Many oncoproteins and growth factors utilize D1 as a common downstream effector. In other words, D1 sensitizes the cells to various extracellular mitogenic stimuli, such as estrogen and estrogen receptors in the case of breast cancer cells. Much of the D1's role in cell cycle progression is exerted via binding to CDK4 or CDK6 to form a holoenzyme, which phosphorylates the pRb protein to drive cell proliferation. Therefore, inhibition of CDK4 or CDK6 is presumably a good strategy for cancer therapy because cancers are characterized by uncontrolled proliferation and frequently by increased D1-CDK4 activities 58,59
. We use NPCD, a novel indolocarbazole-derived inhibitor of CDK4 and CDK6, to test this therapeutic principle and explore the pathways downstream of the CDK4 inhibition. This test is needed because PD0332991 is so far the only CDK4/6 inhibitor that has entered into clinical trials for its chemotherapeutic potential. Our results show that NPCD, which has not been studied for its effect in cells, can indeed arrest breast cancer cell lines at G1 phase and cause cell death with IC50 around 3-8 µM when it is administered as a single, one-time, dose. Although a stronger potency is conceivable if multiple administrations were given to maintain the NPCD level otherwise metabolically decreased, the IC50 at 3-8 µM for cellular effects is still much higher than the reported IC50 for the in vitro
inhibition of the CDK4 (45-75 nM) or CDK2 (270 nM) enzymatic activity 30,31,35
. There are several possible reasons for this discrepancy: 1) Live cells may continue producing CDK proteins once the existing CDKs are inactivated by NPCD, due to a conceivable negative feedback mechanism. 2) Live cells have other NPCD targets that also consume NPCD. At least, CDK4, CDK6 and CDK2 will simultaneously consume NPCD, thus raising the IC50 for the cellular effects. 3) A certain portion of NPCD may not be internalized into the cells while some other NPCD molecules may be metabolized. 4) The IC50 for growth inhibition and cell death should be much higher than the IC50 for enzymatic activity of CDK proteins, because the remaining activities of CDK4, CDK6 and CDK2 may be sufficient to maintain cell proliferation or viability. Besides these explanations, however, the much higher IC50 may reflect that NPCD has other cellular targets that are accountable for the observed growth inhibition and cell death, since many chemical drugs have diverse targets and effects. Probably, p27kip1 and p21cip1 are among these non-CDK targets, as discussed below. These issues deserve further explorations.
A wonderful surprise given by our results is the long-lasting growth inhibition of breast cancer cells, especially MCF7 cells, because clonogenic survival assay shows that while there are many cells that survived the NPCD treatment they cannot propagate as fast as non-treated controls to form sizable colonies even 10-15 days after the cessation of the treatment. Another reported long-lasting CDK inhibitor is R-roscovitine (CYC202), which has a relatively specific inhibitory activity towards cyclin E-CDK2 and shows a long-lasting arrest of polycystic kidney disease in animals 60
. Whether such long-lasting growth inhibition is a trait of G1 phase CDK inhibitors and what is the underlying mechanism remain to be further studied.
A basic finding of the present study is that NPCD has relatively similar efficacy on five human breast cancer cell lines, although these cells have different properties of tumor biology pertaining to such as p53, estrogen receptor, metastatic statuses, etc. Actually, even non-malignant MCF10A cells show only a small difference in the sensitivity. Use of non-transformed epithelial cell lines such as MCF10A as “normal control” in the studies of chemoresponse has disadvantages. One that is often neglected is that these cell lines require different culture conditions, such as glucocorticoid and other supplements (hEGF, insulin, cholera toxin, etc), for an optimal growth. When these cells are cultured in a routine condition used for most epithelial-derived cancer cell lines, they are more fragile than cancer cells and thus easier to die. Actually, MCF10A is not much less sensitive to some other chemotherapeutical agents either (unpublished data). If all cells are cultured in a special medium for MCF10A, the data may not be used broadly since most cancer cells are not routinely cultured with many supplements and glucocorticoid that may influence the effects of the agent to be tested. For this reason, we culture all cells in routine DMEM medium and serum without additional supplements that may affect data interpretation, although it is not optimal for MCF10A.
