Background

PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.

Results

Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.

Conclusions

We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.

Background

Currently, no specific immunohistochemical markers are available to differentiate primary mucinous epithelial ovarian cancer (MOC) from adenocarcinomas originating at other sites that have metastasised to the ovary, which may have an impact on patient management and prognosis.

Aim

To investigate the expression of two intestinal markers, galectin 4 and meprin α, in mucinous carcinomas of the ovary and gastrointestinal tract.

Methods

Using immunohistochemical analysis, the expression of galectin 4 and meprin α was investigated in 10 MOCs and in 38 mucinous adenocarcinomas of colon, pancreas, stomach and appendix, the most common sites of origin of ovarian metastases.

Results

Total cytoplasmic galectin 4 expression was relatively consistent between the different carcinomas. Membranous meprin α expression was significantly lower in MOCs compared with gastrointestinal carcinomas. Moreover, meprin α expression showed greater discrimination between the ovarian and gastrointestinal carcinomas than the cytokeratins CK7 and CK20, the current standard immunohistochemical markers used to determine the tissue origin of mucinous carcinomas involving the ovaries.

Conclusions

Meprin α is a useful additional marker in differentiating primary from secondary mucinous adenocarcinomas of the ovary.

Aberrant Wnt signalling is implicated in numerous human cancers, and understanding the effects of modulation of pathway members may lead to the development of novel therapeutics. Expression of secreted frizzled related protein 4 (SFRP4), an extracellular modulator of the Wnt signalling pathway, is progressively lost in more aggressive ovarian cancer phenotypes. Here we show that recombinant SFRP4 (rSFRP4) treatment of a serous ovarian cancer cell line results in inhibition of β-catenin dependent Wnt signalling as measured by TOP/FOP Wnt reporter assay and decreased transcription of Wnt target genes, Axin2, CyclinD1 and Myc. In addition, rSFRP4 treatment significantly increased the ability of ovarian cancer cells to adhere to collagen and fibronectin, and decreased their ability to migrate across an inflicted wound. We conclude that these changes in cell behaviour may be mediated via mesenchymal to epithelial transition (MET), as rSFRP4 treatment also resulted in increased expression of the epithelial marker E-cadherin, and reduced expression of Vimentin and Twist. Combined, these results indicate that modulation of a single upstream gatekeeper of Wnt signalling can have effects on downstream Wnt signalling and ovarian cancer cell behaviour, as mediated through epithelial to mesenchymal plasticity (EMP). This raises the possibility that SFRP4 may be used both diagnostically and therapeutically in epithelial ovarian cancer.

Background

There are several high throughput approaches to identify methylated genes in cancer. We utilized one such recently developed approach, MIRA (methylated-CpG island recovery assay) combined with CpG island arrays to identify novel genes that are epigenetically inactivated in breast cancer.

Results

Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of COBRA and sequencing of bisulphite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumours; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumours, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal, prostate). We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers.

Conclusion

The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic therapy.