Activation of RAS Signalling is Associated with Altered Cell Adhesion in Phaeochromocytoma
Abstract
:1. Introduction
2. Results
2.1. hPheo1 is Heterozygous for NRAS Q61K and Expresses the Mutant Allele
2.2. Downregulating NRAS in hPheo1 Cells Leads to Upregulation of Genes Involved in Cellular Adhesion
2.3. Effects on Cellular Adhesion
2.4. Effects on Proliferation
2.5. Molecular Subtype-Specific Gene Expression Patterns
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Cell Culture
5.2. DNA Extraction, Exome and Sanger Sequencing
5.3. RNA Extraction and Quantification
5.4. NRAS Knockdown with siRNA (siNRAS Treatment)
5.5. Western Blot
5.6. Microarray Analysis
5.7. Real-Time Reverse Transcriptase Quantitative PCR
5.8. Patient Cohort Analyses
5.9. Proliferation
5.10. Functional Adhesion Studies
5.11. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Castinetti, F.; Taieb, D.; Henry, J.F.; Walz, M.; Guerin, C.; Brue, T.; Conte-Devolx, B.; Neumann, H.P.; Sebag, F. Management of Endocrine Disease: Outcome of adrenal sparing surgery in heritable pheochromocytoma. Eur. J. Endocrinol. 2016, 174, R9–R18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rossitti, H.M.; Soderkvist, P.; Gimm, O. Extent of surgery for phaeochromocytomas in the genomic era. Br. J. Surg. 2018, 105, e84–e98. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Crona, J.; Lamarca, A.; Ghosal, S.; Welin, S.; Skogseid, B.; Pacak, K. Genotype-phenotype correlations in pheochromocytoma and paraganglioma. Endocr. Relat. Cancer 2019. [Google Scholar] [CrossRef] [PubMed]
- John, H.; Ziegler, W.H.; Hauri, D.; Jaeger, P. Pheochromocytomas: Can malignant potential be predicted? Urology 1999, 53, 679–683. [Google Scholar] [CrossRef]
- Goffredo, P.; Sosa, J.A.; Roman, S.A. Malignant pheochromocytoma and paraganglioma: A population level analysis of long-term survival over two decades. J. Surg. Oncol. 2013, 107, 659–664. [Google Scholar] [CrossRef] [PubMed]
- Hamidi, O.; Young, W.F., Jr.; Iniguez-Ariza, N.M.; Kittah, N.E.; Gruber, L.; Bancos, C.; Tamhane, S.; Bancos, I. Malignant Pheochromocytoma and Paraganglioma: 272 Patients Over 55 Years. J. Clin. Endocrinol. Metab. 2017, 102, 3296–3305. [Google Scholar] [CrossRef]
- Hescot, S.; Curras-Freixes, M.; Deutschbein, T.; van Berkel, A.; Vezzosi, D.; Amar, L.; de la Fouchardiere, C.; Valdes, N.; Riccardi, F.; Do Cao, C.; et al. Prognosis of Malignant Pheochromocytoma and Paraganglioma (MAPP-Prono Study): A European Network for the Study of Adrenal Tumors Retrospective Study. J. Clin. Endocrinol. Metab. 2019, 104, 2367–2374. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Burnichon, N.; Vescovo, L.; Amar, L.; Libe, R.; de Reynies, A.; Venisse, A.; Jouanno, E.; Laurendeau, I.; Parfait, B.; Bertherat, J.; et al. Integrative genomic analysis reveals somatic mutations in pheochromocytoma and paraganglioma. Hum. Mol. Genet. 2011, 20, 3974–3985. [Google Scholar] [CrossRef] [PubMed]
- Dahia, P.L.; Ross, K.N.; Wright, M.E.; Hayashida, C.Y.; Santagata, S.; Barontini, M.; Kung, A.L.; Sanso, G.; Powers, J.F.; Tischler, A.S.; et al. A HIF1alpha regulatory loop links hypoxia and mitochondrial signals in pheochromocytomas. PLoS Genet. 2005, 1, 72–80. [Google Scholar] [CrossRef] [Green Version]
- Lopez-Jimenez, E.; Gomez-Lopez, G.; Leandro-Garcia, L.J.; Munoz, I.; Schiavi, F.; Montero-Conde, C.; de Cubas, A.A.; Ramires, R.; Landa, I.; Leskela, S.; et al. Research resource: Transcriptional profiling reveals different pseudohypoxic signatures in SDHB and VHL-related pheochromocytomas. Mol. Endocrinol. 2010, 24, 2382–2391. [Google Scholar] [CrossRef] [Green Version]
