Antitumorigenic effects of a mutant of the heparin affin regulatory peptide on the U87 MG glioblastoma cell line.

Glioblastoma is the most common primary brain tumor in human adults. Since existing treatments are not effective enough, novel therapeutic targets must be sought. The heparin-binding growth factor, heparin affin regulatory peptide (HARP), also known as pleiotrophin (PTN), could potentially represent such a target. We have previously shown that a mutant protein, HARPDelta111-136, which lacks HARP's C-terminal 26 amino acids, acts as a dominant negative HARP effector by heterodimerizing with the wild-type growth factor. The aim of our study was to evaluate the potential inhibitory activity of HARPDelta111-136 on the U87 MG human glioblastoma cell line. By overexpressing the truncated form of HARP in stably established clones of U87 MG cells, we observed an inhibition of proliferation under both anchorage-dependent and anchorage-independent conditions. We confirmed these results in an in vivo subcutaneous tumor xenograft model. In addition, we found that HARPDelta111-136 inhibited cell proliferation in a paracrine manner. Analysis of key cellular pathways revealed a decrease of cell adhesion in U87 MG cells that overexpressed the mutant protein, which could explain this inhibitory effect. A replication-defective adenovirus model that encoded HARPDelta111-136 supported a putative antiproliferative role for the truncated protein in vitro and in vivo. Interestingly, HARPDelta111-136 was also able to abolish angiogenic activity in HUVEC proliferation and in a Matrigel plug assay. These results demonstrate that considering its antiproliferative and angiostatic effects, HARPDelta111-136 could be of great interest when used in conjunction with standard treatments.

Inhibition of the mitogenic, angiogenic and tumorigenic activities of pleiotrophin by a synthetic peptide corresponding to its C-thrombospondin repeat-I domain.

Pleiotrophin (PTN), is a heparin-dependent growth factor involved in angiogenesis and tumor growth. PTN contains a thrombospondin repeat-I (TSR-I) motif in its two beta-sheet domains that are involved in its binding to heparin and its neurite outgrowth activity. Based on the importance of the binding of PTN to heparin in its dimerization and biological activities, we have designed two synthetic peptides, P(13-39) and P(65-97) corresponding to a part of the N-terminal and C-terminal TSR-I motif of PTN, respectively. P(65-97) inhibited the mitogenic, tumorigenic and angiogenic activities of PTN, as well as the mitogenic and an angiogenic activity of fibroblast growth factor-2 (FGF-2). However, P(65-97) had no effect on the mitogenic activity of epidermal growth factor, which does not bind heparin. P(65-97) but not P(13-39) inhibited the binding of PTN and to a lesser extent of FGF-2 to heparin using an immunoassay and an optical biosensor assay and bound directly to heparin with a K(d) of 120 nM. These findings suggest that P(65-97), containing amino acids 65-97 of the TSR-I motif of the C-terminal domain of PTN, inhibits the activities of PTN and FGF-2 by virtue of its ability to bind heparin very effectively and so compete with the growth factors for their polysaccharide co-receptor.

Dominant-stable beta-catenin expression causes cell fate alterations and Wnt signaling antagonist expression in a murine granulosa cell tumor model.

Wnt/beta-catenin signaling is normally involved in embryonic development and tissue homeostasis, and its misregulation leads to several forms of cancer. We have reported that misregulated Wnt/beta-catenin signaling occurs in ovarian granulosa cell tumors (GCT) and have created the Catnb(flox(ex3)/+);Amhr2(cre/+) mouse model, which expresses a dominant-stable mutant of beta-catenin in granulosa cells and develops late-onset GCT. To study the mechanisms leading to GCT development, gene expression analysis was done using microarrays comparing Catnb(flox(ex3)/+);Amhr2(cre/+) ovaries bearing pretumoral lesions with control ovaries. Overexpressed genes identified in Catnb(flox(ex3)/+);Amhr2(cre/+) ovaries included the Wnt/beta-catenin signaling antagonists Wif1, Nkd1, Dkk4, and Axin2, consistent with the induction of negative feedback loops that counteract uncontrolled Wnt/beta-catenin signaling. Expression of the antagonists was localized to cells forming the pretumoral lesions but not to normal granulosa cells. Microarray analyses also revealed the ectopic expression of bone markers, including Ibsp, Cdkn1c, Bmp4, and Tnfrsf11b, as well as neuronal/neurosecretory cell markers, such as Cck, Amph, Pitx1, and Sp5. Increased expression of the gene encoding the cytokine pleiotrophin was also found in Catnb(flox(ex3)/+);Amhr2(cre/+) ovaries and GCT but was not associated with increased serum pleiotrophin levels. In situ hybridization analyses using GCT from Catnb(flox(ex3)/+);Amhr2(cre/+) mice revealed that Wnt/beta-catenin antagonists and neuronal markers localized to a particular cell population, whereas the bone markers localized to a distinct cell type associated with areas of osseous metaplasia. Together, these results suggest that misregulated Wnt/beta-catenin signaling alters the fate of granulosa cells and that the GCT that arise in Catnb(flox(ex3)/+);Amhr2(cre/+) mice result from the clonal expansion of metaplastic cells.