Vast epidemiological data supports a causative role for estrogens in the development of human breast cancer. Though significant progress has been made in the identification and characterization of a large number of proteins that participate in estrogen signaling, the precise molecular mechanisms that control specific cellular responses to endogenous and exogenous ligands of the ER signaling pathway are complex and not completely understood. However, increased understanding of hormone action suggests that the cell type and/or co-regulator levels dictate the ultimate response to ligand. The present study was initiated to examine the cellular and molecular consequences of AIB1 overexpression.
Our previous report demonstrated that the majority of ER-positive breast cancer cell lines have correspondingly high levels of endogenous AIB1 expression (four out of five cell lines analyzed); therefore, the number of suitable cell lines in which to generate stable overexpression of AIB1 are limiting [8]. Of interest, transfection of the AIB1 cDNA into the ER-positive, low AIB1-expressing cell line, T47-D, demonstrated a cell-lethal phenotype and no subclones with high levels of AIB1 expression were generated (data not shown).
Likewise, transfection of AIB1 into MDA-MB-436 breast cancer cells appears to be lethal since all but one of the surviving subclones failed to demonstrate expression of the transfected AIB1. The 436.1 line discussed in this study, however, has integrated the exogenous AIB1 cDNA and maintained high levels of AIB1 mRNA and protein expression. Most likely, 436.1 cells have acquired additional alterations which support its survival.
Though the parental MDA-MB-436 cells are ER-α negative, transient transfection of ERα was capable of inducing expression of an ERE-regulated reporter gene in the presence of estrogen, indicating these cells contain sufficient amounts of co-regulatory factors required for estrogen inducibility. Furthermore, since the levels of ER-α could be experimentally modulated in transient tranfection studies, this derivative cell line provided a useful model to compare the effects of low-level, constitutive versus high-level expression of AIB1 in the presence or absence of varying concentrations of ER-α.
Immunostaining patterns of AIB1 and CBP included diffuse fluorescent staining as well as large, bright, dot-like foci (Fig. 3). Although the punctate-staining pattern of AIB1 in 436.1 cells resembled CBP containing PML (promyelocytic leukemia protein) oncogenic domain (POD)-like structures [17, 18], the AIB1 dots appeared in only a small percentage of the population (< 2%) and typically did not overlap with CBP. Analyses of populations derived from single cell isolates revealed that the punctate staining was not a clonal variant. Similarly, Baumann et al used GFP-fused to GRIP1 to investigate its intracellular distribution in HeLa cells and observed both diffuse and focal accumulation of GRIP1 [19]. CBP staining, on the other hand, was consistently diffuse throughout the nucleus in MDA-MB-436 cells but appeared to accumulate in PML-like bodies in the AIB1-transfected 436.1 cells. These results are consistent with observations made recently by Boisvert et al [20]; several cell lines do not accumulate CBP into PML bodies, but transfection of a GFP-CBP fusion protein into these cells leads to accumulation of CBP into distinct PML nuclear bodies. Taken together, these observations imply that increased amounts of AIB1 and CBP have caused the redistribution of these proteins into nuclear foci in 436.1 cells. Perhaps the levels of CBP and AIB1 are below the level required for recruitment to nuclear bodies in the non-transfected MDA-MB-436 cells. The mechanisms for upregulation of CBP in 436.1 cells remain elusive, but it will be interesting to determine if overexpression of AIB1 plays a direct role in the transcriptional regulation of CBP, or if levels of CBP are sensitized to and somehow adjusted with levels of AIB1, or vise versa.
While the overall level of reporter gene activity was dramatically increased in 436.1 cells, perhaps the most striking observation is in the right in the shift of the estrogen dose-response curve in 436.1 cells. The repositioning of the E2 dose-response curve to the right in 436.1 cells was somewhat unexpected since several studies with other receptors have demonstrated a left shift in the dose-response curve with the addition of p160 proteins, including TIF2 and SRC-1 [21–24]. However, there is no evidence that the total levels of transactivation and the position of the dose-response curve are linked. Studies with glucocorticoid [21, 25–28] and progesterone [22, 28] receptors show that the two phenomena can be totally disconnected. Increased total transactivation might be expected if the concentrations of two coactivators (AIB1 and CBP) are increased, but this does not predict the effects of these changes on the dose-response curve and EC50.
One explanation for these observations is squelching, where increased levels of AIB1 compete for some limiting factor that is crucial for nuclear receptor transactivation, such as CBP. Because we have also demonstrated that the levels of CBP are significantly elevated in the 436.1 cells it is unlikely that the level of CBP is limiting. However, AIB1 and CBP could be competing for a common factor that is now limiting due to AIB1 and CBP abundance [24]. Also, cell specific differences, which have caused some coactivators and corepressors to be apparently inactive [21, 22] or to have opposite activities [22, 28], may be important.
