Induction of a Proinflammatory Program in Normal Human Thyrocytes by the RET͞PTC1 Oncogene

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 microarray 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 RET͞PTC1 or RET͞PTC1-Y451F mutant. (Magniﬁcation ϫ100.) (C) representative housekeeping gene. For details, see Supporting Proliferative capacity of parental and RET͞PTC1- and RET͞PTC1-Y451F- Materials and Methods. expressing thyrocytes. Each point represents the mean of eight independent replicates Ϯ SD. (D) Biochemical analysis of parental and RET͞PTC1-infected Detection of Chemokines by ELISAs and Gelatinase Activity Assay. thyrocytes. Cell extracts were analyzed with the indicated antisera. IP, The supernatants of parental and RET͞PTC1-expressing thy- immunoprecipitate. ␤-Catenin is shown as a control for protein loading. rocytes, maintained in serum-free medium for 24 h, were collected and analyzed. The amounts of CCL2, CCL20, CXCL8, ͞ ͞ and CXCL12 released in the supernatant were quantified by RET PTC1-Y451F mutant. Tyr-451 of RET PTC1 corre- ͞ sponds to RET-Y1062 multidocking site, previously demon- ELISA with a commercial kit by R & D Systems. MMP2 MMP9 ͞ activity was quantified by the gelatinase assay kit (Chemicon). strated necessary for the transforming activity of RET PTC oncogenes in NIH͞3T3 cells (15). Mass populations of thyro- ͞ ͞ Migration Assay. Cell migration of parental and RET͞PTC1- and cytes stably expressing RET PTC1 or RET PTC1-Y451F and RET͞PTC1-Y451F-expressing thyrocytes was evaluated in the parental thyrocytes were used for all this study. ͞ presence͞absence of CXCL12 and of the CXCR4 inhibitor Thyrocytes expressing RET PTC1-Y451F mutant and paren- AMD3100, as detailed in Supporting Materials and Methods. tal thyrocytes showed a similar morphology that differed mark- edly from that of thyrocytes expressing RET͞PTC1 (Fig. 1B). In Tissue Samples. Thyroid samples, 3 normal and 21 tumor speci- fact, whereas parental thyrocytes, as well as those expressing mens [17 PTC and 4 follicular thyroid carcinomas (FTC)], were RET͞PTC1-Y451F, are flat and growth contact inhibited, the selected for this study and characterized as detailed in Supporting thyrocytes expressing the fully functional oncogene showed a Materials and Methods. ‘‘transformed phenotype,’’ being more refractile and growing without contact inhibition. In addition, they showed the char- Results acteristic nuclear envelope irregularity and chromatin clearing of Biological and Biochemical Characterization of Normal Human Thyroid human PTCs as described in ref. 22. The nuclear morphology of Follicular Cells Ectopically Expressing RET͞PTC1 Oncogene. To study human thyrocytes expressing RET͞PTC1-Y451F was identical the molecular mechanisms triggered by RET͞PTC oncogenes to to that of the uninfected ones (A.F., M.G.B., and A.G., unpub- transform thyroid cells, we exogenously expressed RET͞PTC1 lished results). (13) oncogene (Fig. 1A) in normal human thyroid cells. Cultured Uninfected and RET͞PTC1-infected thyrocytes were moni- primary thyrocytes from a healthy thyroid were infected with tored for cell growth. The thyrocytes expressing RET͞PTC1- pLNCX retroviral vector expressing RET͞PTC1 or, as control, Y451F and the parental cells showed similar low proliferative

14826 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503039102 Borrello et al. Downloaded by guest on October 1, 2021 capacity, whereas those expressing fully functional RET͞PTC1 oncoprotein grew rapidly with a doubling time of 36 h (Fig. 1C). When RET͞PTC1-expressing thyrocytes were treated with the RET kinase inhibitor RPI-1 (24), their proliferative capacity became similar to that of parental cells (data not shown). These results confirm the growth-promoting effect of RET͞PTC1 and the relevance of Tyr-451 in the transmission of proliferative signals in our human thyrocyte model. To biochemically characterize cells