Breast cancer is the most frequent cancer in women and represents the second leading cause of cancer death in women. Since the discovery of estrogen receptor (ER) as the modulator of estrogen action, ER has evolved to be one of the most effective targets for breast cancer therapy. Currently available methods for interfering with estrogen action are comprised of ‘antiestrogens’ that either compete with estrogens for binding to ER or reduce the circulating ER levels and ‘aromatase inhibitors’ that inhibit estrogen biosynthesis. However, despite being the ideal treatment of choice for patients with ER-expressing breast cancer, these current ER-targeting therapies have a main limitation related with resistance. Half of the breast cancers expressing hormone receptors are inherently resistant to the current endocrine therapy, and most initially responsive breast tumors acquire resistance. The anticancer drug XR5944 was originally developed as a topoisomerase inhibitor and subsequently shown to be a transcription inhibitor. It has demonstrated exceptional anticancer activity both in vitro and in vivo and was significantly more potent than traditional topoisomerase inhibitors. The solution structure of the XR5944/DNA complex recently obtained in our lab indicates that XR5944 has a novel DNA binding mode. XR5944 bis-intercalates at the 5'-(TpG):(CpA) site, with the carboxamide aminoalkyl linker lying in the major groove of the DNA. Such binding in the major groove is significant since most DNA binding drugs bind in the minor groove, while transcription factors generally bind in the major groove. The DNA bis-intercalating site of XR5944 at 5'-(TpG):(CpA), which is found in the consensus DNA binding site of ER, appears to be highly specific. Thus, we tested the ability of XR5944 to inhibit ER activity both in vitro and in cultured cells. In electrophoretic mobility shift assays, it is seen that the DNA binding of recombinant ERα protein, as well as ER from nuclear extracts, is inhibited by XR5944 in a dose-dependent manner. In luciferase reporter assays, XR5944 inhibited the reporter gene expression from an estrogen response element-containing promoter, but not from a basal promoter sequence that lacks any cis-acting elements. In contrast, the RNA polymerase inhibitor, actinomycin D, inhibits the transcription from both the above-mentioned promoters. The specificity of XR5944 activity is displayed by a separate reporter assay in which the transactivation of reporter gene expression by Sp1 proteins was not inhibited by XR5944. Collectively, these data suggest that XR5944 is capable of specifically inhibiting the binding of ER to its consensus DNA sequence and its subsequent activity. This represents a novel mechanism of ER inhibition, which may be useful in overcoming resistance to current antiestrogens.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA