Tumor hepatocytes and basement membrane–Producing cells specifically express two different forms of the endostatin precursor, collagen XVIII, in human liver cancers

Abstract

Endostatin is an endogenous inhibitor of angiogenesis and tumor growth in mice, which may be generated by proteolytic cleavage of collagen XVIII. In normal tissues, 2 variants of the endostatin precursor, namely the SHORT and LONG forms, regulate tissue specificity. We analyzed 53 human liver biopsies (18 hepatocellular carcinomas, 16 metastases of colorectal cancer, 3 cholangiocarcinomas, and 16 controls) by RNA dot blots, double-labeling immunohistochemistry, and in situ hybridization, using common and variant-specific probes. Tumor hepatocytes expressed the LONG form, whereas cholangiocarcinoma cells expressed the SHORT form, which was deposited in tumor basement membranes. Metastatic colorectal carcinoma cells did not express collagen XVIII. In the stromal compartment of primary and metastatic cancers, myofibroblasts and vascular endothelial cells expressed the SHORT form. Both basement membrane components, collagen IV and the SHORT collagen XVIII form, were codistributed and their mRNA levels strongly correlated (R ‫؍‬ .75, P < .001). In addition, freshly isolated human hepatocytes expressed the LONG form and culture-activated stellate cells the SHORT form. Moreover, the full-length LONG form is a plasma protein. Thus, the LONG form is a hepatocyte-specific variant, and the SHORT form is a major component of the tumor extracellular matrix in primary and metastatic liver cancers. Collagen XVIII is the precursor of endostatin, an angiogenesis inhibitor that suppresses tumor growth in murine models. 1,2 It is a component of most epithelial and vascular basement membranes [3][4][5] and is expressed at high levels by human hepatocytes in normal and fibrotic livers. 6 In normal human tissues, 2 forms of collagen XVIII, respectively designated the SHORT and LONG variants, are differentially expressed: the SHORT form is ubiquitously found in basement membranes 3 ; the LONG form is almost exclusively found in the liver, expressed at remarkably high levels by normal human hepatocytes, 6,7 and regulated by liver-enriched transcription factors in similarity with other liver-specific genes. 8 The SHORT and LONG forms of collagen XVIII originate from 2 alternate promoters and have amino-terminal noncollagenous domains of 303 and 493 amino acids (Fig. 1). However, they share all the downstream sequences, including the 312-amino acid C-terminal noncollagenous domain containing the angiogenesis inhibitor, endostatin. 7 Malignant tumors may generate endostatin by proteolytic cleavage of the C-terminal domain of collagen XVIII. 5,9,10 However, the presence of the endostatin precursor, collagen XVIII, in the extracellular matrix of tumors remains to be demonstrated. We have shown that hepatocytes express high and stable levels of collagen XVIII in normal liver, active and quiescent fibrosis, 6 and small hepatocellular carcinomas, 11 whereas liver myofibroblasts increase their collagen XVIII expression by 13-fold in active liver fibrogenesis. 6 We thus hypothesized that hepatocytes and activated stellate cells may express diffe...

Structural Basis for the Functions of Endogenous Angiogenesis Inhibitors

“…Collagen XVIII is ubiquitous and highly conserved among vertebrates, Caenorhabditis elegans, and Drosophila. It is a triple-helical hybrid molecule bearing several heparan sulfate side chains, and a major proteoglycan of endothelial and epithelial BM (Halfter et al 1998;Musso et al 2001). The carboxy-terminal NC1 of collagen XVIII includes a trimerization region and a hinge region that provides protease-sensitive sites and antiangiogenic fragments, such as endostatin.…”

Chronic liver injury, TGF-β, and cancer

“…"True" angiogenesis also may occur with budding of largevessel endothelium in areas of injury. The expression of pro-angiogenic factors (e.g., VEGF) (Ankoma-Sey et al, 1998) and angiogenesis inhibitors (Musso et al, 2001) has been documented in liver, although their involvement in injury repair is unclear.…”

Blocking Wnt signaling by SFRP-like molecules inhibits in vivo cell proliferation and tumor growth in cells carrying active β-catenin

“…Variant 1 is a ubiquitous structural basement membrane component (Saarela et al, 1998). Variant 2 (V2) is a plasma protein produced mainly in the liver (Musso et al, 2001). Variant 3 (V3) is expressed at very low levels during Xenopus embryogenesis (Elamaa et al, 2002), in human fetal (Elamaa et al, 2003), normal adult and tumor liver tissues (Quelard et al, 2008), and is not detected in metastatic CRCs (Musso et al, 2001).…”

“…Variant 2 (V2) is a plasma protein produced mainly in the liver (Musso et al, 2001). Variant 3 (V3) is expressed at very low levels during Xenopus embryogenesis (Elamaa et al, 2002), in human fetal (Elamaa et al, 2003), normal adult and tumor liver tissues (Quelard et al, 2008), and is not detected in metastatic CRCs (Musso et al, 2001). Proteolytic processing of V3 releases V3Nter, an aminoterminal glycoprotein containing a frizzled CRD, which locates at the cell surface in cancer cells and matches the three-dimensional structures of frizzled 8 and SFRP-3 CRDs (Quelard et al, 2008).…”