infected with the two RET͞PTC1 oncogenes, the expression and tyrosine phosphorylation status of RET͞PTC1 as well as the activation of known RET downstream effectors were analyzed by Western blot, as shown in Fig. 1D. The expression levels of RET͞PTC1 and RET͞PTC1-Y451F proteins in infected cells were comparable, and both proteins were tyrosine-phosphorylated. As expected, SHC proteins were constitutively tyrosine phosphorylated in RET͞PTC1-expressing cells but not in cells expressing RET͞ PTC1 lacking Tyr-451, the SHC docking site, and in parental thyrocytes. SHC is known to play a crucial role in the transforming activity induced by oncogenic RET͞PTC proteins in different cell environments (15). AKT was not activated in uninfected thyrocytes, whereas strong AKT activation was achieved in thyrocytes expressing RET͞PTC1 and, to a lesser extent, in those expressing RET͞PTC1-Y451F. ERK1 phosphorylation was slightly increased in RET͞PTC1-expressing cells compared with the other cells. Phospholipase C␥ (PLC␥) protein was Tyr-phosphorylated in both RET͞PTC1- and RET͞ PTC1-Y451F-expressing cells, as expected, but not in parental thyrocytes, whereas p38 phosphorylation was not induced by either of the RET͞PTC proteins (data not shown). Overall, the biochemical analysis confirmed that the analyzed thyrocyte populations expressed comparable amounts of WT and mutant RET͞PTC1 and that the RET͞PTC1 oncogene activates transforming intracellular pathways involving SHC, PLC␥, ERK1, and AKT.

RET͞PTC1 Expression Induces Inflammatory Molecules in Thyrocytes. The gene expression profiles of infected cells compared with parental thyrocytes was analyzed on human genome set U133 Fig. 2. Expression plot of the inflammation͞invasion-related genes regu- GeneChips to detect genes induced in human thyroid cells on lated by RET͞PTC1 oncogene. Shown is unsupervised hierarchical clustering RET͞PTC1-mediated transformation. Systematic comparison of analysis of genes related to inflammation͞invasion in uninfected thyrocytes or genes present in RET͞PTC1-expressing cells vs. parental thyro- in thyrocytes expressing RET͞PTC1 or RET͞PTC1-Y451F. Each row represents cytes or RET͞PTC1-Y451F-expressing cells was performed. one probe set corresponding to one gene. The dendrogram illustrates the degree of similarity between the expression profiles of the three cell types. Using a cutoff of 2-fold, we identified 1,258 and 612 regulated ͞ ͞ Different colors in the rectangles represent different levels of MAS 5-derived genes in RET PTC1- and RET PTC1-Y451F-expressing cells, signals after per chip and per gene normalization (Gene Spring, Agilent respectively, compared with uninfected thyrocytes. Comparison Technologies, Palo Alto, CA). The color bar beneath the dendrogram repre- of the RET͞PTC1 with RET͞PTC1-Y451F mutant expressing sents the average expression values for this subset of genes. cells showed 1,161 regulated genes, thus suggesting that the multidocking site is responsible for the majority of RET͞PTC transcriptional effects. We found 320 genes, i.e., Ϸ1͞4th of those some cases, both the ligand and the corresponding receptor were regulated by RET͞PTC1-WT, regulated also by RET͞PTC1- induced, suggesting an autocrine loop similar to the chemokine

Y451F, although to a lesser extent. Among the changes induced receptor 4 (CXCR4) and its ligand chemokine ligand 12 MEDICAL SCIENCES by RET͞PTC1, we observed a prominent up-regulation of genes (CXCL12) and osteopontin͞secreted phosphoprotein 1 (OPN͞ related to inflammation and tumor invasion. As listed in Table SPP1) and one of its receptors (CD44), UPA and UPAR. Almost 1, which is published as supporting information on the PNAS all of the genes up-regulated in RET͞PTC1-cells compared with web site, and shown in Fig. 2, nine chemokines and three uninfected thyrocytes were also up-regulated when compared chemokine receptors were induced. In addition, stimulating