Despite recent advances in treatment, HER2/
neu positive or negative breast cancer continues to be a major cause of death in women. α-TEA is an orally active semi-synthetic analog of vitamin E, which has demonstrated anti-tumor activity against several breast cancers
in vitro and
in vivo. Trastuzumab (Herceptin
®), a humanized antibody that targets the extracellular domain of HER2/
neu, has become standard of care for the treatment of HER2/
neu over-expressing early stage and metastatic breast cancer. However, trastuzumab is effective in only a small percentage of patients with high HER2/
neu
+ tumors resulting in only a 15-30% response rate [
18,
21], which can be significantly increased to 50-80% by the addition of chemotherapeutic drugs [
40]. In this study we evaluated a novel combination therapy modality consisting of oral α-TEA and HER2/
neu-specific antibody. Our data demonstrate the capability of α-TEA to efficiently kill mouse mammary and human breast cancer cell lines irrespective of the expression level of HER2/
neu. This finding suggests that HER2/
neu expression is not required for α-TEA-mediated tumor cytotoxicity and that HER2/
neu-independent pathways may be operative in α-TEA-mediated killing of HER2/
neu-expressing tumor cells. Furthermore, the ability of α-TEA to kill breast cancer cells irrespective of HER2/
neu status suggests that it could be effective for treating HER2/
neu
+ as well as HER2/
neu
- breast cancers. This finding led us to hypothesize that combining α-TEA with trastuzumab will result in an enhanced antitumor response. Our data demonstrate a direct correlation between HER2/
neu expression and susceptibility to HER2/
neu-specific antibody. The MMC and MDA-MB-453 cell lines exhibiting high HER2/
neu expression were susceptible to HER2/
neu antibody treatment. Combining sub-optimal doses of α-TEA (20 μM for MMC, and 10 μM for MDA-MB-453) with HER2/
neu-specific antibody resulted in an enhanced cytotoxic effect against the HER2/
neu
+ cell lines that was absent in the HER2/
neu
- cell lines (ANV and MDA-MB-231). This result suggests that lower doses of α-TEA may be therapeutic if combined with HER2/
neu-specific antibody. The modest efficacy of the combination treatment
in vitro is not surprising since the major
in vitro mechanism of trastuzumab is thought to be growth inhibition, while the major
in vivo mechanism of trastuzumab anti-tumor activity, antibody-dependent cell-mediated cytotoxicity [
22,
41] and cross-priming by antigen-presenting cells (APC), are lacking in the
in vitro system. This is corroborated by our results that trastuzumab
in vitro did not induce apoptosis in MDA-MB-453 tumor cells but reduced the levels of activated AKT which is an important survival and growth signal, suggesting that the main trastuzumab effect
in vitro is anti-proliferative. In contrast, α-TEA treatment
in vitro resulted in significant induction of apoptosis but also cooperated with trastuzumab to further decrease activated AKT levels, providing a mechanism of the enhanced tumor cell reduction in the α-TEA+trastuzumab combination treatment. Our finding of increased apoptosis and decreased proliferation in tumor tissue after α-TEA and/or trastuzumab treatment is also consistent with the notion that the major mechanism of anti-tumor efficacy of the combination treatment
in vivo is the result of the accumulation of apoptotic cell death and restricted proliferation.
Using a xenograft model of human breast cancer, we demonstrated for the first time that the combination of α-TEA plus trastuzumab led to complete tumor regression in all treated animals compared to 50% regression in mice that received trastuzumab alone. The enhancement of the
in vivo anti-tumor response in the combination group reinforces the
in vitro studies using HER2/
neu
+ mouse mammary and human breast cancer cell lines. An important aspect of the combination therapy is that anti-tumor activity was detected using a trastuzumab dose that is within the dose range administered to HER2/
neu
+ breast cancer patients [
39], highlighting the translational potential of this novel combination therapy. Evaluation of HER2/
neu expression at an intermediate time point during the treatment regimen revealed no modulation of HER2/
neu expression. This finding suggests that ongoing α-TEA or α-TEA+trastuzumab treatment of established tumors does not impair HER2/
neu expression, allowing for continued concurrent treatment of such tumors with HER2/
neu-specific antibody.
The anti-tumor effect in the α-TEA, trastuzumab and the combination groups correlated with increased levels of in situ apoptosis and decreased proliferation in the tumors compared to the c group. The combination of α-TEA+trastuzumab resulted in the highest level of in situ apoptosis and the lowest level of proliferating cells that correlated with the faster rate of tumor regression in the combination group compared to the trastuzumab alone group. These data suggest that induction of apoptosis and reduction of tumor cell proliferation is a major mechanism of breast cancer suppression by α-TEA+trastuzumab therapy in vivo.