CUB Domain-containing Protein 1 Expression Research Articles

CUB Domain-containing Protein 1 (CDCP1) Is Down-regulated by Active Hexose-correlated Compound in Human Pancreatic Cancer Cells.

We have previously reported that treatment of pancreatic cancer cells with active hexose-correlated compound (AHCC), an extract of a basidiomycete mushroom, decreases the levels of tumor-associated proteins including heat-shock protein 27 (HSP27), heat shock factor 1 (HSF1) and sex-determining region Y-box 2 (SOX2). The transmembrane glycoprotein, CUB domain-containing protein 1 (CDCP1) has been reported to be up-regulated in various cancers, and be associated with invasion and metastasis. The aim of this study was to examine the effect of AHCC on the expression of CDCP1 in KLM1-R cells. Gemcitabine-resistant pancreatic cancer cells (KLM1-R) were treated with AHCC (10 mg/ml) for 48 h. Western blot analysis of cell extracts with anti-CDCP1 or anti-actin antibodies was performed to assess the expression of CDCP1. Expression of CDCP1 was reduced by AHCC treatment of KLM1-R cells, whereas expression of actin was not affected. The ratio of intensities of CDCP1/actin in AHCC-treated KLM1-R cells was significantly suppressed (p<0.05) compared to untreated cells. AHCC down-regulated CDCP1 expression and inhibited the malignant progression of pancreatic cancer cells.

Abstract B096: CDCP1, a potential noninvasive biomarker in circulating tumor cells for treatment of prostate cancer with FAK inhibitors

Abstract Background: Tumor metastasis requires dynamic changes in cell adhesion mediated through beta 1-integrin and focal adhesion kinase (FAK). In particular, FAK is responsible for counteracting anoikis and promoting survival of CTCs. The Cub-Domain Containing Protein–1 (CDCP1) is a transmembrane glycoprotein expressed in epithelial cells that regulates cell adhesion. CDCP1 loss leads to activation of FAK in nonadherent cancer cells (na-PCC) (Spassov, 2011). As we demonstrated, another consequence of loss of CDCP1 is the inhibition of inside-out activation of beta 1-integrin in na-PCC, suggesting that FAK activation can occur in the absence of active beta 1-integrin. The main goal of this project is to determine how FAK becomes activated in na-PCC upon CDCP1 silencing and to establish that CDCP1 loss sensitizes to treatment with FAK inhibitors. Methods: CTCs were collected from nine patients with castrate-resistant prostate cancer (CRPC) (Schehr, 2016). CTC mRNA was eluted and used for CDCP1 qPCR. CDCP1 was stably silenced in DU145 with short hairpins (Spassov, 2013). In the anoikis assay, 5 x 105 DU145 shControl and shCDCP1 cells were suspended on HEMA coated plates for up to 6 hrs. Western blot membranes were probed for pMAPK1/2, MAPK1/2, FAKpY397, FAKpY925, and FAK. For MTT assays, 5,000 DU145 shControl and shCDCP1 were incubated for 24 hrs on HEMA-coated 96-well plates in regular culture medium +/- Saracatinib, FAKi-PF573228 (kinase domain inhibitor) and FAKi-LGA (FAT domain inhibitor) and/or MEKi-PD98059. A kinase dead mutant of PIP5K-D316A from the Addgene PIP5K plasmid was generated by site directed mutagenesis. Results: We find reduced CDCP1 expression in CTCs from patients with CRPC. Six patients did not express detectable CDCP1 mRNA in CTCs, 1 patient demonstrated CDCP1 expression at the limit of detection, and in 2 patients CDCP1 mRNA levels were above the limit of detection. Silencing CDCP1 in DU145 and PC3 cells results in 3.4-fold higher proliferation rates and 4.4-fold greater anchorage-independent growth rates. CDCP1-negative tumors grew in 100% of mice, versus 30% growth of CDCP1 positive control tumors. In na-PCC, CDCP1 silencing triggered FAK activation, as demonstrated by de novo pY397 and pY925. In addition, 73% of CDCP1-negative cells expressed FAK-stimulated pMAPK. FAK activation required a factor in human plasma and involved a trypsin-sensitive receptor in na-PCC. CDCP1 loss also conferred increased sensitivity to treatment with the PF573228 FAK-kinase inhibitor. At the 4uM concentration 98.1% of CDCP1-positive cells survived compared to 32.9% of CDCP1-silenced cells. An inhibitor treatment matrix showed a synergistic treatment effect of dual targeting of SRC, FAK, and MEK with up to 3-fold increased cytotoxicity of FAKi+SRCi or FAKi+MEKi. The largest treatment effect was observed when all 3 inhibitors were combined. To better understand the role of the FAK-MEK pathway in anoikis, a novel C-terminal FAT domain inhibitor was investigated and compared to PF573228. To determine the mechanism of FAK activation in na-PCC, we expressed a dominant negative PIP5K-D316A mutant. Expression of this mutant is expected to reduce CDCP1-induced FAK activation and also diminish SRC activity. Conclusion: We discovered a synergistic treatment effect between FAK and SRC and/or MEK inhibition in CDCP1-negative nonadherent cells. Expression of pFAK and pMAPK downstream of CDCP1 loss overcomes anoikis and depends on a factor in human plasma. By silencing beta 1-integrin and activating FAK, CDCP1-negative cancer cells may gain easy access to the circulation and survive during the CTC phase of cancer dissemination. CDCP1 loss may serve as a biomarker to identify patients who benefit from treatment with FAK inhibitors. Funding: This work was supported by Department of Defense Synergistic Idea Development Award W81XWH-08-1-0268. Citation Format: Sara Pollan, Jamie Sperger, Joshua Lang, Fangjin Huang, Jie Zheng, Colm Morrissey, Anne E. Cress, Danislav Spassov, Mark Moasser, William Carter, Beatrice Knudsen. CDCP1, a potential noninvasive biomarker in circulating tumor cells for treatment of prostate cancer with FAK inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B096.