with RET͞PTC1-Y451F-cells, as shown in Fig. 2. factors for macrophages or granulocytes, M-CSF, GM-CSF, and This finding is supported by the dendrogram at the top of Fig. G-CSF were all up-regulated. Other molecules involved in 2, which highlights the similarity of uninfected thyrocytes with angiogenesis and invasion, such as six different metal- RET mutant-infected cells. loproteinases (MMPs), the transmembrane protease dipeptidyl Real-time Q-PCR (TaqMan gene expression assay) was per- peptidase IV (DPPIV), and the urokinase-type plasminogen formed to confirm expression of representative molecules of the activator (UPA) and its receptor (UPAR), were all up-regulated, different classes of induced genes. In particular, the gene ex- whereas the tissue inhibitors of MMP2 and MMP3 (TIMP2 and pression levels of CXCL12, CCL2, SPP1, TIMP3, GM-CSF, TIMP3) were down-regulated. Moreover, the adhesion mole- MMP9, UPA, CXCL8, CCL20, CXCR4, IL1B, and SELL were cules L-selectin (SELL), P-selectin (SELP), intracellular adhe- analyzed (Fig. 6, which is published as supporting information on sion molecule 1 (ICAM1), and neural and neuronal cell adhesion the PNAS web site). Real-time Q-PCR results confirmed the molecules (NCAM and NRCAM, respectively) were induced. In microarray data. To avoid the possibility of an effect specific to

Borrello et al. PNAS ͉ October 11, 2005 ͉ vol. 102 ͉ no. 41 ͉ 14827 Downloaded by guest on October 1, 2021 expressing thyrocytes significantly migrated in response to the specific ligand CXCL12 in a dose-dependent manner, whereas thyroid cells lacking Tyr-451 and parental thyrocytes were completely unresponsive to the chemokine (Fig. 3 B and C). In line with these results, RET͞PTC1-cells exposed to CXCL12 showed a polarized morphology (Fig. 3E). To test the specificity of the CXCL12-induced chemotaxis in RET͞PTC1-expressing thyrocytes, AMD3100, a specific antagonist to CXCR4, was used. Cell treatment with AMD3100 almost completely inhibited migration in response to CXCL12 (Fig. 3D).

Expression Analysis of RET͞PTC1-Induced Genes in PTC and FTC Spec- imens. The expression of a set of genes induced by RET͞PTC1 in the in vitro experimental model was tested in a panel of thyroid carcinomas and normal thyroid samples. The genes were selected among those most differentially expressed and for being representative of different functional classes. RNAs from 17 PTCs, 4 FTCs, and 3 normal thyroid samples were analyzed. The expression of genes encoding CCL20, CXCR4, SELL, GM-CSF, MMP9, UPA, and SPP1 was significantly higher (P Ͻ 0.05 or P Ͻ 0.01) in PTC specimens compared with normal thyroid samples, thus confirming the presence ‘‘in vivo’’ of molecules identified by the ‘‘in vitro’’ model (Fig. 4). The median of CCL2 and CXCL8 expression tended to be higher in PTC compared with normal thyroid; however, because of the high dispersion of expression levels in PTC specimens and the small number of normal thyroid samples, the differences are not statistically significant (Fig. 4). Moreover, the median expression levels of CCL20, CCL2, CXCL8, SELL, GM-CSF, IL1B, and UPA genes were significantly higher (P Ͻ 0.05 or P Ͻ 0.01) in PTC specimens also when compared with FTCs. PTC and FTC have an identical origin, but molecular and clinical features are different. To explain differences in expression levels among the clinical Fig. 3. Expression of inﬂammation͞invasion-related molecules. (A) Analysis PTC cases (see Fig. 4), we correlated them with clinical and of chemokine production by ELISA and of MMP activity. One representative molecular parameters (Table 2, which is published as supporting experiment of three independent experiments is shown. (B) Spontaneous and information on the PNAS web site). Seven patients displayed ͞ CXCL12-induced migration of uninfected and RET PTC1- or RETPTC-Y451F- RET͞PTC rearrangements including RET͞PTC1 (three cases) infected thyrocytes. Results are