Trop-1 is a 38 kDa transmembrane type I glycoprotein,1–3 that is encoded by a single-copy gene (TROP1/TACSTD1) in man (h), mouse (m), and rat.3–5 The extracellular domain of Trop-1 contains an EGF-like domain and a thyroglobulin repeat2, 3 that plays a role in regulating cell-cell adhesion via a 2-step homophilic oligomerization.6 Homotetramerization occurs first intracellularly; tetramers subsequently bind cognate oligomers at the surface of adjacent cells.6, 7 Notably, the Trop-1 protein is expressed by a large fraction of human cancers,1, 8 among them breast tumors9, 10 (ms. in prep.), squamous lung cancers,11 and most colon carcinomas,12 suggesting a role in tumor development.
To identify fundamental, conserved functional features of Trop-1 in transformed cells, we searched for evolutionarily conserved structure, expression patterns and function by gene cloning, DNA array and serial analysis of gene expression (SAGE), Northern and Western blotting, flow cytometry, and immunohistochemistry of sequential stages of tumor progression in experimental systems and in man. Our findings demonstrate that Trop-1 expression plays a direct growth-stimulatory role at early stages of tumor development and identify Trop-1 as a marker of stem cells in normal tissues and in cancer.
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- MATERIALS AND METHODS
To identify fundamental, conserved functional roles of Trop-1 in cell transformation, we searched for evolutionarily conserved structure, expression patterns, and function by gene cloning, DNA array and SAGE analysis, Northern and Western blotting, flow cytometry, and immunohistochemistry analysis of sequential stages of tumor progression in experimental systems and in man.
Sequence comparison of the murine Trop1 with its human homolog demonstrated high conservation of the coding sequence and an exact match of exon-intron boundaries. The 17E5 mTrop1 map region was shown to correspond in localization and structure to the human 2p21 band where the hTROP1 gene resides,4 consistent with a parallel selective pressure for the conservation of the structure/function of the gene in the 2 species.
The promoter regions of the TROP genes share key transcription factor binding sites (Sp1, AP-1, deltaEF1, and MZF1), suggesting a common regulatory scheme in mouse and man, for a parallel expression pattern in the 2 species. This was indeed shown to be the case by DNA microarray, SAGE, and Northern blot analysis.8 Parallel expression levels of the mTrop1 transcript and protein were observed in most cases. An exception was the small intestine, where the expression of the Trop-1 protein was considerably lower than that of the corresponding mRNA. This suggested a regulated utilization of the TROP1 mRNA for protein synthesis, eg, for a rapid induction of expression of the protein under specific functional requests.
Of interest, Trop-1 was found expressed by totipotent ES cells and by tissues at very early stages of differentiation (eg, embryonic stem cells, endodermal precursors, urogenital sinus). This expression pattern suggested a requirement for Trop-1 expression in progenitor/early-stage differentiated cells. The expression of Trop-1 by germinal cells46 and by progenitor cells of diverse epithelial, eg, epidermis, breast, pancreas, liver,1, 47–49 and hematopoietic50 tissues is consistent with this model.
The findings above and the presence of Trop-1 molecules in highly proliferating tissues (eg, early embryo, fetal and adult colon, breast during pregnancy) suggested that the requirement for Trop-1 expression resided in its capacity to stimulate cell growth. This was shown to be true in epithelial cell transfectants. The induced increase in growth rate was similar for the murine and human gene transfectants, consistently with a conserved structure of the 2 molecules,2, 3, 5, 51 and of the corresponding signaling mechanisms in man and mouse. siRNA silencing assays demonstrated the requirement of TROP1 expression for sustained tumor cell growth and survival. A direct role in tumor development in vivo was demonstrated by the aggressive growth TROP1-expressing cells in immunosuppressed animals. More in detail, analysis of cells at various stages of transformation showed very little expression of Trop-1 in fresh keratinocytes, at variance with the high levels of expression in their immortalized counterparts. Of interest, Trop-1 expression is induced by transformation in vitro by the SV-40 T antigen or activated ras,1 indicating that it is associated with early events of cell immortalization and transformation. Consistently, membrane and cytoplasmic staining for the Trop-1 protein were detected in focal hyperplasia/low-grade dysplasia in intestinal glands, which are precursors of neoplastic lesions in ApcMin mice.45 Similarly, preneoplastic lesions of the stomach in man showed a dramatic induction of hTrop-1, at stark variance with the absence of expression in the normal gastric mucosa. A link with early/causal stages of tumor progression is supported by the expression of Trop-1 in stem cells, eg, totipotent ES cells, which can give rise to tumors with wide differentiative capacity like teratomas.52 The analysis of breast tumor cells according to patterns of expression of CD24, CD44, and Trop-1/ESA was consistent with this model, as cancer stem cells were found to reside only in the population expressing CD44 and Trop-1/ESA.53
Taken together, our findings identify Trop-1 as a marker of proliferating cells in normal tissues and in cancer, and as a novel determinant of cell growth at early stages of tumor development. They also candidate hTrop-1 as a novel target of diagnostic/screening procedures for early cancer detection.