CUB domain-containing protein 1 (CDCP1), a novel type-1 transmembrane glycoprotein, has dysregulated expression in several cancers including kidney cancer. Significantly, CDCP1 has increased expression in kidney cancer samples and little if any expression in normal kidney tissue. CDCP1 expression has been linked with poor prognosis in kidney cancer patients as it has been associated with metastasis and shorter disease specific survival. Although it is clear that CDCP1 is up-regulated in kidney cancer, the role the protein plays in the progression of this disease is uncertain.
Our hypothesis is that CDCP1 is functionally involved in the metastasis of kidney cancer and our aim is to elucidate the role of this protein in in vitro processes associated with cancer metastasis. We therefore examined the effects of up and down regulation of CDCP1 on the ability of kidney cancer cell lines to proliferate, migrate, adhere or form colonies.
Kidney cancer is a significant health issue as it accounts for 2% of all cancers as well as 2% of all cancer related deaths in Australia [1,2]. Organ confined kidney cancer is mainly asymptomatic and hence difficult to diagnose early [3,4]. Furthermore, the variable and unpredictable course of kidney cancer makes disease prognosis difficult. In addition, although radical nephrectomy, the mainstay treatment is effective for the majority of patients up to 40% of patients will have disease recurrence [5,6]. Compounding this issue radiation- and chemo- therapy are ineffective against advanced kidney cancer and immunotherapy generally has poor response rates [7,1,8].
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Recently, CUB domain-containing protein 1 (CDCP1) has been identified as a novel type-1 transmembrane glycoprotein that is dysregulated in several cancers including kidney cancer [9,10,6,11]. Using a microarray-based screening approach, Awakura et al., (2008) demonstrated that increased CDCP1 expression is associated with poor prognosis in kidney cancer. These workers also showed by immunohistochemical analysis that CDCP1 expression is heavily associated with metastasis (33.5% of 230 renal cell carcinoma cases) and shorter disease free survival. Therefore, although it is clear that CDCP1 is up-regulated in kidney cancer, there is no information on whether CDCP1 is functionally involved in the progression of this cancer.
CDCP1 is a cell surface protein also known as SIMA 135 , gp140 , and Trask  and has been assigned the cluster of differentiation (CD) designation CD318 . As shown in Figure 1, CDCP1 has a molecular weight of 135kDa and contains a 29 residue amino-terminal signal peptide, extracellular (636 amino acids), transmembrane (21 amino acids) and cytoplasmic (150 amino acids) domains. The extracellular domain consists of three CUB-like (complement protein subcomponents Clr/Cls, urchin embryonic growth factor and bone morphogenic protein 1) domains [15,16]. CUB-like domains have immunoglobulin like folds involved in protein-protein, protein-carbohydrate and cell-cell interactions [12,17,18,16,19]. The cytoplasmic domain contains a short hexalysine stretch and five tyrosine residues at least 3 of which are phosphorylated by Src family kinases (SFKs) [12,20,21,10,18,6,22]. According to Uekita et al., (2007 and 2008) and Alvares et al., (2008), tyrosine residue Tyr734 is tyrosine phosphorylated by the SFKs. Benes et al., (2005) also showed that SFKs initiate phosphorylation of CDCP1 at Tyr734 which promotes further phosphorylation of Tyr743 and Tyr762. Benes et al., (2005) have also indicated that phosphorylation of Tyr762 results in formation of a CDCP1-Src-PKCδ complex. This is further supported by Uekita et al., (2007) who demonstrated that CDCP1 is the docking protein between SFKs and PKCδ. CDCP1 also contains 12 sites for N-glycosylation which accounts for deviations between the theoretical molecular weight, 90.1kDa, and the actual molecular weight, 135kDa of CDCP1 [23,13].
Figure 1: CDCP1 structural features. CDCP1 contains an amino terminal signal peptide, extracellular region which consists of three CUB-like (CUB-L) domains, a transmembrane domain and a cytoplasmic domain which contains five tyrosine residues.
