There are two complementary aspects of this report; one translational, the other mechanistic. On the practical side, we asked if combination of low concentrations of 1,25D with COX inhibitors is likely to be useful in the prevention or treatment of human myeloid leukemia, and if so, would selective COX-1 or COX-2 inhibitors be more effective than the non-selective inhibitors previously reported to synergize with 1,25D in HL60 cells.11,30
We found that leukemia cell lines other than HL60 also exhibit enhanced differentiation, and arrest in G1
phase of the cell cycle, when exposed to combinations of 1,25D and non-specific COX inhibitors. Of note, ex vivo leukemic cell samples freshly obtained from two patients, also respond to these combinations, particularly ASA/1,25D (, and data not shown). While a more extensive series of patients needs to be accrued, this finding points to a possible success in future translational studies. The greater synergy obtained with the nonspecific inhibitor ASA than with other compounds tested suggests that both the constitutive COX-1 and COX-2 enzymes need to be inhibited to enhance the differentiating action of 1,25D, although a minor effect also was seen with the specific COX-2 inhibitor DuP-697 (). Specific COX-2 inhibitors have recently been reported to have serious side effects when administered to patients, especially on the cardiovascular system, such as myocardial infarctions, strokes, and hypertension.34,35
Thus, even though ASA at high dosage can cause gastrointestinal ulcers and renal toxicity,36
ASA would appear to be a COX inhibitor of choice to combine with 1,25D for prevention of myeloid leukemia in high risk populations, as it is both effective in the present studies, and safe in moderate doses.
The mechanistic rationale for combining a COX inhibitor with 1,25D in cancer therapy may be provided by the reported inhibition of the prostaglandin pathway by 1,25D37,38
and its involvement in 1,25D signaling,39
so that COX inhibitors can enhance this effect by blocking COX enzyme activity. However, how 1,25D can initiate signals that impinge on the transcriptional machinery involved in the regulation of the prostaglandin pathway is not known. We, and others, have previously provided extensive evidence that MAPK pathways are involved in the control of transcription factors, such as C/EBPs, which upregulate programs required for monocytic differentiation,40
and that Raf1 is a critical nodal control point for 1,25D-induced differentiation of human leukemia cells, consistent with its activation under all conditions tested to date that lead to 1,25D-induced differentiation.16,17,41,42
We have also shown that the MEK/ERK module, classically downstream from Raf1, is modulated in a biphasic manner following exposure of the cells to 1,25D.31
It was proposed that the initial exposure to 1,25D directly upregulates the expression of a scaffold protein KSR1 through a VDRE element in its promoter,41
and this increases the efficiency of growth factor and cytokine signals generated at the cell surface. Thus, KSR1, and perhaps also the more recently discovered hKSR2,43
provide a platform that binds Raf1, MEK1 and ERK1/2, and facilitates the phosphorylation of MEK1 by Raf1.44,45
However, it is also known that an excess of a scaffold protein actually inhibits the reactions that it facilitates when at lower concentrations, as Raf1, MEK1 and ERK may bind to different KSR1 molecules when these are numerous, thus impeding phosphorylation of ERK1/2.46
In the 1,25D differentiation system this then leads to reduced P-ERK 1/2 levels, consistent with the results obtained here (), and offering an explanation for the G1
block observed when the cells are exposed to ASA/1,25D combinations (). Thus, it appears that COX inhibitors act on the MAPK pathway upstream of Raf1, perhaps by inhibition of EGFR signaling by growth factors, as shown for an ASA derivative,47
while downstream of Raf1 the signals are similar, though more intense, to the signals provided by 1,25D when it is administered as a single agent. Future studies of the anti-proliferative and differentiation-inducing effects of COX inhibitor/1,25D combinations on leukemic cells should therefore be principally directed to the upstream molecular events that regulate the expression and activation of Raf1.
In summary, this report adds to the growing evidence that combinations of 1,25D with COX inhibitors can retard emergence and progression of malignant clones in various organs, such as the prostate38
The studies presented have also suggested that the paradigm of the Raf-MEK-ERK-RSK cascade in mammalian cells, which was principally based on work with fibroblasts (reviewed in refs. 49
), may require revision when other cell types are considered.