Induction of a Proinflammatory Program in Normal Human Thyrocytes by the RET͞PTC1 Oncogene
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Induction of a proinflammatory program in normal human thyrocytes by the RET͞PTC1 oncogene Maria Grazia Borrello*†, Luisella Alberti*†, Andrew Fischer‡, Debora Degl’Innocenti*, Cristina Ferrario*§, Manuela Gariboldi*§, Federica Marchesi¶ʈ, Paola Allavena¶ʈ, Angela Greco*, Paola Collini**, Silvana Pilotti**, Giuliana Cassinelli††, Paola Bressan*, Laura Fugazzola‡‡, Alberto Mantovani¶ʈ§§¶¶, and Marco A. Pierotti*§¶¶ Departments of Experimental Oncology, Research Units *3 and ††14, and **Pathology, Istituto Nazionale Tumori, 20133 Milan, Italy; ‡Department of Pathology, University of Massachusetts, Worcester, MA 01605; §Fondazione Italiana per la Ricerca sul Cancro, Institute of Molecular Oncology Foundation, 20139 Milan, Italy; ¶Department of Immunology and Cell Biology, Mario Negri Institute, 20157 Milan, Italy; ‡‡Institute of Endocrine Sciences, Ospedale Maggiore, 20122 Milan, Italy; §§Institute of General Pathology, University of Milan, 20133 Milan, Italy; and ʈIstituto Clinico Humanitas, 20089 Rozzano, Italy Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and approved August 22, 2005 (received for review April 13, 2005) Rearrangements of the RET receptor tyrosine kinase gene gener- PTC is the most frequent thyroid malignancy in humans. ating RET͞PTC oncogenes are specific to papillary thyroid carci- Rearrangements of the RET receptor tyrosine kinase (RTK) noma (PTC), the most frequent thyroid tumor. Here, we show that gene, caused by chromosomal inversions or translocations, is a the RET͞PTC1 oncogene, when exogenously expressed in primary frequent genetic event (Ϸ30%) in PTC (12). These rearrange- normal human thyrocytes, induces the expression of a large set of ments mediate fusion of the tyrosine kinase-encoding domain of genes involved in inflammation and tumor invasion, including RET with heterologous genes, leading to the generation of those encoding chemokines (CCL2, CCL20, CXCL8, and CXCL12), chimeric RET͞PTC oncogenes. All RET͞PTCs, differing for the chemokine receptors (CXCR4), cytokines (IL1B, CSF-1, GM-CSF, and 5Ј donor gene involved, display ligand-independent constitutive G-CSF), matrix-degrading enzymes (metalloproteases and uroki- dimerization and activation. RET͞PTC1, the H4-RET fusion nase-type plasminogen activator and its receptor), and adhesion (13), is one of the prevalent variants. The multidocking site molecules (L-selectin). This effect is strictly dependent on the Tyr-1062 (Tyr-451 in RET͞PTC1) in the C-terminal region of presence of the RET͞PTC1 Tyr-451 (corresponding to RET Tyr-1062 RET, interacting with a number of transduction molecules and multidocking site). Selected relevant genes (CCL20, CCL2, CXCL8, involved in almost all downstream pathways activated by RET CXCR4, L-selectin, GM-CSF, IL1B, MMP9, UPA, and SPP1͞OPN) were (14), was demonstrated necessary for the transforming activity of found up-regulated also in clinical samples of PTC, particularly RET͞PTC oncogenes (15). Alternative pathogenetic events have those characterized by RET͞PTC activation, local extrathyroid been found in PTC and include chromosomic rearrangement spread, and lymph node metastases, when compared with normal involving TRKA (12) or BRAF point mutations or rearrangement thyroid tissue or follicular thyroid carcinoma. These results, dem- (16–18). onstrating that the RET͞PTC1 oncogene activates a proinflamma- PTCs are unique among epithelial tumors in that they appear tory program, provide a direct link between a transforming human to develop into a full-fledged malignancy in one step without any oncogene, inflammation, and malignant behavior. apparent benign preinvasive counterpart. It is well established that the translocation generating RET͞PTCs is an early event chemokines ͉ inflammation ͉ papillary thyroid carcinoma ͉ gene expression playing a causative role in the pathogenesis of a significant fraction of PTC. In fact, RET͞PTC expression has been found in Ϸ everal lines of evidence suggest a strong association between 40% of occult PTCs (19). In PC-Cl3 rat thyroid epithelial cells, ͞ Schronic inflammation and increased susceptibility to neo- RET PTCs induce morphological transformation (20) and lead plastic transformation and cancer development (1–4). to the development of thyroid tumors resembling human PTC in ͞ A number of studies in murine tumor models have demon- transgenic mice (21). Furthermore, RET PTC1 expression is strated the protumoral role of inflammatory mediators at dis- sufficient to cause, in primary cultures of normal human thyro- tinct phases of malignant progression (5–8). Conversely, inhibi- cytes, changes in the nuclear envelope