Common Co-activation of AXL and CDCP1 in EGFR-mutation-positive Non-Small Cell Lung Cancer Associated With Poor Prognosis

Epidermal growth factor receptor (EGFR)-mutation-positive non-small cell lung cancer (NSCLC) is incurable, despite high rates of response to EGFR tyrosine kinase inhibitors (TKIs). We investigated receptor tyrosine kinases (RTKs), Src family kinases and focal adhesion kinase (FAK) as genetic modifiers of innate resistance in EGFR-mutation-positive NSCLC. We performed gene expression analysis in two cohorts (Cohort 1 and Cohort 2) of EGFR-mutation-positive NSCLC patients treated with EGFR TKI. We evaluated the efficacy of gefitinib or osimertinib with the Src/FAK/Janus kinase 2 (JAK2) inhibitor, TPX0005 in vitro and in vivo. In Cohort 1, CUB domain-containing protein-1 (CDCP1) was an independent negative prognostic factor for progression-free survival (hazard ratio of 1.79, p=0.0407) and overall survival (hazard ratio of 2.23, p=0.0192). A two-gene model based on AXL and CDCP1 expression was strongly associated with the clinical outcome to EGFR TKIs, in both cohorts of patients. Our preclinical experiments revealed that several RTKs and non-RTKs, were up-regulated at baseline or after treatment with gefitinib or osimertinib. TPX-0005 plus EGFR TKI suppressed expression and activation of RTKs and downstream signaling intermediates. Co-expression of CDCP1 and AXL is often observed in EGFR-mutation-positive tumors, limiting the efficacy of EGFR TKIs. Co-treatment with EGFR TKI and TPX-0005 warrants testing.

Cub domain-containing protein 1 negatively regulates TGF-β signaling and myofibroblast differentiation.