We propose a hypothetical model to explain the increase in total transactivation and the overall right-shift in the dose-response curve, as schematized in Figure 6. In this model, the dose-response curve depends on the presence of a ternary complex composed of AIB1, CBP, and a third, unknown nuclear receptor complex participant, depicted as X. In MDA-MB-436 cells, the levels of AIB1, CBP, and factor X, are at concentrations sufficient to form ternary AIB1/CBP/X complexes that support the observed dose-response curve. The absence or dramatic reduction of such a ternary AIB1/CBP/X complex in 436.1 cells (due to increased levels of AIB1 and CBP and limiting levels of X) might result in a very different EC50. In fact, by the law of mass action, elevated levels of AIB1 and CBP would be expected to cause increased levels of the dimeric complexes (i.e., AIB1/X, AIB1/CBP, and CBP/X). Each of these may cause increased transactivation but not permit the same positioning of the dose-response curve as in MDA-MB-436 cells, resulting in a relative increase in the EC50 and a right shift in the dose-response curve. The greatly decreased levels in 436.1 cells of the ternary AIB1/CBP/X complex, which has no effect on the level of total transactivation, would now have only minimal effects of the positioning of the dose-response curve.
Additional support for this model comes from several lines of evidence. First, convincing evidence for a limiting factor other than p160 coactivators has been demonstrated by Lopez et al. [23]. These investigators have shown that squelching between different receptors cannot be relieved by supplying excess amounts of CBP or p160 coactivator and suggest there is an unknown factor that is necessary for receptor coactivation. Similarly, Kino et al reported that the amount of CBP that is required to see a decrease in glucocorticoid receptor (GR) transactivation is cell dependent [29]. The presence of a limiting complex component that participates in the positioning of the dose-response curve has been established by Chen et al [24]. Here, the effects of modulators (GR, TIF2, or a cis-acting element in the reporter gene) were additive when all components were limiting, but no further effect was obtained if saturating amounts of any one modulator was present. Second, there is a well established physical interaction between CBP/p300 and p160 coactivators [13, 30–32], and several groups have proposed that CBP and other coregulators, e.g., p160s, exist in the cytoplasm in preformed complexes [33, 34].
Candidate genes whose altered expression might contribute to the phenotypic features of the 436.1 cells, such as cell survival, multinucleated morphology, and transcriptional properties were identified by cDNA microarray technology. Comparison of RNAs from MDA-MB-436 versus 436.1 revealed a number of profound changes in the expression of genes with a variety of functions, including cell adhesion, communication, maintenance of cell shape, apoptosis, and cell cycle control. For example, cyclin D3, an important regulator of endomitosis and polyploidization in megakaryocytes [35], was upregulated 3.75-fold in 436.1 cells, suggesting upregulation of cyclin D3 contributed to the altered morphological features observed in these cells. An 8.2-fold upregulation of the apoptosisinhibitor 2 gene may have facilitated the survival of these cells. Downregulation of NFkB and C/EBPβ cDNAs (ratios of 0.3 and 0.5, respectively) and a number of interleukins whose gene expression are regulated by these transcription factors (Table 1) possibly indicates a shift away from NFkB and C/EBPβ signaling to transcriptional activities mediated by other transcription factors. Finally, none of the general transcription factors, including TFIIA (ratio = 1.1), TFIIB (ratio = 1.0), TFIID (ratio=.53), TFIIE (ratio = 1.25), TFIIF polypeptides 1 and 2 (ratio = 0.63, 0.94), and TFIIH (ratio = 1.16) showed increased expression in 436.1 cells, supporting the idea that AIB1 and CBP overexpression play a significant role in the observed increase in basal and E2-induced reporter gene activity in 436.1 cells. Future studies are necessary to confirm these observations and to determine which are genes regulated by transcriptional complexes containing AIB1.
In summary, we have characterized an AIB1-transfected cell line that displays high levels of AIB1 expression. The data presented here support a role for AIB1 in the estrogen-dose response and also suggest that, apart from ER-mediated pathways, AIB1 contributes to a number of signaling events which influence cell cycle, morphology, and survival. Future efforts directed at determining how overexpression of AIB1 affects cell behavior in the presence or absence of specific hormone receptors should shed light onto specific signaling pathways that are altered as a consequence of AIB1 amplification in breast cancer.