Progesterone metabolites regulate induction, growth, and suppression of estrogen- and progesterone receptor-negative human breast cell tumors

Abstract

Introduction: Of the nearly 1.4 million new cases of breast cancer diagnosed each year, a large proportion is characterized as hormone receptor negative, lacking estrogen receptors (ER) and/or progesterone receptors (PR). Patients with receptor-negative tumors do not respond to current steroid hormone-based therapies and generally have significantly higher risk of recurrence and mortality compared with patients with tumors that are ER- and/or PR-positive. Previous in vitro studies had shown that the progesterone metabolites, 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP), respectively, exhibit procancer and anticancer effects on receptor-negative human breast cell lines. Here in vivo studies were conducted to investigate the ability of 5αP and 3αHP to control initiation, growth, and regression of ER/PR-negative human breast cell tumors.

Methods: ER/PR-negative human breast cells (MDA-MB-231) were implanted into mammary fat pads of immunosuppressed mice, and the effects of 5αP and 3αHP treatments on tumor initiation, growth, suppression/regression, and histopathology were assessed in five separate experiments. Specific radioimmunoassays and gas chromatography-mass spectrometry were used to measure 5αP, 3αHP, and progesterone in mouse serum and tumors.

Results: Onset and growth of ER/PR-negative human breast cell tumors were significantly stimulated by 5αP and inhibited by 3αHP. When both hormones were applied simultaneously, the stimulatory effects of 5αP were abrogated by the inhibitory effects of 3αHP and vice versa. Treatment with 3αHP subsequent to 5αP-induced tumor initiation resulted in suppression of further tumorigenesis and regression of existing tumors. The levels of 5αP in tumors, regardless of treatment, were about 10-fold higher than the levels of 3αHP, and the 5αP:3αHP ratios were about fivefold higher than in serum, indicating significant changes in endogenous synthesis of these hormones in tumorous breast tissues.

Conclusions: The studies showed that estrogen/progesterone-insensitive breast tumors are sensitive to, and controlled by, the progesterone metabolites 5αP and 3αHP. Tumorigenesis of ER/PR-negative breast cells is significantly enhanced by 5αP and suppressed by 3αHP, the outcome depending on the relative concentrations of these two hormones in the microenvironment in the breast regions. The findings show that the production of 5αP greatly exceeds that of 3αHP in ER/PR-negative tumors and that treatment with 3αHP can effectively block tumorigenesis and cause existing tumors to regress. The results provide the first hormonal theory to explain tumorigenesis of ER/PR-negative breast tissues and support the hypothesis that a high 3αHP-to-5αP concentration ratio in the microenvironment may foster normalcy in noncancerous breast regions. The findings suggest new diagnostics based on the relative levels of these hormones and new approaches to prevention and treatment of breast cancers based on regulating the levels and action mechanisms of anti- and pro-cancer progesterone metabolites.

Figure 1

Conversion of progesterone to 3α-dihydroprogesterone (3αHP) and 5α-dihydroprogesterone (5αP). In vitro studies have shown that both ER/PR-positive and -negative human breast tissues and cell lines are able to convert progesterone to 3αHP and 5αP by the actions of 3α-hydroxysteroid oxidoreductase (3α-HSO) and 5α-reductase, respectively.

Figure 2

Opposing in vitro effects of 5αP and 3αHP on proliferation of MDA-MB-231 cells used in the in vivo (xenograft) studies. Cells were seeded at 4 × 10⁴ cells per dish, allowed to attach for 24 hours, and then treated for 72 hours without (C, control) or with 10^-6 M 5αP and/or 3αHP, and proliferation was determined by cell counts. Data are presented as cell number (mean and SEM; n = 4). **P < 0.01, ***P < 0.001 for the indicated comparisons or versus the control.

Figure 3

ER/PR-negative breast cell tumor induction and growth are regulated by 5αP and 3αHP. (A) Tumor induction and growth are stimulated by 5αP. MDA-MB-231 cells were implanted in mammary fat pads of 11 mice (day 0, inset); 3 days before (day -3), five mice were injected with vehicle (control; black open circles), and six were injected with 5αP (red, solid circles). Data points represent size (mm³; mean ± SEM) of tumors that developed of a total number of mice per treatment (bracketed values), and the experiment was terminated on day 40. *Significantly different from controls at P < 0.05. (B) Tumor induction and growth are stimulated by 5αP and inhibited by 3αHP. Twenty-four mice were divided into four groups of six mice each, and MDA-MB-231 cells were implanted on day 0 (inset). Two days before (day -2) and on day 12, mice were injected with either vehicle (control; black open circles), 5αP (red, solid circles), 3αHP (blue inverted triangles), or 5αP+3αHP (green squares). The 5αP-treated mice were terminated on day 46 because of tumor burden, and the other mice were terminated on day 59. Data points represent size (mm³; mean ± SEM) of tumors that developed of a total number of mice per treatment (bracketed values). Significantly different from controls at *P < 0.05 and ***P < 0.001.

