In vivo cleaved CDCP1 promotes early tumor dissemination via complexing with activated β1 integrin and induction of FAK/PI3K/Akt motility signaling

Abstract

Specific cleavage of the transmembrane molecule, CUB domain-containing protein-1 (CDCP1), by plasmin-like serine proteases induces outside-in signal transduction that facilitates early stages of spontaneous metastasis leading to tumor cell intravasation, namely cell escape from the primary tumor, stromal invasion and transendothelial migration. We identified active β1 integrin as a biochemical and functional partner of the membrane-retained 70-kDa CDCP1 fragment, newly generated from its full-length 135-kDa precursor though proteolytic cleavage by serine proteases. Both in cell cultures and in live animals, active β1 integrin complexed preferentially with functionally activated, phosphorylated 70-kDa CDCP1. Complexing of β1 integrin the 70-kDa with CDCP1 fragment induced intracellular phosphorylation signaling, involving focal adhesion kinase-1 (FAK) and PI3 kinase (PI3K)-dependent Akt activation. Thus, inhibition of FAK/PI3K activities by specific inhibitors as well as short-hairpin RNA downregulation of β1 integrin significantly reduced FAK/Akt phosphorylation under conditions where CDCP1 was processed by serine proteases, indicating that FAK/PI3K/Akt pathway operates downstream of cleaved CDCP1 complexed with β1 integrin. Furthermore, this complex-dependent signaling correlated positively with high levels of tumor cell intravasation and dissemination. Correspondingly, abrogation in vivo of CDCP1 cleavage either by unique cleavage-blocking monoclonal antibody 10-D7 or by inhibition of proteolytic activity of plasmin-like serine proteases with aprotinin prevented β1 integrin/CDCP1 complexing and downstream FAK/Akt signaling concomitant with significant reduction of stromal invasion and spontaneous metastasis. Therefore, β1 integrin appears to serve as a motility-regulating partner mediating cross-talk between proteolytically cleaved, membrane-retained CDCP1 and members of FAK/PI3K/Akt pathway. This CDCP1 cleavage-induced signaling cascade constitutes a unique mechanism, independent of extracellular matrix remodeling, whereby a proteolytically cleaved CDCP1 regulates in vivo locomotion and metastasis of tumor cells through β1 integrin partnering. Our findings indicate that CDCP1 cleavage, occurring at the apex of a β1 integrin/FAK/PI3K/Akt signaling cascade, may represent a therapeutic target for CDCP1-positive cancers.

Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1

CDCP1 cleavage facilitates spontaneous metastasis of PC cells in an orthotopic mouse model of human prostate cancer. (a) Analysis of CDCP1 expression and CDCP1 cleavage in PC-hi/diss cells in culture and in orthotopic primary tumors. Left panel, western blot analysis performed under non-reducing conditions with polyclonal anti-CDCP1 antibody on cell lysates from control EDTA-treated cultures (lane 1) or cultures pre-treated either with 20 μg/ml control IgG (lane 2) or CDCP1 mAb 10-D7 (lane 3), followed by 30-min exposure to 500 nM plasmin. Right panel, prostate tumors were initiated in immunodeficient mice by orthotopic implantation of PC-hi/diss cells. The mice with developing tumors were inoculated intraperitoneally, 2 times per week, with 100 μg control IgG (lane 4) or mAb 10-D7 (lane 5). Western blot analysis of CDCP1 was performed on lysates of primary tumors excised from mice euthanized at 4–5 weeks after surgery. The full-length 135-kDa and cleaved 70-kDa CDCP1 species are indicated in kDa on the left. The position of molecular weight markers is indicated on the right. (b) Effects of mAb 10-D7 treatment on tumor growth and intraperitoneal spread. Treatment with CDCP1 cleavage-blocking mAb 10-D7 does not affect overall appearance of prostate xenografts (top), but dramatically reduced the extent of mesenterium colonization (bottom). Prostate tumor xenografts are delineated with white lines, while tumor cell colonies on the mesenterium are indicated with arrows. (c) Anti-CDCP1 cleavage-blocking mAb does not affect tumorigenesis. Primary tumors from the IgG- and mAb 10-D7-treated mice were excised and weighed to determine the effect of mAb 10-D7 treatment on tumor growth. (d) Anti-CDCP1 cleavage-blocking mAb inhibits spontaneous metastasis of PC-hi/diss cells. Lungs, liver, brain and bone marrow were harvested and processed by Alu-qPCR to determine the number of metastasized human tumor cells within host tissues. Data are presented as means±s.e.m. determined from two independent experiments, involving 8–10 mice per treatment. * and **, P<0.05, one-tailed and two-tailed unpaired Student’s t-tests, respectively. (e) Immunohistochemical analysis of murine lung tissue for CDCP1-positive human cells. Consecutive paraffin sections of lung tissue harvested from the mice bearing orthotopically implanted PC-hi/diss tumors treated in vivo with control mouse IgG or mAb 10-D7, were immunostained with mAb 41-2 against human CDCP1 or control IgG. The small nodules of positively stained human cells (dark brown) are indicated by black arrowheads.

