Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a therapeutically challenging disease with poor survival rates, owing to late diagnosis and early dissemination. These tumors frequently undergo perineural invasion, spreading along nerves regionally and to distant sites. The RET receptor tyrosine kinase is implicated in increased aggressiveness, local invasion, and metastasis in multiple cancers, including PDAC. RET mediates directional motility and invasion towards sources of its neurotrophic factor ligands, suggesting that it may enhance perineural invasion of tumor cells towards nerves. RET is expressed as two main isoforms, RET9 and RET51, which differ in their protein interactions and oncogenic potentials, however, the contributions of RET isoforms to neural invasion have not been investigated. In this study, we generated total RET and isoform-specific knockdown PDAC cell lines and assessed the contributions of RET isoforms to PDAC invasive spread. Our data show that RET activity induces cell polarization and actin remodeling through activation of CDC42 and RHOA GTPases to promote directional motility in PDAC cells. Further, we show that RET interacts with the adaptor protein TKS5 to induce invadopodia formation, enhance matrix degradation and promote tumor cell invasion through a SRC and GRB2-dependent mechanism. Finally, we show that RET51 is the predominant isoform contributing to these RET-mediated invasive processes in PDAC. Together, our work suggests that RET expression in pancreatic cancers may enhance tumor aggressiveness by promoting perineural invasion, and that RET expression may be a valuable marker of invasiveness, and a potential therapeutic target in the treatment of these cancers.
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
Saad AM, Turk T, Al-Husseini MJ, Abdel-Rahman O. Trends in pancreatic adenocarcinoma incidence and mortality in the United States in the last four decades; a SEER-based study. BMC Cancer. 2018;18:688.
Ilic M, Ilic I. Epidemiology of pancreatic cancer. World J Gastroenterol. 2016;22:9694–705.
Huang L, Jansen L, Balavarca Y, Babaei M, van der Geest L, Lemmens V, et al. Stratified survival of resected and overall pancreatic cancer patients in Europe and the USA in the early twenty-first century: a large, international population-based study. BMC Med. 2018;16:125.
Vincent A, Herman J, Schulick R, Hruban RH, Goggins M. Pancreatic cancer. Lancet. 2011;378:607–20.
Marchesi F, Piemonti L, Mantovani A, Allavena P. Molecular mechanisms of perineural invasion, a forgotten pathway of dissemination and metastasis. Cytokine Growth Factor Rev. 2010;21:77–82.
Liebig C, Ayala G, Wilks JA, Berger DH, Albo D. Perineural invasion in cancer: a review of the literature. Cancer. 2009;115:3379–91.
Hirai I, Kimura W, Ozawa K, Kudo S, Suto K, Kuzu H, et al. Perineural invasion in pancreatic cancer. Pancreas. 2002;24:15–25.
Liang D, Shi S, Xu J, Zhang B, Qin Y, Ji S, et al. New insights into perineural invasion of pancreatic cancer: More than pain. Biochim Biophys Acta. 2016;1865:111–22.
Amit M, Na’ara S, Fridman E, Vladovski E, Wasserman T, Milman N, et al. RET, a targetable driver of pancreatic adenocarcinoma. Int J Cancer. 2019;144:3014–22.
Amit M, Na’ara S, Leider-Trejo L, Binenbaum Y, Kulish N, Fridman E, et al. Upregulation of RET induces perineurial invasion of pancreatic adenocarcinoma. Oncogene. 2017;36:3232–9.
Ceyhan GO, Demir IE, Altintas B, Rauch U, Thiel G, Muller MW, et al. Neural invasion in pancreatic cancer: a mutual tropism between neurons and cancer cells. Biochem Biophys Res Commun. 2008;374:442–7.
Ceyhan GO, Giese NA, Erkan M, Kerscher AG, Wente MN, Giese T, et al. The neurotrophic factor artemin promotes pancreatic cancer invasion. Ann Surg. 2006;244:274–81.
Gil Z, Cavel O, Kelly K, Brader P, Rein A, Gao SP, et al. Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves. J Natl Cancer Inst. 2010;102:107–18.