The effect of NPCD is associated with decreased phosphorylation of the Rb protein at Ser780, Ser807 and ser811 as expected. Of the cell lines studied, MB231 is so-called triple-negative, i.e. absence of estrogen receptor, progesterone receptor and Her-2 receptor expression. Triple-negativity is a special category of breast cancer that is notorious for its refractoriness to various chemotherapies and for its worse prognosis 61
, but MB231 cells are still relatively sensitive to NPCD. However, MB231 cells express Rb protein whereas many other triple-negative cells are basal-cell like and lack Rb expression 62
. Several studies have shown that Rb-proficient cells of breast cancer 47
and other cancer types 63,64
are more sensitive to PD0332991 than the Rb-deficient counterparts. Probably, Rb-deficient breast cancer cells are relatively resistant to NPCD as well, which deserves exploration later. However, in the Rb-proficient cell lines, the protein level of Rb usually varies little and is thus used by some investigators as loading control in western blot assay 54,55
. As expected, we did not detect a significant difference in the Rb level among the cell lines studied, nor a correlation between the sensitivity to NPCD and the Rb protein level. We intend to consider that a complete lack of Rb may affect the response of cells to CDK4 inhibitors but in the Rb-proficient cells its protein level may not significantly influence the response.
Finn et al reported recently that PD0332991 is more effective on high D1 breast cancer cells 47
, and Dai et al also report that D1 overexpression increases the susceptibility of human U266 myeloma cells to CDK inhibitors 65
. We found that the two relatively less sensitive cell lines, T47D and GI101Ap, express a relatively lower level of D1 and/or CDK4 compared with the other three more-sensitive cell lines, which is in line with the observations of Finn et al 47
and Dai et al 65
and dovetails with the assumption that NPCD has its effect by targeting the D1-CDK4 holoenzyme. Considering that D1 may impose resistance to other chemotherapeutic agents onto pancreatic cancer and probably also onto other cancers, as reviewed by us 1
, CDK4 inhibitors may be prescribed especially to those cancers that show high D1-CDK4 activities or D1-CDK4 imposed chemotherapeutic resistance.
There are very few studies on how CDK4/6 inhibitory compounds affect the expression of D1, CDK4 and other G1 regulatory genes, and PD0332991 does not seem to have a consistent effect on D1 protein level 66
. We therefore put specific efforts on this issue. MCF7 and MCF15 seem to represent a group of breast cancer cells that show decreases in both D1 and CDK4 upon treatment with NPCD, whereas MB231 may represent another type of breast cancer cells that shows induction of D1 with unchanged CDK4 levels. CDK6 is expressed at relatively low level in these breast cancer cells and does not show a consistent pattern of changes in response to NPCD treatment. Although D1 is considered an oncogene that drives cell proliferation, it can also cause apoptosis in certain undefined situations 1
. Questions that remain to be answered include how D1 chooses to drive proliferation or cell death and whether this decision is made under influence of CDK4/6 status. Moreover, it is also unclear whether CDK4 alone has dual-functions, i.e. can cause apoptosis as well. We infer that D1, by binding to CDK4/6, may protect breast cancer cells from drug-induced cell death 1
. However, once the D1-CDK4 activity is inhibited by NPCD and conceivably also PD0332991 or other CDK4 inhibitory compounds, the D1 and CDK4 proteins become “useless” and are thus degraded via unknown mechanisms. Without D1-CDK4 sustaining their survival, the cells thus undergo growth arrest and demise. This may be the situation occurring in the cell type represented by MCF7 and MCF15 cells. In the cell type presented by MB231, in which NPCD cannot decrease or even induces D1 and CDK4, the abundant, but kinase-dead, D1-CDK4 holoenzyme may directly promote cell death since it is no longer able to phosphorylate the Rb to drive cell cycle progression. This intriguing hypothesis needs to be tested later.
Phosphorylation of D1 protein at Thr286 by GSK-3β is supposed to trigger proteasomal degradation of D1 2
. The abundance of Thr286-phosphorylated D1 is unexpectedly decreased by NPCD in both the cytoplasmic and nuclear fractions of MCF7 cells, which opposes our original expectation that the Thr286 phosphorylation should be increased, not decreased, in MCF7 cells as a mechanism underlying the decreased level of D1 protein 72 hours post NPCD treatment as seen in figure . Therefore, phosphorylation at Thr286 is insufficient to degrade D1 protein in MCF7 and probably also MCF15 cells. Aspirin has been shown to rapidly degrade D1 protein by activation of p38 mitogen-activated protein kinase (MAPK), resulting in apoptosis of the cells 50
. There are some other compounds with therapeutic potential that can also cause D1 protein degradation, such as STG28 67
, silibinin 68
, fucoxanthin 69
and metformin 70,71
. Of these compounds, STG28 is known to degrade D1 protein via a novel mechanism independent of Thr286 phosphorylation 67
, whereas it is unclear whether degradation of D1 by the other compounds requires phosphorylation at Thr286. Therefore, the unexpected finding that a pronounced decrease in Thr286-phosphorylation is not companied with an increase in D1 protein level in MCF7 cells is of interest and deserves further studies for the underlying mechanism.