and chromatin diagnostic tion of selected proinflammatory cytokines (e.g., TNF, IL1) or for PTC (22). of inflammation-related transcription factors (e.g., NF-B) has These observations prompted us to use primary human thy- resulted in reduced susceptibility to carcinogenesis and slower rocytes as an in vitro model to study the RET oncogene- tumor development in experimental tumor models (7–9). dependent molecular mechanisms driving follicular thyroid cells to malignant transformation and to challenge the concept of its The persistent release of inflammatory molecules may affect MEDICAL SCIENCES tumor progression in a variety of ways, for instance by increasing direct role in triggering an inflammatory program. ͞ tumor cell proliferation and resistance to apoptosis, by promot- In this study, we provide evidence that the RET PTC1 trans- ing angiogenesis and stroma remodeling, and by inhibiting the forming oncogene, exogenously expressed in primary normal establishment of a protective antitumor immunity (4). Tumor- human thyrocytes, regulates through its multidocking site, Tyr- associated cells of the innate and adaptive immunity play a 451, the expression of a distinct set of genes involved in inflam- pivotal role in this context, as they release a whole array of proinflammatory mediators (10). This paper was submitted directly (Track II) to the PNAS office. Indeed, it has been estimated that up to 20% of all tumors arise Abbreviations: PTC, papillary thyroid carcinoma; RTK, receptor tyrosine kinase; FTC, follic- from conditions of persistent inflammation mainly because of ular thyroid carcinomas; UPA, urokinase-type plasminogen activator; UPAR, UPA receptor; chronic infections or autoimmune diseases (1). For example, the MMPs, metalloproteases; Q-PCR, quantitative PCR. autoimmune inflammatory disorders, chronic Hashimoto’s thy- Data deposition: The gene expression data have been deposited at the ArrayExpress roiditis and Graves’ disease, are both associated with an in- database (accession no. E-MEXP-429). creased incidence of papillary thyroid carcinoma (PTC) (11). On †M.G.B. and L.A. contributed equally to this work. the other hand, tumor cells themselves may support their own ¶¶To whom correspondence may be addressed. E-mail: [email protected] growth and invasive phenotype through direct expression of or [email protected]. inflammation-related molecules (2). © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503039102 PNAS ͉ October 11, 2005 ͉ vol. 102 ͉ no. 41 ͉ 14825–14830 Downloaded by guest on October 1, 2021 mation and tumor invasion. The in vivo relevance of in vitro identified profiles was validated by analysis of PTC specimens. Overall, these results demonstrate a direct connection between a transforming human oncogene and a distinct inflammatory program in primary human cells. Materials and Methods Cell Cultures and Retroviral Infections. Primary cultures of normal human thyroid cells were infected with retroviral vectors con- taining RET͞PTC1 or RET͞PTC1-Y451F short isoforms as detailed in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. Cell Growth Assay. Cell growth was evaluated by sulforhodamine B colorimetric assay as detailed in Supporting Materials and Methods. Protein Extraction and Western Blotting. Mass populations of G418- selected thyrocytes stably expressing RET͞PTC1 or RET͞ PTC1-Y451F as well as parental thyrocytes were processed for Western blot analysis as described in Supporting Materials and Methods. RNA Extraction and Microarray Analysis. RNA extraction and puri- fication and cDNA amplification were performed as detailed in Supporting Materials and Methods. Human genome set U133 GeneChips in duplicate (Affymetrix, Santa Clara, CA) were used. The data were analyzed with Affymetrix microarray suite version 5 (MASv5), and statistical analyses were performed by using software designed in our institute (Institute of Molecular Oncology Foun- dation, Milan) (23). All of the microarray data is deposited in the ArrayExpress database (accession no. E-MEXP-429). Real-Time Quantitative PCR (Q-PCR). Total RNA used for microar- ray experiments and from 24 thyroid specimens was used to Fig. 1. Characterization of normal human thyroid follicular cells ectopically analyze the expression of a total of 12 genes: CCL2, CCL20, expressing RET͞PTC1 oncogene. (A) Schematic representation of RET͞PTC1 CXCL8, CXCL12, CXCR4, SELL, GM-CSF, IL1B, MMP9, oncogene short isoform. A portion of H4 donor gene is juxtaposed to RET tyrosine kinase (TK) intracellular portion. The Y451 docking site is marked. (B) TIMP3, UPA, and SPP1 (labeled with 6-carboxyfluorescein). Phase-contrast micrographs of human thyroid cells either uninfected or in- H18S (labeled with VIC, Applied Biosystems) was assayed as a fected with