Estrogen (E-2) supports breast cancer cell growth but suppresses invasiveness and both actions are antagonized by anti-estrogens. As a consequence, anti-estrogen treatment may increase the invasive potential of estrogen receptor (ER)+ tumor cell sub-populations that are endocrine resistant due to HER2 amplification. Either transactivation or transrepression by E-2/ER could lead to both up- and down-regulation of many genes. Inhibition of the transactivation function of ER is adequate to inhibit E-2-dependent growth. However, the impact of inhibiting E-2-dependent transactivation vs. transrepression by ER on regulation of invasiveness by E-2 is less clear. Here we dissect the roles of ER-mediated transactivation and transrepression in the regulation of invasiveness of ER+/HER2+ breast cancer cells by E-2. Knocking down the general ER co-activators CBP and p300 prevented activation by E-2 of its classical target genes but did not interfere with the ability of E-2 to repress its direct target genes known to support invasiveness and tumor progression; there was also no effect on invasiveness or the ability of E-2 to regulate invasiveness. On the other hand, overexpression of a co-repressor binding site mutant of ER (L372R) prevented E-2-dependent transrepression but not transactivation. The mutant ER abrogated the ability of E-2 to suppress invasiveness. E-2 can partially down-regulate HER2 but knocking down HER2 below E-2-regulated levels did not affect invasiveness or the ability of E-2 to regulate invasiveness, although it did inhibit growth. Therefore, in ER+/HER2+ cells, the E-2-dependent transrepression by ER rather than its transactivation function is critical for regulation of invasiveness and this is independent of HER2 regulation by E-2. The findings suggest that selective inhibitors of transactivation by ER may be more beneficial in reducing tumor progression than conventional anti-estrogens that also antagonize E-2-dependent transrepression. (C) 2015 Elsevier Inc. All rights reserved.