DEVELOPMENTAL DYNAMICS 244:99–109, 2015 DOI: 10.1002/DVDY.24242
REVIEWS
Trop2: From Development to Disease Annie R.A. McDougall,1,2,3* Mary Tolcos,1,2 Stuart B. Hooper,1,2 Timothy J. Cole,3 and Megan J. Wallace1,2 a
1
The Ritchie Centre, MIMR-PHI Institute of Medical Research, Monash University, Clayton, Vic, Australia Department of Obstetrics and Gynaecology, Monash University, Clayton, Vic, Australia 3 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic, Australia
DEVELOPMENTAL DYNAMICS
2
Background: Trop2 was first discovered as a biomarker of invasive trophoblast cells. Since then most research has focused on its role in tumourigenesis because it is highly expressed in the vast majority of human tumours and animal models of cancer. It is also highly expressed in stem cells and in many organs during development. Results: We review the multifaceted role of Trop2 during development and tumourigenesis, including its role in regulating cell proliferation and migration, self-renewal, and maintenance of basement membrane integrity. We discuss the evolution of Trop2 and its related protein Epcam (Trop1), including their distinct roles. Mutation of Trop2 leads to gelatinous drop-like corneal dystrophy, whereas over-expression of Trop2 in human tumours promotes tumour aggressiveness and increases mortality. Although Trop2 expression is sufficient to promote tumour growth, the surprising discovery that Trop2-null mice have an increased risk of tumour development has highlighted the complexity of Trop2 signaling. Recently, studies have begun to identify the mechanisms underlying TROP2’s functions, including regulated intramembrane proteolysis or specific interactions with integrin b1 and claudin proteins. Conclusions: Understanding the mechanisms underlying TROP2 signaling will clarify its role during development, aid in the development of better cancer treatments and unlock a promising new direction in regenerative medicine. Developmental C 2014 Wiley Periodicals, Inc. Dynamics 244:99–109, 2015. V Key words: Tacstd2; EpCAM; cell proliferation; cell migration; lung development; cancer Submitted 7 October 2014; First Decision 8 December 2014; Accepted 11 December 2014; Published online 19 December 2014
Introduction TROP2 was first identified as a cell surface marker for trophoblast cells (Lipinski et al., 1981), which are highly invasive cells that originate from the outer layer of the blastocyst, aiding implantation and forming a large portion of the placenta. Since its discovery in the placenta, a large body of work has implicated TROP2 as a major tumorigenic factor and recent studies suggest that it also has a role in organ development and stem cell maintenance. Given the important links between tumourogenesis and organogenesis, it is likely that TROP2 will emerge as an important factor in embryonic and fetal development in the future.
Trop2 Gene and Protein Trop2 (trophoblast antigen 2) is also known as tumour-associated calcium signal transducer 2 (tacstd2), membrane component 1 surface marker 1 (M1S1), epithelial glycoprotein 1 (EGP1), and gastrointestinal antigen 733-1 (GA733-1). The Trop2 gene has been sequenced in chickens and in numerous mammalian species, including mice, rats, humans, a variety of non-human primates (Linnenbach et al., 1993), and sheep (Sozo et al., 2006; McDougall Grant sponsor: National Health and Medical Research Council; Grant sponsor: Victorian Government’s Operational Infrastructure Support Program.. *Correspondence to: Dr. Annie R.A. McDougall, The Ritchie Centre, MIMRPHI Institute of Medical Research, 27–31 Wright Street, Clayton. Vic. 3168, Australia. E-mail: [email protected]
et al., 2011). Trop2 is an intronless gene that encodes a 323 amino acid, 35–49 kDa protein, comprised of a large extracellular domain, a single transmembrane domain, and a short intracellular tail (Linnenbach et al., 1989; Basu et al., 1995; Fornaro et al., 1995). The TROP2 protein has a 30-amino acid signal peptide and 244amino acid extracellular domain, containing twelve cysteine residues and four potential N-linked glycosylation sites (Linnenbach et al., 1989). Within the cysteine-rich region, TROP2 contains a thyroglobulin type-1 repeat domain and a putative epidermal growth factor-like (EGF) domain (Linnenbach et al., 1993; Novinec et al., 2006). Thyroglobulin type-1 repeats are cysteine-rich motifs with variable length and strictly conserved positions for cysteine residues. They occur in a number of functionally unrelated proteins such as thyroglobulin, insulin-like growth factor binding proteins, testican, and the major histocompatibility complex class II-associated invariant chains (Mihelic and Turk, 2007). The functional role of the thyroglobulin repeats is not fully understood; however, in some proteins they can act as an inhibitor of cysteine proteases (Mihelic and Turk, 2007). The thyroglobulin type-1 repeat in insulin-like growth factor binding proteins (IGFBPs) is involved in the binding of IGF-II to the binding proteins. Directly upstream from the TROP2 thyroglobulin type-1 repeat domain is a putative EGF-like domain. This sequence data suggest that TROP2 may be activated by growth factors. Indeed, TROP2 can bind to Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24242/abstract C 2014 Wiley Periodicals, Inc. V
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100 MCDOUGALL ET AL.
