BACKGROUND: The local invasion of tumor cells into the surrounding tissue is the first and most critical step of the metastatic cascade. Cells can invade either collectively, or individually. Individual cancer cell invasion can occur in the mesenchymal or amoeboid mode, which are mutually interchangeable. This plasticity of individual cancer cell invasiveness may represent an escape mechanism for invading cancer cells from anti-metastatic treatment. METHODS: To identify new signaling proteins involved in the plasticity of cancer cell invasiveness, we performed proteomic analysis of the amoeboid to mesenchymal transition with A375m2 melanoma cells in a 3D Matrigel matrix. RESULTS: In this screen we identified PKCα as an important protein for the maintenance of amoeboid morphology. We found that the activation of PKCα resulted in the mesenchymal-amoeboid transition of mesenchymal K2 and MDA-MB-231 cell lines. Consistently, PKCα inhibition led to the amoeboid-mesenchymal transition of amoeboid A375m2 cells. Next, we showed that PKCα inhibition resulted in a considerable decrease in the invading abilities of all analyzed cancer cell lines. CONCLUSIONS: Our results suggest that PKCα is an important protein for maintenance of the amoeboid morphology of cancer cells, and that downregulation of PKCα results in the amoeboid to mesenchymal transition. Our data also suggest that PKCα is important for both mesenchymal and amoeboid invasiveness, making it an attractive target for anti-metastatic therapies.

Current affiliation Microscopy Unit Institute of Experimental Medicine The Czech Academy of Sciences Prague Czech Republic

Department of Cell Biology Laboratory of Cancer Cell Invasion Charles University Prague Prague Czech Republic

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Kopfstein L, Christofori G. Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment. Cell Mol Life Sci. 2006;63(4):449–68. doi: 10.1007/s00018-005-5296-8. PubMed DOI PMC

Farooqui R, Fenteany G. Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement. J Cell Sci. 2005;118(Pt 1):51–63. doi: 10.1242/jcs.01577. PubMed DOI

Friedl P, Gilmour D. Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol. 2009;10(7):445–57. doi: 10.1038/nrm2720. PubMed DOI

Friedl P, Noble PB, Walton PA, Laird DW, Chauvin PJ, Tabah RJ, et al. Migration of coordinated cell clusters in mesenchymal and epithelial cancer explants in vitro. Cancer Res. 1995;55(20):4557–60. PubMed

Friedl P. Prespecification and plasticity: shifting mechanisms of cell migration. Curr Opin Cell Biol. 2004;16(1):14–23. doi: 10.1016/j.ceb.2003.11.001. PubMed DOI

Pankova K, Rosel D, Novotny M, Brabek J. The molecular mechanisms of transition between mesenchymal and amoeboid invasiveness in tumor cells. Cell Mol Life Sci. 2010;67(1):63–71. doi: 10.1007/s00018-009-0132-1. PubMed DOI PMC

Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995;81(1):53–62. doi: 10.1016/0092-8674(95)90370-4. PubMed DOI

Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992;70(3):401–10. doi: 10.1016/0092-8674(92)90164-8. PubMed DOI

Tolde O, Rosel D, Vesely P, Folk P, Brabek J. The structure of invadopodia in a complex 3D environment. Eur J Cell Biol. 2010;89(9):674–80. doi: 10.1016/j.ejcb.2010.04.003. PubMed DOI

Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase) Science. 1996;273(5272):245–8. doi: 10.1126/science.273.5272.245. PubMed DOI

Mierke CT, Rosel D, Fabry B, Brabek J. Contractile forces in tumor cell migration. Eur J Cell Biol. 2008;87(8-9):669–76. doi: 10.1016/j.ejcb.2008.01.002. PubMed DOI PMC

Sahai E, Marshall CJ. ROCK and Dia have opposing effects on adherens junctions downstream of Rho. Nat Cell Biol. 2002;4(6):408–15. doi: 10.1038/ncb796. PubMed DOI

