Single Dose of a Small Molecule Leads to Complete Regressions of Large Breast Tumors in Mice

Abstract

Patients with estrogen receptor α positive (ERα+) breast cancer typically undergo surgical resection, followed by 5-10 years of treatment with adjuvant endocrine therapy. This prolonged intervention is associated with a host of undesired side effects that reduce patient compliance, and ultimately therapeutic resistance and disease relapse/progression are common. An ideal anticancer therapy would be effective against recurrent and refractory disease with minimal dosing; however, there is little precedent for marked tumor regression with a single dose of a small molecule therapeutic. Herein we report ErSO-TFPy as a small molecule that induces quantitative or near-quantitative regression of tumors in multiple mouse models of breast cancer with a single dose. Importantly, this effect is robust and independent of tumor size with eradication of even very large tumors (500-1500 mm³) observed. Mechanistically, these tumor regressions are a consequence of rapid induction of necrotic cell death in the tumor and are immune cell independent. If successfully translated to human cancer patients, the benefits of such an anticancer drug that is effective with a single dose would be significant.

Figure 1

ErSO-TFPy anticancer activity is potent and TRPM4-dependent. (A) Chemical structures of ErSO, ErSO-DFP, and ErSO-TFPy. (B) ErSO-TFPy IC₅₀ values (nM) in a panel of breast cancer cell lines. Cell viability measured via Alamar blue fluorescence at 72 h. (n ≥ 2). (C) ErSO and ErSO-TFPy induce activation of the a-UPR. MCF-7 cells were treated with ErSO or ErSO-TFPy (0–450 nM) for 6 h. Cells were harvested, lysed, and Western blotted for a-UPR markers (20 μg loaded, n = 3). (D) Western blot showing TRPM4 expression (20 μg loaded, n = 3). (E) Dose–response curves of ErSO-TFPy in MCF-7 parental and MCF-7 TRPM4 KO cells at 24–168 h (n ≥ 2). Cell viability measured via Alamar blue fluorescence, Raptinal (100 μM) used as a quantitative dead control. (F) ErSO-TFPy induces TRPM4-dependent cell swelling. Cells were treated with vehicle or ErSO-TFPy (1 μM) for 2 h following harvesting and measurement of cell diameter (n = 3). Statistical significance calculated relative to DMSO using unpaired Student t test; **** P ≤ 0.0001, ns = not significant.

Figure 2

Comparison of ErSO-TFPy to clinical drugs for breast cancer. (A) ErSO-TFPy was compared with breast cancer drugs in MCF-7 and MCF-7 ESR1^mut cell lines. CD-FBS = Charcoal stripped FBS. E2 = Estrogen. IC₅₀ values calculated by measuring cell viability via Alamar blue fluorescence at 24 or 120 h (n ≥ 2), Raptinal (100 μM) used as dead control. IC₅₀ values under 100 nM colored green, values between 100 nM and 1000 nM colored yellow gold, and values above 1000 nM colored red. (B) MCF-7 cells treated with compound for 120 h (5 days) (unless noted otherwise) and cell death measured via Trypan blue exclusion assay (n = 3). Statistical significance calculated relative to DMSO using unpaired one-way ANOVA; **** P ≤ 0.0001, ns = not significant. Descriptions of therapeutics and cell lines provided in boxes.

Figure 3

Multiple intravenous doses of ErSO-TFPy are efficacious in mouse models. (A) MCF-7 tumors were established in athymic nude mice implanted with an estrogen pellet. When tumors reached an average size of 300 mm³, mice were randomly assigned to groups, and treatment began (8 mice/group). Fulvestrant (5 mg/mouse) given subcutaneously (q7dx3). Tumor volume measured over time. (B) Mouse weight over time. (C) ST941 tumors (ESR1^mut (Y537S)) were established in athymic nude mice without estrogen supplementation. Indicated dosing began when tumors reached ∼220 mm³ (8 mice/group). Tumor volume measured over time. (D) Percent (%) tumor change at conclusion of study (day 19–21). Both studies were performed by South Texas Accelerated Research Therapeutics. Statistical significance calculated relative to vehicle using two-way ANOVA with Dunnett correction. * P ≤ 0.05, **** P ≤ 0.0001, ns = not significant.

Figure 4

A single dose of ErSO-TFPy induces complete tumor regressions in mouse models. (A) MCF-7 ESR1^mut (D538G) tumors were established in athymic nude mice without estrogen supplementation (≥4 mice/group). Vehicle and ErSO-TFPy groups dosed once intravenously at day 0, indicated by the black arrow. Fulvestrant (5 mg/mouse) given subcutaneously (q7dx3), indicated by pink arrows. Tumor volume measured over time. Picture of representative mouse at days 0 and 47 after a single ErSO-TFPy (50 mg/kg) treatment. (B) Mice from (A) previously treated with vehicle were given a single dose of ErSO-TFPy (50 mg/kg) at day 53. (C) BT-474 tumors were established in athymic nude mice implanted with an estrogen pellet (0.5 mg/pellet, 90-day release). Mice dosed intravenously with vehicle or ErSO-TFPy (50 mg/kg) once at day 0 (≥5 mice/group), indicated by black arrow. Pictures of representative mice at days 0 and day 35. (D) Mice with large tumors from (C) previously treated with vehicle (n = 4) were given ErSO-TFPy (50 mg/kg) at day 28. Statistical significance calculated relative to vehicle using two-way ANOVA with Dunnett correction. **** P ≤ 0.0001, ns = not significant.

Figure 5

A single dose of ErSO-TFPy induces rapid necrosis in tumors, followed by lymphocyte infiltration. (A) MCF-7 ESR1^mut (D538G) tumors were established in athymic nude mice (4 mice/group). Tumors were collected at indicated time following a single treatment with vehicle or ErSO-TFPy (50 mg/kg) intravenously. Average tumor mass for vehicle-treated mice at day 1 was ∼450 mg. (B) Representative H&E-stained regions (days 1, 3, 5). (C) Representative F4/80 images showing macrophage infiltrate over time. (D) ImageJ quantification of Ki67+ (proliferation marker), cleaved Casp3+ (apoptosis marker), Ly6gG+ (neutrophil marker), CD11c+ (dendritic cell marker), and F4/80+ (macrophage marker) populations within viable regions of the tumor at day 1. Statistical significance calculated relative to vehicle using two-way ANOVA with Tukey correction. **** P ≤ 0.0001, ns = not significant.