Acetylation of Steroidogenic Acute Regulatory Protein Sensitizes 17β-Estradiol Regulation in Hormone-Sensitive Breast Cancer Cells

Abstract

An imbalance in estrogen signaling is a critical event in breast tumorigenesis. The majority of breast cancers (BCs) are hormone-sensitive; they majorly express the estrogen receptor (ER+) and are activated by 17β-estradiol (E2). The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in steroid biosynthesis. The dysregulation of the epigenetic machinery, modulating E2 levels, is a primary occurrence for promoting breast tumorigenesis. StAR expression, concomitant with E2 synthesis, was reported to be aberrantly high in human and mouse hormone-dependent BC cells compared with their non-cancerous counterparts. However, the mechanism of action of StAR remains poorly understood. We discovered StAR as an acetylated protein and have identified a number of lysine (K) residues that are putatively acetylated in malignant and non-malignant breast cells, using LC-MS/MS (liquid chromatography-tandem mass spectrometry), suggesting they differently influence E2 synthesis in mammary tissue. The treatment of hormone-sensitive MCF7 cells with a variety of histone deacetylase inhibitors (HDACIs), at therapeutically and clinically relevant doses, identified a few additional StAR acetylated lysine residues. Among a total of fourteen StAR acetylomes undergoing acetylation and deacetylation, K111 and K253 were frequently recognized either endogenously or in response to HDACIs. Site-directed mutagenesis studies of these two StAR acetylomes, pertaining to K111Q and K253Q acetylation mimetic states, resulted in increases in E2 levels in ER+ MCF7 and triple negative MB-231 BC cells, compared with their values seen with human StAR. Conversely, these cells carrying K111R and K253R deacetylation mutants diminished E2 biosynthesis. These findings provide novel and mechanistic insights into intra-tumoral E2 regulation by elucidating the functional importance of this uncovered StAR post-translational modification (PTM), involving acetylation and deacetylation events, underscoring the potential of StAR as a therapeutic target for hormone-sensitive BC.

Keywords: E2 biosynthesis; HDAC inhibitors; StAR; acetylation; breast cancer; therapeutics.

Figure 1

A schematic representation of intron and exon structures (numbered as I–VI) of the StAR gene illustrating approximate positions of the identified acetylated lysine residues. Both the NH₂-terminal mitochondrial targeting sequence and COOH-terminal StAR/cholesterol interacting regions are depicted. Acetylated StAR lysine residues, highlighted in red and black, are identified endogenously and in response to HDACIs, respectively, using LC-MS/MS.

Figure 2

A schematic representation of WT-hStAR protein structure containing K111 (A) and K253 (B) residues using the PyMOL Molecular Graphics System. Molecular visualizations of the structure of WT-hStAR containing K111 and K253 (left panels), along with K → Q and K → R substitutions at K111Q and K253Q (middle panels) and K111R and K253R (right panels), are depicted in magenta with yellow oval shapes. NH₂-terminal α-helix (amino acid, aa69–92) is outlined in blue that is followed by anti-parallel β-sheet in cyan (aa97–101, 107–112, and 118–126 interspaced by β-hairpin regions). This leads to two α-helixes separated by short loop structures in green (aa129–138 and 140–144) and mixed beta sheets (aa153–159 and 164–171), which are followed by anti-parallel beta sheets connected by loops in yellow (aa182–192 and 197–199) and in orange (aa202–203; 216–217; 219–220; 224–230; 233–243; 245–246), with the COOH-terminal α-helix in red (aa252–275). The cholesterol structure within the cavity of the StAR protein is delineated in dark blue.

Figure 3

Assessment of WT-hStAR, K111Q, K111R, K253Q, and K253R in E2 levels in MCF7 (A) and MB-231 (B) cells. These cells were transfected with EV, WT-hStAR, and different K → Q and K → R mutants, as specified, by Lipofectamine 3000 reagent. Following 48 h of transfection, culture media from different groups were analyzed for E2 levels and expressed as pg/mg protein from three to four independent experiments. Simultaneously, cells were subjected to whole cell extract preparation and representative immunoblots illustrate StAR protein expression using 70 μg of total protein. β-actin expression was assessed as loading controls in both panels. Note two different scales in Y-axes for MCF7 (A) and MB-231 (B) cells. *, p < 0.05; **, p < 0.01, ***, p < 0.001 vs. EV or other groups, as indicated.

Figure 4

A schematic representation depicting the potential mechanisms associated with StAR acetylation and deacetylation events, involving cholesterol trafficking and utilization in mammary tissue. The availability of free cholesterol from cholesterol esters is an important event in steroid hormone biosynthesis, in which hormone-sensitive lipase (HSL) plays a key role. The StAR protein regulates steroid biosynthesis by controlling the intramitochondrial transport of cholesterol. The first steroid, pregnenolone, is formed at the mitochondria by the action of the cytochrome P450scc enzyme. Pregnenolone is then converted to various steroid hormones, including androgens and estrogen/E2, by tissue-specific enzymes. Acetylation of StAR increases its biological activity and results in the overproduction of E2 synthesis for promoting breast tumorigenesis (left panel). Alternatively, a StAR deacetylation event, involving the inefficient transport of cholesterol, suppresses E2 biosynthesis for preventing tumor growth and survival (right panel).