Rosel D, Brabek J, Tolde O, Mierke CT, Zitterbart DP, Raupach C, et al. Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces. Mol Cancer Res. 2008;6(9):1410–20. doi: 10.1158/1541-7786.MCR-07-2174. PubMed DOI

Wyckoff JB, Pinner SE, Gschmeissner S, Condeelis JS, Sahai E. ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo. Curr Biol. 2006;16(15):1515–23. doi: 10.1016/j.cub.2006.05.065. PubMed DOI

Friedl P, Wolf K. Plasticity of cell migration: a multiscale tuning model. J Cell Biol. 2010;188(1):11–9. doi: 10.1083/jcb.200909003. PubMed DOI PMC

Sanz-Moreno V, Marshall CJ. The plasticity of cytoskeletal dynamics underlying neoplastic cell migration. Curr Opin Cell Biol. 2010;22(5):690–6. doi: 10.1016/j.ceb.2010.08.020. PubMed DOI

Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, et al. Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J Cell Biol. 2003;160(2):267–77. doi: 10.1083/jcb.200209006. PubMed DOI PMC

Janostiak R, Janoštiak R, Tolde O, Brůhová Z, Novotný M, Hanks SK, et al. Tyrosine phosphorylation within the SH3 domain regulates CAS subcellular localization, cell migration, and invasiveness. Mol Biol Cell. 2011;22(22):4256–67. doi: 10.1091/mbc.E11-03-0207. PubMed DOI PMC

R Core Team . R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2013.

Bornancin F, Parker PJ. Phosphorylation of protein kinase C-alpha on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state. J Biol Chem. 1997;272(6):3544–9. doi: 10.1074/jbc.272.6.3544. PubMed DOI

Gysin S, Imber R. Replacement of Ser657 of protein kinase C-alpha by alanine leads to premature down regulation after phorbol-ester-induced translocation to the membrane. Eur J Biochem. 1996;240(3):747–50. doi: 10.1111/j.1432-1033.1996.0747h.x. PubMed DOI

Nakashima S. Protein kinase C alpha (PKC alpha): regulation and biological function. J Biochem. 2002;132(5):669–75. doi: 10.1093/oxfordjournals.jbchem.a003272. PubMed DOI

Konopatskaya O, Poole AW. Protein kinase Calpha: disease regulator and therapeutic target. Trends Pharmacol Sci. 2010;31(1):8–14. doi: 10.1016/j.tips.2009.10.006. PubMed DOI PMC

Du HF, Ou LP, Yang X, Song XD, Fan YR, Tan B, et al. A new PKCalpha/beta/TBX3/E-cadherin pathway is involved in PLCepsilon-regulated invasion and migration in human bladder cancer cells. Cell Signal. 2014;26(3):580–93. doi: 10.1016/j.cellsig.2013.11.015. PubMed DOI

Hoshino D, Jourquin J, Emmons SW, Miller T, Goldgof M, Costello K, et al. Network analysis of the focal adhesion to invadopodia transition identifies a PI3K-PKCalpha invasive signaling axis. Sci Signal. 2012;5(241):2002964. doi: 10.1126/scisignal.2002964. PubMed DOI PMC

Lin CC, Lee IT, Wu WB, Liu CJ, Hsieh HL, Hsiao LD, et al. Thrombin mediates migration of rat brain astrocytes via PLC, Ca(2)(+), CaMKII, PKCalpha, and AP-1-dependent matrix metalloproteinase-9 expression. Mol Neurobiol. 2013;48(3):616–30. doi: 10.1007/s12035-013-8450-6. PubMed DOI

Shu Q, Hu ZL, Huang C, Yu XW, Fan H, Yang JW, et al. Orexin-A promotes cell migration in cultured rat astrocytes via Ca2 + -dependent PKCalpha and ERK1/2 signals. PLoS One. 2014;9(4):2014. doi: 10.1371/journal.pone.0095259. PubMed DOI PMC

