Maintenance of DNA methylation in the promoter region of genes can lead to heritable epigenetic silencing of expression with consequences similar to mutational deletions (10
). Antitumor activity of IFNs, commonly used in the treatment of metastatic RCC and melanoma, depends on induction of gene expression in cancer cells, immune cells, and cells regulating angiogenesis(1
). Hypothesizing that unsatisfactory response rates of melanoma and RCC to IFN (about 15%) are in part due to epigenetic silencing of genes, the role of a tumor suppressor gene that is frequently silenced in both malignancies (15
) was examined in vitro. Treatment of three cell lines (2 renal and one melanoma) that were resistant to apoptosis induction by high doses of IFN-α2 and IFN-β (500 U/ml) with the DNA demethylating nucleoside analogue 5-AZA-dC overcame resistance to IFN-induced apoptosis (fig.1A–C) and reactivated expression of RASSF1A ().
5-AZA-dC is a nucleoside analogue, that after incorporation into DNA, inhibits DNMT1 by covalent binding (10
). DNMT1 is thus trapped and not available at the DNA replication fork to copy methylation patterns from mother to daughter strand resulting in demethylation upon cell division. The covalent binding of the 190 KD DNMT1 protein to DNA, however, also results in DNA damage and thus 5-AZA-dC may have effects in cells, independent of its DNA demethylating activity (29
). Sensitization to IFN-induced apoptosis was observed at 5-AZA-dC doses that did not result in apoptosis alone () associated with reactivation of RASSF1A mRNA expression (). More importantly specific inhibition of DNMT1 by oligonucleotide antisense (DNMT1 AS) similarly reactivated RASSF1A and overcame resistance to apoptosis induction by IFNs (–). Compared to mismatch control oligonucleotide, transfection reagent alone, and media alone, DNMT1 AS did not induce ISGs (, and data not shown) suggesting that its effect on IFN resistance was due to DNMT1 depletion () and associated DNA demethylation (). These results furthermore support that at least in RCC cells, which were more amenable to downregulation of DNMT1 by AS than studied melanoma cells, DNMT1 was critical for silencing of genes.
Containing a diacylglycerol and a rasGTP binding domain but no catalytic activity, RASSF1A has influenced function of binding partners, including the E1A-regulated transcription factor p120 (E4F) (31
), the proapoptotic kinase MST1 (20
), the scaffold protein CNK1 (22
), and cdc20, an activator of anaphase promoting complex (24
). Interaction of RASSF1A with cdc20 regulated mitotic progression (21
) and apoptosis resulted when RASSF1A was coexpressed with the scaffold protein CNK1 (22
). RASSF1A directed MST1 to the cell membrane, where MST1 can be activated influencing apoptosis (20
). Since it was activated by IFNs after RASSF1A re-expression (), MST1 may be contributory to the apoptotic effects of IFNs. Interestingly IFNs also increased protein expression of RASSF1A after DNMT1 depletion and in cells with baseline expression (). The exact mechanism of RASSF1A protein regulation by IFNs will need to be studied in future experiments but real-time RT-PCR results suggest post-transcriptional events (data not shown).
In DNMT1 depleted ACHN cells, selective suppression of RASSF1A by siRNA reduced apoptosis in response to IFN from 63.9 +/− 9.19% to 35 +/− 4.1% (mean +/− SD) (). Rationalizing that cells sensitive to apoptosis induction by IFNs might express RASSF1A, WM9 cells (27
) were studied. Without treatment RASSF1A was expressed, IFN increased expression (, C), and RASSF1A siRNA reduced IFN-β induced apoptosis from 60.45 +/− 3.18% to 39.2 +/− 3.11% accompanied by reduction of RASSF1A protein expression (). Conversely, in ACHN cells lentiviral expression of RASSF1A protein to levels comparable to the one achieved by DNA demethylation overcame resistance to IFN-induced apoptosis ().
Recent evidence has suggested requirement of RASSF1A for death receptor-induced Bax conformational change and apoptosis. RASSF1A enabled Bax apoptotic signaling by relieving an inactivating intramolecular conformation of a necessary partner molecule of Bax, BH3-like protein modulator of apoptosis-1 (MAP-1) (18
). Apo2L/TRAIL was induced by IFN-β (50 U/ml) in ACHN cells, 20–25 fold as determined by real-time RT-PCR (data not shown), and TRAIL neutralizing antibody inhibited IFN-induced apoptosis of RASSF1A expressing cells (). Accordingly RASSF1A markedly sensitized ACHN cells to Apo2L/TRAIL-induced cell death ().
On the other hand normal kidney epithelial (NKE) cells did not undergo programmed cell death in response to IFN or TRAIL despite expression of RASSF1A and no synergism of 5-AZA-dC with either drug was observed (, , ). Real-time RT-PCR before and after 4 days of 5-AZA-dC treatment revealed greater than 400 fold higher TRAIL decoy receptor 1 expression in NKE compared to ACHN cells (). TRAIL decoy receptors bind Apo2L/TRAIL without transmission of apoptotic signals into the cell (33
). Thus RASSF1A overcame resistance to IFN-induced apoptosis at least in part by sensitization to Apo2L/TRAIL and strong TRAIL decoy receptor expression might protect certain non-malignant RASSF1A expressing cells from cell death induction by IFN or Apo2L/TRAIL.
Promoter hypermethylation of interferon stimulated genes (ISGs) including DAPK (mainly lymphoid malignancies), XAF1 (gastric), and IRF7 (fibrosarcoma), as well as of genes essential for IFN apoptotic signaling like TRAIL R1 and caspase 8 (lung), have been identified in cell lines and /or biopsy specimens (6
). While not described as frequently hypermethylated in renal cancer, reactivation of such genes could have contributed to sensitization of ACHN cells to IFN-induced apoptosis after DNMT1 depletion. However, except for one ISG of unknown function, IFI27, no other ISGs or genes known to be essential for IFN apoptotic signaling (25
), were increased in expression by DNMT1 AS treatment in ACHN cells, as determined by U133A Affymetrix cRNA array (data not shown). Although TRAIL R1 and TRAIL R2 were not represented on the array, quantitative RT-PCR did not identify evidence for their reactivation by DNMT1 AS (data not shown). However among the 137 genes increased at least twofold in DNMT1 AS over MM treated cells, 8 had roles in apoptosis and also may have contributed to overcoming resistance to IFN-induced programmed cell death by influencing apoptotic pathways (small GTPase ARHGDIB), inhibition of NF-κB (NFKBIA, IER3, C8FW), or other mechanisms (PHLDA1, NAC, STK17A, ASC) (data not shown).
IFN-α2 has increased survival of patients with metastatic RCC in randomized trials, albeit only for several weeks to months (1
). Prolongation of disease-free and possibly overall survival has resulted when IFN-α2 has been administered to melanoma patients for high risk primary disease (1
). Resistance of RCC and melanoma cells to the apoptosis-inducing effects of IFN-α2 and IFN-β was overcome by inhibition of DNA methyltransferase 1 (DNMT1) (, ). This was at least in part due to reactivation of the tumor suppressor gene RASSF1A (, ), that is frequently silenced by DNA methylation in RCC and melanoma (15
), and as shown herein up-regulated in expression by IFNs (). By targeting DNMT1 in RCC and melanoma, clinical antitumor effects of IFNs may be augmented through reactivation of silenced RASSF1A and possibly by reactivation of other heterogeneously silenced genes in IFN pathways.