In addition to SFKs and PKCδ interactions, there are reports which propose that 135kDa CDCP1 is processed via interactions with proteolytic enzymes such as matriptase, trypsin and other unknown endogenous tryptic serine proteases to the ~80kDa form. According to Brown et al., (2004) the outcome of the proteolytic conversion of full length CDCP1 causes phosphorylation of the short ~80kDa form. In addition, there have been reports which suggest that cell de-adhesion causes phosphorylation of the ~80kDa form of CDCP1, while re-adhesion results in de-phosphorylation
A number of reports demonstrate that CDCP1 is involved in signal transduction across the cell surface membrane and suggest that aberrant CDCP1 activity facilitates cancer development . For example the CDCP1-Src-PKCδ complex described above plays a role in the control of anoikis resistance [24,25]. Anoikis is a type of apoptosis which is triggered when a cell detaches from the extracellular matrix and loses cell survival signals generated from this interaction [24,17]. The ability of a malignant cell to evade this form of cell death is critical in cancer progression. Uekita et al., (2007) reported phosphorylation of CDCP1 regulated resistance to anoikis in lung cancer cells and Uekita et al. (2008) have also shown that over expression of CDCP1 facilitates anchorage-independent growth of gastric carcinoma. Deryugina et al.,(2009) also confirm these findings revealing that CDCP1 serves a an anti-apoptotic molecule which assists cell survival during metastasis. Clearly the ability of a malignant cell to evade this form of programmed cell death is critical in cancer metastasis.
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Therefore our hypothesis was that CDCP1 is functionally involved in the metastasis of kidney cancer. The purpose of this study was to elucidate the role of this protein in in vitro processes associated with cancer metastasis. We therefore examined the effects of up and down regulation of CDCP1 on the ability of kidney cancer cell lines to acquire a tumourigenic phenotype using proliferation, migration, adhesion and soft agar colony formation assays which are markers of metastatic potential.
Materials and Methods
Cell Culture and reagents
All cell lines were acquired from the American Type Culture Collection. HEK293, ACHN and A704 cell lines were cultured in DMEM (Invitrogen) and cell lines 786-O and 769-P were cultured in RPMI-1040 Medium (Invitrogen) supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin (100 units per ml) and incubated at 37ËšC in an atmosphere of 5% CO2. Cells were forced into suspension with 0.5mM EDTA in 1X phosphate buffered saline (PBS).
A704 and HEK293 cell lines were transfected with 700ng of the pcDNA 3.1 expression constructs: pcDNA -CDCP1-wild type, pcDNA -CDCP1-R368A single cleavage site mutant, pcDNA -CDCP1- R368A -K369A double cleavage site mutant (these mutants were designed to prevent cleavage of CDCP1 by proteases) and pcDNA 3.1 empty vector using Lipofectamine 2000 reagent (Invitrogen). G418 (InvivoGen) resistant colonies were selected and expanded to produce monoclonal stable cell lines. Cells over- expressing CDCP1 were identified by Western blot and flow cytometry analysis using rabbit anti-CDCP1 polyclonal antibody and our mouse anti-CDCP1 monoclonal antibody 10D7 respectfully.
769-P and ACHN cell lines were transfected with 700ng of the pcDNA 6.2 GW/±EmGFP-miR knock down constructs and 786-O cell line was transfected with 2000ng of the pcDNA 6.2 GW/±EmGFP-miR knock down constructs using Lipofectamine 2000 reagent (Invitrogen). The constructs used were Block-it-negative (off target control) and Block-it-CDCP1 . Blasticidin resistant colonies were selected and expanded to produce monoclonal stable cell lines. Cells with reduced CDCP1 expression were identified by quantitative Real time PCR (qRT PCR) and Western blot analysis using rabbit anti-CDCP1 polyclonal antibody (Cell Signaling Technology).
