Abstract
Tumor metastasis depends on the dynamic regulation of cell adhesion through β1-integrin. The Cub-Domain Containing Protein-1, CDCP1, is a transmembrane glycoprotein which regulates cell adhesion. Overexpression and loss of CDCP1 have been observed in the same cancer types to promote metastatic progression. Here, we demonstrate reduced CDCP1 expression in high-grade, primary prostate cancers, circulating tumor cells and tumor metastases of patients with castrate-resistant prostate cancer. CDCP1 is expressed in epithelial and not mesenchymal cells, and its cell surface and mRNA expression declines upon stimulation with TGFβ1 and epithelial-to-mesenchymal transition. Silencing of CDCP1 in DU145 and PC3 cells resulted in 3.4-fold higher proliferation of non-adherent cells and 4.4-fold greater anchorage independent growth. CDCP1-silenced tumors grew in 100% of mice, compared to 30% growth of CDCP1-expressing tumors. After CDCP1 silencing, cell adhesion and migration diminished 2.1-fold, caused by loss of inside-out activation of β1-integrin. We determined that the loss of CDCP1 reduces CDK5 kinase activity due to the phosphorylation of its regulatory subunit, CDK5R1/p35, by c-SRC on Y234. This generates a binding site for the C2 domain of PKCδ, which in turn phosphorylates CDK5 on T77. The resulting dissociation of the CDK5R1/CDK5 complex abolishes the activity of CDK5. Mutations of CDK5-T77 and CDK5R1-Y234 phosphorylation sites re-establish the CDK5/CDKR1 complex and the inside-out activity of β1-integrin. Altogether, we discovered a new mechanism of regulation of CDK5 through loss of CDCP1, which dynamically regulates β1-integrin in non-adherent cells and which may promote vascular dissemination in patients with advanced prostate cancer.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Wortmann A, He Y, Deryugina EI, Quigley JP, Hooper JD. The cell surface glycoprotein CDCP1 in cancer—insights, opportunities, and challenges. IUBMB Life. 2009;61:723–30.
Siva AC, Kirkland RE, Lin B, Maruyama T, McWhirter J, Yantiri-Wernimont F, et al. Selection of anti-cancer antibodies from combinatorial libraries by whole-cell panning and stringent subtraction with human blood cells. J Immunol Methods. 2008;330:109–19.
Siva AC, Wild MA, Kirkland RE, Nolan MJ, Lin B, Maruyama T, et al. Targeting CUB domain-containing protein 1 with a monoclonal antibody inhibits metastasis in a prostate cancer model. Cancer Res. 2008;68:3759–66.
Deryugina EI, Conn EM, Wortmann A, Partridge JJ, Kupriyanova TA, Ardi VC, et al. Functional role of cell surface CUB domain-containing protein 1 in tumor cell dissemination. Mol Cancer Res. 2009;7:1197–211.
Fukuchi K, Steiniger SC, Deryugina E, Liu Y, Lowery CA, Gloeckner C, et al. Inhibition of tumor metastasis: functional immune modulation of the CUB domain containing protein 1. Mol Pharm. 2010;7:245–53.
Zarif JC, Lamb LE, Schulz VV, Nollet EA, Miranti CK. Androgen receptor non-nuclear regulation of prostate cancer cell invasion mediated by Src and matriptase. Oncotarget. 2015;6:6862–76.
Casar B, He Y, Iconomou M, Hooper JD, Quigley JP, Deryugina EI. Blocking of CDCP1 cleavage in vivo prevents Akt-dependent survival and inhibits metastatic colonization through PARP1-mediated apoptosis of cancer cells. Oncogene. 2012;31:3924–38.
Yang L, Dutta SM, Troyer DA, Lin JB, Lance RA, Nyalwidhe JO, et al. Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer. Oncotarget. 2015;6:43743–58.
Sandvig K, Llorente A. Proteomic analysis of microvesicles released by the human prostate cancer cell line PC-3. Mol Cell Proteom. 2012;11:M111.012914.
Minciacchi VR, Zijlstra A, Rubin MA, Di Vizio D. Extracellular vesicles for liquid biopsy in prostate cancer: where are we and where are we headed? Prostate Cancer Prostatic Dis. 2017;20:251–8.