Fig. 1. Homology between EpCAM and TROP2 proteins. Alignment of the human TROP2 (NP_002353) and EpCAM (NP_002354) protein sequences. TROP2 and EpCAM share 49% sequence identity and 67% sequence similarity. The highest homology is observed in the thyroglobulin repeat domain (yellow) and the single transmembrane region (red). The intracellular tails of TROP2 and EpCAM have differing domains, suggesting differences in intracellular signaling mechanisms. Trop2 contains four N-glycosylation sites (dark blue), whereas EpCAM only contains 3 N-glycosylation sites, 2 of which are conserved between both proteins. TROP2 contains a HIKE domain (green) that contains a potential PIP2 binding site (underlined) and a serine (Ser303; bold) that can be phosphorylated by PKC. The intracellular tail of EpCAM contains two putative a-actinin binding sites (light blue), as well as a putative PDZ-binding site (double underline). Alignment was performed using Clustal W2. Asterisks (*) indicate homologous amino acids; colons (:) indicate conserved substitutions; periods (.) indicate semi-conserved substitutions.
IGF-I causing an inhibition of IGF-1R signaling suggesting that TROP2 may regulate IGF signaling by sequestering IGF-I and preventing it from activating its receptor (Lin et al., 2012). No other activators of TROP2 have been identified. TROP2 has a single 23-residue transmembrane region and a short (26 amino acid) cytoplasmic region (Linnenbach et al., 1989). The cytoplasmic tail of the TROP2 protein shows structural and sequence similarities to a HIKE domain (Ciccarelli et al., 2000) and contains a serine residue (S303) that can be phosphorylated by protein kinase C (PKC) (Basu et al., 1995) and a phosphatidylinositol 4,5-bisphosphate (PIP2) binding site (El Sewedy et al., 1998). HIKE sequences are candidates for binding to pleckstrinhomology domains, which are found in the b subunit of G proteins, kinases, ankyrin, and kinesin (Alberti, 1999). Calmodulin (CaM), a calcium signalling protein, can bind to the HIKE domain in G proteins, regulating the binding of other proteins. Both neurogranin and neuromodulin also contain HIKE-like CaM binding sequences that bind phosphatidylinositol (PI) and are phosphorylated by PKC (Alberti, 1999). The presence of the HIKE domain together with the PIP2 binding site and the serine phosphorylated by PKC, therefore, support a role for TROP2 in calcium signalling.
Evolution of the tacstd Gene Family Trop2 is a member of the tacstd gene family, which has only one other family member: tacstd1, also known as trophoblast antigen 1 (Trop1), gastrointestinal antigen 2 (GA733-2), and most commonly known as epithelial cell adhesion molecule (Epcam). TROP2 shares 49% sequence identity and 67% similarity with EpCAM, which is a 314–amino acid, 35-kDa protein (Szala et al., 1990) (Fig. 1). The most highly conserved regions are the thyroglobulin repeat domain and the transmembrane region. Unlike, Trop2, which is intronless, Epcam consists of nine coding exons; Exons 1–6 encode the extracellular domain, exon 7 the transmembrane region, and exons 8–9 the intracellular tail (Balzar
et al., 1999). The cysteine positions and distributions of hydrophilic and hydrophobic residues in the thyroglobulin repeat domain are conserved between TROP2 and EpCAM. However, EpCAM only has three N-glycosylation sites, while Trop2 has four, two of which are conserved (Schnell et al., 2013a). The intracellular tail of EpCAM contains two possible a-actinin binding sites, involved in EpCAMs cell adhesion properties (Balzar et al., 1998), as well as a putative PDZ domain; PDZ domains are important for anchoring transmembrane proteins to the cell cytoskeleton (Schnell et al., 2013a). The difference in the intracellular tails suggests that EpCAM and Trop2 may initiate different intracellular signaling pathways, which may explain the differences in their functions and distribution, as discussed throughout this review. In zebrafish, there is only one tacstd gene, which is orthologous to Epcam (Villablanca et al., 2006). Trop2 is thought to have arisen from Epcam by a retrotransposition event after the divergence of fish and tetrapods, but prior to the divergence of avian and mammalian lineages (Linnenbach et al., 1993; Villablanca et al., 2006). Zebrafish Tacstd protein has 37.2% identity and 60.5% similarity to human EpCAM and 35.97% identity and 62.71% similarity to human TROP2 (Villablanca et al., 2006). The promoter regions of Epcam and Trop2 are unrelated, which likely explains the differences in their expression patterns as discussed in this review.
