting that E2F may act as a modulator of squamous differentiation. We therefore examined the effects of E2F d/n in a differentiation-insensitive SCC cell line. We found that treatment with the differentiating agent, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or expression of E2F d/n alone had no effect on differentiation markers. However, a combination of E2F d/n + TPA induced the expression of differentiation markers. Combined, these data indicate that E2F may play a key role in keratinocyte differentiation. These data also illustrate the unique potential of anti-E2F therapies in arresting proliferation and inducing differentiation of SCCs.

Clinical response after intradermal immature dendritic cell vaccination in metastatic melanoma is associated with immune response to particulate antigen.

Metastatic melanoma is poorly responsive to treatment, and immunotherapeutic approaches are potentially beneficial. Predictors of clinical response are needed to identify suitable patients. We sought factors associated with melanoma-specific clinical response following intradermal vaccination with autologous melanoma peptide and particulate hepatitis B antigen (HBsAg)-exposed immature monocyte-derived dendritic cells (MDDC). Nineteen patients with metastatic melanoma received a maximum of 8, 2-weekly vaccinations of DC, exposed to HBsAg in addition to autologous melanoma peptides. A further 3 patients received an otherwise identical vaccine that did not include HBsAg. Patients were assessed 1-2 monthly for safety, disease volume, and cellular responses to HBsAg and melanoma peptide. There was no significant toxicity. Of 19 patients receiving HBsAg-exposed DC, 9 primed or boosted a cellular response to HBsAg, and 10 showed no HBsAg response. HBsAg-specific responses were associated with in vitro T cell responses to melanoma peptides and to phytohemagglutinin (PHA). Zero out of 10 non-HBsAg-responding and 4/9 HBsAg-responding patients achieved objective melanoma-specific clinical responses or disease stabilization - 1 complete and 2 partial responses and 1 case of stable disease ( P=0.018). Development of melanoma-specific cellular immunity and T cell responsiveness to mitogen were greater in the group of patients responding to HBsAg. Therefore stimulation of an immune response to nominal particulate antigen was necessary when presented by melanoma peptide-exposed immature DC, to achieve clinical responses in metastatic melanoma. Since general immune competence may be a determinant of treatment response, it should be assessed in future trials on DC immunotherapy.