Spassov DS, Wong CH, Wong SY, Reiter JF, Moasser MM. Trask loss enhances tumorigenic growth by liberating integrin signaling and growth factor receptor cross-talk in unanchored cells. Cancer Res. 2013;73:1168–79.
Dong Y, He Y, de Boer L, Stack MS, Lumley JW, Clements JA, et al. The cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor-mediated cell migration. J Biol Chem. 2012;287:9792–803.
Spassov DS, Wong CH, Moasser MM. Trask phosphorylation defines the reverse mode of a phosphotyrosine signaling switch that underlies cell anchorage state. Cell Cycle. 2011;10:1225–32.
DeVries-Seimon TA, Ohm AM, Humphries MJ, Reyland ME. Induction of apoptosis is driven by nuclear retention of protein kinase C delta. J Biol Chem. 2007;282:22307–14.
Spassov DS, Baehner FL, Wong CH, McDonough S, Moasser MM. The transmembrane Src substrate Trask is an epithelial protein that signals during anchorage deprivation. Am J Pathol. 2009;174:1756–65.
Wright HJ, Arulmoli J, Motazedi M, Nelson LJ, Heinemann FS, Flanagan LA, et al. CDCP1 cleavage is necessary for homodimerization-induced migration of triple-negative breast cancer. Oncogene. 2016;35:4762–72.
Alvares SM, Dunn CA, Brown TA, Wayner EE, Carter WG. The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1. Biochim Biophys Acta. 2008;1780:486–96.
Casar B, Rimann I, Kato H, Shattil SJ, Quigley JP, Deryugina EI. In vivo cleaved CDCP1 promotes early tumor dissemination via complexing with activated beta1 integrin and induction of FAK/PI3K/Akt motility signaling. Oncogene. 2014;33:255–68.
Takagi J, Petre BM, Walz T, Springer TA. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell. 2002;110:599–611.
Moser M, Legate KR, Zent R, Fässler R. The tail of integrins, talin, and kindlins. Science. 2009;324:895–9.
Shattil SJ, Kim C, Ginsberg MH. The final steps of integrin activation: the end game. Nat Rev Mol Cell Biol. 2010;11:288–300.
Schwartz MA, Schaller MD, Ginsberg MH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol. 1995;11:549–99.
Provasi D, Negri A, Coller BS, Filizola M. Talin-driven inside-out activation mechanism of platelet αIIbβ3 integrin probed by multimicrosecond, all-atom molecular dynamics simulations. Proteins. 2014;82:3231–40.
Ginsberg MH. Integrin activation. BMB Rep. 2014;47:655–9.
Jin JK, Tien PC, Cheng CJ, Song JH, Huang C, Lin SH, et al. Talin1 phosphorylation activates β1 integrins: a novel mechanism to promote prostate cancer bone metastasis. Oncogene. 2015;34:1811–21.
Hsu FN, Chen MC, Chiang MC, Lin E, Lee YT, Huang PH, et al. Regulation of androgen receptor and prostate cancer growth by cyclin-dependent kinase 5. J Biol Chem. 2011;286:33141–9.
Strock CJ, Park JI, Nakakura EK, Bova GS, Isaacs JT, Ball DW, et al. Cyclin-dependent kinase 5 activity controls cell motility and metastatic potential of prostate cancer cells. Cancer Res. 2006;66:7509–15.
Sato K, Zhu YS, Saito T, Yotsumoto K, Asada A, Hasegawa M, et al. Regulation of membrane association and kinase activity of Cdk5-p35 by phosphorylation of p35. J Neurosci Res. 2007;85:3071–8.
Gao C, Negash S, Guo HT, Ledee D, Wang HS, Zelenka P. CDK5 regulates cell adhesion and migration in corneal epithelial cells. Mol Cancer Res. 2002;1:12–24.
Nakano N, Nakao A, Ishidoh K, Tsuboi R, Kominami E, Okumura K, et al. CDK5 regulates cell-cell and cell-matrix adhesion in human keratinocytes. Br J Dermatol. 2005;153:37–45.
Bosutti A, Qi J, Pennucci R, Bolton D, Matou S, Ali K, et al. Targeting p35/Cdk5 signalling via CIP-peptide promotes angiogenesis in hypoxia. PLoS ONE. 2013;8:e75538.
Tsai LH, Delalle I, Caviness VS, Chae T, Harlow E. p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature. 1994;371:419–23.
