The molecular mechanisms underlying erlotinib resistance in breast cancer have not been well defined. In our screening of breast cancer cell lines, down-regulation of CDK2 after treatment with erlotinib showed association with erlotinib sensitivity. Moreover, our study provides that erlotinib sensitivity is causally linked with CDK2 activity, indicating that erlotinib sensitivity depends, at least in part, on CDK2 activity. Some reports have indicated that the growth-inhibitory effect induced by EGFR-TKIs in sensitive cell lines depends mainly on G1
cell cycle arrest (1
); others have shown CDK2 activity to be down-regulated after treatment with an EGFR-TKI (12
). However, this is the first report establishing cause and effect relationship between erlotinib sensitivity and CDK2 activity.
The erlotinib sensitivity of breast cancer cell lines has clinical relevance for several reasons. The limited clinical activity of EGFR-TKIs in breast cancer was also echoed by the findings of this in vitro
study. Because EGFR-TKIs were designed to block EGFR-TK phosphorylation, EGFR expression was previously thought to be a molecular marker of the effectiveness of EGFR-TKIs. However, in vitro
and in vivo
preclinical studies (4
) as well as phase I and II clinical studies (6
) suggest that the efficacy of EGFR-TKIs is not directly related to EGFR expression levels. Our own findings indicated a lack of association between the expression of EGFR and p-EGFR and sensitivity to erlotinib, although we did find that erlotinib blocked EGFR phosphorylation even in erlotinib-resistant cell lines.
Other evidence suggests that HER2 overexpression indicates sensitivity to EGFR-TKIs (5
). Although one recent article reported that erlotinib directly blocked HER2 kinase and downstream signaling events in cells that did not express EGFR (27
), our Western blotting results indicated that HER2 expression level (high in SK-BR-3, BT-474, MDA-MB-453, and MDA-MB-361 cells) was not associated with erlotinib sensitivity. Further, HER2 expression level in SK-BR-3 and BT-474 cells, noted to be sensitive to erlotinib, did not change after erlotinib treatment (data not shown).
Some investigators have investigated the downstream signaling pathways of EGFR and have suggested that down-regulated activity of ERK1/ERK2, mitogen-activated protein kinase, and phosphatidylinositol 3′-kinase/Akt after treatment with EGFR-TKIs might serve as a marker of drug response (6
). However, firm conclusions about which of these two pathways (if either) is relevant for measuring the efficacy of EGFR-TKIs have not been drawn. Some studies have found down-regulation of the ERK1/ERK2 mitogen-activated protein kinase signal in both EGFR-TKI-sensitive and EGFR-TKI-resistant cancer cell lines, suggesting that ERK1/ERK2 mitogen-activated protein kinase activation is not a reliable marker of EGFR-TKI -induced inhibition of proliferation (5
), and some showed that Akt activity could not predict erlotinib sensitivity (5
). In our study, no association was found between erlotinib sensitivity and the expression of Akt, p-Akt, ERK1/ERK2, or p-ERK1/ERK2, as detected by Western blotting, after a 72-h exposure to erlotinib. Although molecular defects in the EGFR signaling molecules of the Akt and ERK1/ERK2 mitogen-activated protein kinase pathway cannot be excluded, more complex mechanisms probably underlie the effect of EGFR-TKIs (5
The CDK inhibitor p27 is a potent negative regulator of the cell cycle involved in both EGFR and non-EGFR signaling pathways, and it is directly upstream of CDK2. p27 inactivation, by down-regulation of its expression or its exclusion from the nucleus, has been implicated in human carcinogenesis (30
). Cytoplasmic sequestration of p27 has been reported in breast carcinomas (32
), and loss of p27 function has been proposed as a marker of malignancy (30
). Previous reports have suggested that p27 could be a predictive marker of response to EGFR-TKIs (10
). In our study, the erlotinib-sensitive cell lines showed translocation of p27 from the cytoplasm to the nucleus, but the erlotinib-resistant cell lines did not, despite increased p27 expression levels. Our finding that knocking down p27 protein only partially blocked the effects of erlotinib may reflect insufficient transfection efficiency or the participation of other molecules in the erlotinib effect. However, our results do indicate that p27 is an important factor in how erlotinib affects cells, although erlotinib-induced up-regulation of p27 by itself is insufficient to block CDK2 activity.
This is also the first report that p27 cytoplasmic localization is related to resistance to EGFR-TKIs. In other words, “mislocalization” of p27, and the subsequent continuous activation of CDK2, may be an important mechanism of resistance to EGFR-TKIs. Cells in which the Akt pathway was activated subsequent to PTEN deficiency were recently reported to be naturally resistant to the effect of EGFR-TKIs (35
). In addition, several mechanisms of p27 mislocalization have been proposed, some of which involve its phosphorylation at various sites. For example, phosphorylation of serine 10 by the hKIS kinase promotes the export of p27 from the nucleus (38
), whereas phosphorylation of threonine 157 by Akt impairs the import of p27 into the nucleus (32
). However, future studies will be necessary to identify the phosphorylation site of p27 that is related to its translocation after erlotinib treatment. We can also speculate that phosphorylation status of p27 may have affected the overall efficacy of erlotinib even among the erlotinib-sensitive cell lines.
We found that blocking CDK2 activity with DN-CDK2 led to increased sensitivity to erlotinib in the EGFR-overexpressing cell line MDA-MB-468. This finding supports the concept that CDK2 is a target of erlotinib, at least in some cell lines. However, DN-CDK2 had no effect on other three erlotinib-resistant cell lines (MDA-MB-361, MDA-MB-453, and MCF-7; Supplementary Fig. S5D
EGFR expression has been reported to be necessary, if not sufficient, for cell lines to be sensitive to EGFR-TKIs (11
). The mechanisms by which DN-CDK2 affected MDA-MB-468 cells but not other cell lines are unclear; however, one possible explanation may be that MDA-MB-468 overexpressed EGFR but MDA-MB-361, MDA-MB-453, and MCF-7 cells did not (as shown by our Western blotting results). Another possibility is that CDK2 down-regulation requires other molecular effects such as p27 up-regulation, p-ERK1/ERK2 down-regulation, as seen in MDA-MB-468 cells, to increase the sensitivity of erlotinib-resistant cell lines. Alternatively, EGFR signaling pathways may be more intact in MDA-MB-468 cells than in other cell lines, as suggested by our findings that p-ERK1/ERK2 were down-regulated and p27 was up-regulated in MDA-MB-468 cells but not in other cell lines. We found that erlotinib blocked EGFR phosphorylation in both EGFR-expressing sensitive cell lines and EGFR-expressing resistant cell lines (). In the EGFR-expressing erlotinib-sensitive cell lines, CDK2 was suppressed, perhaps subsequent to the movement of p27 into the nucleus; however, CDK2 was not suppressed, and p27 did not move into the nucleus, in EGFR-expressing resistant cell lines. These results indicate that suppression of EGFR, by itself, was not sufficient for cells to be sensitive to erlotinib. In other words, both blockade of EGFR phosphorylation and down-regulation of CDK2 are required for cells to be sensitive to erlotinib. Our findings suggest that targeting CDK2 in EGFR-overexpressing erlotinib-resistant cells could be fruitful. Future studies are necessary to clarify the role of CDK2 and that of molecular effects upstream of CDK2 by erlotinib treatment.
In summary, our results showed that erlotinib did have some effect on breast cancer cells independent of the level of EGFR and HER2 expression; that suppression of CDK2 activity correlated with erlotinib activity regardless of the upstream molecular effects; and that p27 subcellular localization is critical for erlotinib sensitivity.