Fibroblasts are thought to be the prime cell type for producing and secreting extracellular matrix (ECM) proteins in the connective tissue. The profibrotic cytokine transforming growth factor-β1 (TGF-β1) activates and transdifferentiates fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts, which exhibit increased ECM secretion, in particular collagens. Little information, however, exists about cell-surface molecules on fibroblasts that mediate this transdifferentiation process. We recently identified, using unbiased cell-surface proteome analysis, Cub domain-containing protein 1 (CDCP1) to be strongly downregulated by TGF-β1. CDCP1 is a transmembrane glycoprotein, the expression and role of which has not been investigated in lung fibroblasts to date. Here, we characterized, in detail, the effect of TGF-β1 on CDCP1 expression and function, using immunofluorescence, FACS, immunoblotting, and siRNA-mediated knockdown of CDCP1. CDCP1 is present on interstitial fibroblasts, but not myofibroblasts, in the normal and idiopathic pulmonary fibrosis lung. In vitro, TGF-β1 decreased CDCP1 expression in a time-dependent manner by impacting mRNA and protein levels. Knockdown of CDCP1 enhanced a TGF-β1-mediated cell adhesion of fibroblasts. Importantly, CDCP1-depleted cells displayed an enhanced expression of profibrotic markers, such as collagen V or α-SMA, which was found to be independent of TGF-β1. Our data show, for the very first time that loss of CDCP1 contributes to fibroblast to myofibroblast differentiation via a potential negative feedback loop between CDCP1 expression and TGF-β1 stimulation.

ADAM9 enhances CDCP1 by inhibiting miR-1 through EGFR signaling activation in lung cancer metastasis.

MicroRNAs (miRNAs), which are endogenous short noncoding RNAs, can regulate genes involved in important biological and pathological functions. Therefore, dysregulation of miRNAs plays a critical role in cancer progression. However, whether the aberrant expression of miRNAs is regulated by oncogenes remains unclear. We previously demonstrated that a disintegrin and metalloprotease domain 9 (ADAM9) promotes lung metastasis by enhancing the expression of a pro-migratory protein, CUB domain containing protein 1 (CDCP1). In this study, we found that this process occurred via miR-1 down-regulation. miR-1 expression was down-regulated in lung tumors, but increased in ADAM9-knockdown lung cancer cells, and was negatively correlated with CDCP1 expression as well as the migration ability of lung cancer cells. Luciferase-based reporter assays showed that miR-1 directly bound to the 3′-untranslated region of CDCP1 and inhibited its translation. Treatment with a miR-1 inhibitor restored CDCP1 protein levels and enhanced tumor cell mobility. Overexpression of miR-1 decreased tumor metastases and increased the survival rate in mice. ADAM9 knockdown reduced EGFR signaling and increased miR-1 expression. These results revealed that ADAM9 down-regulates miR-1 via activating EGFR signaling pathways, which in turn enhances CDCP1 expression to promote lung cancer progression.

Effect of HIF-2α on regulating CDCP1 to promote hepatocellular carcinoma metastasis

Objective: To investigate the effect of hypoxia inducible factor 2α (HIF-2α) on regulating CUB domain-containing protein 1 (CDCP1) and its role in hepatocellular carcinoma metastasis. Methods: HIF-2α-knocked down and HIF-2α-stably overexpressing cells (MHCC97H) were prepared by small interfering RNA (siRNA) and lentivirus transfection, respectively. The expression of CDCP1 protein and mRNA in the above cells was detected by western blot and real-time PCR. The effect of HIF-2α on cell invasion ability was determined by Transwell assay. Furthermore, immunohistochemical staining was performed to detect the expression of CDCP1 in human HCC tissue samples. Results: Both HIF-2α and CDCP1 were induced under hypoxic conditions. The activation of CDCP1 under hypoxic conditions was dependent on the expression of HIF-2α.When HIF-2α was overexpressed, the mRNA level of CDCP1 was greatly upregulated (5.92±0.28, P<0.05). When HIF-2α was knocked down by siRNA for 48 h and 72 h, the expression of CDCP1 was significantly downregulated (48 h: 0.25±0.04; 72 h: 0.18±0.02, all P<0.05). Moreover, analysis of human HCC samples showed that CDCP1 expression was correlated with tumor-free survival (P<0.05). Conclusions: The results of this study indicate that the expression of CDCP1 is regulated by HIF-2α and is correlated with the progression of HCC. Inhibition of HIF-2α/CDCP1 may play certain inhibitory role in the metastasis of HCC.