You S, Knudsen BS, Erho N, Alshalalfa M, Takhar M, Al-Deen Ashab H, et al. Integrated classification of prostate cancer reveals a novel luminal subtype with poor outcome. Cancer Res. 2016;76:4948–58.
Das L, Anderson TA, Gard JM, Sroka IC, Strautman SR, Nagle RB, et al. Characterization of laminin binding integrin internalization in prostate cancer cells. J Cell Biochem. 2017;118:1038–49.
Sperger JM, Strotman LN, Welsh A, Casavant BP, Chalmers Z, Horn S. et al. Integrated analysis of multiple biomarkers from circulating tumor cells enabled by exclusion-based analyte isolation. Clin Cancer Res. 2016;23:746–756.
Putzke AP, Ventura AP, Bailey AM, Akture C, Opoku-Ansah J, Celiktaş M, et al. Metastatic progression of prostate cancer and E-cadherin regulation by zeb1 and SRC family kinases. Am J Pathol. 2011;179:400–10.
Schehr JL, Schultz ZD, Warrick JW, Guckenberger DJ, Pezzi HM, Sperger JM, et al. High specificity in circulating tumor cell identification is required for accurate evaluation of programmed death-ligand 1. PLoS ONE. 2016;11:e0159397.
Kimura H, Morii E, Ikeda JI, Ezoe S, Xu JX, Nakamichi N, et al. Role of DNA methylation for expression of novel stem cell marker CDCP1 in hematopoietic cells. Leukemia. 2006;20:1551–6.
Tun HW, Marlow LA, von Roemeling CA, Cooper SJ, Kreinest P, Wu K, et al. Pathway signature and cellular differentiation in clear cell renal cell carcinoma. PLoS ONE. 2010;5:e10696.
Cantu E, Lederer DJ, Meyer K, Milewski K, Suzuki Y, Shah RJ, et al. Gene set enrichment analysis identifies key innate immune pathways in primary graft dysfunction after lung transplantation. Am J Transplant. 2013;13:1898–904.
Spassov DS, Wong CH, Sergina N, Ahuja D, Fried M, Sheppard D, et al. Phosphorylation of Trask by Src kinases inhibits integrin clustering and functions in exclusion with focal adhesion signaling. Mol Cell Biol. 2011;31:766–82.
Calderwood DA, Campbell ID, Critchley DR. Talins and kindlins: partners in integrin-mediated adhesion. Nat Rev Mol Cell Biol. 2013;14:503–17.
Hisanaga S, Saito T. The regulation of cyclin-dependent kinase 5 activity through the metabolism of p35 or p39 Cdk5 activator. Neurosignals. 2003;12:221–9.
Chang KH, Multani PS, Sun KH, Vincent F, de Pablo Y, Ghosh S, et al. Nuclear envelope dispersion triggered by deregulated Cdk5 precedes neuronal death. Mol Biol Cell. 2011;22:1452–62.
Wortmann A, He Y, Christensen ME, Linn M, Lumley JW, Pollock PM, et al. Cellular settings mediating Src substrate switching between focal adhesion kinase tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) tyrosine 734. J Biol Chem. 2011;286:42303–15.
Stahelin RV, Kong KF, Raha S, Tian W, Melowic HR, Ward KE, et al. Protein kinase Cθ C2 domain is a phosphotyrosine binding module that plays a key role in its activation. J Biol Chem. 2012;287:30518–28.
Benes CH, Wu N, Elia AE, Dharia T, Cantley LC, Soltoff SP. The C2 domain of PKCdelta is a phosphotyrosine binding domain. Cell. 2005;121:271–80.
Li W, Yu JC, Shin DY, Pierce JH. Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity. J Biol Chem. 1995;270:8311–8.
Uekita T, Jia L, Narisawa-Saito M, Yokota J, Kiyono T, Sakai R. CUB domain-containing protein 1 is a novel regulator of anoikis resistance in lung adenocarcinoma. Mol Cell Biol. 2007;27:7649–60.
Uekita T, Tanaka M, Takigahira M, Miyazawa Y, Nakanishi Y, Kanai Y, et al. CUB-domain-containing protein 1 regulates peritoneal dissemination of gastric scirrhous carcinoma. Am J Pathol. 2008;172:1729–39.
