In this study, we provided evidence that p27 cytoplasmic localization is associated with erlotinib resistance in breast cancer. Inhibition of S10 phosphorylation of p27 led to p27 nucleus accumulation and enhanced the anti-proliferative effect of erlotinib against breast cancer. Our findings elucidated the molecular mechanisms that explain why EGFR TKIs have minimum activity in breast cancer. Here we report for the first time to use KIS in vivo siRNA delivery technique in combination with erlotinib to enhance the EGFR TKI sensitivity.
p27 is an important negative regulator of the mammalian cell cycle. The activity of p27 depends on the subcellular localization of p27. Even though phosphorylation status of p27 at T157, T187, and S10 plays a role in p27 localization, we found that only S10 p27 phosphorylation increased in response to erlotinib treatment, indicating that S10 phosphorylation is involved in erlotinib resistance in breast cancer. The effects produced by KIS siRNA knockdown on growth and anchorage-independent growth are only partial. Therefore, other mechanisms may contribute to erlotinib resistance. We speculate that other types of phosphorylated p27 may play roles in erlotinib resistance. Our current study involved testing only three phosphorylation sites of p27 (S10, T157, and T187), but other phosphorylation sites of p27 may be important for regulating p27 localization and stability. For example, p27 can be phosphorylated by the oncogenic kinase Src at tyrosine (Y) 74 and Y88, and this phosphorylation reduces the p27-mediated inhibition of cyclin E-CDK2 (33
). Phosphorylation of p27 at T198 by Akt leads to p27 cytoplasmic mislocalization (18
). Therefore, currently we are also focusing on other phosphorylation sites on p27 to further elucidate the molecular mechanism of EGFR-TKI resistance.
HER2/ErbB2 is another member of the ErbB family of receptor tyrosine kinases. Overactivation of HER2 plays a critical role in the progression of human breast cancer (34
). Targeting of HER2 by anti-HER2 antibodies such as trastuzumab is now widely used in the treatment of patients with HER2-positive breast cancer (35
). Even though trastuzumab showed relevant clinical activity against HER2-positive breast cancer, the overall rate of response rates to trastuzumab as a single agent is only 15% to 30% (36
). Resistance to trastuzumab therapy remains a significant clinical problem. p27 is also a downstream molecule of the HER2 pathway and is involved in trastuzumab resistance. It was reported recently that activation of MET receptor tyrosine kinase contributes to trastuzumab resistance, as Met protects cells against trastuzumab by abrogating p27 induction (37
). By inhibiting Akt, trastuzumab can inhibit KIS expression and therefore inhibit KIS-induced nuclear export of p27 (21
). Therefore, our study suggests that one way to overcome trastuzumab resistance is to modulate S10 phosphorylation and subcellular localization of p27.
Lapatinib is a dual TKI that inhibits both EGFR and HER2. We previously reported that the major activity of lapatinib in EGFR/HER2-positive breast cancer cells is through its anti-HER2 activity and not through anti-EGFR activity (26
). In this study, our results provided further evidence to support this finding. When we used lapatinib to treat SUM149 cells, which express EGFR but not HER2, we found that EGFR blockage by lapatinib was enhanced by KIS depletion. In contrast, KIS depletion did not enhance lapatinib’s activity in cells that expressed both EGFR and HER2, suggesting that the activity of lapatinib in such cells is mainly through HER2.
In the current study, we tested the role of S10 phosphorylation of p27 in erlotinib sensitivity not only in cells that overexpress both EGFR and HER2, but also in cells that overexpress EGFR but not HER2. EGFR is reported to be overexpressed in > 60% of basal-like breast cancers (38
), and EGFR
gene amplification is also found in a subset of basal-like tumors (40
). Basal-like breast cancer often has a so-called triple-negative phenotype—in other words, it lacks expression of estrogen receptor, progesterone receptor, and HER2 (41
). Triple-negative breast cancer is the most aggressive form of primary breast cancer, and the majority of these tumors cannot be managed effectively with existing targeted treatments (trastuzumab and hormonal treatments) (43
). EGFR expression is associated with early relapse and poor survival in triple-negative breast cancer, suggesting that EGFR might be a promising target in this type of disease (45
). Several studies have reported the use of cetuximab, a humanized monoclonal antibody against EGFR, in treatment of the basal-like tumor type (46
). Another EGFR TKI, gefitinib, enhanced response to other chemotherapeutic drugs such as carboplatin and docetaxel (47
). Therefore, even though erlotinib induces clinical responses in only a small proportion of breast cancer patients, there is still reason to believe that inhibition of the EGFR pathway can have substantial activity against triple-negative breast cancer. Identifying the molecules involved in and necessary for resistance to EGFR TKIs will enable us to develop clinically relevant therapeutic approaches by making EGFR a relevant target for breast cancer. In this study we tested the impact of p27 phosphorylation on erlotinib’s activity in 2 triple-negative EGFR-expressing breast cancer cells MDA-MB-231 and MDA-MB-468. We found that inhibiting S10 phosphorylation of p27 enhanced the sensitivity of erlotinib to these cells, suggesting that this effect is not restricted to a few cell lines but is a universal phenomenon.
It is still unclear whether there is some relationship between pS10-p27 expression and erlotinib sensitivity in breast cancer cells. To study whether erlotinib sensitivity depends on p27 phosphorylation in breast cancer cells, we tested the basal expression levels of pS10-p27 in a panel of breast cancer cell lines including erlotinib-sensitive cell lines SUM149 and KPL-4 (32
), erlotinib-moderate-sensitive cell lines SK-BR-3 and BT-474 (25
), and erlotinib-resistant cell lines MDA-MB-231 and MDA-MB-468 (25
). The basal expression level of pS10-p27 is low in most of the cell lines and is not correlates with erlotinib sensitivity (data not shown). The underlying mechanism linking EGFR and KIS is also not clear. To investigate whether the KIS expression level is regulated by EGFR activation, we activated EGFR by EGF stimulation in BT-474 and SK-BR-3 cells and then tested KIS expression. We found that KIS expression did not change significantly after EGFR activation (Supplementary Fig. S2
). In a future study, we will focus on detecting whether KIS can regulate the EGFR pathway.
Acquired resistance to EGFR TKIs in non–small-cell lung cancer commonly occurs after continuous drug administration. It is reported that MET amplification is involved in this acquired resistance (48
). Therefore, targeting of MET may enhance the sensitivity of non–small cell lung cancer to EGFR TKIs. However, even though MET was found to be highly activated in cetuximab-resistant cells, inhibition of MET activity did not sensitize cetuximab-resistant cells to cetuximab. The reason why MET inhibition does not restore cetuximab sensitivity is still unknown. Because MET’s ligand hepatocyte growth factor induces cell cycle progression in medulloblastoma cells in a p27- and Cdk2-dependent manner (49
), we speculate that modulating p27 directly may be more powerful than MET inhibition to abrogate the acquired resistance to EGFR TKIs and is thus worthy of prospective clinical investigation.
Overall, our study demonstrated that p27 phosphorylation at S10 plays a critical role in breast cancer sensitivity to erlotinib. Combining EGFR TKIs with siRNA knockdown of KIS, which leads to inhibition of S10 phosphorylation of p27, enhanced erlotinib activity both in vitro and in vivo in breast cancer. We expect this combination therapy to be potentially translatable to clinical use, where it may ultimately improve the efficacy of EGFR-TKIs for women with advanced breast cancer that is resistant to EGFR-TKIs.