Trop2 in Health and Disease The Role of Trop2 During Embryonic and Fetal Development Loss-of-function studies in zebrafish demonstrate a role for the ancestral gene tacstd in development. The embryonic posterior lateral line is a superficial sensory organ system that originates from a primordium that migrates towards the tail of the zebrafish
TROP2: FROM DEVELOPMENT TO DISEASE 101
(Tsujikawa et al., 1999; Lu et al., 2005; McDougall et al., 2011; Trerotola et al., 2013a). To date, the role of Trop2 during embryonic and fetal development has only been studied in any detail in the developing lung, intestines, and kidney. Epcam is also widely expressed in epithelial cells of many tissues during development, as well as the placenta and has a vital role in embryonic development. Epcam knockout mice are embryonic lethal, with defects in placental development, overall fetal growth, and failure of neural tube closure (Nagao et al., 2009).
DEVELOPMENTAL DYNAMICS
Trop2 in the Developing Lung
Fig. 2. Differential regulation of Trop2 and Epcam expression during normal fetal lung development. Mean (6 SEM) Trop2 (black squares) and Epcam (open circles) mRNA levels during the pseudoglandular (dark grey bar, embryonic day 17; E17, n¼10), canalicular (light grey bar, E18, n¼8; E19, n¼7; and E20, n¼6), and saccular (black bar, E20 and E21, n¼6) stages of lung development in fetal rats. Epcam expression does not change during normal fetal lung development. In contrast, Trop2 expression significantly (P
REVIEWS
Trop2: From Development to Disease Annie R.A. McDougall,1,2,3* Mary Tolcos,1,2 Stuart B. Hooper,1,2 Timothy J. Cole,3 and Megan J. Wallace1,2 a
1
The Ritchie Centre, MIMR-PHI Institute of Medical Research, Monash University, Clayton, Vic, Australia Department of Obstetrics and Gynaecology, Monash University, Clayton, Vic, Australia 3 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic, Australia
DEVELOPMENTAL DYNAMICS
2
Background: Trop2 was first discovered as a biomarker of invasive trophoblast cells. Since then most research has focused on its role in tumourigenesis because it is highly expressed in the vast majority of human tumours and animal models of cancer. It is also highly expressed in stem cells and in many organs during development. Results: We review the multifaceted role of Trop2 during development and tumourigenesis, including its role in regulating cell proliferation and migration, self-renewal, and maintenance of basement membrane integrity. We discuss the evolution of Trop2 and its related protein Epcam (Trop1), including their distinct roles. Mutation of Trop2 leads to gelatinous drop-like corneal dystrophy, whereas over-expression of Trop2 in human tumours promotes tumour aggressiveness and increases mortality. Although Trop2 expression is sufficient to promote tumour growth, the surprising discovery that Trop2-null mice have an increased risk of tumour development has highlighted the complexity of Trop2 signaling. Recently, studies have begun to identify the mechanisms underlying TROP2’s functions, including regulated intramembrane proteolysis or specific interactions with integrin b1 and claudin proteins. Conclusions: Understanding the mechanisms underlying TROP2 signaling will clarify its role during development, aid in the development of better cancer treatments and unlock a promising new direction in regenerative medicine. Developmental C 2014 Wiley Periodicals, Inc. Dynamics 244:99–109, 2015. V Key words: Tacstd2; EpCAM; cell proliferation; cell migration; lung development; cancer Submitted 7 October 2014; First Decision 8 December 2014; Accepted 11 December 2014; Published online 19 December 2014
Introduction TROP2 was first identified as a cell surface marker for trophoblast cells (Lipinski et al., 1981), which are highly invasive cells that originate from the outer layer of the blastocyst, aiding implantation and forming a large portion of the placenta. Since its discovery in the placenta, a large body of work has implicated TROP2 as a major tumorigenic factor and recent studies suggest that it also has a role in organ development and stem cell maintenance. Given the important links between tumourogenesis and organogenesis, it is likely that TROP2 will emerge as an important factor in embryonic and fetal development in the future.