Abstract 1625: CUB domain-containing protein 1 (CDCP1) promotes metastatic phenotype in pancreatic ductal adenocarcinoma

Abstract CUB domain-containing protein 1 (CDCP1) is a transmembrane glycoprotein that is overexpressed and highly tyrosine phosphorylated in multiple cancer types including pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease. CDCP1 has been implicated in the promotion of metastases, namely cancer cell migration, invasion, and resistance to anoikis. In addition, prior studies in other cancer cell lines have shown that hypoxia inducible factors (HIF-1α and HIF-2α), which are key regulators of metastasis, induce both expression of CDCP1 and phosphorylation at tyrosine 734. The role of CDCP1 and the mechanisms that regulate CDCP1 still need further elucidation in PDAC. In this study, we demonstrated that CDCP1 is highly expressed and tyrosine phosphorylated in PDAC cell lines MiaPaCa2, PaTu89902, and Panc-1 cell lines compared to the non-tumorigenic pancreatic ductal cell line HPNE. shRNA knockdown of CDCP1 showed a reduction in the phosphorylation of Src and PKC-delta. To determine whether HIF regulates CDCP1 in PDAC, we used PDAC cell lines that stably express shRNAs targeting HIF-1α and HIF-2α. Hypoxia induced an increase in CDCP1 expression in a HIF-1α-dependent fashion. Additionally, CDCP1 knockdown in PaTu8902, a highly metastatic PDAC line, impaired cancer cell invasion by transwell invasion assay but did not show resistance to anoikis by soft agar assay. To validate our data in vivo, we performed intra-pancreatic injection of shCDCP1 PaTu8902 cells and scramble control transfected with GFP/luciferase into nude mice. Live imaging demonstrated that 2 weeks after initial injection resulted in decreased tumor establishment in mice injected with the shCDCP1 cell line compared to control. We propose that CDCP1 expression and tyrosine phosphorylation in PDAC is mediated by hypoxia inducible factors and that CDCP1 confers invasive properties in vitro and may play a role in initial tumor establishment in vivo. Based on these results, CDCP1 is a promising novel target for PDAC. Citation Format: Sunnie Kim, Brooke Emerling, Benjamin Hopkins, Ryan Loughran, Salva Naranjos, Lewis Cantley. CUB domain-containing protein 1 (CDCP1) promotes metastatic phenotype in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1625.

Cell line and patient-derived xenograft models reveal elevated CDCP1 as a target in high-grade serous ovarian cancer

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.

Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer.

CUB-domain-containing protein 1 (CDCP1) is a trans-membrane protein regulator of cell adhesion with a potent pro-migratory function in tumors. Given that proteolytic cleavage of the ectodomain correlates with outside-in oncogenic signaling, we characterized glycosylation in the context of cellular processing and expression of CDCP1 in prostate cancer. We detected 135 kDa full-length and proteolytic processed 70 kDa species in a panel of PCa cell models. The relative expression of full-length CDCP1 correlated with the metastatic potential of syngeneic cell models and an increase in surface membrane expression of CDCP1 was observed in tumor compared to adjacent normal prostate tissues. We demonstrated that glycosylation of CDCP1 is a prerequisite for protein stability and plasma membrane localization, and that the expression level and extent of N-glycosylation of CDCP1 correlated with metastatic status. Interestingly, complex N-linked glycans with sialic acid chains were restricted to the N-terminal half of the ectodomain and absent in the truncated species. Characterization of the extracellular expression of CDCP1 identified novel circulating forms and revealed that extracellular vesicles provide additional processing pathways. Employing immunoaffinity mass spectrometry, we detected elevated levels of circulating CDCP1 in patient urine with high-risk disease. Our results establish that differential glycosylation, cell surface presentation and extracellular expression of CDCP1 are hallmarks of PCa progression.

