Previous cell culture models on the impact of growth factor receptor signaling have shown both over-expression or gene amplification of ErbB-receptors in breast cancers can confer a tamoxifen-resistant phenotype, yet others have shown that acquired resistance to tamoxifen in breast cancer cells was not a result in changes in ErbB-receptors.(30;31;35;38;39;50-52
) In order to address whether a naturally-occurring, constitutively active EGFR variant has any impact on the dependence of breast cancer cells on estrogen and their response to anti-estrogens, we used an isogenic MDA-MB-361 breast cancer cell line system. We clearly demonstrated that expressing EGFRvIII in MDA-MB-361 cells promote these estrogen-dependent, tamoxifen-sensitive cells to become estrogen-independent and tamoxifen-resistant in vivo
( and ). However, these cells still maintained their ability to respond to estrogen-induced tumor growth (). Various mechanisms can contribute to an estrogen-independent and tamoxifen-resistant phenotype, such as loss of PgR expression in breast cancer resulting from a loss or non-functioning ERα, low circulating levels of estrogen, hypermethylation of the PgR promoter, loss of the PgR gene locus, excessive growth factor signaling, increased ERα activity by SERMS or by growth factor simulation at the cell membrane, and altered ERα co-regulators.(53
) Preclinical and clinical studies have recently suggested that PgR negativity in ER-positive breast cancer may be a marker of hyperactive growth factor signaling rather than a result of a nonfunctional ER signaling pathways, as previously suggested.(53;54
) Our study provides direct evidence that suppressed/reduced PgR levels may derive from the hyperactivity in the signaling cascade generated by constitutively activated EGFRvIII. Our model system suggests that a constitutive activation of the EGFR receptor in high ErbB-2 expressing breast cancer cells leads to partial ERα loss and a significant reduction in PgR expression which may contribute to an estrogen-independent and tamoxifen-resistant phenotype (). Conversely, a similar isogenic EGFRvIII breast cancer model using MCF-7 cells, which do not become estrogen-independent nor tamoxifen-resistant in vivo
, showed an increase in PgR expression without any changes in ERα expression (). In this context, PgR loss may be a marker of an active EGFRvIII/ErbB2 signaling network and a conferred estrogen-independent, tamoxifen- resistant phenotype. Furthermore, a similar observation was seen in our immunohistochemical analysis of invasive clinical breast cancer specimens. EGFRvIII expression was found to be significantly correlated to loss of PgR expression in invasive human breast cancer (). Although our immunohistochemistry results only showed a significant association of EGFRvIII expression with loss of PgR expression (), it is important to mention that the trend for ERα in the same set of breast cancer tissue samples is very similar to that of PgR expression. However, further investigation would be needed to confirm these results with a lager sample size.
Early studies have shown that increased MAPK activation may result in a decrease in ERα protein levels as was shown in MCF-7 cells which had hyperactivation of MAPK due to the over-expression of EGFR or ErbB-2 and inhibition of MAPK activity in ERα-negative breast cancer cells resulted in re-expression of ERα.(40;42
) Growth factor stimulation has been shown to down-regulate PgR expression by inhibition of PgR gene transcription mediated by the PI3K/Akt/mTOR pathway and not by altered ERα levels or activity.(54
) However, MDA-MB-361/vIII cells had enhanced activation of the pro-survival and proliferative, Akt and MAPK pathways (). This synergistic effect by both a MAPK-induced down-regulation of ERα and a possible decrease in ERα activity combined with increased activation of the PI3K/Akt/mTOR may result in reduced PgR levels, an indicator for tumor aggressiveness and tamoxifen-resistance. Conversely, transactivation with EGFRvIII and other growth factor receptors, such as IGF receptors, has not been explored in breast cancer and may be critical in the potent oncogenic properties of the EGFRvIII receptor. In future studies, the use of Akt and MAPK inhibitors may help use dissect whether these two signaling pathways have any role in the changes in ERα and PgR protein levels as well as other genomic and non-genomic activities of ERα which lead to the estrogen-independent, tamoxifen-resistant phenotype of the MDA-MB-361/vIII cells.
