Synergistic control of sex hormones by 17β-HSD type 7: a novel target for estrogen-dependent breast cancer

Abstract

17β-hydroxysteroid dehydrogenase (17β-HSD) type 1 is known as a critical target to block the final step of estrogen production in estrogen-dependent breast cancer. Recent confirmation of the role of dyhydroxytestosterone (DHT) in counteracting estrogen-induced cell growth prompted us to study the reductive 17β-HSD type 7 (17β-HSD7), which activates estrone while markedly inactivating DHT. The role of DHT in breast cancer cell proliferation is demonstrated by its independent suppression of cell growth in the presence of a physiological concentration of estradiol (E2). Moreover, an integral analysis of a large number of clinical samples in Oncomine datasets demonstrated the overexpression of 17β-HSD7 in breast carcinoma. Inhibition of 17β-HSD7 in breast cancer cells resulted in a lower level of E2 and a higher level of DHT, successively induced regulation of cyclinD1, p21, Bcl-2, and Bik, consequently arrested cell cycle in the G(0)/G(1) phase, and triggered apoptosis and auto-downregulation feedback of the enzyme. Such inhibition led to significant shrinkage of xenograft tumors with decreased cancer cell density and reduced 17β-HSD7 expression. Decreased plasma E2 and elevated plasma DHT levels were also found. Thus, the dual functional 17β-HSD7 is proposed as a novel target for estrogen-dependent breast cancer by regulating the balance of E2 and DHT. This demonstrates a conceptual advance on the general belief that the major role of this enzyme is in cholesterol metabolism.

Keywords: 17β-hydroxysteroid dehydrogenase type 7; breast cancer; steroid enzyme inhibition; xenograft tumor.

Figure 1

Cytostatic effect of INH7/INH1 in MCF-7 cells. (A) Cell proliferation assay of MCF-7 treated with INH7 or INH1. Data are reported as % of DNA synthesis vs. control (100%). Each point represents the mean of experiments carried out in quadruplicate (mean ± SD). Statistical significance by Student's t-test: *P < 0.05 vs. control (Ctrl). (B) Cell cycle analysis of MCF-7 cells treated with INH7 or INH1. Data are reported as % of living cells (G₀/G₁, S, and G₂/M cells = 100%). Each number represents the mean of experiments carried out in triplicate (mean ± SD). Statistical significance (P < 0.05) by Student's t-test is highlighted in bold box in table. (C) E2 and DHT formation in MCF-7 cells treated with INH7 or INH1. Each bar represents the mean of experiments carried out in quadruplicate (mean ± SD). Statistical significance by Student's t-test: *P < 0.05 vs. control (Ctrl); **P < 0.001 vs. Ctrl. (D) Cyclin D1, p21, and PCNA protein expression determined by western blot in MCF-7 cells treated with INH7. Data are reported as mean ± SD (n = 3) of the individual experiments. Statistical significance by Student's t-test: *P < 0.05 vs. control (Ctrl); **P < 0.001 vs. Ctrl. (E) 17β-HSD1/7 protein expression determined by western blot in MCF-7 cells treated with INH1 and INH7, respectively. Data are reported as mean ± SD (n = 3) of the individual experiments. Significant differences by Student's t-test: *P < 0.05 vs. control (Ctrl).

Figure 2

Apoptotic effect of INH7/INH1 in MCF-7 cells. (A) Cell viability of MCF-7 treated with INH7 or INH1 by MTT assay. Each point represents the mean of experiments carried out in quadruplicate (mean ± SD). Statistical significance by Student's t-test: *P < 0.05 vs. Ctrl. **P < 0.001 vs. Ctrl. (B) Cell apoptosis of MCF-7 treated with INH7 or INH1. Each number represents the mean of experiments carried out in triplicate (mean ± SD). Statistical significance (P < 0.05) by Student's t-test is highlighted in bold box in table. Flow cytometry panels represent the distribution of each cell type after treatment. (C) Expression of Bcl-2, Bik, and pro-caspase 7 in INH7-treated MCF-7 cells. Data are reported as mean ± SD (n = 3) of the individual experiments. Statistical significance by Student's t-test: *P < 0.05 vs. Ctrl; **P < 0.001 vs. Ctrl.

Figure 3

In vivo study of INH7-treated xenograft MCF-7 breast tumor. (A) Regression of xenograft tumor growth in OVX nude mice by INH7. The growth regression curves represent the decrease in tumor volume in response to INH7 treatment. Black arrow represents the date of treatment interruption (Day 21). Blue arrow represents the treatment starting again on Day 28. Statistical significance by one-way ANOVA assay is shown by *P < 0.05. Tumor weight bar graph represents the shrinkage of tumor by INH7. Statistical significance by T-test is shown by *P < 0.05. (B) Pathohistological analysis of INH7-treated MCF-7 xenograft tumors. Representative images show histological tumor sections stained by hematoxylin–eosin–saffron (HES Stain) and Masson's Trichrome (MT Stain). The table reports the pathohistological scores of cancer cell organization and tumor stroma quantity. Bar graph represents cancer cell density within tissue. Statistical significance by Student's t-test: *P < 0.05. Scale bar, 40 µm. (C) Immunohistological analysis of 17β-HSD7 expression level within xenograft tumor tissue. Representative images of immune-reactivity by DAB (brown) reveal 17β-HSD7 expression within tumor tissue. Bar graph represents quantification results of immune-reactivity of 17β-HSD7 with ImageJ software. Statistical significance by Student's t-test: *P < 0.05. Scale bar, 100 µm. (D) The effect of INH7 on plasma E2 and DHT concentrations (left) and estrogen-responsive organ (uterus and vagina) weights (right) after treatment with INH7. Statistical significance by Student's t-test: *P < 0.05 vs. control (Ctrl).

Figure 4

Integrative analysis of 17β-HSD7 overexpression in neoplastic breast vs. normal breast type. (A) A box plot shows relative 17β-HSD7 expression in TCGA breast datasets grouped by cancer and normal tissue. The box reflects the interquartile range, the whiskers reflect the 10%–90% range, and the dots reflect the minimum and maximum values. Legend code indicates different cancer/normal type described in B. Horizontal axis units are normalized expression values (standard deviation above or below the medium per array). Data are normalized to facilitate inter-study comparison. The box plot was downloaded from ONCOMINE (Rhodes et al., 2004). (B) Comparison of 17β-HSD7 overexpression in different cancer types vs. normal breast tissue. P-value indicates the difference between two group comparisons. Fold change indicates the fold difference in 17β-HSD7 overexpression in cancer type vs. normal breast tissue.

Figure 5

Schematic mechanisms of INH7 toward ER⁺ breast cancer cells. Inhibition of 17β-HSD7 by INH7 blocks E2 formation from E1 and inhibits DHT conversion into the weak estrogen metabolite (3β-diol). Furthermore, blockage of E2 conversely attenuates the expression of 17β-HSD7 itself. A decrease in E2 weakens the estrogenic effect through ER and an increase in DHT strengthens androgenic effect through AR, synergistically leading to post-receptor gene transcription culminating in cytostatic and/or cytotoxic effects.