Zeng Q, Cheng Y, Zhu Q, Yu Z, Wu X, Huang K, et al. The relationship between overexpression of glial cell-derived neurotrophic factor and its RET receptor with progression and prognosis of human pancreatic cancer. J Int Med Res. 2008;36:656–64.
Ito Y, Okada Y, Sato M, Sawai H, Funahashi H, Murase T, et al. Expression of glial cell line-derived neurotrophic factor family members and their receptors in pancreatic cancers. Surgery. 2005;138:788–94.
Mulligan LM. GDNF and the RET receptor in cancer: new insights and therapeutic potential. Front Physiol. 2019;9:1873.
Lian EY, Maritan SM, Cockburn JG, Kasaian K, Crupi MJ, Hurlbut D, et al. Differential roles of RET isoforms in medullary and papillary thyroid carcinomas. Endocr Relat Cancer. 2017;24:53–69.
Kohno T, Ichikawa H, Totoki Y, Yasuda K, Hiramoto M, Nammo T, et al. KIF5B-RET fusions in lung adenocarcinoma. Nat Med. 2012;18:375–7.
Morandi A, Plaza-Menacho I, Isacke CM. RET in breast cancer: functional and therapeutic implications. Trends Mol Med. 2011;17:149–57.
Moodley S, Lian EY, Crupi MJF, Hyndman BD, Mulligan LM. RET isoform-specific interaction with scaffold protein Ezrin promotes cell migration and chemotaxis in lung adenocarcinoma. Lung Cancer. 2020;142:123–31.
Tahira T, Ishizaka Y, Itoh F, Sugimura T, Nagao M. Characterization of ret proto-oncogene mRNAs encoding two isoforms of the protein product in a human neuroblastoma cell line. Oncogene. 1990;5:97–102.
Myers SM, Eng C, Ponder BA, Mulligan LM. Characterization of RET proto-oncogene 3’ splicing variants and polyadenylation sites: a novel C-terminus for RET. Oncogene. 1995;11:2039–45.
Besset V, Scott RP, Ibanez CF. Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase. J Biol Chem. 2000;275:39159–66.
Boulay A, Breuleux M, Stephan C, Fux C, Brisken C, Fiche M, et al. The Ret receptor tyrosine kinase pathway functionally interacts with the ERalpha pathway in breast cancer. Cancer Res. 2008;68:3743–51.
Ibanez CF. Structure and physiology of the RET receptor tyrosine kinase. Cold Spring Harb Perspect Biol. 2013;5:a009134.
Richardson DS, Rodrigues DM, Hyndman BD, Crupi MJ, Nicolescu AC, Mulligan LM. Alternative splicing results in RET isoforms with distinct trafficking properties. Mol Biol Cell. 2012;23:3838–50.
Crupi MJ, Yoganathan P, Bone LN, Lian E, Fetz A, Antonescu CN, et al. Distinct temporal regulation of RET isoform internalization: roles of clathrin and AP2. Traffic. 2015;16:1155–73.
Tsui-Pierchala BA, Ahrens RC, Crowder RJ, Milbrandt J, Johnson EM Jr. The long and short isoforms of Ret function as independent signaling complexes. J Biol Chem. 2002;277:34618–25.
Hyndman BD, Crupi MJF, Peng S, Bone LN, Rekab AN, Lian EY, et al. Differential recruitment of E3 ubiquitin ligase complexes regulates RET isoform internalization. J Cell Sci. 2017;130:3282–96.
Crupi MJF, Maritan SM, Reyes-Alvarez E, Lian EY, Hyndman BD, Rekab AN, et al. GGA3-mediated recycling of the RET receptor tyrosine kinase contributes to cell migration and invasion. Oncogene. 2020;39:1361–77.
Griseri P, Garrone O, Lo Sardo A, Monteverde M, Rusmini M, Tonissi F, et al. Genetic and epigenetic factors affect RET gene expression in breast cancer cell lines and influence survival in patients. Oncotarget. 2016;7:26465–79.
Bhinge K, Yang L, Terra S, Nasir A, Muppa P, Aubry MC, et al. EGFR mediates activation of RET in lung adenocarcinoma with neuroendocrine differentiation characterized by ASCL1 expression. Oncotarget. 2017;8:27155–65.