HIF-2α regulates CDCP1 to promote PKCδ-mediated migration in hepatocellular carcinoma.

Overexpression of CUB domain-containing protein 1 (CDCP1), a transmembrane glycoprotein and major substrate of Src family kinases (SFKs), always indicates unfavorable outcomes in various cancers. The characteristics of CDCP1 in hepatocellular carcinoma (HCC) have not been assessed. Most recently, CDCP1 was identified as a specific target gene of HIF-2α in clear cell renal carcinoma (CC-RCC). However, considering the role of HIF-2α in the progression of HCC is highly controversial, it is necessary to figure out whether HIF-2α and CDCP1 play a significant part in the metastasis of HCC. Our results showed that HIF-2α and CDCP1 were both induced by hypoxia, and the activation of CDCP1 was HIF-2α dependent. CDCP1 was governed by HIF-2α at mRNA and protein levels in HCC cell lines. Moreover, knocking down of HIF-2α not only inhibited cell invasion but also impaired the expression of Tyr(311) phosphorylation of protein kinase Cδ (PKCδ) which is a downstream factor of CDCP1 and has been reported to induce malignant migration in various tumors. Analysis of human HCC samples showed a negative correlation of CDCP1 expression with disease-free survival, and CDCP1 was an independent prognostic factors of disease-free survival. Taken together, these data demonstrated that HIF-2α could promote HCC cell migration by regulating CDCP1, and targeting HIF-2α-CDCP1-PKCδ pathway might be effective to inhibit HCC metastasis.

Abstract 4154: Large oncosomes derived from the aggressive prostate cancer sub-line, DU145R80, can modify the biological behavior of the parental DU145 cells

Abstract We have recently established a zoledronic acid-resistant prostate cancer cell line, DU145R80,which exhibits higher invasive capability compared to parental DU145 cells, and epithelial-to-mesenchymal transition (EMT)[Milone MR, Cell Death Dis. 2013]. To investigate the mechanism by which the cells become invasive, we compared DU145R80 and DU145 cells by a proteomic approach, unveiling a signaling network that links the interior of the nucleus to changes in cytoskeleton dynamics and to the extracellular matrix, dictating prostate cancer aggressiveness [Milone MR Oncotarget 2014].Cytoskeletal modifications allow cells to gain a more migratory/invasive behavior and to interact with the surrounding microenvironment triggering different kind of biological phenomena, including the formation of large oncosomes (LO), a bioactive class of extracellular vesicles (EVs) [Di Vizio D, Cancer Res. 2009]. In this study, we found a higher amount of spontaneously shed LO along with an increased gelatinase activity from DU145R80 cells compared to the parental counterpart. By applying low speed and discontinuous gradient ultracentrifugation to both DU145 and DU145R80 cell media, we obtained a pure preparation of LO, floating at a 1.15 g/mL density fraction and positive for LO markers such as as Cav-1, Ck18, GAPDH. Moreover, we treated DU145 parental cells with pure preparations of LO from either DU145 or DU145R80 cells and performed an invasion assay, showing that LO from the resistant, aggressive DU145R80 cell line increase the invading ability of DU145. Treatment of DU145 cells with LO originating from the parental cell line itself did not result in increased invasinevess, suggesting a specific role for EVs from aggressive cells. In addition, in order to investigate LO content, we focused our attention on a cell surface glycoprotein, CUB domain-containing protein 1 (CDCP-1), putatively linked to the network of proteins identified in DU145R80 cells and whose role in cancer is still under debate. Our results demonstrate that CDCP-1 expression is significantly reduced in DU145R80 compared to DU145 cells, suggesting a tumor-protective role for the protein in prostate cancer. We also observed, for the first time, that the CDCP-1 expression pattern displayed by both cell lines was reflected in their derived LO. These data intriguingly suggest that CDCP-1 may change its expression pattern upon modifications in tumor cells properties, playing a different role in different stages of cancer development. Overall, these findings highlight LO as a new biological component in the development of aggressive features by prostate cancer cells, connecting molecular alterations to changes in the ability to interact with the surrounding environment, and suggested new prognostic markers and/or therapeutical targets. Citation Format: Chiara Ciardiello, Valentina R. Minciacchi, Mariana Reis-Sobreiro, Maria R. Milone, Biagio Pucci, Rita Lombardi, Francesca Bruzzese, Dolores Di Vizio, Alfredo Budillon. Large oncosomes derived from the aggressive prostate cancer sub-line, DU145R80, can modify the biological behavior of the parental DU145 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4154. doi:10.1158/1538-7445.AM2015-4154