Cyclin E and CDK2 protein levels also show obvious changes in response to NPCD treatment in some cell lines. However, in most occasions the changes show a reciprocal pattern, i.e. a decrease in cyclin E is companied by an increase in CDK2 and vice versa. It is possible that an NPCD-induced decrease in cyclin E or CDK2 may trigger a negative feedback mechanism to increase the level of its partner. It is conceivable that such a reciprocal change may help stabilizing the cyclin E-CDK2 activity. Therefore, the cyclin E-CDK2 holoenzyme may not be a primary target of NPCD for its chemotherapeutic effects.
Another unexpected finding in our study is the decrease in the p27kip1 protein in all three NPCD-treated cell lines, i.e. MCF7, MB231 and MCF15 cells. P21cip1 protein level is also decreased in MCF7 and MCF15 cells, although not in MB231 cells. These results are unexpected because most chemotherapeutic agents induce, not decrease, these CDK inhibitory proteins. For instance, anticancer activities of R-roscovitine (a CDK2 inhibitor) 72
, inositol hexphosphate 73
, Silibinin 68
and ZD1839 74
are associated with increased p27kip1 and/or p21cip1. On the other hand, lost expression of these CDK inhibitory genes, especially the p27kip1, is suggested as a common reason for the resistance of cancer cells to certain chemotherapeutic agents 75,76
. Therefore, NPCD may represent a new category of chemotherapeutic compounds that compensate for the weakness of those agents that require p27cip1 or p21cip1 to elicit their therapeutic effects. However, whether and how decreases of these CDK inhibitory proteins contribute to NPCD-induced cell death of cancer cells are unknown. Probably p27kip1 and p21cip1 are also cellular targets of NPCD, besides G1 phase CDKs, as a reason for a much higher IC50 for the cellular effects as discussed above. Increased abundance of the D1-CDK4 complex is known to trap more p27kip1 and/or p21cip1 in the complex, indirectly preventing cyclin E-CDK2 holoenzyme from inhibition by these CDK inhibitors. As the result, cells are enhanced to complete the G1-S phase transit and accelerate the S phase. Our results raise an intriguing question as to whether decreases in p27kip1 and/or p21cip1 per se
can cause apoptosis when D1-CDK4 holoenzyme is either decreased or inactivated. This question is valid for at least two reasons: 1) p27kip1 or p21cip1 is actually required for assembling the cyclin-CDK complex and bringing the complex into the nucleus because neither D1 nor CDK4 has a canonical nuclear localization sequence whereas p27kip1 or p21cip1 has 77,78
. 2) These proteins can also function as oncogenes to promote tumor formation or to render cancer cells resistant to anticancer therapies under certain situations 79-82
. Whether PD0332991 and other CDK4/6 inhibitory compounds also decrease the protein levels of p21cip1 and p27kip1 is another interesting question waiting for an answer.
The mRNA levels of p27kip1, p21cip1, D1, CDK4, CDK2 and cyclin E are induced by NPCD at concentrations around the IC50 in different breast cancer cell lines, which is a surprise to us because we expected that NPCD might also have certain inhibitory effects on CDK7 and CDK9, just like many other CDK inhibitory compounds, although this has not really been studied. CDK7 and CDK9 are required for transcriptional elongation of mRNAs, and thus their inhibition results in decrease in the mRNA levels of most, if not all, genes 83
. Therefore, the increases in the mRNA levels of many genes observed herein indirectly suggest that NPCD may not be very potent in inhibition of CDK7 and CDK9. However, a weaker inhibition may still occur, at least in certain cells, since NPCD at 8 µM seems to decrease the levels of all the genes in MCF15 cells (fig ). Further study on the enzymatic activity is required to clarify this issue.
In summary, we show for the first time that the CDK4 inhibitory compound NPCD can cause long-lasting growth arrest and cell death of breast cancer cell lines at an IC50 of 3-8 µM. An expected decrease in the Rb phosphorylation by D1-CDK4/6 and an unexpected decrease in p27kip1 protein level may be part of the underlying mechanism, although the D1 and CDK4 protein levels show different changes among different cell lines treated with NPCD.