- Qin, N.; de Cubas, A.A.; Garcia-Martin, R.; Richter, S.; Peitzsch, M.; Menschikowski, M.; Lenders, J.W.; Timmers, H.J.; Mannelli, M.; Opocher, G.; et al. Opposing effects of HIF1alpha and HIF2alpha on chromaffin cell phenotypic features and tumor cell proliferation: Insights from MYC-associated factor X. Int. J. Cancer 2014, 135, 2054–2064. [Google Scholar] [CrossRef]
- Welander, J.; Soderkvist, P.; Gimm, O. Genetics and clinical characteristics of hereditary pheochromocytomas and paragangliomas. Endocr. Relat. Cancer 2011, 18, R253–R276. [Google Scholar] [CrossRef] [Green Version]
- Crona, J.; Taieb, D.; Pacak, K. New Perspectives on Pheochromocytoma and Paraganglioma: Toward a Molecular Classification. Endocr. Rev. 2017, 38, 489–515. [Google Scholar] [CrossRef] [PubMed]
- Fishbein, L.; Leshchiner, I.; Walter, V.; Danilova, L.; Robertson, A.G.; Johnson, A.R.; Lichtenberg, T.M.; Murray, B.A.; Ghayee, H.K.; Else, T.; et al. Comprehensive Molecular Characterization of Pheochromocytoma and Paraganglioma. Cancer Cell 2017, 31, 181–193. [Google Scholar] [CrossRef] [PubMed]
- Eisenhofer, G.; Lenders, J.W.; Timmers, H.; Mannelli, M.; Grebe, S.K.; Hofbauer, L.C.; Bornstein, S.R.; Tiebel, O.; Adams, K.; Bratslavsky, G.; et al. Measurements of plasma methoxytyramine, normetanephrine, and metanephrine as discriminators of different hereditary forms of pheochromocytoma. Clin. Chem. 2011, 57, 411–420. [Google Scholar] [CrossRef]
- Berends, A.M.A.; Eisenhofer, G.; Fishbein, L.; Horst-Schrivers, A.; Kema, I.P.; Links, T.P.; Lenders, J.W.M.; Kerstens, M.N. Intricacies of the Molecular Machinery of Catecholamine Biosynthesis and Secretion by Chromaffin Cells of the Normal Adrenal Medulla and in Pheochromocytoma and Paraganglioma. Cancers (Basel) 2019, 11, 1121. [Google Scholar] [CrossRef] [Green Version]
- Stenman, A.; Welander, J.; Gustavsson, I.; Brunaud, L.; Backdahl, M.; Soderkvist, P.; Gimm, O.; Juhlin, C.C.; Larsson, C. HRAS mutation prevalence and associated expression patterns in pheochromocytoma. Genes Chromosomes Cancer 2016, 55, 452–459. [Google Scholar] [CrossRef] [Green Version]
- Ghayee, H.K.; Bhagwandin, V.J.; Stastny, V.; Click, A.; Ding, L.H.; Mizrachi, D.; Zou, Y.S.; Chari, R.; Lam, W.L.; Bachoo, R.M.; et al. Progenitor cell line (hPheo1) derived from a human pheochromocytoma tumor. PLoS ONE 2013, 8, e65624. [Google Scholar] [CrossRef] [Green Version]
- Dammann, R.; Schagdarsurengin, U.; Seidel, C.; Trumpler, C.; Hoang-Vu, C.; Gimm, O.; Dralle, H.; Pfeifer, G.P.; Brauckhoff, M. Frequent promoter methylation of tumor-related genes in sporadic and men2-associated pheochromocytomas. Exp. Clin. Endocrinol. Diabetes 2005, 113, 1–7. [Google Scholar] [CrossRef] [PubMed]
- Sanchez-Vega, F.; Mina, M.; Armenia, J.; Chatila, W.K.; Luna, A.; La, K.C.; Dimitriadoy, S.; Liu, D.L.; Kantheti, H.S.; Saghafinia, S.; et al. Oncogenic Signaling Pathways in The Cancer Genome Atlas. Cell 2018, 173, 321–337. [Google Scholar] [CrossRef] [Green Version]
- Schlisio, S.; Kenchappa, R.S.; Vredeveld, L.C.; George, R.E.; Stewart, R.; Greulich, H.; Shahriari, K.; Nguyen, N.V.; Pigny, P.; Dahia, P.L.; et al. The kinesin KIF1Bbeta acts downstream from EglN3 to induce apoptosis and is a potential 1p36 tumor suppressor. Genes Dev. 2008, 22, 884–893. [Google Scholar] [CrossRef] [Green Version]
