The hnRNP A/B proteins are among the most abundant RNA-binding proteins, forming the core of the ribonucleoprotein complex that associates with nascent transcripts in eukaryotic cells. There are several paralogs in this subfamily, each of which is subject to alternative transcript splicing and post-translational modifications. The structural diversity of these proteins generates a multitude of functions that involve interactions with DNA or, more commonly, RNA. They also recruit regulatory proteins associated with pathways related to DNA and RNA metabolism, and appear to accompany transcripts throughout the life of the mRNA. We have highlighted here recent progress in elucidation of molecular mechanisms underlying the roles of these hnRNPs in a wide range of nuclear processes, including DNA replication and repair, telomere maintenance, transcription, pre-mRNA splicing, and mRNA nucleo-cytoplasmic export.

The CUB domain-containing protein 1 (CDCP1) is a transmembrane molecule that recently has been implicated in cancer progression. In this study we have established a novel mechanism for initiation of CDCP1-mediated signaling in vivo and demonstrated that specific 135→70 kDa processing of cell surface CDCP1 by extracellular serine proteases is a prerequisite for CDCP1-dependent survival of cancer cells during metastasis. The in vivo cleavage of CDCP1 triggers a survival program involving recruitment of Src and PKCδ, Src-mediated phosphorylation of cell surface-retained 70 kDa CDCP1, activation of Akt, and suppression of PARP1-induced apoptosis. We demonstrate in vivo that phosphorylated Src, PKCδ and Akt, all constitute activated elements of a CDCP1 signaling axis during tissue colonization of tumor cells. Preventing the in vivo cleavage of CDCP1 with unique anti-CDCP1 antibodies, serine protease inhibitors or genetic modulation of the cleavage site in the CDCP1 molecule completely abrogated survival signaling associated with the 70 kDa CDCP1 and induced PARP1 cleavage and PARP1-mediated apoptosis, ultimately resulting in substantial inhibition of tissue colonization by tumor cells. The lack of CDCP1 cleavage in the lung tissue of plasminogen knockout mice along with a coordinated reduction in tumor cell survival in a lung retention model and importantly the rescue of both by in vivo supplied plasmin, indicated that plasmin is the crucial serine protease executing in vivo cleavage of cell surface CDCP1 during early stages of lung colonization. Together, our findings indicate that in vivo blocking of CDCP1 cleavage upstream of CDCP1-induced pro-survival signaling provides a potential mechanism for therapeutic intervention into metastatic disease.

IntroductionHeterogeneous nuclear ribonucleoprotein particle proteins (hnRNPs) are abundant, multi-tasking proteins that play a central role in RNA metabolism. They are involved in packaging nascent hnRNA, alternative RNA splicing, mRNA export from the nucleus, and cytoplasmic trafficking, stability and translation (Dreyfuss et al., 2002;Krecic and Swanson, 1999;Shyu and Wilkinson, 2000;Weighardt et al., 1996). They also have an as yet poorly defined role in telomere maintenance.More than 20 hnRNPs, many of which are characterised by possession of RNA-recognition motifs (RRMs), have been identified. The hnRNPs A/B are the major components of the 40S particles that package hnRNA. This packaging was originally envisaged to mimic that of histones in nucleosomes but this analogy appears to be inappropriate as the levels of the core proteins are not stoichiometric: they differ markedly between cell types, and their association with RNA is dependent on the nucleic acid sequence (Dreyfuss et al., 1993). However, given the role of these core particles in hnRNA packaging one might anticipate a correlation between the abundance of their proteins and the transcriptional activity in the cell. This is borne out experimentally: there is a marked difference in the concentration of hnRNP A1 between resting or slowly dividing cells and rapidly dividing cells. In the latter, hnRNPs A1 and A2 have been proposed to be present at similar levels. But hnRNP A1, which is abundant in a range of human, hamster and mouse proliferating cells, is present at markedly lower levels in confluent or resting cells, whereas hnRNP A2 is less affected (Celis et al., 1986;LeStourgeon, 1978).The levels of hnRNPs A/B differ not only between proliferating and resting cells: some also fluctuate during the cell cycle (Leser and Martin, 1987;Minoo et al., 1989). In HeLa cells hnRNPs A2 and B1 are synthesized in the G1 phase, and their levels fall in G2 and M phases, with hnRNP B1 protein level falling more markedly . The relative and absolute levels of these two proteins also differ markedly between tissues, both being particularly abundant in rat brain, testis, lung, spleen and ovary (Kamma et al., 1999;Ma et al., 2002).Many genes show a correlation between strong expression in proliferating cancer cells and the fluctuations in the protein level across the cell cycle (Dreyfuss et al., 1993;Whitfield et al., 2002). The levels of hnRNP A/B proteins are of particular interest as it has been suggested that upregulation of some members of this protein family is associated, as either a cause or consequence, with cellular proliferation and cancer. The hnRNP A2 and its longer B1 isoform are expressed at an early stage in a variety of tumours and have been proposed as early markers for cancer, especially lung cancer (Fielding et al., 1999;Mulshine et al., 2002;Pino et al., 2003;Sueoka et al., 1999;Whitfield et al., 2002;Zhou et al., 1996) and possibly breast cancer (Zhou et al., 2001a). The upregulation of hnRNP A2/B1 in cancer parallels its expression in lung dev...

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 ...

Our findings highlight CDCA3 as a prognostic factor and potential novel therapeutic target in NSCLC through inhibition of tumor growth and promotion of tumor senescence.

SummaryIn the last few years dysregulated expression of the cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) has been associated with several cancers and this cell surface molecule has been recognized both as a tumor marker and as a potential target to disrupt progression of cancer. Here we summarize what is known about CDCP1 including its structural features, expression in normal and cancerous tissues, and the in vitro experiments and studies in animal models that have provided the key insights into its potential role in tumor formation and metastasis in humans. We conclude by highlighting opportunities and challenges in targeting CDCP1 in cancer.2009 IUBMB IUBMB Life, 61(7): [723][724][725][726][727][728][729][730] 2009

Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.

Background:Development of targeted therapies for high-grade serous ovarian cancer (HGSC) remains challenging, as contributing molecular pathways are poorly defined or expressed heterogeneously. CUB-domain containing protein 1 (CDCP1) is a cell-surface protein elevated in lung, colorectal, pancreas, renal and clear cell ovarian cancer.Methods:CUB-domain containing protein 1 was examined by immunohistochemistry in HGSC and fallopian tube. The impact of targeting CDCP1 on cell growth and migration in vitro, and intraperitoneal xenograft growth in mice was examined. Three patient-derived xenograft (PDX) mouse models were developed and characterised for CDCP1 expression. The effect of a monoclonal anti-CDCP1 antibody on PDX growth was examined. Src activation was assessed by western blot analysis.Results:Elevated CDCP1 was observed in 77% of HGSC cases. Silencing of CDCP1 reduced migration and non-adherent cell growth in vitro and tumour burden in vivo. Expression of CDCP1 in patient samples was maintained in PDX models. Antibody blockade of CDCP1 significantly reduced growth of an HGSC PDX. The CDCP1-mediated activation of Src was observed in cultured cells and mouse xenografts.Conclusions:CUB-domain containing protein 1 is over-expressed by the majority of HGSCs. In vitro and mouse model data indicate that CDCP1 has a role in HGSC and that it can be targeted to inhibit progression of this cancer.