CTGF is a secreted matricellular protein with very complex biology. It has been shown to modulate many signaling pathways leading to cell adhesion and migration, angiogenesis, myofibroblast activation, and extracellular matrix deposition and remodeling, which together lead to tissue remodeling and fibrosis. It has been reported in the literature that inhibition of CTGF expression by siRNA prevents CCl4-induced liver fibrosis and can reverse fibrosis when administered after significant collagen deposition is observed. A monoclonal antibody to CTGF that is currently in clinical development (FG-3019) has demonstrated the ability to reverse vascular stiffening and improve cardiac function in a rat model of diabetic complications. FG-3019 has also exhibited activity in a murine radiation-induced pulmonary fibrosis model. When FG-3019 was administered to mice after a significant radiation-induced increase in lung density could be observed by CT imaging, the density of the lungs was observed to decrease over the period during which the antibody was administered and to remain stable after therapy had ceased. When considered together, these data indicate that inhibition of CTGF can prevent and reverse the process of fibrosis.

Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant desmoplasia and poor tissue perfusion. These features are proposed to limit the access of therapies to neoplastic cells and blunt treatment efficacy. Indeed, several agents that target the PDA tumor microenvironment promote concomitant chemotherapy delivery and increased antineoplastic response in murine models of PDA. Prior studies could not determine whether chemotherapy delivery or microenvironment modulation per se were the dominant features in treatment response, and such information could guide the optimal translation of these preclinical findings to patients. To distinguish between these possibilities, we used a chemical inhibitor of cytidine deaminase to stabilize and thereby artificially elevate gemcitabine levels in murine PDA tumors without disrupting the tumor microenvironment. Additionally, we used the FG-3019 monoclonal antibody (mAb) that is directed against the pleiotropic matricellular signaling protein connective tissue growth factor (CTGF/CCN2). Inhibition of cytidine deaminase raised the levels of activated gemcitabine within PDA tumors without stimulating neoplastic cell killing or decreasing the growth of tumors, whereas FG-3019 increased PDA cell killing and led to a dramatic tumor response without altering gemcitabine delivery. The response to FG-3019 correlated with the decreased expression of a previously described promoter of PDA chemotherapy resistance, the X-linked inhibitor of apoptosis protein. Therefore, alterations in survival cues following targeting of tumor microenvironmental factors may play an important role in treatment responses in animal models, and by extension in PDA patients.

Laminin-5, a major adhesive ligand for epithelial cells, undergoes processing of its ␥2 and ␣3 chains. This study investigated the mechanism of laminin-5 processing by keratinocytes. BI-1 (BMP-1 isoenzyme inhibitor-1), a selective inhibitor of a small group of astacin-like metalloproteinases, which includes bone morphogenetic protein 1 (BMP-1), mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mammalian Tolloid-like 2 (mTLL-2), inhibited the processing of laminin-5 ␥2 and ␣3 chains in keratinocyte cultures in a dose-dependent manner. In a proteinase survey, all BMP-1 isoenzymes processed human laminin-5 ␥2 and ␣3 chains to 105-and 165-kDa fragments, respectively. In contrast, MT1-MMP and MMP-2 did not cleave the ␥2 chain of human laminin-5 but processed the rat laminin ␥2 chain to an 80-kDa fragment. An immunoblot and quantitative PCR survey of the BMP-1 isoenzymes revealed expression of mTLD in primary keratinocyte cultures but little or no expression of BMP-1, mTLL-1, or mTLL-2. mTLD was shown to cleave the ␥2 chain at the same site as the previously identified BMP-1 cleavage site. In addition, mTLD/BMP-1 null mice were shown to have deficient laminin-5 processing. Together, these data identify laminin-5 as a substrate for mTLD, suggesting a role for laminin-5 processing by mTLD in the skin.Proteolysis of the extracellular matrix is emerging as a key mechanism in processes such as wound healing and tumor metastasis (1, 2). Although most studies have investigated the role of serine proteases and matrix metalloproteases, members of the astacin and ADAM (a disintegrin and metalloprotease) families have also been implicated in this process (1, 2). Laminin-5, the major component of epithelial basement membranes, is a heterotrimeric protein consisting of ␣3, ␤3, and ␥2 subunits (3, 4). Laminin-5 undergoes extracellular proteolysis of the ␣3 chain from a 200-to a 165-kDa form and of the ␥2 chain from a 155-to a 105-kDa form (5). Through its interaction with ␣ 3 ␤ 1 (6, 7), ␣ 6 ␤ 4 (8), and ␣ 2 ␤ 1 integrins (9), laminin-5 supports epithelial cell adhesion (3, 10), and migration (11, 12).Several proteases have been implicated in laminin-5 processing. Exogenous addition of matrix metalloprotease 2 (MMP-2) 1 cleaved the ␥2 subunit of rat laminin-5 (12). A subsequent study suggested that membrane type 1 matrix metalloprotease (MT1-MMP) may play a role in cleaving laminin-5 (13). Cleavage of laminin-5 by plasmin converted the ␣3 chain into the 165-kDa form observed in human breast and rat epithelial cells and capable of nucleating hemidesmosomes (14). Bone morphogenetic protein 1 (BMP-1) has also been implicated in laminin-5 proteolysis. N-terminal sequencing of the 105-kDa ␥2 chain obtained from human keratinocytes revealed a cleavage site that matched the minimal consensus sequence of this metalloprotease (15). In vitro studies demonstrated that BMP-1 cleaved the recombinant ␥2 short arm at the predicted site and that the enzyme cleaved both the ␣3 and ␥2 chains of whole laminin-5 to generate characterist...