- Welander, J.; Andreasson, A.; Juhlin, C.C.; Wiseman, R.W.; Backdahl, M.; Hoog, A.; Larsson, C.; Gimm, O.; Soderkvist, P. Rare germline mutations identified by targeted next-generation sequencing of susceptibility genes in pheochromocytoma and paraganglioma. J. Clin. Endocrinol. Metab. 2014, 99, E1352–E1360. [Google Scholar] [CrossRef]
- Prior, I.A.; Lewis, P.D.; Mattos, C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012, 72, 2457–2467. [Google Scholar] [CrossRef] [Green Version]
- Wilzen, A.; Rehammar, A.; Muth, A.; Nilsson, O.; Tesan Tomic, T.; Wangberg, B.; Kristiansson, E.; Abel, F. Malignant pheochromocytomas/paragangliomas harbor mutations in transport and cell adhesion genes. Int. J. Cancer 2016, 138, 2201–2211. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Buffet, A.; Morin, A.; Castro-Vega, L.J.; Habarou, F.; Lussey-Lepoutre, C.; Letouze, E.; Lefebvre, H.; Guilhem, I.; Haissaguerre, M.; Raingeard, I.; et al. Germline Mutations in the Mitochondrial 2-Oxoglutarate/Malate Carrier SLC25A11 Gene Confer a Predisposition to Metastatic Paragangliomas. Cancer Res. 2018, 78, 1914–1922. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Evenepoel, L.; Helaers, R.; Vroonen, L.; Aydin, S.; Hamoir, M.; Maiter, D.; Vikkula, M.; Persu, A. KIF1B and NF1 are the most frequently mutated genes in paraganglioma and pheochromocytoma tumors. Endocr. Relat. Cancer 2017, 24, L57–L61. [Google Scholar] [CrossRef] [PubMed]
- Liberzon, A.; Subramanian, A.; Pinchback, R.; Thorvaldsdottir, H.; Tamayo, P.; Mesirov, J.P. Molecular signatures database (MSigDB) 3.0. Bioinformatics 2011, 27, 1739–1740. [Google Scholar] [CrossRef]
- Liberzon, A.; Birger, C.; Thorvaldsdottir, H.; Ghandi, M.; Mesirov, J.P.; Tamayo, P. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 2015, 1, 417–425. [Google Scholar] [CrossRef] [Green Version]
- Naba, A.; Clauser, K.R.; Ding, H.; Whittaker, C.A.; Carr, S.A.; Hynes, R.O. The extracellular matrix: Tools and insights for the “omics” era. Matrix Biol. 2016, 49, 10–24. [Google Scholar] [CrossRef]
- Tam, W.L.; Lu, H.; Buikhuisen, J.; Soh, B.S.; Lim, E.; Reinhardt, F.; Wu, Z.J.; Krall, J.A.; Bierie, B.; Guo, W.; et al. Protein kinase C alpha is a central signaling node and therapeutic target for breast cancer stem cells. Cancer Cell 2013, 24, 347–364. [Google Scholar] [CrossRef] [Green Version]
- Walia, V.; Elble, R.C. Enrichment for breast cancer cells with stem/progenitor properties by differential adhesion. Stem Cells Dev. 2010, 19, 1175–1182. [Google Scholar] [CrossRef] [PubMed]
- Morata-Tarifa, C.; Jimenez, G.; Garcia, M.A.; Entrena, J.M.; Grinan-Lison, C.; Aguilera, M.; Picon-Ruiz, M.; Marchal, J.A. Low adherent cancer cell subpopulations are enriched in tumorigenic and metastatic epithelial-to-mesenchymal transition-induced cancer stem-like cells. Sci. Rep. 2016, 6, 18772. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Raaijmakers, M.I.; Widmer, D.S.; Maudrich, M.; Koch, T.; Langer, A.; Flace, A.; Schnyder, C.; Dummer, R.; Levesque, M.P. A new live-cell biobank workflow efficiently recovers heterogeneous melanoma cells from native biopsies. Exp. Dermatol. 2015, 24, 377–380. [Google Scholar] [CrossRef] [PubMed]
- Humphries, M.J. Cell-substrate adhesion assays. Curr. Protoc. Cell Biol. 2001. Chapter 9, Unit 9.1. [Google Scholar] [CrossRef]
- Hellewell, A.L.; Rosini, S.; Adams, J.C. A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix. J. Vis. Exp. 2017, 19, e55051. [Google Scholar] [CrossRef] [Green Version]