Trop2 Gene and Protein Trop2 (trophoblast antigen 2) is also known as tumour-associated calcium signal transducer 2 (tacstd2), membrane component 1 surface marker 1 (M1S1), epithelial glycoprotein 1 (EGP1), and gastrointestinal antigen 733-1 (GA733-1). The Trop2 gene has been sequenced in chickens and in numerous mammalian species, including mice, rats, humans, a variety of non-human primates (Linnenbach et al., 1993), and sheep (Sozo et al., 2006; McDougall Grant sponsor: National Health and Medical Research Council; Grant sponsor: Victorian Government’s Operational Infrastructure Support Program.. *Correspondence to: Dr. Annie R.A. McDougall, The Ritchie Centre, MIMRPHI Institute of Medical Research, 27–31 Wright Street, Clayton. Vic. 3168, Australia. E-mail: [email protected]
et al., 2011). Trop2 is an intronless gene that encodes a 323 amino acid, 35–49 kDa protein, comprised of a large extracellular domain, a single transmembrane domain, and a short intracellular tail (Linnenbach et al., 1989; Basu et al., 1995; Fornaro et al., 1995). The TROP2 protein has a 30-amino acid signal peptide and 244amino acid extracellular domain, containing twelve cysteine residues and four potential N-linked glycosylation sites (Linnenbach et al., 1989). Within the cysteine-rich region, TROP2 contains a thyroglobulin type-1 repeat domain and a putative epidermal growth factor-like (EGF) domain (Linnenbach et al., 1993; Novinec et al., 2006). Thyroglobulin type-1 repeats are cysteine-rich motifs with variable length and strictly conserved positions for cysteine residues. They occur in a number of functionally unrelated proteins such as thyroglobulin, insulin-like growth factor binding proteins, testican, and the major histocompatibility complex class II-associated invariant chains (Mihelic and Turk, 2007). The functional role of the thyroglobulin repeats is not fully understood; however, in some proteins they can act as an inhibitor of cysteine proteases (Mihelic and Turk, 2007). The thyroglobulin type-1 repeat in insulin-like growth factor binding proteins (IGFBPs) is involved in the binding of IGF-II to the binding proteins. Directly upstream from the TROP2 thyroglobulin type-1 repeat domain is a putative EGF-like domain. This sequence data suggest that TROP2 may be activated by growth factors. Indeed, TROP2 can bind to Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24242/abstract C 2014 Wiley Periodicals, Inc. V
99
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100 MCDOUGALL ET AL.
Fig. 1. Homology between EpCAM and TROP2 proteins. Alignment of the human TROP2 (NP_002353) and EpCAM (NP_002354) protein sequences. TROP2 and EpCAM share 49% sequence identity and 67% sequence similarity. The highest homology is observed in the thyroglobulin repeat domain (yellow) and the single transmembrane region (red). The intracellular tails of TROP2 and EpCAM have differing domains, suggesting differences in intracellular signaling mechanisms. Trop2 contains four N-glycosylation sites (dark blue), whereas EpCAM only contains 3 N-glycosylation sites, 2 of which are conserved between both proteins. TROP2 contains a HIKE domain (green) that contains a potential PIP2 binding site (underlined) and a serine (Ser303; bold) that can be phosphorylated by PKC. The intracellular tail of EpCAM contains two putative a-actinin binding sites (light blue), as well as a putative PDZ-binding site (double underline). Alignment was performed using Clustal W2. Asterisks (*) indicate homologous amino acids; colons (:) indicate conserved substitutions; periods (.) indicate semi-conserved substitutions.