Sachdev S, Bu Y, Gelman IH. Paxillin-Y118 phosphorylation contributes to the control of Src-induced anchorage-independent growth by FAK and adhesion. BMC Cancer. 2009;9:12.
Razorenova OV, Finger EC, Colavitti R, Chernikova SB, Boiko AD, Chan CK, et al. VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKC{delta}-driven migration. Proc Natl Acad Sci USA. 2011;108:1931–6.
Benes CH, Poulogiannis G, Cantley LC, Soltoff SP. The SRC-associated protein CUB domain-containing protein-1 regulates adhesion and motility. Oncogene. 2012;31:653–63.
Liu H, Ong SE, Badu-Nkansah K, Schindler J, White FM, Hynes RO. CUB-domain-containing protein 1 (CDCP1) activates Src to promote melanoma metastasis. Proc Natl Acad Sci USA. 2011;108:1379–84.
Liu Y, Belkina NV, Graham C, Shaw S. Independence of protein kinase C-delta activity from activation loop phosphorylation: structural basis and altered functions in cells. J Biol Chem. 2006;281:12102–11.
O’Hare MJ, Kushwaha N, Zhang Y, Aleyasin H, Callaghan SM, Slack RS, et al. Differential roles of nuclear and cytoplasmic cyclin-dependent kinase 5 in apoptotic and excitotoxic neuronal death. J Neurosci. 2005;25:8954–66.
Uekita T, Sakai R. Roles of CUB domain-containing protein 1 signaling in cancer invasion and metastasis. Cancer Sci. 2011;102:1943–8.
Hofmann J. Protein kinase C isozymes as potential targets for anticancer therapy. Curr Cancer Drug Targets. 2004;4:125–46.
Kawauchi T. Cell adhesion and its endocytic regulation in cell migration during neural development and cancer metastasis. Int J Mol Sci. 2012;13:4564–90.
Ganguly KK, Pal S, Moulik S, Chatterjee A. Integrins and metastasis. Cell Adh Migr. 2013;7:251–61.
Spassov DS, Wong CH, Harris G, McDonough S, Phojanakong P, Wang D, et al. A tumor-suppressing function in the epithelial adhesion protein Trask. Oncogene. 2012;31:419–31.
Ohlsson L, Israelsson A, Öberg Å, Palmqvist R, Stenlund, H, Hammarström, ML, et al. Lymph node CEA and MUC2 mRNA as useful predictors of outcome in colorectal cancer. Int J Cancer. 2012;130:1833–43.
Mamat S, Ikeda J, Enomoto T, Ueda Y, Rahadiani N, Tian T, et al. Prognostic significance of CUB domain containing protein expression in endometrioid adenocarcinoma. Oncol Rep. 2010;23:1221–7.
Sawada G, Takahashi Y, Niida A, Shimamura T, Kurashige J, Matsumura T, et al. Loss of CDCP1 expression promotes invasiveness and poor prognosis in esophageal squamous cell carcinoma. Ann Surg Oncol. 2014;21:S640–7.
Kato H, Liao Z, Mitsios JV, Wang HY, Deryugina EI, Varner JA, et al. The primacy of β1 integrin activation in the metastatic cascade. PLoS ONE. 2012;7:e46576.
Parvani JG, Galliher-Beckley AJ, Schiemann BJ, Schiemann WP. Targeted inactivation of β1 integrin induces β3 integrin switching, which drives breast cancer metastasis by TGF-β. Mol Biol Cell. 2013;24:3449–59.
Twardowski PW, Beumer JH, Chen CS, Kraft AS, Chatta GS, Mitsuhashi M, et al. A phase II trial of dasatinib in patients with metastatic castration-resistant prostate cancer treated previously with chemotherapy. Anticancer Drugs. 2013;24:743–53.
Dickreuter E, Eke I, Krause M, Borgmann K, van Vugt MA, Cordes N. Targeting of β1 integrins impairs DNA repair for radiosensitization of head and neck cancer cells. Oncogene. 2016;35:1353–62.
Hoffmann A, Lannert H, Brischwein K, Pipp FC, Reindl J, Groll K, et al. inventors; Merck Patent GmbH, Assignee. Anti-alpha-V integrin antibody for the treatment of prostate cancer. 2017; US Patent 9555110, International PCT/EP2012/000548.