- Welander, J.; Andreasson, A.; Brauckhoff, M.; Backdahl, M.; Larsson, C.; Gimm, O.; Soderkvist, P. Frequent EPAS1/HIF2alpha exons 9 and 12 mutations in non-familial pheochromocytoma. Endocr. Relat. Cancer 2014, 21, 495–504. [Google Scholar] [CrossRef] [Green Version]
- Flynn, A.; Dwight, T.; Harris, J.; Benn, D.; Zhou, L.; Hogg, A.; Catchpoole, D.; James, P.; Duncan, E.L.; Trainer, A.; et al. Pheo-Type: A Diagnostic Gene-expression Assay for the Classification of Pheochromocytoma and Paraganglioma. J. Clin. Endocrinol. Metab. 2016, 101, 1034–1043. [Google Scholar] [CrossRef]
- Oudijk, L.; de Krijger, R.R.; Rapa, I.; Beuschlein, F.; de Cubas, A.A.; Dei Tos, A.P.; Dinjens, W.N.; Korpershoek, E.; Mancikova, V.; Mannelli, M.; et al. H-RAS mutations are restricted to sporadic pheochromocytomas lacking specific clinical or pathological features: Data from a multi-institutional series. J. Clin. Endocrinol. Metab. 2014, 99, E1376–E1380. [Google Scholar] [CrossRef] [Green Version]
- Karnoub, A.E.; Weinberg, R.A. Ras oncogenes: Split personalities. Nat. Rev. Mol. Cell Biol. 2008, 9, 517–531. [Google Scholar] [CrossRef] [Green Version]
- Mo, S.P.; Coulson, J.M.; Prior, I.A. RAS variant signalling. Biochem. Soc. Trans. 2018, 46, 1325–1332. [Google Scholar] [CrossRef] [Green Version]
- Shain, A.H.; Yeh, I.; Kovalyshyn, I.; Sriharan, A.; Talevich, E.; Gagnon, A.; Dummer, R.; North, J.; Pincus, L.; Ruben, B.; et al. The Genetic Evolution of Melanoma from Precursor Lesions. N. Engl. J. Med. 2015, 373, 1926–1936. [Google Scholar] [CrossRef] [PubMed]
- Saito, H.; Yoshida, T.; Yamazaki, H.; Suzuki, N. Conditional N-rasG12V expression promotes manifestations of neurofibromatosis in a mouse model. Oncogene 2007, 26, 4714–4719. [Google Scholar] [CrossRef] [Green Version]
- Cimino, P.J.; Gutmann, D.H. Neurofibromatosis type 1. Handb. Clin. Neurol. 2018, 148, 799–811. [Google Scholar] [CrossRef] [PubMed]
- Welander, J.; Larsson, C.; Backdahl, M.; Hareni, N.; Sivler, T.; Brauckhoff, M.; Soderkvist, P.; Gimm, O. Integrative genomics reveals frequent somatic NF1 mutations in sporadic pheochromocytomas. Hum. Mol. Genet. 2012, 21, 5406–5416. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Foty, R.A.; Steinberg, M.S. Differential adhesion in model systems. Wiley Interdiscip. Rev. Dev. Biol. 2013, 2, 631–645. [Google Scholar] [CrossRef]
- Ye, X.; Weinberg, R.A. Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression. Trends Cell Biol. 2015, 25, 675–686. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Eskandarpour, M.; Huang, F.; Reeves, K.A.; Clark, E.; Hansson, J. Oncogenic NRAS has multiple effects on the malignant phenotype of human melanoma cells cultured in vitro. Int. J. Cancer 2009, 124, 16–26. [Google Scholar] [CrossRef]
- Cervera, A.M.; Apostolova, N.; Crespo, F.L.; Mata, M.; McCreath, K.J. Cells silenced for SDHB expression display characteristic features of the tumor phenotype. Cancer Res. 2008, 68, 4058–4067. [Google Scholar] [CrossRef] [Green Version]
- D’Antongiovanni, V.; Martinelli, S.; Richter, S.; Canu, L.; Guasti, D.; Mello, T.; Romagnoli, P.; Pacak, K.; Eisenhofer, G.; Mannelli, M.; et al. The microenvironment induces collective migration in SDHB-silenced mouse pheochromocytoma spheroids. Endocr. Relat. Cancer 2017, 24, 555–564. [Google Scholar] [CrossRef] [Green Version]