Site-directed mutagenesis (SDM) (Agilent Technologies, US) was utilised according to the manufacturers protocol to produce the CDCP1 cleavage mutant constructs. The two templates used for SDM were pcDNA 3.2 CDCP1 - R368A (single cleavage mutation) in which the Arginine at position 368 was changed to an Alanine and pcDNA 3.2 CDCP1 - R368A K369A (double cleavage mutation) which Arginine at position 368 was changed to an Alanine and also the Lysine at position 369 changed to an Alanine. These cleavage mutations inhibit the cleavage of CDCP1 by matriptase and other proteases and thus the production of the 80KDa CDCP1 product. QIAprep spin miniprep system was used according to the manufacturer's protocol to purify the vector from the super-competent cells. Purified vector was sequenced to determine which clone had the correct sequence which was then transfected into A704 and HEK293 cells.
Western blot analysis
The expression of CDCP1 in the transfected cell lines was determined by Western blot analysis. Total cellular lysates of the transfected cells were collected in lysis buffer (10mM tris pH 8.0, 150mM NaCl, 1% Triton X-100, 1x protease inhibitors, 10mM sodium fluoride and 2mM sodium orthovanadate) and scraped on ice. Total protein concentration was determined using BCA protein assay (Peirce, Rockford, IL) and 50µg of each lysate were separated in reduced conditions on 10% SDS polyacrylamide gels and transferred to a nitrocellulose membrane. Non- specific binding sites were blocked with 5% skim milk Tris buffered saline (TBS T) (0.1%T) and the membrane probed with rabbit anti-CDCP1 monoclonal antibody (Cell Signaling Technology). The membrane was further probed with a horseradish peroxidase conjugated secondary antibody (Abcam) and equal loading was determined using a GAPDH or β-Tubulin antibody (Abcam). The protein was detected by chemiluminescence and visualised by autoradiography.
The expression of CDCP1 on the over-expressing CDCP1 cells' surface was analysed using flow cytometry (Beckman Coulter FC500 Flow Cytometer). Whole cells were incubated with our anti-CDCP1 monoclonal antibody 10D7 , then with Alexa Fluor 488 Goat anti- Mouse IgG (H+L) and analysed in the flow cytometer.
Cells were cultured on glass coverslips and fixed in 4% paraformaldehyde. Cells were stained with 10D7 mouse monoclonal antibody and were not permeabilised. Controls samples included cells stained with secondary antibody only. Glass cover slips were mounted on glass slides using Prolong Gold Antifade. Cells were visualised using a Leica SP5 spectral scanning confocal microscope (Leica, Sydney, Australia).
RNA isolation, cDNA synthesis and quantitative real- time PCR
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RNA isolation, cDNA synthesis and quantitative real- time PCR was performed exactly as previously described in (S.E Perry et al 2007) and used to determine level of CDCP1 knockdown in the 769-P clones.
Quantitaive real-time PCR was performed on the ABI 7300 Real time PCR system (Applied Biosystems) and using the 7300 system SDS software (Applied Biosystems). The following CDCP1 primers were designed: Forward (5'-CATCGCAGCGGTGGGAGGTG-3') and reverse (5'-CCACAGCGGGGCCCTTGTTT-3'). The housekeeping gene used as the reference gene was hypoxanthine phosphoribosyltransferase 1 (HPRT1).
CDCP1 was immunoprecipitated from cell lysates (~3.5 mg) with rabbit anti-CDCP1 antibody (Cell Signaling Technology) or rabbit IgG as a control using Protein A agarose immobilized protein (Roche diagnostics GmBH, Mannheim, Germany). Precipitated proteins were eluted with SDS buffer, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. Western blot analysis was then used to detect for immunoprecipitated protein using the following antibodies: rabbit anti-FAK, anti-Src, anti-PKCδ and anti-CDCP1 using the above mentioned protocol.