Antal CE, Hudson AM, Kang E, Zanca C, Wirth C, Stephenson NL, et al. Cancer-associated protein kinase C mutations reveal kinase’s role as tumor suppressor. Cell. 2015;160:489–502.
Blake RA, Garcia-Paramio P, Parker PJ, Courtneidge SA. Src promotes PKCdelta degradation. Cell Growth Differ. 1999;10:231–41.
Glaser KB, Sung A, Bauer J, Weichman BM. Regulation of eicosanoid biosynthesis in the macrophage. Involvement of protein tyrosine phosphorylation and modulation by selective protein tyrosine kinase inhibitors. Biochem Pharmacol. 1993;45:711–21.
Yang J, Zheng Z, Yan X, Li X, Liu Z, Ma Z. Integration of autophagy and anoikis resistance in solid tumors. Anat Rec. 2013;296:1501–8.
Li X, Wu JB, Li Q, Shigemura K, Chung LW, Huang WC. SREBP-2 promotes stem cell-like properties and metastasis by transcriptional activation of c-Myc in prostate cancer. Oncotarget. 2016;7:12869–84.
Schiewer MJ, Goodwin JF, Han S, Brenner JC, Augello MA, Dean JL, et al. Dual roles of PARP-1 promote cancer growth and progression. Cancer Discov. 2012;2:1134–49.
Knudsen BS, Zhao P, Resau J, Cottingham S, Gherardi E, Xu E, et al. A novel multipurpose monoclonal antibody for evaluating human c-Met expression in preclinical and clinical settings. Appl Immunohistochem Mol Morphol. 2009;17:57–67.
Orchard-Webb DJ, Lee TC, Cook GP, Blair GE. CUB domain containing protein 1 (CDCP1) modulates adhesion and motility in colon cancer cells. BMC Cancer. 2014;14:754.
Rai AC, Singh I, Singh M, Shah K. Expression of ZAT12 transcripts in transgenic tomato under various abiotic stresses and modeling of ZAT12 protein in silico. Biometals. 2014;27:1231–47.
Acknowledgements
We heartily acknowledge Wen-Chin Huang for assistance in animal surgery, L. Chung for support in in vivo experiments, and to C. Pospisil for cloning PKCδ mutant with inactive kinase into vector. We thank the patients and their families, Robert Vessella, Eva Corey, Celestia Higano, Bruce Montgomery, Peter Nelson, Paul Lange, Martine Roudier, and Lawrence True for their contributions to the University of Washington Medical Center Prostate Cancer Donor Rapid Autopsy Program supported by funding by the Pacific Northwest Prostate Cancer SPORE (P50CA97186) and the Richard M. LUCAS Foundation. We are thankful to Jennifer Kitchel and the Biobank and Translational Research Core at Cedars-Sinai Medical Center for xenograft embedding, tissue processing, and IHC staining. This work was supported by Department of Defense Synergistic Idea Development Award W81XWH-08-1-0268 and start-up funding from Cedars-Sinai Medical Center.
Author information
Authors and Affiliations
Contributions
SP performed experiments and wrote the paper. FH performed computational experiments found in Fig. 1A, B, D. JS and JL contributed the qPCR analysis of CTCs found in Fig. 1C. CM provided tissues from rapid autopsies of patients with metastatic prostate cancers. AC provided scientific input on regulation of β1-integrin. NB provided scientific input on TGF-beta 1 in prostate cancer. DS and MM generated and contributed the short hairpins for knockdown of CDCP1. WC independently discovered CDCP1 and initiated the project. SRS performed CDK5 kinase assays found in Fig. 6C and Supplementary Fig. 6A. KS contributed prediction models of protein phosphorylation. BK provided the conceptual framework and wrote the paper.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Pollan, S.G., Huang, F., Sperger, J.M. et al. Regulation of inside-out β1-integrin activation by CDCP1. Oncogene 37, 2817–2836 (2018). https://doi.org/10.1038/s41388-018-0142-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41388-018-0142-2
This article is cited by
-
Influence of extracellular matrix composition on tumour cell behaviour in a biomimetic in vitro model for hepatocellular carcinoma
Scientific Reports (2023)
-
Substrate-biased activity-based probes identify proteases that cleave receptor CDCP1
Nature Chemical Biology (2021)
-
Extracellular matrix and its therapeutic potential for cancer treatment
Signal Transduction and Targeted Therapy (2021)