A panel of neu expressing BALB/c mammary tumor cell lines, TUBO, Bam1a, Bam IR-5, and D2F2/neu, have been established to represent three types of Her-2+
human breast cancers. In a portion of Her-2+
breast cancers Trastuzumab monotherapy inhibits tumor growth (11
). TUBO and Bam1a cells which are highly sensitive to anti-neu antibody and RTKI would represent these responsive cancer cells. In Bam IR-5 cells, neu L726I mutation induced by gefitinib corresponds to Her-2 L726F mutation in human breast cancer cells that are resistant to another RTKI lapatinib (38
). Therefore, Bam IR-5 represents treatment induced drug-resistant tumor cells. The majority of Her-2 expressing human tumors have intrinsic resistance to Trastuzumab because only 11-23% of metastatic breast cancer patients respond to antibody monotherapy (11
). D2F2/neu which are refractory to Her-2 targeted therapeutics without prior selection is a representative of these resistant cells. When developing Her-2 based immunotherapy or vaccines, all three types of Her-2/neu+
tumors should be tested to fully assess treatment efficacy. The propensity to develop cerebral metastases after Trastuzumab treatment suggests therapy-induced genetic alterations or the selection of aggressive tumor cells (15
), warranting vaccines that can initiate or amplify Her-2 immunity to control drug resistant tumor cells.
Although TUBO and Bam1a are both derived from spontaneous NeuT mammary tumors and they express comparable levels of neu, more chromosomal aberrations are found in TUBO cells, including the translocation of chromosome 4 which has been reported in other neu induced tumors (40
). It is not clear whether the disparities between TUBO and Bam1a cells indicate heterogeneity of neu induced tumors or changes induced during tissue culture selection. However, increased chromosomal aberrations in TUBO cells correlate with their reduced sensitivity to gefitinib when compared with Bam1a cells, suggesting the use of alternative survival and growth signaling pathways in TUBO cells.
Bam IR-5 cells established by prolonged culture in gefitinib have developed CCAs, NCCAs, trisomies and monosomies which were not detected in the parent Bam1a cells. The point mutation of leucine 726 to isoleucine 726 in the ATP binding pocket of the neu tyrosine kinase domain may change the binding of gefitinib to Her-2 and mediate their resistance to drug and antibody treatment, although other genetic changes such as those revealed by SKY analysis may also contribute to the resistance.
Neu transfected D2F2/neu cells are resistant to drug and antibody treatment both in vitro
and in vivo
, showing their independence of neu in survival and proliferation. The common origin of D2F2 and D2F2/neu cells is evidenced by the shared chromosome 11 and 16 translocations which occur in some hormone-induced tumors (41
). It is of interest that expression of neu resulted in a less aberrant SKY profile, suggesting the transfection and expansion of a cell with less aberration.
Whether gefitinib resistance is a result of drug selection as in Bam IR-5 or intrinsic resistance as in D2F2/neu, these cells are also resistant to antibody treatment, indicating shared escape mechanism. But, all test cells are rejected by pcytneu. vaccination which induces CTL without Ab response. We reported previously using pcytE2 vaccinated mice that depletion of CD8, but not CD4 T cells significantly reduced anti-tumor immunity, showing the induction of CTL which mediated tumor rejection (37
). Therefore, a strong CTL response to the complete repertoire of neu peptides abolishes tumor growth regardless of their sensitivity to neu targeted therapy. When a strong humoral response is induced without effector T cells, as in T cell depleted, pneuTM immunized mice, only drug sensitive cells were completely rejected, with partial protection against drug resistant cells. In patients whose tumors are refractory to drug and antibody therapy, induction of comprehensive immunity by active vaccination will be critical to their long term protection.
Treatment with gefitnib increased neu expression. Since neu activation is required to induce receptor endocytosis and degradation, inactivation of the kinase domain by gefitinib binding may trigger a compensatory increase in Her-2/neu surface expression. Binding of Her-2+
breast cancer cells with Trastuzumab has been associated with decreased Her-2 surface expression (42
) with increased internalization and degradation of Her-2 (43
). Priming of endogenous Her-2 immunity and increased tumor cell lysis by CTL has also been associated with Trastuzumab treatment of breast cancer (44
). In Bam1a and TUBO cells, treatment with anti-neu mAb reduced neu expression, Akt phosphorylation, and cell proliferation. Anti-neu immunity may also be enhanced by anti-neu antibody binding to tumor cells.
It is of note that even D2F2/neu cells which are resistant to antibody in vitro are partially inhibited by vaccine induced antibodies in vivo, suggesting effector mechanisms such as ADCC in tumor growth control. D2F2/neu cells that survive immune attack in vivo completely lost neu expression, indicating the selection of anitgen negative tumor cells in vaccinated hosts, whereas Bam IR-5 cells maintained neu expression in immunized hosts, showing their continued dependence on neu for tumor outgrowth.
Regarding the specificity and safety of Her-2/neu vaccination, mice immunized with Her-2/neu DNA could not reject D2F2 cells which did not express Her-2/neu (not shown), showing specific recognition of Her-2/neu by DNA vaccination (). Little to no binding of human epidermal growth factor receptor (EGFR, Her-1) transiently expressed on 3T3 cells was detected with Her-2 immune sera (not shown). Immunized mice do not show any apparent abnormality for at least one year except for their anti-Her-2/neu immunity (not shown) further showing the specificity of Her-2/neu DNA vaccination. The complete blood count of untreated and DNA vaccinated Her-2 Tg mice did not show obvious differences (not shown). Therefore, the induction of Her-2/neu specific immunity without noticeable toxicity supports the efficacy of Her-2/neu DNA vaccine.
Taken together, we have demonstrated the benefit of DNA vaccination in the management of diverse tumors that exhibit differential sensitivity or resistance to Her-2 targeted therapies. Anti-neu antibody can suppress tumor growth either directly or through mechanisms such as ADCC and may amplify T cell immunity. A strong Her-2/neu-specific T cell response continues to be the goal of Her-2 vaccination as drug-sensitive and -resistant tumors are both controlled by T cells.