expressed as the mean number of migrated ͞ cells counted in 10 microscope high-power ﬁelds. (C) Dose–response migra- and RET PTC3 (four cases). TRKA rearrangement was present tion of RET͞PTC1-infected thyrocytes to CXCL12. (D) Effect of the CXCR4 in one case, and BRAF mutations were present in six cases. Three inhibitor AMD3100 (1 ␮g͞ml) on CXCL12-induced (100 ng͞ml) chemotaxis. (E) cases did not have RET͞TRKA rearrangements or mutations in Phase-contrast micrographs of RET͞PTC1-infected thyroid cells, treated with BRAF (17). Clinicopathological parameters {sex, age, PTC CXCL12 (100 ng͞ml) or medium for 3 h. subtype (25), tumor side, and TNM [description of primary tumor (T), the extent of the regional lymph node metastasis (N), and distant metastasis (M)] classification (26)} for each patient one patient’s sample of cells, we have performed real-time are indicated (Table 2). Tumor samples displaying RTK rear- Q-PCR analyses also on thyroid cultures from a distinct donor rangements (RET͞PTCs and TRK) and extrathyroid spread and infected with the same RET͞PTC1 or RET͞PTC1-Y451F- metastasis to regional lymph nodes (pT4N1) tended to express expressing retroviruses. The RET͞PTC1-expressing thyrocytes higher levels of CCL2, CXCR4, SELL, GM-CSF, MMP9, and from the two donors showed comparable results for all genes UPA genes (Table 2 and Fig. 4), suggesting a role for these genes except TIMP3 (data not shown). in locoregional spread. Further studies in larger case lists are To confirm gene expression at the protein level, supernatants needed to validate this (these) inflammatory signature(s) as a of the three cell cultures were measured by ELISA for the correlate of molecular pathogenesis and clinical aggressiveness. chemokines CCL2, CCL20, CXCL8, and CXCL12. In line with the gene expression analysis, cells infected with RET͞PTC1 Discussion actively produced a large amount of inflammatory chemokines, In this study, we show that the RET͞PTC1 oncogene, exog- as shown in Fig. 3A. CXCL8 protein was strongly induced by enously expressed in normal primary human thyrocytes, directly RET͞PTC1, being increased Ϸ70-fold, whereas both CCL2 and induces the up-modulation of several inflammatory genes, in- CCL20 chemokines were increased Ϸ10-fold. The levels of cluding chemokines and their receptors, cytokines, matrix- CXCL12 showed lower but reproducible increase. degrading enzymes, and adhesion molecules. As schematically Of the several MMPs whose expression increased after RET͞ depicted in Fig. 5, these molecules may affect tumor progression PTC1 infection (listed in Table 1), we tested the functional and invasion of surrounding tissues. activity of MMP2 and MMP9. Fig. 3A shows that, in the There is accumulating evidence that the complex chemokine supernatants of RET͞PTC1-infected cells, gelatinase activity is network present in human tumors influences both extent and much higher compared with the other cell cultures. phenotype of immune cell infiltration as well as several tumor The strong up-regulation of CXCR4 in RET͞PTC1- properties. Here, we show that RET͞PTC1 induces the expres- expressing cells prompted us to analyze the functional activity of sion of both CXCR4 and CXCL12, thus suggesting a possible this chemokine receptor in chemotaxis assays. RET͞PTC1- autocrine loop. The CXCR4͞CXCL12 axis may be involved in

14828 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503039102 Borrello et al. Downloaded by guest on October 1, 2021 Fig. 4. Real-time Q-PCR analysis of selected genes in normal and tumor specimens. The results are given as relative expression (mRNA expression normalized for 18S rRNA levels). Medians and P values are indicated. As determined by using the Student’s t test with unequal variance, **, P Ͻ 0.05; ***, P Ͻ 0.01; #, P ϭ 0.05. N, normal thyroid; ƒ, PTC from patients characterized by RTK (RET͞PTC1, RET͞PTC3, and TRK) rearrangements and pT4N1 presentation.