Figure 2

Blocking of CDCP1 cleavage inhibits dissemination of PC cells in chick embryo CAM model for spontaneous metastasis. (a–c) Analysis of tumor growth and dissemination. PC-hi/diss cells were grafted onto the CAM of chick embryos (2 ×106 per embryo) in the presence of 50 μg control IgG or mAb 10-D7 or 0.1 TIU aprotinin. Developing tumors were treated additionally on days 2 and 4 with topical applications of corresponding agents. On day 7, the levels of tumor cell intravasation to the CAM (a) and metastasis to the liver (b) were quantified by Alu-qPCR. Primary tumors were excised and weighed to determine the effect of treatments on tumor growth (c). Presented are means±s.e.m. determined in pooled data from three independent experiments, each employing from 24–36 embryos per treatment variant. ***P<0.001. (d) Analysis of CDCP1 cleavage. Portions of primary tumors were lysed and analyzed for the status of CDCP cleavage by immunoprecipitation with anti-CDCP1 mAb 41–2 and western blotting under non-reducing conditions with poly-clonal anti-CDCP1 antibody. Lane 1, analysis of CDCP1 in the cells inoculated onto the CAM. Lanes 2–4, analysis of CDCP1 in primary CAM tumors treated with control IgG (lane 2), mAb 10-D7 (lane 3) or aprotinin (lane 4). The full-length 135-kDa and cleaved 70-kDa CDCP1 species are indicated on the right. The position of molecular weight markers is indicated in kDa on the left. Asterisk in lane 2 indicates the lower portion of the protein band containing 150-kDa IgG crossreacting with the secondary antibody.

Figure 3

Kinetic analysis of PC cell dissemination in the chick embryo CAM model. (a) Time-course analysis of tumor cell dissemination. PC-hi/diss cells were grafted on the CAM of chick embryos. Primary tumors, distal portions of the CAM and the liver were harvested at the indicated time points to determine tumor weight and levels of CAM intravasation and liver metastasis by Alu-qPCR. (b–c) Effects of delays in CDCP1 cleavage-blocking treatments on PC cell dissemination. PC-hi/diss cells were grafted on the CAM of chick embryos and allowed to develop for 6 days, at which time point primary tumors were treated topically with 50 μg control IgG or mAb 10-D7 or 0.5 TIU aprotinin. The levels of CAM intravasation (left), liver metastasis (middle) and weight of primary tumors (right) were determined on day 7 (b), or on day 7 and day 8 (c). Presented are means±s.e.m. determined in pooled data from three independent experiments employing from 18 to 28 embryos per treatment variant. ***P<0.001. (d) Analysis of CDCP1 in primary CAM tumors. CDCP1 was immunoprecipitated with mAb 41–2 from primary CAM tumors treated with control IgG (lane 1), mAb 10-D7 (lane 2) or aprotinin (lane 3) as described in (b) and harvested the following day (day 7 after cell grafting). Western blot analysis was performed with polyclonal anti-CDCP1 antibody. The full-length 135-kDa and cleaved 70-kDa CDCP1 species are indicated on the right. The position of molecular weight markers is indicated in kDa on the left.

