Abstract
Breakdown of the basement membrane is a key step that precedes tumor invasion, and accumulating evidence suggests a key role for the type II transmembrane proteases (TTSPs) in that process. Overexpression of a TTSP hepsin characterizes many solid cancers, including prostate, breast, and ovarian cancer, and in experimental tumor models, the elevated proteolytic activity of hepsin simultaneously activates several growth factors and cleaves basement membrane protein laminin-332, which is an essential component of the cell-basement membrane junction hemidesmosome. These hepsin-dependent molecular events associate with dramatic loss of basement membrane integrity in mouse tumor models and in three-dimensional (3D) epithelial culture. In particular, the 3D culture systems offer unprecedented possibilities to clarify the mechanistic basis of destructive interactions between out-of-control serine protease activity and the basement membrane structure. Here, we describe how to establish 3D mammary epithelial culture in an exogenous basement membrane-free egg white matrix and provide a protocol for quantitative analysis of the impact of hepsin on laminin-332 and its hemidesmosomal receptor α6-integrin by means of confocal microscopy imaging. These protocols were established to facilitate studies aiming to decipher the exact role of oncogenic proteases in tumor invasion processes and to identify novel therapeutic agents able to intervene these cancer critical processes.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Szabo R, Bugge TH (2011) Membrane-anchored serine proteases in vertebrate cell and developmental biology. Annu Rev Cell Dev Biol 27:213–235. https://doi.org/10.1146/annurev-cellbio-092910-154247
Antalis TM, Buzza MS, Hodge KM et al (2010) The cutting edge: membrane-anchored serine protease activities in the pericellular microenvironment. Biochem J 428(3):325–346. https://doi.org/10.1042/BJ20100046
Hedstrom L (2002) Serine protease mechanism and specificity. Chem Rev 102(12):4501–4524
Murray AS, Varela FA, List K (2016) Type II transmembrane serine proteases as potential targets for cancer therapy. Biol Chem 397(9):815–826. https://doi.org/10.1515/hsz-2016-0131
Tanabe LM, List K (2016) The role of type II transmembrane serine protease-mediated signaling in cancer. FEBS J. https://doi.org/10.1111/febs.13971
Dhanasekaran SM, Barrette TR, Ghosh D et al (2001) Delineation of prognostic biomarkers in prostate cancer. Nature 412(6849):822–826. https://doi.org/10.1038/35090585
Partanen JI, Tervonen TA, Myllynen M et al (2012) Tumor suppressor function of liver kinase B1 (Lkb1) is linked to regulation of epithelial integrity. Proc Natl Acad Sci U S A 109(7):E388–E397. https://doi.org/10.1073/pnas.1120421109
Tervonen TA, Belitskin D, Pant SM et al (2016) Deregulated hepsin protease activity confers oncogenicity by concomitantly augmenting HGF/MET signalling and disrupting epithelial cohesion. Oncogene 35(14):1832–1846. https://doi.org/10.1038/onc.2015.248
Xing P, Li JG, Jin F et al (2011) Clinical and biological significance of hepsin overexpression in breast cancer. J Investig Med 59(5):803–810. https://doi.org/10.2310/JIM.0b013e31821451a1
Matsuo T, Nakamura K, Takamoto N et al (2008) Expression of the serine protease hepsin and clinical outcome of human endometrial cancer. Anticancer Res 28(1A):159–164
Miao J, Mu D, Ergel B et al (2008) Hepsin colocalizes with desmosomes and induces progression of ovarian cancer in a mouse model. Int J Cancer 123(9):2041–2047. https://doi.org/10.1002/ijc.23726
Klezovitch O, Chevillet J, Mirosevich J et al (2004) Hepsin promotes prostate cancer progression and metastasis. Cancer Cell 6(2):185–195. https://doi.org/10.1016/j.ccr.2004.07.008
Li W, Wang BE, Moran P et al (2009) Pegylated kunitz domain inhibitor suppresses hepsin-mediated invasive tumor growth and metastasis. Cancer Res 69(21):8395–8402. https://doi.org/10.1158/0008-5472.CAN-09-1995
Pantel K, Brakenhoff RH (2004) Dissecting the metastatic cascade. Nat Rev Cancer 4(6):448–456. https://doi.org/10.1038/nrc1370
Guan X (2015) Cancer metastases: challenges and opportunities. Acta Pharm Sin B 5(5):402–418. https://doi.org/10.1016/j.apsb.2015.07.005
Tripathi M, Nandana S, Yamashita H et al (2008) Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression. J Biol Chem 283(45):30576–30584. https://doi.org/10.1074/jbc.M802312200
Walko G, Castanon MJ, Wiche G (2015) Molecular architecture and function of the hemidesmosome. Cell Tissue Res 360(3):529–544. https://doi.org/10.1007/s00441-015-2216-6
Bissell MJ, Rizki A, Mian IS (2003) Tissue architecture: the ultimate regulator of breast epithelial function. Curr Opin Cell Biol 15(6):753–762
Debnath J, Brugge JS (2005) Modelling glandular epithelial cancers in three-dimensional cultures. Nat Rev Cancer 5(9):675–688. https://doi.org/10.1038/nrc1695
Debnath J, Muthuswamy SK, Brugge JS (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30(3):256–268
Hoffman AS (2002) Hydrogels for biomedical applications. Adv Drug Deliv Rev 54(1):3–12
Kaipparettu BA, Kuiatse I, Tak-Yee Chan B et al (2008) Novel egg white-based 3-D cell culture system. BioTechniques 45(2):165–168., 170-161. https://doi.org/10.2144/000112883
Acknowledgments
We would like to acknowledge all the members of the Klefström laboratory for their critical input in developing this protocol. Special thanks to Tiina Raatikainen and Tarja Välimäki for technical assistance. Biomedicum Imaging Unit and Biomedicum Functional Genomics Unit are acknowledged for core services and technical support. This study was supported by funding from the Academy of Finland, TEKES, Finnish Cancer Organizations, Doctoral Program in Biomedicine, and University of Helsinki.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Pant, S.M., Belitskin, D., Ala-Hongisto, H., Klefström, J., Tervonen, T.A. (2018). Analyzing the Type II Transmembrane Serine Protease Hepsin-Dependent Basement Membrane Remodeling in 3D Cell Culture. In: Cal, S., Obaya, A. (eds) Proteases and Cancer. Methods in Molecular Biology, vol 1731. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7595-2_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7595-2_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7594-5
Online ISBN: 978-1-4939-7595-2
eBook Packages: Springer Protocols