A-M Cleton-Jansen

E-cadherin transcriptional downregulation by promoter methylation but not mutation is related to epithelial-to-mesenchymal transition in breast cancer cell lines

Using genome-wide expression profiling of a panel of 27 human mammary cell lines with different mechanisms of E-cadherin inactivation, we evaluated the relationship between E-cadherin status and gene expression levels. Expression profiles of cell lines with E-cadherin (CDH1) promoter methylation were significantly different from those with CDH1 expression or, surprisingly, those with CDH1 truncating mutations. Furthermore, we found no significant differentially expressed genes between cell lines with wild-type and mutated CDH1. The expression profile complied with the fibroblastic morphology of the cell lines with promoter methylation, suggestive of epithelial–mesenchymal transition (EMT). All other lines, also the cases with CDH1 mutations, had epithelial features. Three non-tumorigenic mammary cell lines derived from normal breast epithelium also showed CDH1 promoter methylation, a fibroblastic phenotype and expression profile. We suggest that CDH1 promoter methylation, but not mutational inactivation, is part of an entire programme, resulting in EMT and increased invasiveness in breast cancer. The molecular events that are part of this programme can be inferred from the differentially expressed genes and include genes from the TGFβ pathway, transcription factors involved in CDH1 regulation (i.e. ZFHX1B, SNAI2, but not SNAI1, TWIST), annexins, AP1/2 transcription factors and members of the actin and intermediate filament cytoskeleton organisation.

Peripheral chondrosarcoma progression is accompanied by decreased Indian Hedgehog signalling.

Hedgehog (HH) signalling is important for specific developmental processes, and aberrant, increased activity has been described in various tumours. Disturbed HH signalling has also been implicated in the hereditary syndrome, Multiple Osteochondromas. Indian Hedgehog (IHH), together with parathyroid hormone‐like hormone (PTHLH), participates in the organization of growth plates in long bones. PTHLH signalling is absent in osteochondromas, benign tumours arising adjacent to the growth plate, but is reactivated when these tumours undergo malignant transformation towards secondary peripheral chondrosarcoma. We describe a gradual decrease in the expression of Patched (PTCH) and glioma‐associated oncogene homologue 1 (GLI1) (both transcribed upon IHH activity), and GLI2 with increasing malignancy, suggesting that IHH signalling is inactive and PTHLH signalling is IHH independent in secondary peripheral chondrosarcomas. cDNA expression profiling and immunohistochemical studies suggest that transforming growth factor‐β (TGF‐β)‐mediated proliferative signalling is active in high‐grade chondrosarcomas since TGF‐β downstream targets were upregulated in these tumours. This is accompanied by downregulation of energy metabolism‐related genes and upregulation of the proto‐oncogene jun B. Thus, the tight regulation of growth plate organization by IHH signalling is still seen in osteochondroma, but gradually lost during malignant transformation to secondary peripheral chondrosarcoma and subsequent progression. TGF‐β signalling is stimulated during secondary peripheral chondrosarcoma progression and could potentially regulate the retained activity of PTHLH. Copyright

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

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. Copyright

Mutation Analysis of H3F3A and H3F3B as a Diagnostic Tool for Giant Cell Tumor of Bone and Chondroblastoma.