Ben-Chetrit N, Chetrit D, Russell R, Korner C, Mancini M, Abdul-Hai A, et al. Synaptojanin 2 is a druggable mediator of metastasis and the gene is overexpressed and amplified in breast cancer. Sci Signal. 2015;8:ra7.
Parachoniak CA, Luo Y, Abella JV, Keen JH, Park M. GGA3 functions as a switch to promote Met receptor recycling, essential for sustained ERK and cell migration. Dev Cell. 2011;20:751–63.
Condeelis JS, Wyckoff JB, Bailly M, Pestell R, Lawrence D, Backer J, et al. Lamellipodia in invasion. Semin Cancer Biol. 2001;11:119–28.
Abella JV, Parachoniak CA, Sangwan V, Park M. Dorsal ruffle microdomains potentiate Met receptor tyrosine kinase signaling and down-regulation. J Biol Chem. 2010;285:24956–67.
Murphy DA, Courtneidge SA. The ‘ins’ and ‘outs’ of podosomes and invadopodia: characteristics, formation and function. Nat Rev Mol Cell Biol. 2011;12:413–26.
Parachoniak CA, Park M. Dynamics of receptor trafficking in tumorigenicity. Trends Cell Biol. 2012;22:231–40.
Lamorte L, Royal I, Naujokas M, Park M. Crk adapter proteins promote an epithelial-mesenchymal-like transition and are required for HGF-mediated cell spreading and breakdown of epithelial adherens junctions. Mol Biol Cell. 2002;13:1449–61.
Radhakrishna H, Al-Awar O, Khachikian Z, Donaldson JG. ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements. J Cell Sci. 1999;112:855–66.
Menard L, Parker PJ, Kermorgant S. Receptor tyrosine kinase c-Met controls the cytoskeleton from different endosomes via different pathways. Nat Commun. 2014;5:3907.
Gorelik R, Gautreau A. Quantitative and unbiased analysis of directional persistence in cell migration. Nat Protoc. 2014;9:1931–43.
Mayor R, Carmona-Fontaine C. Keeping in touch with contact inhibition of locomotion. Trends Cell Biol. 2010;20:319–28.
Rottner K, Hall A, Small JV. Interplay between Rac and Rho in the control of substrate contact dynamics. Curr Biol. 1999;9:640–8, S1.
Raftopoulou M, Hall A. Cell migration: Rho GTPases lead the way. Dev Biol. 2004;265:23–32.
Di Martino J, Paysan L, Gest C, Lagree V, Juin A, Saltel F, et al. Cdc42 and Tks5: a minimal and universal molecular signature for functional invadosomes. Cell Adh Migr. 2014;8:280–92.
Courtneidge SA, Azucena EF, Pass I, Seals DF, Tesfay L. The SRC substrate Tks5, podosomes (invadopodia), and cancer cell invasion. Cold Spring Harb Symp Quant Biol. 2005;70:167–71.
Murphy DA, Diaz B, Bromann PA, Tsai JH, Kawakami Y, Maurer J, et al. A Src-Tks5 pathway is required for neural crest cell migration during embryonic development. PLoS ONE. 2011;6:e22499.
Encinas M, Crowder RJ, Milbrandt J, Johnson EM. Tyrosine 981, a novel Ret autophosphorylation site, binds c-Src to mediate neuronal survival. J Biol Chem. 2004;279:18262–9.
Poteryaev D, Titievsky A, Sun YF, Thomas-Crusells J, Lindahl M, Billaud M, et al. GDNF triggers a novel ret-independent Src kinase family-coupled signaling via a GPI-linked GDNF receptor alpha1. FEBS Lett. 1999;463:63–6.
Rajadurai CV, Havrylov S, Zaoui K, Vaillancourt R, Stuible M, Naujokas M, et al. Met receptor tyrosine kinase signals through a cortactin-Gab1 scaffold complex, to mediate invadopodia. J Cell Sci. 2012;125:2940–53.
Paz H, Pathak N, Yang J. Invading one step at a time: the role of invadopodia in tumor metastasis. Oncogene. 2014;33:4193–202.
