In this study, we found that the strong FLT1 suppression observed in choriocarcinoma cells is caused by DNA hypermethylation of its 5’-promoter region, and showed that DNA demethylation by 5azadC rescued sFLT1 production. Furthermore, we demonstrated that the stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo. These results suggest that FLT1 may be a tumor suppressor gene in choriocarcinoma.
Promoter hypermethylation is known to play a key role in the epigenetic silencing of tumor suppressor genes in the development and progression of cancers . In this study, we showed that the FLT1 promoter was hypermethylated in all three choriocarcinoma cell lines. The methylation status of FLT1 has also been reported in other cancer cell lines and tissue samples besides choriocarcinoma. For example, Yamada et al. reported that the promoter and exon 1 of FLT1 were aberrantly methylated in prostate cancer cell lines and tumor tissue samples, whereas benign prostate tissue samples were hypomethylated . Furthermore, Kim et al. reported that the FLT1 promoter showed variable hypermethylation in different cancer cell lines, including colon, stomach, lung, melanocyte, breast, thyroid, and kidney [33, 34]. Among those, the colon, stomach and renal cancer cell lines had more frequent FLT1 hypermethylation than the other cancer cell lines. Methylation of the FLT1 promoter was also found to be significantly higher in the tumor tissues of stomach cancer, colon cancer, hepatocellular carcinoma, and renal cancer, in comparison with normal tissues. Therefore, the methylation status of FLT1 promoter may be useful as a new potential biomarker for cancer diagnosis.
It is widely accepted that HIF-α is stabilized at protein level, then translocates to the nucleus where it forms a heterodimer with HIF-1β under hypoxic or hypoxia-mimicking conditions, inducing the expression of hypoxia-associated genes. During this process, the HIF-α/HIF-1β heterodimer binds the consensus HRE sequence 5’-(A/G)CGTG-3’ of the target genes . We recently discovered that HIF-2α, not HIF-1α, mediates the hypoxia-induced up-regulation of FLT1 expression in choriocarcinoma cell lines (BeWo, JAR, and JEG-3) and primary trophoblasts . The HRE motif contains one CpG dinucleotide, and it has been confirmed that CpG methylation within this sequence prevents the binding of HIF-1α or HIF-2α and subsequently prevents the HIF-mediated transcription of HRE-containing genes . Our results suggest that HRE motifs in FLT1 were methylated, suppressing the binding of HIF-α in all three choriocarcinoma cell lines, as sFLT1 production was enhanced by hypoxic stimulation after 5azadC treatment. A putative HRE motif was reported to exist approximately 1,000 bp upstream of the transcription start site in mouse and human FLT1 genes . The next step is to investigate whether HIF-2α binds to the HRE site when exposed under hypoxic conditions and to assess the CpG methylation status of the HRE site before and after 5azadC treatment.
Previously we reported that both the CRE (cyclic adenosine monophosphate response element) and ETS (E26 transformation-specific) motifs located in the region 90 bp upstream of the transcription start site are important for FLT1 promoter activity in human embryonic kidney 293E1 cells . However, the transcription factors binding with each motif have not been fully determined. The CRE motif 5’-TGACGTCA-3’ also contains one CpG dinucleotide, and CpG methylation of the CRE motif has been reported to inhibit the binding of specific transcription factors and transcriptional activation . In the BeWo, JAR, and JEG-3 cells, DNA methylation was also observed at a CpG dinucleotide located in the CRE motif (position –81 of the FLT1 promoter), and its methylation level was decreased in all these cells compared to before 5azadC treatment (Fig. 4B). By contrast, the CpG dinucleotide in the CRE motif was unmethylated in the human primary choriocarcinoma tissue specimen (Fig. 4B). Further studies are required to confirm whether the CRE motif at this site is important for FLT1 promoter activation in human choriocarcinoma cells.
The first-line treatments for choriocarcinoma are well-established as being either single agent chemotherapy (methotrexate or actinomycin D) or multiagent chemotherapy using etoposide, methotrexate, actinomycin D, cyclophosphamide, and vincristine . However, the systemic administration of anticancer drugs results in nonselective drug distribution and severe toxicity. In this study, stable expression of sFLT1 in choriocarcinoma cells resulted in strong suppression of tumor growth and vascularity in vivo. Therefore, the over-expression of sFLT1 using a DNA demethylating agent could be a novel therapeutic alternative to anticancer drugs in choriocarcinoma. In this study the 5azadC, also known as decitabine, was used as a potent DNA methylation inhibitor . This agent is also used as a drug for the treatment of myelogenous leukemia and solid tumors in lung cancer, esophageal cancer, and pleural mesothelioma [40, 41], however its efficacy is limited by the 35 minutes half-life in plasma of humans . It has been reported the drug delivery systems using nanogels or nanoparticles can overcome this limitation and enhance chemotherapeutic efficacy [42, 43]. Recently, Zhang et al. developed nanoparticles for targeted choriocarcinoma delivery using a synthetic chondroitin sulfate A-binding peptide derived from the VAR2CSA malaria protein, demonstrating the inhibition of tumor growth and metastasis by the nanoparticles when loaded with doxorubicin in JEG-3 choriocarcinoma-bearing mice . Thus, by combining DNA demethylating agents and nanoparticle delivery systems, a novel choriocarcinoma therapy could be offered in the future.