Connective tissue growth factor (CTGF) plays an important role in fibrosis by modulating cell migration and cell growth but may also modify tumor growth and metastasis. Because CTGF is overexpressed in pancreatic ductal adenocarcinoma, we investigated the in vitro effects of CTGF on the proliferation and invasiveness of PANC-1 pancreatic cancer cells and examined the consequences of its in vivo inhibition on the growth and metastasis of these cells using a fully human CTGF-specific monoclonal antibody (FG-3019) in an orthotopic nude mouse model. Although PANC-1 cells expressed relatively high levels of endogenous CTGF mRNA, the addition of CTGF to conditioned medium increased the proliferation and invasiveness of PANC-1 cells. Moreover, transforming growth factor-beta1 caused a further increase in CTGF expression in these cells. In vivo, the twice weekly i.p. administration of FG-3019 decreased tumor growth and metastasis and attenuated tumor angiogenesis and cancer cell proliferation. FG-3019 did not enhance apoptosis and did not attenuate the inhibitory effects of gemcitabine on tumor growth and metastasis. These findings suggest that CTGF may contribute to aberrant autocrine and paracrine pathways that promote pancreatic cancer cell growth, invasion, metastasis, and angiogenesis. Therefore, blocking CTGF actions with FG-3019 may represent a novel therapeutic approach in pancreatic ductal adenocarcinoma.

Pancreatic cancer is highly aggressive and refractory to most existing therapies. Past studies have shown that connective tissue growth factor (CTGF) expression is elevated in human pancreatic adenocarcinomas and some pancreatic cancer cell lines. To address whether and how CTGF influences tumor growth, we generated pancreatic tumor cell lines that overexpress different levels of human CTGF. The effect of CTGF overexpression on cell proliferation was measured in vitro in monolayer culture, suspension culture, or soft agar, and in vivo in tumor xenografts. Although there was no effect of CTGF expression on proliferation in two-dimensional cultures, anchorage-independent growth (AIG) was enhanced. The capacity of CTGF to enhance AIG in vitro was linked to enhanced pancreatic tumor growth in vivo when these cells were implanted s.c. in nude mice. Administration of a neutralizing CTGF-specific monoclonal antibody, FG-3019, had no effect on monolayer cell proliferation, but blocked AIG in soft agar. Consistent with this observation, anti-CTGF treatment of mice bearing established CTGF-expressing tumors abrogated CTGF-dependent tumor growth and inhibited lymph node metastases without any toxicity observed in normal tissue. Together, these studies implicate CTGF as a new target in pancreatic cancer and suggest that inhibition of CTGF with a human monoclonal antibody may control primary and metastatic tumor growth. (Cancer Res 2006; 66(11): 5816-27)