Elevated CDCP1 predicts poor patient outcome and mediates ovarian clear cell carcinoma by promoting tumor spheroid formation, cell migration and chemoresistance.

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.

CUB domain containing protein 1 (CDCP1) modulates adhesion and motility in colon cancer cells.

BackgroundDeregulated expression of the transmembrane glycoprotein CDCP1 (CUB domain-containing protein-1) has been detected in several cancers including colon, lung, gastric, breast, and pancreatic carcinomas. CDCP1 has been proposed to either positively or negatively regulate tumour metastasis. In this study we assessed the role of CDCP1 in properties of cells that are directly relevant to metastasis, namely adhesion and motility. In addition, association between CDCP1 and the tetraspanin protein CD9 was investigated.MethodsCDCP1 and CD9 protein expression was measured in a series of colon cancer cell lines by flow cytometry and Western blotting. Adhesion of Colo320 and SW480 cells was determined using a Matrigel adhesion assay. The chemotactic motility of SW480 cells in which CDCP1 expression had been reduced by RNA interference was analysed using the xCELLigence system Real-Time Cell Analyzer Dual Plates combined with 8 μm pore filters. Detergent-resistant membrane fractions were generated following density gradient centrifugation and the CDCP1 and CD9 protein composition of these fractions was determined by Western blotting. The potential association of the CDCP1 and CD9 proteins was assessed by co-immunoprecipitation.ResultsEngineered CDCP1 expression in Colo320 cells resulted in a reduction in cell adhesion to Matrigel. Treatment of SW480 cells with CDCP1 siRNA reduced serum-induced chemotaxis. CDCP1 and CD9 cell-surface protein and mRNA levels showed a positive correlation in colon cancer cell lines and the proteins formed a low-level, but detectable complex as judged by co-sedimentation of detergent lysates of HT-29 cells in sucrose gradients as well as by co-immunoprecipitation in SW480 cell lysates.ConclusionsA number of recent studies have assigned a potentially important role for the cell-surface protein CDCP1 in invasion and metastasis of a several types of human cancer cells. In this study, CDCP1 was shown to modulate cell-substratum adhesion and motility in colon cancer cell lines, with some variation depending on the colon cancer cell type. CDCP1 and CD9 were co-expressed at the mRNA and protein level and we obtained evidence for the presence of a molecular complex of these proteins in SW480 colon cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-754) contains supplementary material, which is available to authorized users.

CDCP1 identifies a CD146 negative subset of marrow fibroblasts involved with cytokine production.

