Treatment strategies for advanced-stage or recurrent ACC predominantly rely on adjuvant chemotherapeutic regimens that include mitotane. Mitotane remains the most significant single agent shown to affect the disease course of patients with metastatic ACC, with useful clinical remissions of 10 months in up to 30% of patients.6
Perhaps most important, mitotane leads to significantly diminished hormonal hypersecretion in 80% of patients with functional tumors, thereby improving the quality of life for affected patients.5
Furthermore, these effects seem optimal at lower-dosing regimens (≤3 g/d of mitotane), which have the dual benefit of achieving therapeutic serum concentrations (10–14 mg/L) and minimizing adverse reactions.20
However, 70% of patients with ACC do not respond to mitotane treatment, and many patients develop debilitating neurologic and gastrointestinal toxic effects, which attest to the limitations of mitotane therapy in controlling the spread of this aggressive cancer. The use of mitotane in combination with other agents, most notably cisplatin, has only slightly improved outcomes.7
Recently, mounting evidence about the role of epigenetic mechanisms in carcinogenesis has prompted interest in whether agents that reverse these processes can be effective anticancer treatments. Epigenetics refers to reversible changes in gene expression that do not fundamentally mutate the genomic DNA sequence. Hypermethylation of DNA promoter sequences is a classic example of these changes, which usually cause silencing of genes downstream of the affected region.13
Theoretically, the reversal of these changes could lead to reexpression of silenced tumor suppressor genes and inhibit cancer cell progression. Indeed, the relevance of activated DNA methyltransferase to adrenocortical carcinogenesis was first established in mouse models more than a decade ago.21
Decitabine is an inhibitor of DNA methyltransferase, which effectively removes methyl groups from silenced promoter sequences. Decitabine has a dual dose dependent mechanism of action. Lower doses (5μM to 10μM) of decitabine inhibit methylation and reactivate gene expression, whereas higher doses (10μM to 100μM) induce cytotoxic effects via covalent trapping of DNA methyltransferase into DNA.16
Even at low doses, decitabine has been shown to inhibit in vitro growth in several human cancers, including colorectal carcinoma, melanoma, renal cell carcinoma, lung carcinoma, and hepatocellular carcinoma.22–25
Its benefits have been most apparent in hematologic malignant neoplasms, especially myelodysplastic syndrome, for which decitabine has recently been approved by the Food and Drug Administration as first-line therapy.17
We hypothesized that decitabine treatment of NCI H295R cells would have a significant antineoplastic effect. To this aim, we examined the effects of decitabine on ACC cell proliferation, cortisol secretion, and cell invasion, which are 3 hallmarks causing clinical morbidity and mortality in this disease. Using 2 lower doses (0.1μM and 1.0μM), we examined the effects of decitabine on ACC cells at daily intervals for up to 5 days. Decitabine cytostatically inhibited ACC cell proliferation in a time dependent and dose-dependent manner. Cortisol secretion was also attenuated at 5 days after treatment, although only the lower 0.1μM dose seemed to act via a mechanism independent of its growth inhibitory effects. As opposed to the long incubation time that is necessary to observe differences in cell count (probably because of a long doubling time for NCI-H295R cells), the effects of decitabine on invasive behavior were seen after only 24 hours. These findings support the idea that the functional effects of decitabine can be immediate, regardless of the basal growth rate of the target cell.
Our findings about ACC cell proliferation are in agreement with the results of other studies.18,19,26
To our knowledge, we are the first group to demonstrate inhibition of ACC cell invasion by decitabine. Our modified Boyden chamber technique used a commercially available kit with a 2-dimensional barrier composed of reconstituted basement membrane and ECM components. Despite its widespread acceptance, this method for determining cell invasion has well-known limitations. First, despite improved standardization of preparation techniques by manufacturers, reconstituted matrices may contain “contaminants” (such as metalloproteinases) that could affect experimental results. Second, individual tumor cells display significant variability in their ability to adhere to and migrate through ECM, which may not be taken into account by quantifying cells in aggregate. Third, with its 2-dimensional design and components within reconstituted matrices, the Boyden chamber model does not necessarily recapitulate the tumor microenvironment. Newer techniques offer 3-dimensional models or tissue-based cell invasion barriers, which may address these limitations but need to be validated in future experiments.27
At first glance, our findings about cortisol secretion seem to contradict those in a prior study by Liu et al,18
who found that decitabine increased cortisol secretion in NCIH295R cells, possibly via selective regulation of steroidogenic gene expression. However, 2 primary differences between our studies may explain these divergent results. First, Liu et al used a higher dose of decitabine (10μM). The differing mechanism of action at higher doses (as already described) could at least partially account for the discrepancies in our results. Indeed, our results show that only the lower 0.1μM dose led to decreased cortisol secretion independent of decreased cell count. Furthermore, our studies seem to agree that the relative trend of cortisol secretion decreases in proportion to decitabine dose. We speculate that the lower dose of decitabine may affect cortisol expression and secretion, while the higher dose of decitabine may have only a cytotoxic effect, reducing cell count and cortisol secretion.
