Abstract Introduction: Conversion of androgens to estrogens within breast tumors by aromatization plays a pivotal role in the proliferation of tumor cells expressing the estrogen receptor (ER). Testosterone (T) has demonstrated breast cancer (BC) cell proliferation effects in vitro and clinically has been linked to a breast cancer signal in postmenopausal women. Given this link, the increasing number of women using T therapy and its potential association with an increased risk of breast cancer is of concern. However, the androgen receptor (AR) in general, which T binds to, has been postulated to have BC suppressive effects and in certain clinical situations T has been reported to be an effective BC treatment. The ability to decouple these countervailing proliferative and antiproliferative effects of T in BC cell growth is of interest. AVA-291 (d3-testosterone) is a structurally identical, deuterium-substituted isotopologue of T that retains T’s androgen effects and hepatic metabolic profile but is highly resistant to aromatization. In the studies described here, the ER+ BC cell line MCF-7 was used to assess the relative proliferative effects of T or AVA-291 (ranging from 0.1 nM to 3 µM) in vitro. Single 10 nM concentrations of estradiol (E2) and dihydrotestosterone (DHT) were included as positive and negative controls, respectively. Results: Consistent with literature reports, T stimulated MCF-7 cell proliferation in a concentration-dependent manner. The proliferative effects of T were observed starting at 1 nM reaching a maximum effect at 10 nM. In contrast, AVA-291 did not stimulate proliferation over a broad concentration range with a proliferative effect only observed at concentrations ≥1 µM. As expected, the positive control (10 nM E2) demonstrated maximal stimulation and the negative control (10 nM DHT) resulted in no proliferative effect. Conclusions: We demonstrate that T has a proliferative effect on the ER+ BC cell line MCF-7 and that AVA-291, a non-aromatizing form of T, did not stimulate cell proliferation at similar concentrations. These findings confirm prior literature reports on this effect by T and the purported underlying mechanism of androgen aromatization by MCF-7 ER+ BC cells. These results suggest that AVA-291 may be a useful alternative to T in clinical situations where the aromatization of T limits its therapeutic potential, such as hormone replacement therapy in postmenopausal women. Citation Format: Paul M. Tarantino, Judith A. Boice, Pamela A. Trail, Bradford C. Sippy. Effects of AVA-291 (d3-testosterone) versus testosterone on MCF-7 estrogen receptor positive breast cancer cells - lack of aromatization to estradiol leads to a highly differentiated cellular proliferation profile abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2297.
Tarantino et al. (Fri,) studied this question.