It is well known that breast cancer progresses from ER driven to growth factor receptor driven growth. The ErbB receptors, EGFR and ErbB2, as well as the ErbB ligands TGFα
, amphiregulin and heregulin1, which leads to activation of the downstream kinases Akt and Erk have all been found at increased level in endocrine resistant breast tumors and breast cancer cell lines [31
]. Much less is known about the ErbB3 and ErbB4 receptors, and the goal of this study has been to use a large panel of fulvestrant resistant cell lines to evaluate all four receptors including their activation status and expression of the cognate ligands and thus to unravel the involvement of the ErbB receptors in fulvestrant resistant cell growth. The fulvestrant resistant cell lines have reduced expression of the ER compared to parental MCF-7 cells when grown without fulvestrant [6
], and in the experiments presented in this paper in which the resistant cell lines are constantly grown in presence of fulvestrant, the ER expression is extremely low, due to destabilization of the ER upon binding of fulvestrant [34
, data not shown]. Thus, whereas crosstalk between ErbB receptors and ER has been described to be an important mechanism for endocrine resistant cell growth in other model systems [35
], crosstalk cannot be a major mechanism for resistant cell growth in this model system.
Several alterations in expression of the ErbB receptors were observed and the general pattern was increased activation of ErbB3 and EGFR and downregulation of ErbB4 and its activity in the fulvestrant resistant cell lines. Upregulation of EGFR in antiestrogen resistant cell lines has been shown previously [7
], and our data demonstrating that treatment with 1 μM gefitinib preferentially inhibits growth of fulvestrant resistant cell lines support EGFR involvement. Also, Erk activation is severely inhibited by 1 μM gefitinib, showing that Erk and not Akt is activated by EGFR signaling in the resistant cell lines. However, the order of growth inhibition is about 30%, suggesting that EGFR activated by the available endogenous ligands may not alone drive fulvestrant resistant cell growth. Moreover, treatment with 10 μM gefitinib is not specific for the EGFR and we observed a reduction of pErbB3 to an almost undetectable level in all cell lines, indicating that targeting also other receptors may give better growth control.
Thus, we investigated the importance of the increased activation of ErbB3 in resistant cell lines by inhibition of ligand binding by treatment with neutralizing antibodies (Ab5) to ErbB3. Firstly, we demonstrated that Ab5 could inhibit both basal and hrg1β
mediated ErbB3 activation in both MCF-7 and fulvestrant resistant 165R
-5 cells. We then selected three resistant cell lines for treatment with Ab5 and in two of three resistant cell lines, Ab5 treatment resulted in a modest but statistically significant growth inhibition, showing that a highly specific inhibition of ErbB3 can indeed inhibit growth of fulvestrant resistant cell lines. Furthermore, Ab5-treatment reduced the pErbB3 expression to a very low level, indicating that the increased amount of pErbB3 was due to increased activation by endogenous ligand(s). The endogenous production of ligand was confirmed by treating MCF-7 cells with conditioned medium from two resistant cell lines, which resulted in ErbB3 activation, whereas conditioned medium from MCF-7 cells did not have any effect. Lastly, we suggest that this ligand may be hrg2 (α
isoform), which was the only ErbB3 ligand detectable by qPCR and which was also found upregulated in the resistant cell lines at both mRNA and protein level. ErbB3 involvement in antiestrogen resistance is supported by the recent data, in which tamoxifen resistance conferred by ErbB2 overexpression could be reverted by siRNA-mediated knockdown of ErbB3 [36
]. In support of the role of ligand activated ErbB3 for antiestrogen resistance, a recent publication has demonstrated that addition of hrg1β
to a tamoxifen resistant cell line leads to ErbB3/ErbB2 heterodimerization and to decreased sensitivity to tamoxifen [37
]. These data all point to ErbB3 and its cognate ligand as important mediators of antiestrogen resistant cell growth, probably acting in concert with EGFR and ErbB2. Our initial experiments designed to block both EGFR and ErbB3 revealed a more severe growth inhibition by treating 164R
-5 cells with Ab5 and gefitinib, and more experiments with combination therapy also targeting the ErbB2 are in progress.