Oikawa T, Itoh T, Takenawa T. Sequential signals toward podosome formation in NIH-src cells. J cell Biol. 2008;182:157–69.
Burger KL, Learman BS, Boucherle AK, Sirintrapun SJ, Isom S, Diaz B, et al. Src-dependent Tks5 phosphorylation regulates invadopodia-associated invasion in prostate cancer cells. Prostate. 2014;74:134–48.
Stylli SS, Stacey TT, Verhagen AM, Xu SS, Pass I, Courtneidge SA, et al. Nck adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation. J Cell Sci. 2009;122:2727–40.
Jacquemet G, Hamidi H, Ivaska J. Filopodia in cell adhesion, 3D migration and cancer cell invasion. Curr Opin Cell Biol. 2015;36:23–31.
Eddy RJ, Weidmann MD, Sharma VP, Condeelis JS. Tumor cell invadopodia: invasive protrusions that orchestrate metastasis. Trends Cell Biol. 2017;27:595–607.
Itoh Y. Membrane-type matrix metalloproteinases: their functions and regulations. Matrix Biol. 2015;44–46:207–23.
Bakst RL, Wong RJ. Mechanisms of perineural invasion. J Neurol Surg B Skull Base. 2016;77:96–106.
Rodrigues DM, Li AY, Nair DG, Blennerhassett MG. Glial cell line-derived neurotrophic factor is a key neurotrophin in the postnatal enteric nervous system. Neurogastroenterol Motil. 2011;23:e44–56.
Honma Y, Araki T, Gianino S, Bruce A, Heuckeroth RO Jr, Johnson E, et al. Artemin is a vascular-derived neurotropic factor for developing sympathetic neurons. Neuron. 2002;35:267–82.
Suter-Crazzolara C, Unsicker K. GDNF is expressed in two forms in many tissues outside the CNS. NeuroReport. 1994;5:2486–8.
Chermenina M, Schouten P, Nevalainen N, Johansson F, Oradd G, Stromberg I. GDNF is important for striatal organization and maintenance of dopamine neurons grown in the presence of the striatum. Neuroscience. 2014;270:1–11.
Nevalainen N, Chermenina M, Rehnmark A, Berglof E, Marschinke F, Stromberg I. Glial cell line-derived neurotrophic factor is crucial for long-term maintenance of the nigrostriatal system. Neuroscience. 2010;171:1357–66.
Meka DP, Muller-Rischart AK, Nidadavolu P, Mohammadi B, Motori E, Ponna SK, et al. Parkin cooperates with GDNF/RET signaling to prevent dopaminergic neuron degeneration. J Clin Invest. 2015;125:1873–85.
Drinkut A, Tillack K, Meka DP, Schulz JB, Kugler S, Kramer ER. Ret is essential to mediate GDNF’s neuroprotective and neuroregenerative effect in a Parkinson disease mouse model. Cell Death Dis. 2016;7:e2359.
Kramer ER, Liss B. GDNF-Ret signaling in midbrain dopaminergic neurons and its implication for Parkinson disease. FEBS Lett. 2015;589:3760–72.
Leong HS, Robertson AE, Stoletov K, Leith SJ, Chin CA, Chien AE, et al. Invadopodia are required for cancer cell extravasation and are a therapeutic target for metastasis. Cell Rep. 2014;8:1558–70.
Williams KC, Cepeda MA, Javed S, Searle K, Parkins KM, Makela AV, et al. Invadopodia are chemosensing protrusions that guide cancer cell extravasation to promote brain tropism in metastasis. Oncogene. 2019;38:3598–615.
He S, Chen CH, Chernichenko N, He S, Bakst RL, Barajas F, et al. GFRalpha1 released by nerves enhances cancer cell perineural invasion through GDNF-RET signaling. Proc Natl Acad Sci USA. 2014;111:E2008–17.
Cavel O, Shomron O, Shabtay A, Vital J, Trejo-Leider L, Weizman N, et al. Endoneurial macrophages induce perineural invasion of pancreatic cancer cells by secretion of GDNF and activation of RET tyrosine kinase receptor. Cancer Res. 2012;72:5733–43.