Second, our preparation of decitabine (unlike that by Liu et al) involved the use of DMSO vehicle to prevent rapid degradation of the unstable molecule. In theory, the benefit of our approach was longer duration of decitabine action, which may be more clinically relevant to longer-course treatment protocols used in patients. The disadvantage was the incorporation of another drug into our experiments and the associated possibility of a confounding effect, despite our use of DMSO concentrations well below thresholds that are known to have functional effects in other in vitro models.28–30
We believe that we effectively controlled for this factor by standardizing decitabine to proper DMSO vehicle control specimens in our experiments.
In addition to demonstrating the functional effects of low-dose decitabine on NCI-H295R cells, we also sought to evaluate its effects on several genes at 11q13. This chromosomal region seems relevant in adrenocortical carcinogenesis, with studies10,11
showing that loss of heterozygosity at 11q13 is found in 70% to 100% of sporadic ACCs. A previous microarray-based study12
identified 6 genes on 11q13 that were underexpressed in ACC and demonstrated high diagnostic accuracy for distinguishing benign from malignant tumors. We tested the effects of decitabine on these genes using quantitative RTPCR, with the hypothesis that decitabine could recover gene expression if methylation had a role in silencing any of these genes. For these experiments, we used decitabine at 1.0μM because of the marked effects on ACC cell proliferation and cell invasion that we observed at this dose, along with the indirect inhibitory effects on cortisol secretion. Of 6 genes, NDUFS8
showed significantly recovered expression after decitabine treatment, suggesting that hypermethylation may have a gene silencing role in ACC. However, future studies using methylation-specific techniques are needed to definitively establish methylation patterns of these genes in tumor samples.
The clinical relevance of NDUFS8
remains unknown. NDUFS8
encodes a subunit protein of a critical enzyme in the mitochondrial respiratory chain, and mutations of this gene are associated with Leigh disease.31,32
encodes the antioxidant enzyme peroxiredoxin, which has genome-protective properties in response to oxidative stressors.33
Underexpression of neither NDUFS8
has been shown in other cancer models,34–38
suggesting that dysregulation of these genes may be specific to adrenocortical carcinogenesis. However, further study is needed to determine the functional effects of modifying expression of these genes individually in the absence of other confounding factors. Moreover, the mechanisms behind the seemingly paradoxical downregulation of gene expression after decitabine treatment (such as that of DDB1
in our experiments), as well as the ways in which restoration of other genes interacts with these inhibitory effects, are unknown and require further investigation.
A cautionary note must be given about attempts to mold the results of our in vitro experiments to clinical relevance. The effects of decitabine on ACC cells are undoubtedly different depending on whether the drug is administered via culture medium or via in vivo intravenous or subcutaneous routes. Decitabine’s success in inhibiting hematogenous cancers both in patients as well as in cell cultures may in part be due to similar immersive drug exposures in the bloodstream and in culture medium. This similarity may not necessarily translate as well for solid organ tumors. Indeed, early evidence in the 1980s showed a disappointing lack of demethylating agent activity on solid organ cancers.16
Nevertheless, we believe that decitabine holds promise as a therapy for patients with ACC for several reasons. First, the earlier findings were limited by adverse effects secondary to higher drug doses and by limited treatment durations.16
More recent trials13,17
in other cancers reported superior results using low-dose and longer duration drug regimens. Second, studies18,19,21,26,39,40
using in vitro and in vivo models demonstrated the relevance of DNA promoter methylation in adrenocortical carcinogenesis. Third, decitabine has already been approved for use in humans, which should theoretically streamline its path to clinical trials.
In conclusion, low-dose decitabine exhibits significant antineoplastic effects in human ACC cells, possibly by recovering expression ofNDUFS8 and PRDX5. Considering that decitabine is already approved by the Food and Drug Administration for hematologic malignant neoplasms and because ACC is an orphan disease for which there is no effective chemotherapy to date for locally advanced and metastatic ACC, future studies evaluating the clinical efficacy of decitabine should be considered based on our results.