In contrast to ErbB3, the molecular function of ErbB4 in antiestrogen resistance has not been described at all and its role in breast cancer cell lines is not yet clear. ErbB4 mRNA is downregulated by estradiol in MCF-7 cells [38
] and we also found that both ErbB4 mRNA and protein were clearly estrogen downregulated in our MCF-7 cells (data not shown). Therefore, it could be expected that long-term fulvestrant treatment would act opposite and increase the ErbB4 level. On the contrary, all resistant cell lines had counteracted this direct effect of fulvestrant and displayed a strong downregulation of both ErbB4 protein and activity, indicating an antiproliferative role of ErbB4. Also, the decreased level of ErbB4 in resistant cells may facilitate heterodimerization of ErbB3 with EGFR and ErbB2. Several groups have published that ErbB4 mediates growth inhibition and cell differentiation in the ER positive cell lines MCF-7 and T47D [39
], while others have found that ErbB4 promotes growth in these cell lines [41
]. In breast tumors, membranous ErbB4 is most often found to correlate with expression of the ER and with a more favorable prognosis both alone [43
] and when co-expressed with ErbB3 [45
The role for ErbB2 in growth of our resistant cell lines is not completely clear. Firstly, the level of pErbB2 is not elevated in the resistant cell lines and the gefitinib-mediated growth inhibition in the resistant cell lines is not concomitant with a general reduction of pErbB2. Secondly, we have shown that treatment of MCF-7, 182R
-5 and 164R
-7 cells with 10 μg/ml of the monoclonal ErbB2 antibody trastuzumab does not have an effect on growth [46
; data not shown]. However, as trastuzumab does not inhibit ErbB2 dimerisation [47
], we cannot exclude a role for ErbB2 as co-receptor for the activated EGFR and ErbB3 in the resistant cell lines.
Turning to the downstream kinases, we have previously shown that active Akt is required for growth of our resistant cell lines [9
]. Interestingly, the strong Ab5 mediated inhibition of the basal pErbB3 level had no effect on either Erk or Akt phosphorylation, even though the hrg1 and hrg2 mediated induction of pAkt was totally blocked by Ab5. Thus, ErbB3 activated by addition of exogenous recombinant ligand signals to Akt, whereas ErbB3 activated by endogenous ligand must signal to other yet unidentified downstream targets. A possible explanation for the inability of Ab5 and gefitinib to inhibit basal pAkt in our cell lines could be due to the fact that MCF-7 cells harbor amplification of the PIK3CA gene as well as carry the E545K mutation [48
]. Also, inhibition of either the PI-3K by Wortmannin or Akt directly by the Akt inhibitor SH-6 results in downregulation of pAkt and inhibition of the fulvestrant resistant cell growth [9
]. Furthermore, we found that treatment with the Erk inhibitor U0126 exerted a dose dependent and preferential growth inhibition of the resistant cell lines, supporting that the EGFR mediated activation of Erk is important for growth of the resistant cell lines.
These data are in concordance with other studies showing that activation of Akt and Erk is necessary for growth in both fulvestrant and tamoxifen resistant cell lines [8
]. Also, in tamoxifen treated patients, increased pAkt and pErk expression have been correlated to early relapse [51
In conclusion, we have demonstrated several alterations within the ErbB system upon acquiring resistance to fulvestrant, including activation of ErbB3, EGFR and Erk, upregulation of hrg2 and downregulation of ErbB4. The activation of ErbB3 in resistant cells occurred via production of autocrine growth factors e.g. hrg2, and abolishment of ligand binding to ErbB3 inhibited growth of resistant cells. The importance of EGFR in resistant cell growth is supported by the preferential inhibition of growth with gefitinib and with U0126. Thus, resistant cell growth involves both ErbB3 and EGFR, and the severely reduced expression of ErbB4 may be instrumental by facilitating heterodimer formation of ErbB3 with EGFR and ErbB2 in resistant cells. In order to obtain complete growth suppression of fulvestrant resistant cells, concurrent treatment with fulvestrant and compounds targeting ErbB3 and EGFR, and presumably also ErbB2, may be required.