In vitro expanded bone marrow stromal cells contain at least two populations of fibroblasts, a CD146/MCAM positive population, previously reported to be critical for establishing the stem cell niche and a CD146-negative population that expresses CUB domain-containing protein 1 (CDCP1)/CD318. Immunohistochemistry of marrow biopsies shows that clusters of CDCP1+ cells are present in discrete areas distinct from areas of fibroblasts expressing CD146. Using a stromal cell line, HS5, which approximates primary CDCP1+ stromal cells, we show that binding of an activating antibody against CDCP1 results in tyrosine-phosphorylation of CDCP1, paralleled by phosphorylation of Src Family Kinases (SFKs) Protein Kinase C delta (PKC-δ). When CDCP1 expression is knocked-down by siRNA, the expression and secretion of myelopoietic cytokines is increased. These data suggest CDCP1 expression can be used to identify a subset of marrow fibroblasts functionally distinct from CD146+ fibroblasts. Furthermore the CDCP1 protein may contribute to the defining function of these cells by regulating cytokine expression.

CUB-domain containing protein 1 represses the epithelial phenotype of pancreatic cancer cells

The prognosis of pancreatic cancer is dismal due to the frequent metastasis and invasion to surrounding organs. Numerous molecules are involved in the malignant behavior of pancreatic cancer cells, but the entire process remains unclear. Several reports have suggested that CUB-domain containing protein-1 (CDCP1) is highly expressed in pancreatic cancer, but its impact on the invasive growth and the upstream regulator remain elusive. To clarify the role of CDCP1 in pancreatic cancer, we here examined the effects of CDCP1 knockdown on the cell behaviors of pancreatic cancer cells. Knockdown of CDCP1 expression in Panc-1 resulted in reduced cellular migration accompanied by the increased expression of E-cadherin and decreased expression of N-cadherin. Knockdown of CDCP1 attenuated the spheroid formation and resistance against gemcitabine, which are some of the cancer stem cell-related phenotypes. Bone morphogenetic protein 4 (BMP4) was found to induce CDCP1 expression via the extracellular signal regulated kinase pathway, suggesting that CDCP1 has a substantial role in the BMP4-induced epithelial-mesenchymal transition. These results indicate that CDCP1 represses the epithelial phenotype of pancreatic cancer cells.

Covering the Cover

Covering the Cover

Identification of CDCP1 as a hypoxia-inducible factor 2α (HIF-2α) target gene that is associated with survival in clear cell renal cell carcinoma patients

CUB domain-containing protein 1 (CDCP1) is a transmembrane protein that is highly expressed in stem cells and frequently overexpressed and tyrosine-phosphorylated in cancer. CDCP1 promotes cancer cell metastasis. However, the mechanisms that regulate CDCP1 are not well-defined. Here we show that hypoxia induces CDCP1 expression and tyrosine phosphorylation in hypoxia-inducible factor (HIF)-2α-, but not HIF-1α-, dependent fashion. shRNA knockdown of CDCP1 impairs cancer cell migration under hypoxic conditions, whereas overexpression of HIF-2α promotes the growth of tumor xenografts in association with enhanced CDCP1 expression and tyrosine phosphorylation. Immunohistochemistry analysis of tissue microarray samples from tumors of patients with clear cell renal cell carcinoma shows that increased CDCP1 expression correlates with decreased overall survival. Together, these data support a critical role for CDCP1 as a unique HIF-2α target gene involved in the regulation of cancer metastasis, and suggest that CDCP1 is a biomarker and potential therapeutic target for metastatic cancers.

Structural Requirements for Cub Domain Containing Protein 1 (CDCP1) and Src Dependent Cell Transformation

Cub domain containing protein 1 (CDCP1) is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762) abolished transformation, and mutation of a palmitoylation motif (C689,690G) strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.