Figure 4

Inhibition of CDCP1 cleavage diminishes carcinoma cell escape from primary tumors, stromal invasion and transendothelial migration. (a) Cell escape and invasion in the intramesodermal microtumor model. Fluorescent-labeled PC-hi/diss cells (green) were inoculated directly into CAM mesoderm. Developing microtumors were treated twice, on days 2 and 4, with control IgG or mAb 10-D7. On day 6, the embryos were inoculated intravenously with rhodamine-tagged LCA to highlight the vasculature (red). Imaging of CAM microtumors and vasculature were performed at ×10 magnification. Dotted lines depict microtumor border and invasion distances covered by escaped tumor cells. Scale bar, 50 μm. (b) Quantification of tumor cell escape and invasion. Mean invasion distance (±s.e.m.) covered by escaped cells from the edge of the microtumor (left graph) and the mean number (±s.e.m.) of cells escaped from the microtumor (right graph) were determined in acquired images. * and ***, respectively, P<0.05 and <0.001. (c) Analysis of CDCP1 cleavage status in the CAM tissue. Following microscopy, mesodermal microtumors with adjacent stromal tissue were dissected from the CAM of embryos treated with control IgG or mAb 10-D7 as described in (a). CDCP1 was immunoprecipitated from tissue lysates with mAb 41–2 and analyzed by western blotting with polyclonal anti-CDCP1 antibody. The full-length 135-kDa and cleaved 70-kDa CDCP1 species are indicated on the right. The position of molecular weight markers is indicated in kDa on the left. (d) Transendothelial migration of PC cells. Fluorescent-tagged PC-hi/diss cells were pre-treated with 20 μg/ml IgG or mAb 10-D7, detached either with EDTA or trypsin, and placed into Transwell inserts with endothelial monolayers pre-grown on the Matrigel-covered undersurfaces. Tumor cells that crossed endothelial barriers were quantified after 2 days by flow cytometry. Data are means±s.e.m. from a representative experiment (out of three independent experiments) performed in triplicate. ** and ***, respectively, P<0.01 and <0.001, unpaired two-tailed Student’s t-tests. Inset, western blot analysis of CDCP1 immunoprecipitated from the transmigrated cells recovered from outer chambers 2 days after cells were pre-treated with IgG and EDTA (lane 1), IgG and trypsin (lane 2), or mAb 10-D7 and trypsin (lane 3), and placed into inserts. CDCP1 species are indicated on the right.

Figure 5

Proteolytic cleavage of CDCP1 induces Src-dependent CDCP1 phosphorylation, β1 integrin complexing with the 70-kDa CDCP1, and intracellular phosphorylation signaling involving FAK and Akt activation. (a) In vitro plasmin-executed CDCP1 cleavage results in complexing of β1 integrin with the 70-kDa CDCP1. PC-hi/diss cells were pre-treated with 20 μg/ml control mouse IgG or mAb 10-D7, and then treated with 500 nM plasmin. CDCP1 was immunoprecipitated from cell lysates with mAb 41–2. Eluted proteins were analyzed by western blotting under reducing conditions with either polyclonal anti-CDCP1 antibody, polyclonal antibody recognizing phosphorylated CDCP1 or mAbs against human β1 or α2 integrin subunits. (b) Complexing of activated β1 integrin with the 70-kDa CDCP1 requires Src-dependent phosphorylation of cleaved CDCP1. PC-hi/diss cells were pre-treated with 20 μg/ml control mouse IgG or mAb 10-D7 or 0.5 μM SFK inhibitor dasatinib and then treated with EDTA (lane 1) or trypsin/EDTA (lanes 2–4) and lysed. Cleavage status of CDCP1 was confirmed by western blotting of cell lysates with polyclonal anti-CDCP1 antibody. Integrin complexes were precipitated from tissue lysates with the antibody HUTS-4 recognizing activated form of β1 integrin. Eluted proteins were then analyzed for the presence of co-precipitated CDCP1 and phospho CDCP1. Equal levels of precipitated activated β1 integrin are indicated by western blotting with the mAb 17783 recognizing β1 integrin after SDS–PAGE. Position of molecular weight markers in kDa is indicated on the left. (c) Signaling pathways induced by CDCP1 cleavage in vivo in the mouse lung retention model. PC-hi/diss cells were inoculated via tail vein into mice along with 100 μg control mouse IgG or mAb 10-D7. After 24 h, the lungs were excised from euthanized mice and lysed. Tissue lysates were subjected to western blot analysis for CDCP1 cleavage status or immunoprecipitation with anti-activated β1 integrin antibody HUTS-4. Eluted proteins were analyzed for β1 integrin levels with the mAb 17783 and for presence of CDCP1 and phosphorylated CDCP1. In addition, lung tissue lysates were analyzed for the levels of activated (phospho FAK) versus total FAK and activated (phospho Akt) versus total Akt.