Sawai H, Okada Y, Kazanjian K, Kim J, Hasan S, Hines OJ, et al. The G691S RET polymorphism increases glial cell line-derived neurotrophic factor-induced pancreatic cancer cell invasion by amplifying mitogen-activated protein kinase signaling. Cancer Res. 2005;65:11536–44.
Gupton SL, Gertler FB. Filopodia: the fingers that do the walking. Sci STKE. 2007;2007:re5.
Sit ST, Manser E. Rho GTPases and their role in organizing the actin cytoskeleton. J Cell Sci. 2011;124:679–83.
Mattila PK, Lappalainen P. Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol. 2008;9:446–54.
Partridge MA, Marcantonio EE. Initiation of attachment and generation of mature focal adhesions by integrin-containing filopodia in cell spreading. Mol Biol Cell. 2006;17:4237–48.
Arjonen A, Kaukonen R, Ivaska J. Filopodia and adhesion in cancer cell motility. Cell Adh Migr. 2011;5:421–30.
Fischer RS, Lam PY, Huttenlocher A, Waterman CM. Filopodia and focal adhesions: an integrated system driving branching morphogenesis in neuronal pathfinding and angiogenesis. Dev Biol. 2019;451:86–95.
Friedl P, Alexander S. Cancer invasion and the microenvironment: plasticity and reciprocity. Cell. 2011;147:992–1009.
Beaty BT, Condeelis J. Digging a little deeper: the stages of invadopodium formation and maturation. Eur J Cell Biol. 2014;93:438–44.
Hoshino D, Branch KM, Weaver AM. Signaling inputs to invadopodia and podosomes. J Cell Sci. 2013;126:2979–89.
Yang C, Svitkina T. Filopodia initiation: focus on the Arp2/3 complex and formins. Cell Adh Migr. 2011;5:402–8.
Sharma VP, Eddy R, Entenberg D, Kai M, Gertler FB, Condeelis J. Tks5 and SHIP2 regulate invadopodium maturation, but not initiation, in breast carcinoma cells. Curr Biol. 2013;23:2079–89.
Richardson DS, Lai AZ, Mulligan LM. RET ligand-induced internalization and its consequences for downstream signaling. Oncogene. 2006;25:3206–11.
Esseghir S, Todd SK, Hunt T, Poulsom R, Plaza-Menacho I, Reis-Filho JS, et al. A role for glial cell derived neurotrophic factor induced expression by inflammatory cytokines and RET/GFR alpha 1 receptor up-regulation in breast cancer. Cancer Res. 2007;67:11732–41.
Gujral TS, van Veelen W, Richardson DS, Myers SM, Meens JA, Acton DS, et al. A novel RET kinase-beta-catenin signaling pathway contributes to tumorigenesis in thyroid carcinoma. Cancer Res. 2008;68:1338–46.
Hickey JG, Myers SM, Tian X, Zhu SJ, Shaw JLV, Andrew SD. et al. RET-mediated gene expression pattern is affected by isoform but not oncogenic mutation. Genes Chromosomes Cancer. 2009;48:429–40.
Maritan SM, Lian EY, Mulligan LM. An efficient and flexible cell aggregation method for 3D spheroid production. J Vis Exp. 2017;121:e55544.
Acknowledgements
The authors would like to thank Dr. Marco Magalhaes (University of Toronto) for the gift of TKS5 constructs and Dr. Donald Maurice (Queen’s University) for providing human smooth muscle cell lines.
Funding
This work was supported by an operating grant from the Cancer Research Society of Canada (19439) and a Canadian Institutes for Health Research operating grant (MOP-142303 (LMM)) and postdoctoral fellowship (398979 (SM)), and Canadian Graduate Scholarship (SMM) and by Ontario Graduate Scholarships and studentships from the Terry Fox Research Institute Training Program in Transdisciplinary Cancer Research (EYL, SMM).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
About this article
Cite this article
Lian, E.Y., Hyndman, B.D., Moodley, S. et al. RET isoforms contribute differentially to invasive processes in pancreatic ductal adenocarcinoma. Oncogene 39, 6493–6510 (2020). https://doi.org/10.1038/s41388-020-01448-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41388-020-01448-z