CyQUANT NF (No Freeze) Cell proliferation assay (Invitrogen)
The proliferation assay was carried out using the commercially available CyQUANT Cell proliferation assay (Invitrogen) and was completed according to the manufacturer's protocol. Briefly, 1000 (for HEK293 cell line) and 1500 (for 769-P cell line) cells were cultured a 96 well microplate. At 24 hour time points for 5 days, culture medium was removed and CyQuant dye binding solution was added and incubated for 60 minutes and the fluorescence intensity measured on the Polar Star fluorescence microplate reader with excitation at 485nm and emission at 530nm.
Soft agar colony formation assay
Six well plates were coated with medium containing 10% FCS and 0.5% agar (Sigma) and allowed to set. 5000 cells in medium containing 10% FCS and 0.35% agarose (Sigma) was poured onto the coated well, solidified and incubated at 37°C for 3 weeks, with 200 µl of fresh medium added to each well once a week. The number of colonies per 5 fields of view in each well was counted using light microscopy. Untransfected cells were the control and medium/agarose without cells was the blank control.
96 well black Perkin Elmer plates were coated with the following extracellular matrix (ECM) components: collagen 1V, vitronectin, fibronectin, and laminin-1 and blocked with 5% Bovine Serum Albumin (BSA) PBS as described previously (Cell adhesion assays William scott). Briefly, 30000 cells were seeded onto the coated 96 well plate in serum free medium. Cells were allowed to adhere for 60 minutes and after which non- adherent cells were washed off with PBS. CyQuant dye binding solution was used as described above. Blank wells were included to subtract the background binding of CyQuant dye to plastic, while wells coated with ECM components had BSA added in order for the non-specific binding to ECM components to be used as blank control.
Chemotactic Migration assay
100000 cells were placed into uncoated transwell inserts (8µm pores) in serum free media and stimulated to migrate towards medium containing 5% foetal calf serum (FCS) for 24 hours at 37 degrees Celsius and 5% CO2. Migrated cells were detached with 1xtrypsin and the total number of cells migrated was counted using a haemocytometer.
CDCP1 expression in kidney cancer
Recently, CDCP1 has been identified as a novel type-1 transmembrane glycoprotein that is dysregulated in several cancers including kidney cancer [9,10,6,11]. To confirm this observation in kidney cancer, researchers within the Hooper group undertook a Western blot analysis for CDCP1 expression in ten kidney cancer patient samples (Figure 1a) (He and Hooper, unpublished). Consistent with published research ((Hooper et al., 2003; Ikeda et al., 2006; Awakura et al., 2008; Wong et al., 2009) it is clear in Fig 1a. that CDCP1 (135 kDa band) is highly expressed in patients' one to eight and ten kidney tumour samples however it had much lower expression in normal tissue samples taken from the same patient. The result indicates that CDCP1 is up-regulated in kidney cancer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an equal loading control.
A Western blot analysis of CDCP1 expression in kidney cancer cell lines available in the laboratory was also performed (Figure 1b). The cell lines included in this panel were HEK293 which is a transformed embryonic kidney cell line, A704 a non-tumourigenic kidney cancer cell line while ACHN, 786-O and 769-P are tumourigenic kidney cancer cell lines. As revealed in Fig 1b. the tumourigenic kidney cancer cell lines 786-O and 769-P express full length CDCP1 (135 kDa band), while ACHN cell line expresses both full length (135kDa band) and cleaved CDCP1 (~80kDa band). The embryonic kidney cell line HEK293 and the non-tumourigenic kidney cancer cell line A704 do not express CDCP1. From the results acquired, it is apparent these cell lines are valuable as in vitro models to study modulated CDCP1 expression in kidney cancer. Hence these cell lines were utilized as model systems to study the effect up and down regulation of CDCP1 in in vitro assays associated with cancer progression.