Wang C, Wang X, Liang H, Wang T, Yan X, Cao M, et al. miR-203 inhibits cell proliferation and migration of lung cancer cells by targeting PKCalpha. PLoS One. 2013;8(9):2013. PubMed PMC

Wu B, Zhou H, Hu L, Mu Y, Wu Y. Involvement of PKCalpha activation in TF/VIIa/PAR2-induced proliferation, migration, and survival of colon cancer cell SW620. Tumour Biol. 2013;34(2):837–46. doi: 10.1007/s13277-012-0614-x. PubMed DOI

Koivunen J, Aaltonen V, Koskela S, Lehenkari P, Laato M, Peltonen J. Protein kinase C alpha/beta inhibitor Go6976 promotes formation of cell junctions and inhibits invasion of urinary bladder carcinoma cells. Cancer Res. 2004;64(16):5693–701. doi: 10.1158/0008-5472.CAN-03-3511. PubMed DOI

Masur K, Lang K, Niggemann B, Zanker KS, Entschladen F. High PKC alpha and low E-cadherin expression contribute to high migratory activity of colon carcinoma cells. Mol Biol Cell. 2001;12(7):1973–82. doi: 10.1091/mbc.12.7.1973. PubMed DOI PMC

Engers R, Mrzyk S, Springer E, Fabbro D, Weissgerber G, Gernharz CD, et al. Protein kinase C in human renal cell carcinomas: role in invasion and differential isoenzyme expression. Br J Cancer. 2000;82(5):1063–9. doi: 10.1054/bjoc.1999.1043. PubMed DOI PMC

Parsons M, Keppler MD, Kline A, Messent A, Humphries MJ, Gilchrist R, et al. Site-directed perturbation of protein kinase C- integrin interaction blocks carcinoma cell chemotaxis. Mol Cell Biol. 2002;22(16):5897–911. doi: 10.1128/MCB.22.16.5897-5911.2002. PubMed DOI PMC

Podar K, Tai YT, Lin BK, Narsimhan RP, Sattler M, Kijima T, et al. Vascular endothelial growth factor-induced migration of multiple myeloma cells is associated with beta 1 integrin- and phosphatidylinositol 3-kinase-dependent PKC alpha activation. J Biol Chem. 2002;277(10):7875–81. doi: 10.1074/jbc.M109068200. PubMed DOI

Hu JG, Wang XF, Zhou JS, Wang FC, Li XW, Lu HZ. Activation of PKC-alpha is required for migration of C6 glioma cells. Acta Neurobiol Exp. 2010;70(3):239–45. PubMed

Lonne GK, Cornmark L, Zahirovic IO, Landberg G, Jirstrom K, Larsson C. PKCalpha expression is a marker for breast cancer aggressiveness. Mol Cancer. 2010;9(76):1476–4598. PubMed PMC

Haughian JM, Bradford AP. Protein kinase C alpha (PKCalpha) regulates growth and invasion of endometrial cancer cells. J Cell Physiol. 2009;220(1):112–8. doi: 10.1002/jcp.21741. PubMed DOI

Cameron AJ, Procyk KJ, Leitges M, Parker PJ. PKC alpha protein but not kinase activity is critical for glioma cell proliferation and survival. Int J Cancer. 2008;123(4):769–79. doi: 10.1002/ijc.23560. PubMed DOI

Wolf K, Te Lindert M, Krause M, Alexander S, Te Riet J, Willis AL, et al. Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force. J Cell Biol. 2013;201(7):1069–84. doi: 10.1083/jcb.201210152. PubMed DOI PMC

Mehta D, Rahman A, Malik AB. Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function. J Biol Chem. 2001;276(25):22614–20. doi: 10.1074/jbc.M101927200. PubMed DOI