tumor cell proliferation and survival, as well as in the metastatic differentiation and survival (31). As documented by correlative capacity of tumor cells (27). Indeed, we demonstrated that studies, the presence of high numbers of macrophages at the tumor CXCR4-positive thyrocytes migrate in response to CXCL12. site is more frequently associated with poor prognosis (32). Interestingly, all of the components of ELRϩ CXC chemokine Overall, the de novo expression of a large number of chemo- family (CXCL1, -2, -3, -5, -6, and -8), with proangiogenic activity, kines is a feature of RET͞PTC-infected human thyrocytes. This were strongly up-regulated in RET͞PTC1-infected thyrocytes. finding is in line with the work of Melillo et al. (33) describing Because the appropriate chemokine receptor (CXCR2) was not the up-regulation of some chemokines (CXCL1, CXCL10, and expressed (data not shown), a paracrine effect for these chemo- CCL2) and of CXCR4 in a rat thyroid cell line expressing kines could be envisaged. Accordingly, in a mouse model of lung RET͞PTC (33–35) and suggesting a chemokine role in prolif- ϩ cancer, the protumor effect of ELR CXC chemokines was eration and invasion. demonstrated to be mainly because of their action on nontumor The induction of high levels of the serine protease UPA and ϩ cells (28). ELR chemokines could facilitate tumor development UPAR, known to be overexpressed in 80% of PTC, is another through the stimulation of blood vessel growth and recruitment putative autocrine loop triggered by RET͞PTC1 expression. A of inflammatory cells, as recently demonstrated in RAS- number of findings suggest that the UPA system is causally involved transformed HeLa cells (29). at multiple steps in cancer progression, metastasis, and shortened RNA and protein levels of the chemokines CCL2 and CCL20 survival in cancer patients. UPAR has been suggested to be induced ͞ were also up-regulated by RET PTC1. CCL20 expression has been by HGFR͞MET, a protein overexpressed in PTC (36). In this reported to be associated with the presence of immature dendritic paper, we found that RET͞PTC oncogenes induce MET expression cells in PTC, where, in general, they promote tumor progression in thyrocytes, as reported in ref. 36, but that the induction is (30). CCL2 is a major tumor-derived chemoattractant for blood independent from RET-Y1062 multidocking site (data not shown) monocytes (1), and established data demonstrate that CCL2 levels instead necessary for inducing UPA and UPAR. correlate with the macrophage contents of several tumor types (10). Interestingly, at variance with the induction of the MET gene, ͞ In addition, RET PTC1 also induces the expression of myeloid the up-regulation of the majority of inflammatory molecules was CSFs, which not only recruit myeloid cells but also sustain their strictly dependent on the presence of Tyr-451 (the Tyr-1062 multidocking site in proto-RET). Tyr-1062 has been demon- ͞ strated as necessary for the transforming activity of RET PTC MEDICAL SCIENCES oncogenes (15), for the activation of NF-␬B, a key player in inflammation (14), and for induction of CCL2 and GM-CSF by RET͞PTC3 oncoprotein (35). Another putative autocrine loop triggered by RET͞PTC1 expression in our study is SPP1, a multifunctional secreted phosphoprotein, whose overexpression is associated with cell transformation, and one of its receptors, CD44. The expression of SPP1͞CD44 has been shown to be triggered by RET͞PTC oncogenes in rat thyroid cells and to sustain tumor cell proliferation and invasion (37). Several MMPs and the dipeptidyl peptidase IV (DPPIV) were induced by RET͞PTC1. The proposed pathogenic role of MMPs in cancer is tissue remodeling by degrading extracellular matrix (ECM) during invasive tumor growth and neoangiogenesis and, Fig. 5. Inﬂammation͞invasion molecules with speciﬁed functions triggered more recently, in processing molecules such as chemokines, che- by RET͞PTC1 oncogene in primary human thyrocyte. mokine receptors, and cytokines (38). Similarly, the transmem-

