br Fig Synthesis pathways for analogues
Fig. 2. Synthesis pathways for analogues. Analogues 13-16. See text for more details.
3.2. Selected steroid-like SERD candidates promote ER downregulation, bind ER-positive breast tumor ML-162 and block ER-dependent transcription in vitro
We used diﬀerent assays to screen antiestrogen/SERD candidates (see Figs. 1–4), including determination of the eﬀect of antiestrogens on downregulation of ERα protein using PAGE and Western immunoblots (Fig. 5). As shown in the figure, SERD candidates JD128 and 140 were most eﬀective in reducing ER protein levels in ER-positive MCF-7 BC cells in vitro, with the eﬀect of JD128 comparable to that of fulvestrant. Additional studies were also done to assess competitive binding of SERD JD128 in MCF-7 cells (Fig. 5B) and inhibition of ER-dependent transcription in ER-positive T47D BC cells stably transfected with an ER-dependent luciferase reporter gene (Fig. 5C).
The combined results of these studies indicate that JD128 is a promising SERD with ER antagonist activity in ER downregulation, target cell binding and ER-dependent transcription comparable to that of the pure antiestrogen fulvestrant.
3.3. Steroid-like SERD JD128 inhibits human BC progression in vitro and in xenograft models in vivo
Investigations of the properties of SERD JD128 in blocking the progression of human breast tumors in vitro and in vivo. As shown in Fig. 6A, the E2-induced proliferation of several ER-positive BC cells including MCF-7, T47D and ZR75 cells was significantly inhibited by treatment with 10 nM JD128 (all at P < 0.001). This antiproliferative action of JD128 was also found with diﬀerent MCF-7 cell populations that included cells with no HER2-overexpression (MCF-7/PAR), cells
with HER2-overexpression (MCF-7/HER2) and MCF-7 cells with ta-moxifen resistance (MCF-7/TMR).
In Fig. 6B, orally administered JD128 is shown to inhibit the growth of human MCF-7 breast tumor xenografts in vivo in a dose-dependent manner. MCF-7 cells were subcutaneously inoculated in nude mice previously primed with estradiol pellets. When animals developed tu-mors of comparable size, they were randomized to treatment with ve-hicle control (vehicle) or JD128 at 15 and 75 mg/kg once a day by oral gavage. It is important to note that JD128, in contrast to fulvestrant , has potent biologic action in blocking the progression of breast tumors in vivo via an oral route of administration.
3.4. Eﬀects of estrogen and antiestrogens on expansion and activation of human immune MDSC
Emerging findings indicate that E2 can modulate expansion/activity of MDSC [7,8,17]. Since MDSC that often occur in the TME reportedly play a critical role in tumor immune tolerance and cancer progression, we assessed eﬀects of E2 and potential antagonist eﬀects of fulvestrant and JD128 (Fig. 7).
In these studies, we used archival retrospectively-collected bone marrow (BM) cells from de-identified BC patients. The BM cells were purified by established methods and then stimulated with cytokines under conditions specified in Fig. 7. Thereafter, MDSC were detected using established gating strategies by flow cytometry. When compared to MDSC derived from BM cultivated in normal medium containing E2 and cytokines, several findings are apparent: a) MDSC levels are markedly reduced in E2-free medium; b) addition of E2 to E2-depleted medium stimulates significant expansion of MDSC numbers; c) JD128
Fig. 3. Synthesis pathways for analogues. Analogues 17-20. See text for more details.
and fulvestrant each block E2-induced expansion of MDSC, and the eﬀect of JD128 exceeds that of fulvestrant at equivalent doses (all at
P < 0.05; Fig. 7B, top panel). Furthermore, the accumulation of MDSC is known to involve the expansion of immature myeloid cells and ac-tivation/conversion of immature cells to MDSC, a process that appears to be driven at least in part by STAT3 signaling . Importantly, es-trogen is reported to activate such signaling pathways in MDSC via the phosphorylation of STAT3 . Accordingly, antiestrogen JD128 is especially eﬀective in blocking the phosphorylation and activation of STAT3 in G-MDSC subsets, an action that may be crucial for blocking the enhanced immunosuppressive activity of MDSC in BC (Fig. 7B, lower panel).
3.5. Eﬀects of estrogens and antiestrogens on ERα-negative tumor growth in vitro and in vivo
TNBC cells that lack expression of ERα, PR and HER2 amplification were selected for use in experiments to investigate the potential actions of antiestrogens primarily on immune cells in the TME. In mice with implants of E2-insensitive orthotopic tumors, Svoronos et al.  re-ported a significant survival benefit associated with ovariectomy (OVX; estrogen depletion) when compared to non-OVX controls (normal es-trogen levels), while treatment of OVX mice with E2 reversed the