Figure 6

Serine protease-induced Akt signaling regulates stromal invasion and transendothelial migration of tumor cells through complex formation between β1 integrin with cleaved 70-kDa CDCP1. (a) Akt signaling pathway is induced by plasmin-mediated cleavage of CDCP1 in a PI3K-dependent manner. PC-hi/diss cells were treated with 500 nM plasmin in the presence of serine protease inhibitor aprotinin (0.1 TIU/ml), vehicle (PBS-1% dimethyl sulfoxide (DMSO)) or PI3K inhibitor wortmannin (1 μM). CDCP1 was immunoprecipitated from cells lysates and analyzed for CDCP1 cleavage status, CDCP1 phosphorylation and presence of co-precipitated β1 integrin. In addition, whole cell lysates were analyzed for the levels of activated and total Akt. (b) Akt signaling regulates stromal invasion of PC cells. Fluorescent-tagged PC-hi/diss cells were inoculated directly into CAM mesoderm. Developing microtumors were treated twice on days 2 and 4 with 0.1 ml of aprotinin (0.1 TIU/ml), vehicle (PBS-2% DMSO) or wortmannin (2 μM). Invasion distances were determined in digitally acquired images as described in Figure 4. The data are mean±s.e.m. of invasion expressed as percentage of aprotinin-treated conditions (100%), that is, in the absence of serine protease activity. One of three independent experiments is presented. ***P<0.001. (c) Akt signaling pathway, induced in vitro under conditions allowing for activity of plasmin-like serine proteases, regulates transendothelial migration of PC cells. Fluorescent-tagged PC-hi/diss cells were incubated with aprotinin (0.1 TIU/ml), vehicle (PBS-1% DMSO) or wortmannin (1 μM). The cells were then treated with trypsin in the presence of the corresponding agents, washed and placed into Transwell inserts, the undersurface of which was covered by a monolayer of human endothelial cells pre-grown on a layer of Matrigel. Tumor cells were allowed to migrate for 2 days to the outer chamber filled with Dulbecco’s modified Eagle’s medium–5% fetal calf serum and corresponding pre-treatment agents. The number of green fluorescent cells that crossed the endothelial barrier was determined in individual outer chambers by flow cytometry. The data are mean±s.e.m. of migration expressed as percentage of aprotinin-treated conditions (100%), that is, in the absence of serine protease activity. One of four independent experiments is presented. **P<0.01.

