Decreased EXT expression and intracellular accumulation of heparan sulphate proteoglycan in osteochondromas and peripheral chondrosarcomas

Abstract

Mutational inactivation of EXT1 or EXT2 is the cause of hereditary multiple osteochondromas. These genes function in heparan sulphate proteoglycan (HSPG) biosynthesis in the Golgi apparatus. Loss of heterozygosity of the EXT1 locus at 8q24 is frequently found in solitary osteochondromas, whereas somatic mutations are rarely found. We investigated the expression of EXT1 and EXT2 (quantitative RT-PCR) and of different HSPGs (immunohistochemistry) in solitary and hereditary osteochondromas and in cases with malignant progression to secondary peripheral chondrosarcoma, in relation to possible mutations and promoter methylation. The mutation status of patients with multiple osteochondromas correlated with decreased EXT1 or EXT2 expression found in their resected tumours. We could not show somatic point mutations or promoter hypermethylation in 17 solitary tumours; however, EXT1 expression was decreased in 15 cases, whereas EXT2 was not. Intracellular accumulation of syndecan-2 and heparan sulphate-bearing isoforms of CD44 (CD44v3) was found in most tumours, which concentrated in the Golgi apparatus as shown by confocal microscopy. This contrasted with the extracellular expression found in normal growth plates. In conclusion, mutational inactivation of either EXT1 or EXT2 leads to loss of mRNA expression of the corresponding gene. We hypothesize that loss of EXT expression disrupts the function of the EXT1/2 complex in HSPG biosynthesis, resulting in the intracellular accumulation of HSPG core proteins that we found in these tumours.

Secondary peripheral chondrosarcoma evolving from osteochondroma as a result of outgrowth of cells with functional EXT

“…First, the mRNA expression level of EXT1 in osteochondromas is not completely absent, which suggests the presence of cells with functional EXT1 in the tumor. Second, although not significant, the mRNA level of EXT1 seems to increase from low-to high-grade in secondary peripheral chondrosarcomas (Hameetman et al, 2007a). We have also shown that signaling pathways dependent on heparan sulfate, such as fibroblast growth factor and parathyroid hormone-related protein, are inactive in osteochondromas and active in secondary peripheral chondrosarcomas.…”

“…The mutation status from 12 cases has been reported previously (Hameetman et al, 2007a). Mutation screening of EXT1 or EXT2 genes was performed and analyzed as described earlier (Hameetman et al, 2007a;Reijnders et al, 2010).…”

Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma

“…Recently, we showed that in osteochondromas, heparan sulfate proteoglycans are no longer present at the cell surface but accumulate in the cytoplasm. 36 Therefore, it is possible that the lack of heparan sulfate proteoglycans at the cell surface might influence cell adhesion and motility. The random orientation of the cilium in osteochondromas leads to the assumption that osteochondroma cells have impaired movement (Figure 6b) that may underlie the lack of orientation of the cells, which is a morphological characteristic of this tumor.…”

A mouse model of chondrocyte-specific somatic mutation reveals a role for Ext1 loss of heterozygosity in multiple hereditary exostoses

“…It was originally hypothesized that EXT1 and EXT2 are tumor suppressor genes, and the loss of heterozygosity (LOH) at these loci plays a key role in osteochondroma formation (8,9). However, the data from genetic analysis of osteochondromas are equivocal (20)(21)(22)(23). It is also uncertain whether osteochondroma is a true neoplasm, which by definition is derived from clonal expansion of progenitor cells, or whether it represents a focal developmental malformation (20).…”