- Loriot, C.; Domingues, M.; Berger, A.; Menara, M.; Ruel, M.; Morin, A.; Castro-Vega, L.J.; Letouze, E.; Martinelli, C.; Bemelmans, A.P.; et al. Deciphering the molecular basis of invasiveness in Sdhb-deficient cells. Oncotarget 2015, 6, 32955–32965. [Google Scholar] [CrossRef] [Green Version]
- Pacak, K.; Sirova, M.; Giubellino, A.; Lencesova, L.; Csaderova, L.; Laukova, M.; Hudecova, S.; Krizanova, O. NF-kappaB inhibition significantly upregulates the norepinephrine transporter system, causes apoptosis in pheochromocytoma cell lines and prevents metastasis in an animal model. Int. J. Cancer 2012, 131, 2445–2455. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lau, K.S.; Schrier, S.B.; Gierut, J.; Lyons, J.; Lauffenburger, D.A.; Haigis, K.M. Network analysis of differential Ras isoform mutation effects on intestinal epithelial responses to TNF-alpha. Integr. Biol. 2013, 5, 1355–1365. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Leibovich-Rivkin, T.; Liubomirski, Y.; Meshel, T.; Abashidze, A.; Brisker, D.; Solomon, H.; Rotter, V.; Weil, M.; Ben-Baruch, A. The inflammatory cytokine TNFalpha cooperates with Ras in elevating metastasis and turns WT-Ras to a tumor-promoting entity in MCF-7 cells. BMC Cancer 2014, 14, 158. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Naba, A.; Clauser, K.R.; Hoersch, S.; Liu, H.; Carr, S.A.; Hynes, R.O. The matrisome: In silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices. Mol. Cell Proteom. 2012, 11, M111.014647. [Google Scholar] [CrossRef] [Green Version]
- Theveneau, E.; Marchant, L.; Kuriyama, S.; Gull, M.; Moepps, B.; Parsons, M.; Mayor, R. Collective chemotaxis requires contact-dependent cell polarity. Dev. Cell 2010, 19, 39–53. [Google Scholar] [CrossRef] [Green Version]
- Hoadley, K.A.; Yau, C.; Hinoue, T.; Wolf, D.M.; Lazar, A.J.; Drill, E.; Shen, R.; Taylor, A.M.; Cherniack, A.D.; Thorsson, V.; et al. Cell-of-Origin Patterns Dominate the Molecular Classification of 10,000 Tumors from 33 Types of Cancer. Cell 2018, 173, 291–304. [Google Scholar] [CrossRef] [Green Version]
- Futaki, S.; Hayashi, Y.; Yamashita, M.; Yagi, K.; Bono, H.; Hayashizaki, Y.; Okazaki, Y.; Sekiguchi, K. Molecular basis of constitutive production of basement membrane components. Gene expression profiles of Engelbreth-Holm-Swarm tumor and F9 embryonal carcinoma cells. J. Biol. Chem. 2003, 278, 50691–50701. [Google Scholar] [CrossRef] [Green Version]
- Geiger, B.; Yamada, K.M. Molecular architecture and function of matrix adhesions. Cold Spring Harb. Perspect. Biol. 2011, 3. [Google Scholar] [CrossRef] [Green Version]
- Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
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Rossitti, H.M.; Dutta, R.K.; Larsson, C.; Ghayee, H.K.; Söderkvist, P.; Gimm, O. Activation of RAS Signalling is Associated with Altered Cell Adhesion in Phaeochromocytoma. Int. J. Mol. Sci. 2020, 21, 8072. https://doi.org/10.3390/ijms21218072
Rossitti HM, Dutta RK, Larsson C, Ghayee HK, Söderkvist P, Gimm O. Activation of RAS Signalling is Associated with Altered Cell Adhesion in Phaeochromocytoma. International Journal of Molecular Sciences. 2020; 21(21):8072. https://doi.org/10.3390/ijms21218072
Chicago/Turabian StyleRossitti, Hugo M., Ravi Kumar Dutta, Catharina Larsson, Hans K. Ghayee, Peter Söderkvist, and Oliver Gimm. 2020. "Activation of RAS Signalling is Associated with Altered Cell Adhesion in Phaeochromocytoma" International Journal of Molecular Sciences 21, no. 21: 8072. https://doi.org/10.3390/ijms21218072