Specific H3F3A driver mutations and IDH2 mutations were recently described in giant cell tumor of bone (GCTB) and H3F3B driver mutations in chondroblastoma; these may be helpful as a diagnostic tool for giant cell–containing tumors of the bone. Using Sanger sequencing, we determined the frequency of H3F3A, H3F3B, IDH1, and IDH2 mutations in GCTBs (n=60), chondroblastomas (n=12), and other giant cell–containing tumors (n=24), including aneurysmal bone cyst, chondromyxoid fibroma, and telangiectatic osteosarcoma. To find an easy applicable marker for H3F3A mutation status, H3K36 trimethylation and ATRX expression were correlated with H3F3A mutations. In total, 69% of all GCTBs harbored an H3F3A (G34W/V) mutation compared with 0% of all other giant cell–containing tumors (P<0.001), whereas 70% of chondroblastomas showed an H3F3B (K36M) mutation compared with 0% of other giant cell–containing tumors (P<0.001). Diffuse H3K36 trimethylation positivity was more often seen in mutated H3F3A GCTBs compared with other giant cell–containing tumors (P=0.005). ATRX protein expression was not correlated with H3F3A mutation status. Hotspot mutations in IDH1 or IDH2 were absent. Our results show that H3F3A and H3F3B mutation analysis appears to be a highly specific, although less sensitive, diagnostic tool for the distinction of GCTB and chondroblastoma from other giant cell–containing tumors. Although H3K36 trimethylation and ATRX immunohistochemistry cannot be used as surrogate markers for H3F3A mutation status, mutations in H3F3A are associated with increased H3K36 trimethylation, suggesting that methylation at this residue may play a role in the etiology of the disease.

An association between cartilaginous tumours and breast cancer in the national pathology registration in The Netherlands points towards a possible genetic trait.

Breast cancer and cartilaginous tumours (enchondroma and chondrosaroma) were found to occur rather frequently in the same patient. In order to identify a possible association between occurrence of these tumour types, a population‐based study was performed. This was a nation‐wide case–control study, using the Dutch national pathology database. Between 1973 and 1998, the study identified 2295 cases of cartilaginous tumours in female patients and 132 636 females with breast cancer. Of these patients, 61 were diagnosed with both tumour types. To exclude a possible bias due to screening for occult bone metastases in breast cancer patients, a similar analysis was performed for lung cancer, since screening is performed similarly in lung cancer patients. Of 16 559 females diagnosed with lung cancer, only one case with a cartilaginous tumour was found. The odds ratio for a potential association of breast and cartilaginous tumours is 7.62, implicating a 7.62 increased risk for the same female patient having both breast cancer and a cartilaginous tumour. Furthermore, the mean age of onset in patients with breast cancer as the first tumour is nearly 10 years earlier than breast cancer in general, i.e. 51 years versus 60.9 years. The association of breast cancer and cartilaginous tumours and the early age of onset of breast cancer in these patients may suggest a genetic trait. Copyright

Targeting survivin as a potential new treatment for chondrosarcoma of bone

Chondrosarcomas are malignant cartilage-forming bone tumors, which are intrinsically resistant to chemo- and radiotherapy, leaving surgical removal as the only curative treatment option. Therefore, our aim was to identify genes involved in chondrosarcoma cell survival that could serve as a target for therapy. siRNA screening for 51 apoptosis-related genes in JJ012 chondrosarcoma cells identified BIRC5, encoding survivin, as essential for chondrosarcoma survival. Using immunohistochemistry, nuclear as well as cytoplasmic survivin expression was analyzed in 207 chondrosarcomas of different subtypes. Nuclear survivin has been implicated in cell-cycle regulation while cytoplasmic localization is important for its anti-apoptotic function. RT–PCR was performed to determine expression of the most common survivin isoforms. Sensitivity to YM155, a survivin inhibitor currently in phase I/II clinical trial for other tumors, was examined in 10 chondrosarcoma cell lines using viability assay, apoptosis assay and cell-cycle analysis. Survivin expression was found in all chondrosarcoma patient samples. Higher expression of nuclear and cytoplasmic survivin was observed with increasing histological grade in central chondrosarcomas. Inhibition of survivin using YM155 showed that especially TP53 mutant cell lines were sensitive, but no caspase 3/7 or PARP cleavage was observed. Rather, YM155 treatment resulted in a block in S phase in two out of three chondrosarcoma cell lines, indicating that survivin is more involved in cell-cycle regulation than in apoptosis. Thus, survivin is important for chondrosarcoma survival and chondrosarcoma patients might benefit from survivin inhibition using YM155, for which TP53 mutational status can serve as a predictive biomarker.