Singh I, Knezevic N, Ahmmed GU, Kini V, Malik AB, Mehta D. Galphaq-TRPC6-mediated Ca2+ entry induces RhoA activation and resultant endothelial cell shape change in response to thrombin. J Biol Chem. 2007;282(11):7833–43. doi: 10.1074/jbc.M608288200. PubMed DOI

Holinstat M, Mehta D, Kozasa T, Minshall RD, Malik AB. Protein kinase Calpha-induced p115RhoGEF phosphorylation signals endothelial cytoskeletal rearrangement. J Biol Chem. 2003;278(31):28793–8. doi: 10.1074/jbc.M303900200. PubMed DOI

Dovas A, Choi Y, Yoneda A, Multhaupt HA, Kwon SH, Kang D, et al. Serine 34 phosphorylation of rho guanine dissociation inhibitor (RhoGDIalpha) links signaling from conventional protein kinase C to RhoGTPase in cell adhesion. J Biol Chem. 2010;285(30):23296–308. doi: 10.1074/jbc.M109.098129. PubMed DOI PMC

DerMardirossian C, Bokoch GM. GDIs: central regulatory molecules in Rho GTPase activation. Trends Cell Biol. 2005;15(7):356–63. doi: 10.1016/j.tcb.2005.05.001. PubMed DOI

Avalos AM, Valdivia AD, Munoz N, Herrera-Molina R, Tapia JC, Lavandero S, et al. Neuronal Thy-1 induces astrocyte adhesion by engaging syndecan-4 in a cooperative interaction with alphavbeta3 integrin that activates PKCalpha and RhoA. J Cell Sci. 2009;122(Pt 19):3462–71. doi: 10.1242/jcs.034827. PubMed DOI PMC

Dovas A, Yoneda A, Couchman JR. PKCbeta-dependent activation of RhoA by syndecan-4 during focal adhesion formation. J Cell Sci. 2006;119(Pt 13):2837–46. doi: 10.1242/jcs.03020. PubMed DOI

Madigan JP, Bodemann BO, Brady DC, Dewar BJ, Keller PJ, Leitges M, et al. Regulation of Rnd3 localization and function by protein kinase C alpha-mediated phosphorylation. Biochem J. 2009;424(1):153–61. doi: 10.1042/BJ20082377. PubMed DOI PMC

Riento K, Guasch RM, Garg R, Jin B, Ridley AJ. RhoE binds to ROCK I and inhibits downstream signaling. Mol Cell Biol. 2003;23(12):4219–29. doi: 10.1128/MCB.23.12.4219-4229.2003. PubMed DOI PMC

Pinner S, Sahai E. PDK1 regulates cancer cell motility by antagonising inhibition of ROCK1 by RhoE. Nat Cell Biol. 2008;10(2):127–37. doi: 10.1038/ncb1675. PubMed DOI

Hwang YP, Yun HJ, Kim HG, Han EH, Lee GW, Jeong HG. Suppression of PMA-induced tumor cell invasion by dihydroartemisinin via inhibition of PKCalpha/Raf/MAPKs and NF-kappaB/AP-1-dependent mechanisms. Biochem Pharmacol. 2010;79(12):1714–26. doi: 10.1016/j.bcp.2010.02.003. PubMed DOI

Hansen SH, Zegers MM, Woodrow M, Rodriguez-Viciana P, Chardin P, Mostov KE, et al. Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway. Mol Cell Biol. 2000;20(24):9364–75. doi: 10.1128/MCB.20.24.9364-9375.2000. PubMed DOI PMC

Rochelle T, Daubon T, Van Troys M, Harnois T, Waterschoot D, Ampe C, et al. p210bcr-abl induces amoeboid motility by recruiting ADF/destrin through RhoA/ROCK1. FASEB J. 2013;27(1):123–34. doi: 10.1096/fj.12-205112. PubMed DOI

RNA-seq Characterization of Melanoma Phenotype Switch in 3D Collagen after p38 MAPK Inhibitor Treatment