Modulated CDCP1 expression in kidney cell lines
Over-expression of CDCP1 in kidney cell lines
To determine whether up regulation of CDCP1 facilitates kidney cancer progression, kidney cell lines over-expressing CDCP1 needed to be generated. Monoclonal cell lines stably over-expressing CDCP1 were generated from HEK293 cell line, a transformed embryonic kidney cell line which does not endogenously express CDCP1. This was also attempted in the A704 cell line, a non-tumourigenic kidney cancer cell line which does not endogenously express CDCP1, without success. HEK293 cells were transfected with 700ng of the pcDNA 3.1 expression constructs: pcDNA -CDCP1-wild type, pcDNA -CDCP1-R368A single cleavage site mutant, pcDNA -CDCP1- R368A -K369A double cleavage site mutant (these mutants were designed to prevent cleavage of CDCP1 by proteases) and pcDNA 3.1 empty vector which will be used as a control in the in vitro assays. G418 resistant colonies were selected and expanded. Cells stably expressing CDCP1 were identified by Western blot analysis (Figure 2a and Table 1) using rabbit anti-CDCP1 polyclonal primary antibody (Cell Signaling Technology).
As illustrated in Fig 2a from left to right: there are three vector control clones (named VC1, VC2 and VC3) and two clones over-expressing CDCP1 (135kDa band) for each CDCP1 construct: pcDNA -CDCP1-wild type (named W8 and W9), pcDNA -CDCP1-R368A single cleavage site mutant (named S8 and S13) and pcDNA -CDCP1- R368A -K369A double cleavage site mutant (named DC3 and DC13). From this Western blot it is evident that CDCP1 over-expressing HEK293 cells only express full length CDCP1 (135kDa). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an equal loading control.
Results were confirmed by flow cytometry analysis (Figure 2b) using 10D7 mouse monoclonal primary antibody . HeLa cell line which does not endogenously express CDCP1 was used as a negative control. PC3 cell line which endogenously expresses CDCP1 was used as a positive control. Included in the flow cytometry analysis were controls using the HEK293 parental cell line. These controls were HEK293 cells with no antibody staining, with secondary antibody staining only and also staining with both primary and secondary antibody.
Knockdown of CDCP1 expression in Kidney cancer cell lines
To determine whether down-regulation of CDCP1 expression negatively impacts on kidney cancer progression, kidney cells lines with reduced CDCP1 expression needed to be generated. Stable monoclonal cell lines with reduced CDCP1 expression were generated from 769-P cell line, a CDCP1 endogenous expressing, tumourigenic kidney cancer cell line. Generation of monoclonal cell lines with reduced CDCP1 expression in ACHN and 786-O cell lines was not completed due to time constraints. 769-P cell line was transfected with 700ng of the pcDNA 6.2 GW/±EmGFP-miR knock down constructs. The constructs used were Block-it-negative (off target control) and Block-it-CDCP1 . Blasticidin resistant colonies were selected and expanded to produce monoclonal stable cell lines. Cells with reduced CDCP1 expression were identified by Western blot analysis (Figure 3b and Table 1) using rabbit anti-CDCP1 polyclonal antibody (Cell Signaling Technology).
As shown in Fig 3a from left to right, there two Block-it-negative clones (named N10 and N12) and three Block-it-CDCP1 clones (named C13, C26 and C17). The Block-it-CDCP1 clones appear to have markedly reduced CDCP1 expression compared to 769-P parental cell line and the Block-it-negative clones.
As displayed in Fig 3b the clones with reduced CDCP1 expression were confirmed via quantitative Real time PCR (qRT PCR). Clones C13 and C17 had significantly reduced CDCP1 expression (p<0.0001) compared to the parental cell line and also to N10 and N12.
Generation of monoclonal cell lines with modulated CDCP1 expression required the screening via Western blot analysis of numerous clones. This is exemplified in Table 1. Many clones needed to be generated in order for a small number have to been successful in expressing the pcDNA 3.1 and Block-it constructs.