Where Do We Go From Here in the Treatment of ERaBreast Cancer

We have demonstrated the ability to re-express ERa in ERa-breast cancer cells via the inhibition of hyperactive MAPK resulting from overexpression of EGFR or erbB-2 in both established ERa-breast cancer cell lines as well as ERa-tumors. This re-expression of ERa can be achieved via either direct inhibition of MAPK or via inhibition of the upstream growth factor receptor (EGFR or erbB-2) that is driving its hyperactivation. We have also established that the restoration of ERa expression is sufficient to induce anti-estrogen responses in a subset of these ERa-breast cancer cells.

Clearly then, strong signaling via MAPK directly represses ERa expression and ERa signaling is known to repress the expression of both EGFR and erbB-2. It is apparent that in breast cancer, the co-expression of high level signaling from both receptor types is mutually exclusive. A clue for why this co-expression is not tolerated by breast cancer cells comes from our own studies as well as those of others. Upon the generation of our high growth factor signaling transfected lines, they were originally expanded in estrogen-containing media and exhibited a high rate of apop-tosis (El-Ashry et al., 1996). Only upon shut-down of expression of the transfected gene (which occurred if cells were growing in the presence of estrogen) or down-regulation of ER expression by MAPK (as we've shown occurs when cell are grown in the absence of estrogen) did the cells not undergo apoptosis. Complimentary to these data, several investigators have forced the expression of ERa in ERa-breast cancer cell lines like MDA-MB-231 cells and have demonstrated that estrogen now induces apoptosis in these cells with high growth factor receptor signaling (Bayliss et al., 2007; Huang et al., 2003; Sotiriou et al., 2003). Collectively, these data indicate that high level signaling through both receptor systems is not tolerated by cells, although the mechanisms underlying this are not well understood.

The re-expression of ERa in established ERa-breast cancer cell lines had only been previously demonstrated via inhibition of DNA methylation or histone deacetylation in those cell lines in which the ERa promoter has been shown to be methylated (Ferguson et al., 1995; Ottaviano et al., 1994; Zhou et al., 2007). The methylation of ERa promoter is presumably a means of permanent repression secondary to some other down-regulating event. The down-regulation of ERa expression by hyperactive MAPK is a more direct mechanism and is dynamic and reversible - that is the down-regulation is reversed by the inhibition of MAPK activity and occurs again shortly after return of MAPK activity. And as we found with the two cell lines in which the ERa promoter turned out to be hypermethylated, despite the very high levels of MAPK exhibited by these cells and the effectiveness of U0126 in inhibiting MAPK, ERa expression could not be restored. Our data indicates that in addition to hypermethylation of the ERa promoter, hyperactiva-tion of MAPK resulting from overexpression of EGFR or erbB-2 can also be directly responsible for the lack of ERa expression in ERa-tumors. Importantly, this MAPK meditated down-regulation of ERa expression can be targeted to result in re-expression of ERa. In support of our in vitro and ex vivo data, it has recently been shown that in a small study of 10 ERa-/erbB-2+ patients treated for various lengths of time with Herceptin, that 3 of these re-expressed ERa (Munzone et al., 2006). A more recent study by Massarweh et al suggests this mechanism can be exploited in ERa + /erbB-2+ tumors that lose ERa expression during treatment. They found that resistance to estrogen deprivation/fulvestrant in an ERa + /erbB-2+ MCF-7 xenograft model was accompanied by upregulation of MAPK activity and loss of ERa expression, and subsequent co-treatment with Iressa resulted in inhibition of MAPK activity and increased ERa expression (Massarweh et al., 2006).

Regardless of the different potential mechanisms for down-regulating/restoring ERa expression, the re-expressed ERa must be not only functional upon re-expression, that is induce the regulation of estrogen-responsive genes, but must also be able to regulate growth in response to estrogen/anti-estrogens in order to be clinically relevant. In studies where demethylation of the ERa promoter or use of histone deacetylase inhibitors restored ERa expression, this ERa was functional in that it could regulate ERE-luciferase activity as well as the expression of specific estrogen regulated genes such as the progesterone receptor (PR) (Yang et al., 2000, 2001; Zhou et al., 2007). And in our previous studies in our hyperactive MAPK cell line models, re-expresion of ERa upon inhibition of MAPK also restored ERa's transcriptional activity (Bayliss et al., 2007; Holloway et al., 2004; Oh et al., 2001). The ability of the re-expressed ERa upon MAPK inhibition to mediate the growth inhibition of anti-estrogens in both established ERa-breast cancer cell lines and in dissociated tumor cell cultures demonstrates clearly for the potential for a novel therapeutic strategy for ERa-breast cancer. In these studies, three different cell line types were observed. In the cell lines (SUM 229 and SUM 190) which were quite sensitive to MAPK inhibition in terms of growth inhibition, restoration of ERa expression correlated with restoration of response to both 4-hydroxy-tamoxifen and ICI 182,780 (fulvestrant, faslodex), however, in the cell line (SUM 149) which also exhibits hyperactivation of NFkB and RhoC overexpression (Pan et al., 2002; van Golen et al., 2000a, 2000b), the re-expressed ERa was not able to restore responses to either anti-estrogen. A possible explanation for this is the hyperactivation of NFkB in these cells which is known to result in anti-estrogen resistance in breast cancer cells (Campbell et al., 2001; Degraffenried et al., 2004; Nakshatri et al., 1997; Riggins et al., 2005). Thus, even though the MAPK repression of ERa mechanism is operative in these cells and can be targeted to allow for re-expression of ERa, these cells have additional cell signaling alterations that allow them to bypass ERa and remain anti-estrogen resistant although now ERa+. Indeed, these cells were very resistant to growth inhibition induced by MAPK inhibition whereas even modest inhibition of NFkB significantly impacted their proliferation. The third cell line type were those cell lines (SUM 102 and SUM 159) in which MAPK inhibition alone could not restore ERa expression and which turned out to exhibit hyperme-thylation of the ERa promoter. These two cell lines in fact also were representative of the basal genotype of breast cancer as opposed to the luminal genotype. Whether in fact, basal breast cancers are marked by ERa promoter hypermethyla-tion or whether ERa promoter hypermethylation occurs in a subset of basal breast cancers remains to be determined. Understanding these features of basal versus luminal breast cancers is important in terms of understanding the mechanisms of lack of ERa expression. While it is certainly possible that reversible mechanisms such as MAPK mediated down-regulation of ERa are operative in luminal breast cancers and more permanent mechanisms such as hypermethylation of the ERa promoter are operative in basal breast cancers, our data with our primary cultures from ERa-breast tumors would seem to argue against this, at least preliminarily. The number of ERa- luminal breast cancers that are not also erbB-2/Her-2neu over-expressors is not large. And yet, both of the triple negative tumors from which we generated primary cultures exhibited MAPK mediated down-regulation of ERa expression and did not have hypermethylation of the ERa promoter. It would seem too lucky that both of these happened to be representative of the small number of luminal triple negatives that exist. Analyses of the increasing number of primary cultures we have from triple negative breast cancers will help answer this question.

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