Abstract 2154: CDCP1 links RAS and Src signaling pathways as a common effector for the promotion of tumor metastasis

Abstract The oncogenic RAS is one of the most intensively studied proteins which are well recognized as a master regulator of cancer initiation and progression, however rather limited information is available as for their contribution to the cell properties associated with tumor metastasis such as anoikis resistance, cell migration and invasion. We have revealed that CUB domain-containing protein 1 (CDCP1) protein, a substrate of Src family kinase (SFK), in solid cancers is involved in the regulation of anoikis resistance, cell migration and invasion. CDCP1 contributes significantly to the metastatic potential of various cancers including lung, stomach, and pancreatic cancers in a tyrosine phosphorylation dependent manner. It was recently observed that human lung cancer cell lines with K-RAS mutations show significantly higher expression of CDCP1 than those without, while the other common mutations such as EGFR, LKB1 and p53 did not show such relationship. The introduction of the activating mutation of K-RAS clearly induced the expression of CDCP1 and that this enhancement is also affected by the expression levels of Myc protein. Treatment of PD98059, an inhibitor of MEK downstream of Ras, as well as expression of dominant-negative Ras, reduced the expression of CDCP1, and at the same time suppressed cell survival in suspension culture, cell migration and invasion. However, these properties were rescued only when both CDCP1 and Fyn, a member of SFKs were expressed. On the contrary, metastatic potentials caused by expression of activated K-Ras in lung cancer cells was blocked by knockdown of in CDCP1 expression induced by Ras. Using three dimensional organotypic culture assay of human cervical keratinocytes (HCKs) which mimics in vivo tissue invasion of cervical cancers, marked expression of CDCP1 was detected at the site of cell invasion into the collagen raft. The treatment of CDCP1 siRNA or SFKs inhibitor PP2 repressed cell invasion into the collagen raft induced by Ras and Myc. As a summary, it was revealed that expression of CDCP1 is induced by oncogenic Ras, and upregulated CDCP1 triggers cancer invasion and metastasis when phosphorylated by SFKs. Metastatic potential induced by Ras is dependent on the CDCP1 induction, while CDCP1 is not responsive for proliferative change also induced by Ras. These findings demonstrated the central role of CDCP1 as the functional link between RAS and SFKs signaling during the progression of human malignant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2154. doi:1538-7445.AM2012-2154

KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

BackgroundKAI1 was initially identified as a metastasis-suppressor gene in prostate cancer. It is a member of the tetraspan transmembrane superfamily (TM4SF) of membrane glycoproteins. As part of a tetraspanin-enriched microdomain (TEM), KAI1 inhibits tumor metastasis by negative regulation of Src. However, the underlying regulatory mechanism has not yet been fully elucidated. CUB-domain-containing protein 1 (CDCP1), which was previously known as tetraspanin-interacting protein in TEM, promoted metastasis via enhancement of Src activity. To better understand how KAI1 is involved in the negative regulation of Src, we here examined the function of KAI1 in CDCP1-mediated Src kinase activation and the consequences of this process, focusing on HIF-1 α and VEGF expression.MethodsWe used the human prostate cancer cell line PC3 which was devoid of KAI1 expression. Vector-transfected cells (PC3-GFP clone #8) and KAI1-expressing PC3 clones (PC3-KAI1 clone #5 and #6) were picked after stable transfection with KAI1 cDNA and selection in 800 μg/ml G418. Protein levels were assessed by immunoblotting and VEGF reporter gene activity was measured by assaying luciferase activitiy. We followed tumor growth in vivo and immunohistochemistry was performed for detection of HIF-1, CDCP1, and VHL protein level.ResultsWe demonstrated that Hypoxia-inducible factor 1α (HIF-1α) and VEGF expression were significantly inhibited by restoration of KAI1 in PC3 cells. In response to KAI1 expression, CDCP1-enhanced Src activation was down-regulated and the level of von Hippel-Lindau (VHL) protein was significantly increased. In an in vivo xenograft model, KAI1 inhibited the expression of CDCP1 and HIF-1α.ConclusionsThese novel observations may indicate that KAI1 exerts profound metastasis-suppressor activity in the tumor malignancy process via inhibition of CDCP1-mediated Src activation, followed by VHL-induced HIF-1α degradation and, ultimately, decreased VEGF expression.