Finally, cross-talk between EGFRvIII and ERα signaling pathways was further revealed through the altered expression of Bcl-2, which corresponded to PgR expression and potentially transcriptional activity of ERα in the isogenic MCF-7 and MDA-MB-361 cell lines ( and ). Bcl-2 confers a growth advantage to cells by inhibiting apoptosis and inducing chemotherapy resistance and has prognostic significance in certain types of lymphomas.(55-57
) In this respect, Bcl-2 should correlate with more aggressive tumor and resistance to hormonal therapy and chemotherapy. However, the data has been conflicting in breast cancer. Clinical studies have revealed that high levels of Bcl-2 expression are associated with a number of favorable prognostic factors including ER positivity, PgR positivity, low histological grade and well-differentiated tumors.(45;46;49
) Unexpectedly, numerous studies have shown that tumors with high Bcl-2 expression are more responsive to hormone therapy and have more favorable disease-free and overall survival.(58-60
) Furthermore, low levels of BAD expression was associated with relapse in tamoxifen-treated breast cancer patients and Bcl-xL expression was often not associated with breast cancer patient outcomes.(44;49
) We demonstrated that EGFRvIII-expressing breast cancer cells reveal a paradoxical relationship between proteins involved in apoptosis and tamoxifen-response. Tamoxifen-sensitive MCF-7/vIII cells express increased levels of Bcl-2, Bcl-xL, and BAD () as well as increased levels of PgR (), whereas MDA-MB-361/vIII cells are tamoxifen-resistant, had decreased levels of all three apoptotic proteins and a reduction in PgR expression (). The striking correlation between the apoptotic molecules and PgR expression observed in these EGFRvIII transfectants suggests a potentially important role for the apoptosis pathway in EGFRvIII expressing breast cancer cells and their response to estrogen and anti-estrogens. The predictive value of apoptotic proteins, ERα, PgR, and EGFRvIII in breast cancer requires further analysis and may potentially be used to identify breast cancer patients which may benefit from an aggressive chemotherapeutic regimen in place of endocrine therapy.
In conclusion, our study provides a direct evidence that expressing EGFRvIII in high ErbB-2-expressing breast cancer cells induces an estrogen-independent, tamoxifen-resistant phenotype. Unraveling the mechanisms of resistance to tamoxifen- mediated by EGFRvIII provides new insights and implicates that multiple mechanisms are involved in resistance. Acquired resistance to tamoxifen is likely through active EGFRvIII/ErbB-2 signaling networks, and PgR expression is negatively associated with tamoxifen resistance in breast cancer. Based on our MCF-7 and MDA-MB-361 model systems, reduced PgR or loss of PgR expression may be a predictive factor for the outcome of endocrine therapy in EGFRvIII/ErbB-2 expressing breast tumors. Furthermore, while evaluation of the apoptotic status of breast cancer, the regulators of apoptosis, such as Bcl-2, Bcl-xL, and BAD must be considered together with ERα and PgR. Since this EGFR variant is both naturally-occurring and tumor-specific, it may also prove to be a valuable drug-target and potentially a tumor-specific antigen that allows for targeting chemotherapeutic agents to breast cancer cells without causing systemic toxic to breast cancer patients. Although recently it was reported that EGFRvIII expression was not correlated to patient outcomes in breast cancer patients with locoregionally advanced breast cancer, a larger number of breast cancer specimens expressing EGFRvIII as well as a more stringent method of detecting EGFRvIII expression is required to make an accurate assessment of the clinical significance of EGFRvIII expression in breast cancer.(1
) Our previous results have shown that EGFRvIII expression is not high in breast cancer specimens, however, this does not suggest that EGFRvIII is not involved in breast cancer tumorigenesis and progression since EGFRvIII expression can be readily transferred from expressing cells to non-expressing through “oncosomes”, which has been shown to enhance transformation of glioma cells.(17;61
) Pre-clinical studies evaluating the use of other anti-estrogens alone and in combination with therapeutic agents targeting EGFRvIII and/or ErbB-2, such as antibodies or tyrosine kinase inhibitors, is also warranted.