Metastasis tumor-associated protein 2 enhances metastatic behavior and is associated with poor outcomes in estrogen receptor-negative breast cancer.

Metastasis remains a major clinical problem in breast cancer. One family of genes previously linked with metastasis is the metastasis tumor-associated (MTA) family, with members MTA1 enhancing and MTA3 inhibiting cancer metastasis. We have previously found that MTA2 enhances anchorage-independent growth in estrogen receptor α (ERα) breast cancers, and, in combination with other genes, performed as a predictive biomarker in ERα-positive breast cancer. We therefore hypothesized that MTA2 enhances breast cancer progression. To test this, cell growth, soft-agar colony formation, migration, and in vivo metastasis were examined in MTA2-overexpressing and Vector control transfected ERα-negative breast cancer cells. Pathways regulating cell-cell interaction, adhesion, and signaling through the Rho pathway were also investigated. Effects of the inhibition of the Rho pathway using a Rho Kinase inhibitor were assessed in soft-agar colony formation and motility assays in MTA2-overexpressing cells. MTA2 expression was associated with poor prognostic markers, and levels of MTA2 were associated with increased risk of early recurrence in retrospective analyses. MTA2 overexpression was associated with enhanced metastasis, and pathways regulating cell-cell interactions in vitro and in vivo. Most critically, MTA2-enhanced motility could be blocked by inhibiting Rho pathway signaling. We present the novel finding that MTA2 defined a subset of ERα-negative patients with a particularly poor outcome.

A hypersensitive estrogen receptor alpha mutation that alters dynamic protein interactions.

Estrogen receptor alpha (ERalpha) is highly regulated through multiple mechanisms including cell signaling, posttranslational modifications, and protein-protein interactions. We have previously identified a K303R ERalpha mutation within the hinge region of ERalpha. This mutation results in an altered posttranslational regulation and increased in vitro growth in the presence of low estrogen concentrations. We sought to determine if cells expressing this mutant ERalpha would display hypersensitive tumor growth in in vivo athymic ovariectomized nude mice. MCF-7 cells, stably expressing the K303R ERalpha, formed tumors in nude mice faster than cells expressing wild-type ERalpha in the presence of low levels of estrogen. When estrogen was withdrawn, all tumors regressed but half of the K303R ERalpha-expressing tumors became estrogen-independent and regrew. We evaluated potential mechanisms for the observed hypersensitivity. The mutant ERalpha did not demonstrate increased estrogen binding affinity, but did exhibit increased interactions with members of the SRC family of coactivators. The mutant ERalpha demonstrated increased levels and occupancy time on the pS2 promoter. In the presence of the K303R ERalpha, the SRC-3 and p300 coactivators also displayed increased levels and time on the pS2 promoter. The K303R ERalpha has, in part, lost critical negative regulation by the F domain. Collectively, these data demonstrate an important role for the K303R ERalpha mutation in hormonal regulation of tumor growth and estrogen-regulated promoter dynamics in human breast cancer.

Expression of the K303R estrogen receptor-alpha breast cancer mutation induces resistance to an aromatase inhibitor via addiction to the PI3K/Akt kinase pathway.

Aromatase inhibitors (AI) are rapidly becoming the first choice for hormonal treatment of estrogen receptor-alpha (ERalpha)-positive breast cancer in postmenopausal women. However, de novo and acquired resistance frequently occurs. We have previously identified a lysine to arginine transition at residue 303 (K303R) in ERalpha in premalignant breast lesions and invasive breast cancers, which confers estrogen hypersensitivity and resistance to tamoxifen treatment. Thus, we questioned whether resistance to AIs could arise in breast cancer cells expressing the ERalpha mutation. As preclinical models to directly test this possibility, we generated K303R-overexpressing MCF-7 cells stably transfected with an aromatase expression vector. Cells were stimulated with the aromatase substrate, androstenedione, with or without the AI anastrozole (Ana). We found that Ana decreased androstenedione-stimulated growth of wild-type cells, whereas K303R-expressing cells were resistant to the inhibitory effect of Ana on growth. We propose that a mechanism of resistance involves an increased binding between the mutant receptor and the p85alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI3K), leading to increased PI3K activity and activation of protein kinase B/Akt survival pathways. Inhibition of the selective "addiction" to the PI3K/Akt pathway reversed AI resistance associated with expression of the mutant receptor. Our findings suggest that the K303R ERalpha mutation might be a new predictive marker of response to AIs in mutation-positive breast tumors, and that targeting the PI3K/Akt pathway may be a useful strategy for treating patients with tumors resistant to hormone therapy.