Borrello et al. PNAS ͉ October 11, 2005 ͉ vol. 102 ͉ no. 41 ͉ 14829 Downloaded by guest on October 1, 2021 brane protease DPPIV has been implicated in regulating the aggressive clinical behavior (pT4N1 presentation) tended to ex- activity of chemotactic peptides and appears to have a critical role press higher levels of selected inflammatory molecules CCL2, in angiogenesis (39, 40). It is worth mentioning that DPPIV was CXCR4, SELL, GM-CSF, MMP9, and UPA, suggesting a role for recently suggested (41) as a molecular marker for differentiated these molecules in locoregional spread, a pathological characteristic thyroid carcinoma. of PTC. However, further studies in larger case lists are needed to Finally, RET͞PTC1 induced the adhesion molecule L-selectin. validate this inflammatory signature as a correlate of molecular L-selectin plays an important role in homing of lymphocytes to pathogenesis and clinical aggressiveness. lymphoid tissues, and it has also been shown to facilitate cancer In conclusion, the RET͞PTC1 oncogene activates in normal metastasis (42). Thus, induction of L-selectin and CXCR4 by human primary thyrocytes a transcriptional program related to RET͞PTC may arm PTC with molecular tools to disseminate to inflammation. RET͞PTC1-induced genes were also expressed in lymph nodes. The fact that many RET͞PTC-induced molecules are specimens of PTC, particularly those with rearranged RTK and known to interact with each other is in agreement with the scenario pT4N1 presentation, thus validating the in vivo relevance of the in that different components of an inflammatory͞invasive program vitro identified profile. These results establish a direct connection are coordinately induced by this oncogene. between a transforming oncogene involved in the pathogenesis of ͞ Selected molecules induced by RET PTC1 in our in vitro model a human tumor and the activation of a proinflammatory program of PTC were also present in tumor specimens of PTC. In particular, in the primary human cell type originating that tumor. Of note, PTC expression levels of CCL20, CXCR4, SELL, GM-CSF, MMP9, UPA, is associated, in Ϸ30% of cases, with chronic inflammatory thy- and SPP1 were significantly higher in PTC specimens compared roiditis (43). The set of inflammation-related genes activated by with normal thyroid tissues. CCL20, CCL2, CXCL8, SELL, GM- RET͞PTC1 in human thyrocytes is therefore likely to contribute to CSF, IL1B, and UPA expression levels were significantly higher in tumor progression and locoregional metastasis, characteristic of PTC also when compared with FTC specimens. Furthermore, this type of thyroid cancer. among different PTC specimens, enhanced expression of inflammatory genes was more frequently seen in tumors positive for RTK ͞ ͞ We thank Loris De Cecco, James F. Reid, and Simone Minardi for their rearrangement (RET PTC1, RET PTC3, and TRK), rather than in contribution to gene profiling. We also thank Mrs. Maria Grazia Rizzetti BRAF-positive cases. Profiling in PTC tumors carrying RTK rear- for technical assistance and Mrs. Cristina Mazzadi for secretarial help. rangements or BRAF mutations already showed a different set of This work was supported by the Associazione Italiana per la Ricerca sul gene expression in these tumors (17). Finally, tumors with more Cancro.

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