IGF-I causing an inhibition of IGF-1R signaling suggesting that TROP2 may regulate IGF signaling by sequestering IGF-I and preventing it from activating its receptor (Lin et al., 2012). No other activators of TROP2 have been identified. TROP2 has a single 23-residue transmembrane region and a short (26 amino acid) cytoplasmic region (Linnenbach et al., 1989). The cytoplasmic tail of the TROP2 protein shows structural and sequence similarities to a HIKE domain (Ciccarelli et al., 2000) and contains a serine residue (S303) that can be phosphorylated by protein kinase C (PKC) (Basu et al., 1995) and a phosphatidylinositol 4,5-bisphosphate (PIP2) binding site (El Sewedy et al., 1998). HIKE sequences are candidates for binding to pleckstrinhomology domains, which are found in the b subunit of G proteins, kinases, ankyrin, and kinesin (Alberti, 1999). Calmodulin (CaM), a calcium signalling protein, can bind to the HIKE domain in G proteins, regulating the binding of other proteins. Both neurogranin and neuromodulin also contain HIKE-like CaM binding sequences that bind phosphatidylinositol (PI) and are phosphorylated by PKC (Alberti, 1999). The presence of the HIKE domain together with the PIP2 binding site and the serine phosphorylated by PKC, therefore, support a role for TROP2 in calcium signalling.
Evolution of the tacstd Gene Family Trop2 is a member of the tacstd gene family, which has only one other family member: tacstd1, also known as trophoblast antigen 1 (Trop1), gastrointestinal antigen 2 (GA733-2), and most commonly known as epithelial cell adhesion molecule (Epcam). TROP2 shares 49% sequence identity and 67% similarity with EpCAM, which is a 314–amino acid, 35-kDa protein (Szala et al., 1990) (Fig. 1). The most highly conserved regions are the thyroglobulin repeat domain and the transmembrane region. Unlike, Trop2, which is intronless, Epcam consists of nine coding exons; Exons 1–6 encode the extracellular domain, exon 7 the transmembrane region, and exons 8–9 the intracellular tail (Balzar
et al., 1999). The cysteine positions and distributions of hydrophilic and hydrophobic residues in the thyroglobulin repeat domain are conserved between TROP2 and EpCAM. However, EpCAM only has three N-glycosylation sites, while Trop2 has four, two of which are conserved (Schnell et al., 2013a). The intracellular tail of EpCAM contains two possible a-actinin binding sites, involved in EpCAMs cell adhesion properties (Balzar et al., 1998), as well as a putative PDZ domain; PDZ domains are important for anchoring transmembrane proteins to the cell cytoskeleton (Schnell et al., 2013a). The difference in the intracellular tails suggests that EpCAM and Trop2 may initiate different intracellular signaling pathways, which may explain the differences in their functions and distribution, as discussed throughout this review. In zebrafish, there is only one tacstd gene, which is orthologous to Epcam (Villablanca et al., 2006). Trop2 is thought to have arisen from Epcam by a retrotransposition event after the divergence of fish and tetrapods, but prior to the divergence of avian and mammalian lineages (Linnenbach et al., 1993; Villablanca et al., 2006). Zebrafish Tacstd protein has 37.2% identity and 60.5% similarity to human EpCAM and 35.97% identity and 62.71% similarity to human TROP2 (Villablanca et al., 2006). The promoter regions of Epcam and Trop2 are unrelated, which likely explains the differences in their expression patterns as discussed in this review.
Trop2 in Health and Disease The Role of Trop2 During Embryonic and Fetal Development Loss-of-function studies in zebrafish demonstrate a role for the ancestral gene tacstd in development. The embryonic posterior lateral line is a superficial sensory organ system that originates from a primordium that migrates towards the tail of the zebrafish
TROP2: FROM DEVELOPMENT TO DISEASE 101
(Tsujikawa et al., 1999; Lu et al., 2005; McDougall et al., 2011; Trerotola et al., 2013a). To date, the role of Trop2 during embryonic and fetal development has only been studied in any detail in the developing lung, intestines, and kidney. Epcam is also widely expressed in epithelial cells of many tissues during development, as well as the placenta and has a vital role in embryonic development. Epcam knockout mice are embryonic lethal, with defects in placental development, overall fetal growth, and failure of neural tube closure (Nagao et al., 2009).
DEVELOPMENTAL DYNAMICS
Trop2 in the Developing Lung
Fig. 2. Differential regulation of Trop2 and Epcam expression during normal fetal lung development. Mean (6 SEM) Trop2 (black squares) and Epcam (open circles) mRNA levels during the pseudoglandular (dark grey bar, embryonic day 17; E17, n¼10), canalicular (light grey bar, E18, n¼8; E19, n¼7; and E20, n¼6), and saccular (black bar, E20 and E21, n¼6) stages of lung development in fetal rats. Epcam expression does not change during normal fetal lung development. In contrast, Trop2 expression significantly (P