Figure 7

Spontaneous dissemination and stromal invasion of PC cells involves FAK and PI3K/Akt pathways induced in vivo downstream of β1 integrin complexing with the cleaved phosphorylated CDCP1. (a) Spontaneous tumor cell dissemination, but not primary tumor growth, is inhibited by abrogating FAK and Akt activation. PC-hi/diss cells were grafted on the CAM of chick embryos and developing tumors were treated twice with 0.1 ml vehicle (PBS-1% DMSO), PI3K inhibitor wortmannin (1 μM) or FAK inhibitor 14 (10 μM). On day 7, primary tumors were harvested and weighed. Portions of distal CAM and liver were excised, processed and analyzed by Alu-qPCR to determine the number of tumor cells that intravasated into CAM vasculature and metastasized to the liver. Data (means±s.e.m.) are from two independent experiments for each inhibitor employing 13 and 11 embryos treated with wortmannin and FAK inhibitor, respectively, and a total of 31 control embryos. *, **, and *** indicate P<0.05, <0.01, <0.001, respectively. (b) Analysis of β1 integrin complexing with cleaved phosphorylated CDCP1 and activation of Akt signaling. CDCP1 was immunoprecipitated from primary CAM tumors harvested on day 7 from the embryos treated with vehicle or wortmannin as described above in (a). Presence of cleaved and phosphorylated CDCP1 and co-precipitated β1 and α2 integrin subunits was confirmed in eluted complexes by western blotting performed with corresponding specific mAbs. Total CAM tissue lysates were analyzed for the levels of phosphorylated Akt versus total Akt. (c) Analysis of FAK and Akt phosphorylation downstream of complexing between β1 integrin with cleaved CDCP1. β1 integrin was immunoprecipitated from primary CAM tumors harvested on day 7 from the embryos treated with vehicle or FAK inhibitor 14 as described above in (a). Eluted complexes were analyzed for the presence of β1 integrin subunit, cleavage status of CDDP1 and levels of phospho FAK versus total FAK specifically associated with β1 integrin. Total tissue lysates were also analyzed for the levels of phospho FAK and Akt versus corresponding total proteins. (d) Stromal invasion of PC-hi/diss cells depends on the activity of FAK and β1 integrin. Intramesodermal PC-hi/diss microtumors were treated once on day 3 with FAK inhibitor 14 (25 μl of 20 μM solution per bolus) or anti-β1 integrin function-blocking mAb P5D2 (25 μl of 50 μg/ml solution per bolus). Control microtumors were treated with 25 μl vehicle (2% DMSO in PBS). Invasion distances were measured on day 6 after cell grafting. ***P<0.001. Primary microtumors and invasion distances are indicated by dotted lines in representative images on the right. Scale bar, 50 μm.

Figure 8

Proteolytic cleavage of CDCP1 contributes to enhanced tumor cell survival and motility through the FAK and Akt signaling pathways downstream of complex formation between cleaved CDCP1 and β1 integrin. Schematic depicts a sequence of events that follow proteolytic cleavage of the cell surface CDCP1 molecule, consisting of three extracellular CUB domains, a transmembrane domain (TM) and a C-terminal domain (CT) containing several tyrosine residues (Tyr). Specifically, the serine protease plasmin executes singular cleavage of the full-length 135-kDa CDCP1, clipping off a 65-kDa fragment and generating the 70-kDa membrane-retained fragment. This 70-kDa fragment serves as a docking platform for Src family kinases, including Src. If the activity of Src kinase is inhibited by the Src inhibitor dasatinib, the cleaved CDCP1 does not become phosphorylated although inactive Src still docks onto the C-terminus of cleaved 70-kDa CDCP1. If cytoplasmic Src is active (phosphorylated, pSrc), CDCP1-bound pSrc phosphorylates the C-terminus of CDCP1 (70-kDa pCDCP1). Phosphorylation of 70-kDa CDCP1 at Tyr residues facilitates docking of PKCδ, which becomes activated (pPKCδ) by CDCP1-bound active Src. The phosphorylated 70-kDa CDCP1 forms stable complexes with β1 integrins (e.g., α2β1), which in turn enhances phosphorylation of integrin-associated FAK (pFAK) and leads to PI3K-dependent activation of Akt. Inhibition of FAK activity (e.g, by specific FAK inhibitor 14) prevents FAK phosphorylation, leading to diminished phosphorylation of Akt (pAkt). Akt activation downstream of FAK can be abrogated completely through inhibition of the direct activator of Akt, PI3K (e.g, by wortmannin). Ultimately, the inhibition of FAK/PI3K/Akt pathway reduces signaling for cell survival and motility. While CDCP1 cleavage signaling is crucial for tumor cell survival during late stages of metastasis, namely tissue colonization at the secondary sites, cleaved CDCP1 signals for enhanced motility during early stages of spontaneous dissemination, namely cell escape from the primary tumor, stromal invasion and intravasation. Synergistically, these motility-involving processes contribute to enhanced metastatic dissemination of aggressive CDCP1-expressing tumor cells.