The Trop-2 signalling network in cancer growth

Oncogene (2013) 32, 1594–1600 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc SHORT COMMUNICATION The Trop-2 signalling network in cancer growth E Guerra1, M Trerotola1,3, AL Aloisi1, R Tripaldi1, G Vacca1, R La Sorda1, R Lattanzio1, M Piantelli1 and S Alberti1,2 Our findings show that upregulation of a wild-type Trop-2 has a key controlling role in human cancer growth, and that tumour development is quantitatively driven by Trop-2 expression levels. However, little is known about the regulation of expression of the TROP2 gene. Hence, we investigated the TROP2 transcription control network. TROP2 expression was shown to depend on a highly interconnected web of transcription factors: TP63/TP53L, ERG, GRHL1/Get-1 (grainyhead-like epithelial transactivator), HNF1A/TCF-1 (T-cell factor), SPI1/PU.1, WT (Wilms’ tumour)1, GLIS2, AIRE (autoimmune regulator), FOXM1 (forkhead box M1) and FOXP3, with HNF4A as the major network hub. TROP2 upregulation was shown to subsequently drive the expression and activation of CREB1 (cyclic AMP-responsive-element binding protein), Jun, NF-kB, Rb, STAT1 and STAT3 through induction of the cyclin D1 and ERK (extracellular signal regulated kinase)/MEK (MAPK/ERK kinase) pathways. Growth-stimulatory signalling through NF-kB, cyclin D1 and ERK was shown to require an intact Trop-2 cytoplasmic tail. Network hubs and interacting partners are co-expressed with Trop-2 in primary human tumours, supporting a role of this signalling network in cancer growth. Oncogene (2013) 32, 1594–1600; doi:10.1038/onc.2012.151; published online 7 May 2012 Keywords: Trop-2; human tumours; cell growth; cell signalling INTRODUCTION Trop-2 belongs to a unique family of transmembrane glycoproteins,1–3 that has a regulatory role in cell–cell adhesion.4,5 The Trop proteins have structural features that are not related to those of the four classical adhesion molecule families, that is, integrins, cadherins, selectins and Immunoglobulin-CAM.6–8 The extracellular domains of the Trop proteins contain a globular portion with a GA733 type-I domain9 and a thyroglobulin type-IA motif, which are required for Trop homo-multimerisation.5 A cysteine-free region acts as a ‘stem’ to connect the globular moiety of the Trop proteins to their single hydrophobic transmembrane segments. The Trop proteins have a short (25–26 amino-acid-long) intracytoplasmic C-terminal tail, which appears to be devoid of enzymatic domains. Rather, it contains a HIKE-like phosphoinositide-binding motif, which is frequently present in signal transducers and which can act as a docking site for regulatory/effector molecules.2,10,11 We have shown that Trop-2 is overexpressed by most human cancers, as compared with their tissue of origin, and that Trop-2 quantitatively stimulates cancer growth in vitro and in vivo.12 However, little is known about the regulation of expression of the TROP2 gene. Hence, we investigated its regulatory network. TROP2 expression was shown to depend on a highly connected network of transcription factors, which includes TP63/TP53L, ERG, GRHL1/ Get-1 (grainyhead-like epithelial transactivator), HNF1A/TCF-1 (T-cell factor), SPI1/PU.1, WT1 (Wilms’ tumour), GLIS2, autoimmune regulator (AIRE), forkhead box M1 (FOXM1) and FOXP3. TROP2 upregulation was shown to subsequently drive the expression of cancer-growth-regulatory downstream effectors, that is, cyclic AMP-responsive-element binding protein (CREB), Jun, NF-kB, Rb, STAT1 and STAT3,13–17 through the cyclin D1 and ERK (extracellular signal regulated kinase)/MEK (MAPK/ERK kinase) pathways. Signalling by the intact Trop-2 cytoplasmic tail was shown to be required for cell growth, as deletion of the Trop-2 cytoplasmic tail abolishes both modulation of NF-kB, cyclin D1 and pERK, and stimulation of growth. Trop-2 and main network partners appear to be concordantly upregulated in primary human tumours, supporting the in-vivo relevance of the Trop-2 signalling network for cancer growth. RESULTS AND DISCUSSION The Trop-2 signalling network Upregulation of TROP2 was shown to be necessary and sufficient to quantitatively stimulate tumour growth.12 Upregulation of molecules involved in tumourigenesis is often a consequence of gene amplification (for example, HER2/neu).18 Alternatively, inactivating mutations (for example, TP53) can drive compensatory overexpression.19 However, we did not identify instances of mutations12 or of amplification of the TROP2 gene, which indicates that TROP2 overexpression does not arise from structural alterations of the gene itself. Therefore, we sought to identify the network of transcription factors that can modulate the expression of TROP2. To capture actual in-vivo relevance, knockout models were analysed where ablation of specific genes was achieved (7 of 12 as gene replacement by homologous recombination) (Figure 1). DNA microarray analysis demonstrated that TROP2 expression is modulated through the inactivation of TP63/TP53L (a TP53 family member), ERG (of the ETS family), GRHL1/Get-1, lymphoid enhancer factor/TCF-1/HNF1A, SPI1/PU.1, WT1, the Kruppel-like zinc finger transcription factor GLIS2, the autoimmunity regulator transcription factor AIRE, and the forkhead transcription factors FOXM1 and FOXP3 (Figure 1). We then proceeded to determine whether these TROP2modulating transcription factors can take part in a functionally relevant signalling module, and if this has a role in cancer. Ingenuity pathways analysis shows that the TROP2-modulating transcription factors can form a tightly interconnected TROP2- 1 Unit of Cancer Pathology, Department of Oncology and Experimental Medicine and CeSI, Foundation University ‘G. d’Annunzio’, Chieti, Italy and 2Department of Neuroscience and Imaging, University ‘G. d’Annunzio’, Chieti, Italy. Correspondence: Dr S Alberti, Unit of Cancer Pathology, Department of Oncology and Experimental Medicine and CeSI, Foundation University ‘G. d’Annunzio’, CeSI, via Colle dell’ Ara, Chieti Scalo 66100, Italy. E-mail: 3 Current address: Kimmel Cancer Center at Thomas Jefferson University, Philadelphia, PA, USA. Received 21 February 2012; accepted 8 March 2012; published online 7 May 2012 Trop-2 signalling network E Guerra et al 1595 Figure 1. TROP2 transcription network. Map of TROP2-driving transcription factors (centre). Proteins upstream and downstream of Trop-2 were examined using the Ingenuity Pathways Analysis software (version 8.8, Ingenuity Systems, www.ingenuity.com). All of the molecules were mapped onto the Ingenuity Knowledge Base as focus points. Networks of focus molecules were generated by maximising interconnectedness versus random distributions. Networks were ranked by a score based on a hypergeometric distribution and quantified as –log of right-tailed Fisher’s exact test result. The transcription-factor network score was 34 (Fisher’s exact test: 1  10  34). The Trop-2-modulating proteins are in grey and connecting proteins are in (...truncated)