Figure 4

3αHP results in suppression and regression of 5αP-induced ER/PR-negative tumors. (A) 3αHP suppresses ER/PR-negative breast cell tumorigenesis in 5αP-pretreated mice. Fourteen mice were treated with 5αP on day -3 and day 11 and then were divided into two groups. One group (Group I) continued to be treated with 5αP, whereas the other group (Group II) was treated with 3αHP on days 27, 36, and 47 (Inset). One mouse from each group was excluded from the final analysis, as explained under Results. The data are presented as the percentage of mice with tumors at termination. (B) 3αHP results in regression of 5αP-induced ER/PR-negative breast cell tumors. Twenty-four mice with MDA-MB-231 cell implants received injections of 5αP on days 0, 20, and 61 (inset); on day 75, the 14 mice with approximately similar-sized small palpable tumors (18 to 34 mm³) were divided into two groups, consisting of seven mice each, which received a single injection of either vehicle (veh) or 3αHP, and the experiment was terminated 24 days later. Bars represent size (mm³; mean ± SEM) of tumors that developed of a total number of mice per treatment (bracketed values), at the start of treatments (day 75, Initial) and at termination (day 99, Final).

Figure 5

Examples of MDA-MB-231 tumors and tumor histology from 5αP- and 3αHP-treated mice. (A) Large aggressive tumor from a 5αP-treated mouse, with histologic sections showing (B) invasion of rib-cage muscle by the spreading tumor cells, and (C) higher magnification of region with numerous mitoses (arrows). (D) Residual tumor from a 3αHP-treated mouse, with no signs of invasion (E) and showing region of tumor with numerous apoptotic and necrotic cells (F). Formalin-fixed sections (5 μm) stained with hematoxylin and eosin. Scale bars at 1.0 cm for whole tumors (A, D), at 160 µm for (B) and (E), and at 40 µm for (C) and (F).

Figure 6

Hormone levels in serum. (A) Serum hormone levels after treatment. Serum samples (n = 4 to 5) were analyzed 15 to 22 days and 42 days after mice received an injection of vehicle (control), 5αP, or 3αHP. Data points represent nanograms per milliliter (mean ± SEM). (**P < 0.01 compared with respective controls). (B) Hormone levels in serum from 10 control (vehicle only) mice, six of which had developed tumors spontaneously (With tumor), and four that remained without tumors (No tumor). Hormone levels were determined with RIA and are presented as nanograms per milliliter 5αP, 3αHP, and progesterone (Pro) and (C) as the ratio of 5αP to 3αHP. *P < 0.05; **P < 0.01 for the comparison, as indicated or versus the levels in the "No tumor" mice.

Figure 7

Hormone levels in tumors. (A) Effect of hormone treatment on 5αP and 3αHP levels in tumors. Hormone levels in tumors from four vehicle-injected, five 5αP-treated, and four 3αHP-treated mice were determined with RIA, as described in Methods. Hormone levels are presented as nanograms per milliliter 5αP and 3αHP and (B) as the ratio of 5αP to 3αHP. (**P < 0.01 for the comparison between 5αP and 3αHP levels). (C) Levels of 5αP, 3αHP, and progesterone (Pro) in tumors and respective sera from nine mice were determined after extraction with organic solvents and separation by thin-layer chromatography (TLC) as described in Methods. Levels of hormones are presented as nanograms per milliliter (serum) and nanograms per milligram (tumors), and (D), as the ratio of 5αP to 3αHP. **, ***Significantly different from serum levels at P < 0.01 and P < 0.001, respectively.

Figure 8

Summary of the opposing autocrine/paracrine effects of the progesterone metabolites, 5αP and 3αHP, in a stylized ER/PR-negative human breast cell. Evidence presented here shows that a high concentration of 5αP relative to 3αHP, in the microenvironment, promotes initiation and growth of ER/PR-negative human breast cell tumors, whereas a higher concentration of 3αHP, relative to 5αP, suppresses tumorigenesis and promotes normalcy. Progesterone is converted to 3αHP and 5αP in breast cells. Tumorigenic and tumor cells convert more progesterone to 5αP and less to 3αHP than do normal cells. The steroids, being lipophylic, are able to pass out of cells and result in a concentration buildup in the microenvironment. The result is a significant increase in the 5αP-to-3αHP concentration ratio in the microenvironment of tumorigenic cells and within tumorous tissues in comparison with normal (nontumorous) breasts. 3αHP and 5αP bind to specific receptors on the plasma membrane linked to signaling pathways involving PKC, phospholipase C, and Ca²⁺ mobilization (3αHP) and MAPK/Erk1/2 (5αP) and to modulators of gene expression. The cancer-inhibiting actions of 3αHP result in decreased proliferation and detachment of cells, increased apoptosis, and suppression of tumor initiation and growth. The cancer-promoting actions of 5αP have the opposite effects and result in stimulation of tumorigenesis and tumor growth. The evidence suggests that high concentrations of 5αP relative to 3αHP in the microenvironment will promote progression toward neoplasia and tumorigenesis, whereas a low 5αP-to-3αHP concentration ratio favors maintenance of the normal state.