The estrogen receptor (ER) status and the availability of agonists or antagonists of these receptors determine the processes of growth, differentiation, and proliferation of breast cancer cells. Estrogens and anti-estrogenic compounds have been shown to influence breast cancer cell survival/apoptosis via action through the mitochondrial enzyme proline dehydrogenase/proline oxidase (PRODH/POX).
1. Introduction
Breast cancer was the most common malignant neoplasm in women and accounted for 11.7% of all cancers globally. WHO cites obesity as one of the main reasons for the high incidence of the disease. The recent increase in the mortality of breast cancers was due to the COVID-19 pandemic that affected both therapy and prevention of the disease
[1][2]. Although several therapeutic approaches for breast cancer treatment have been established, the role of estrogen receptor (ER) status in the complex regulatory mechanisms driving apoptosis/survival of cancer cells is not fully understood.
The presence of the ER (ER+) in breast cancers increases positive response to anticancer treatment. Moreover, a better prognosis concerns progesterone receptors (PR+) and human epidermal growth factor (HER2+) positive cancers. The absence of ER is a significant risk factor for relapse and shorter life expectancy. Some authors emphasize that at least a two-receptor ER+PR+HER- expansion profile has a better prognosis than a single-receptor profile such as ER+PR-HER- or ER-PR+HER-
[3]. This is probably due to the hormonal reorganization of cellular metabolism driving pro-survival or pro-apoptotic pathways. However, the mechanisms driving apoptosis/survival are not fully understood.
2. Estrogen Receptors Structure, Location and Function
Two distinct estrogen receptor (ER) types, ERα and ERβ, are known to be encoded by two different genes located on two different chromosomes. ERα and ERβ are encoded by ESR1 (chromosome 6, region q24-q27) and ESR2 gene (chromosome 14, region q23.2). The molecular weight of ERα is 67 kDa, the ERβ isoform has 57 kDa
[4]. Both types are composed of 6 functional domains named A–F
[5]. Domains A and B are located at the amino terminal of the protein. The domain AF1 is able to activate gene transcription in the absence of bound ligand (e.g., the estrogen); however, the activation is weak. Domain C is responsible for receptor dimerization and binding of the ligand-receptor complex to a specific sequence on DNA. The D domain is also called the hinge. It has DNA-binding properties, and its sequence is more variable than that of the C domain. Next is the E domain, which contains a hydrophobic pocket structure called the ligand-binding domain (LBD). The E domain also enables dimerization of nuclear receptors. Some receptors also have an F domain, whose role is not fully elucidated (
Figure 1)
[5].
Figure 1. The structure of the estrogen receptor. ERα—Estrogen Receptor α; ERβ—Estrogen Receptor β; AF1—activator of transcription 1; C-DBD—DNA Binding Domain, domain C; D-H—Domain D-hinge; E-LBD—Ligand Binding Domain, domain E; AF-2—activator of transcription 2; NH2—amino-terminus, NH2—terminus, N—terminal end or amine-terminus; COOH—carboxylic terminus.
Non-active ERs occur in the cell cytosol, where they form large complexes with chaperone proteins of the HSP (Heat Shock Proteins) family. In this form, they are still inactive but capable of ligand attachment
[5]. Ligand binding causes dimerization of the receptor. This process is crucial for the formation of a functional transcription factor and the regulation of gene transcription interacting with the Estrogen Response Element (ERE) (
Figure 2). The molecule required for the binding of ER to DNA is FoxA1. It is a critical factor that promotes binding to chromatin
[6].
Figure 2. ER-dependent gene transcription. E2—estradiol; ER—Estrogen Receptor, HSP—Heat Shock Proteins; FoxA1—Forkhead box protein A1; ERE—Estrogen Response Element; mRNA—messenger RNA.
The distribution of ERα and ERβ receptors in tissues and organs varies. In most tissues and organs, both types of estrogen receptors are present, while in some, only one type predominates. In the ovaries, uterus, mammary gland, kidney, adrenal gland, testes, epididymis, pituitary gland, and hypothalamus, ERα expression is higher
[7][8][9] than in the urinary bladder, prostate gland, heart, and liver
[10]. The highest level of ERβ expression was found in the ovary and prostate gland
[11]. An important function of estrogen receptors is transcriptional and post-transcriptional regulation of cellular metabolism
[12]. It has been suggested that ERα is involved in the regulation of cell proliferation, while ERβ evokes anti-proliferative and pro-apoptotic activity
[13][14]. However, ERs comprise also several membranes bound receptors as G protein-coupled estrogen receptor (GPER) and Gq-coupled membrane estrogen receptor (GqmER). Recent studies revealed a functional link between all types of ERs. Interestingly, several oncogenic miRNAs have been shown to modulate the expression of ERs affecting malignant behaviour of cancer cells
[15]. Moreover, a ligand-independent signaling has been reported for ERα through kind of cross-talk with epidermal growth factor or insulin-like growth factor-I
[16][17]. Whether they are involved in PRODH/POX-dependent regulation of apoptosis/survival requires to be explored.
3. Apoptosis
Apoptosis is the process of programmed cell death, important in the development and homeostasis of multicellular organisms
[18]. This process enables the elimination of damaged, old or unnecessary cells. Initiation of the apoptosis pathway is one of the possible cell responses to intracellular or extracellular action of the chemical, physical or biological factors. The external factors that cause cell damage include UV radiation, ionizing radiation, thermal shock, low availability of oxygen and nutrients, drugs, or viral and bacterial infections
[19]. The internal factors are activated by oncogenes, cell cycle defects, deficiency of growth factors, energy, hormonal deregulation, etc.
[20][21]. Factors inducing apoptosis contribute to the development of neurodegenerative and autoimmune diseases, growth defects, and cancer. The disturbed balance between survival and apoptosis is a common feature of cancer cells
[22]. It is also the cause of resistance to chemotherapy, radiotherapy, hormonal and immune therapy
[23].
Apoptosis is a precisely regulated process by several classes of proteins. The most important are caspases (a family of intracellular cysteine proteases). They are divided into initiator, implementing, and inflammation caspases. Another important protein in the apoptosis process is the family of BCL-2 proteins (Bax; Bak, Bid, Bim), which have proapoptotic, antiapoptotic, and regulatory activities
[24].
Several pathways lead to the induction of apoptosis. The extrinsic pathway is initiated by binding a ligand to the death surface receptors
[25]. The intrinsic pathway of apoptosis can be activated by proapoptotic factors released from mitochondria. Apoptogenic molecules that are produced during intracellular stress leads to the increase in permeation of mitochondria. Both pathways stimulate apoptosis through proteolytic cleavage of pro-caspases into active enzymes
[26]. The initiator caspases include caspase-8, -9, -10, whereas caspases-3, -6, and -7 are called effector caspases
[27]. They can disrupt entire cells within a few minutes.
3.1. The Extrinsic Apoptosis Pathway
The extrinsic process of apoptosis is induced in the cell through the signals from other cells activating the death receptor, which initiates a cascade of intracellular effector proteins
[28][29]. Tumor necrosis factor (TNF) is the best-characterized protein that initiates programmed cell death
[30]. The same superfamily includes ligand of TNF family receptors (THANK), lymphotoxin (LT), Fas Ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL), or the Vascular Endothelial Growth Inhibitor (VEGI)
[31]. Some of them contain an intracellular death domain (DD). During protein binding to the receptors of the TNF family, the TRADD (Tumor necrosis factor receptor type 1-associated DEATH domain protein) or FADD (Fas-associated protein with death domain) adapter proteins interact with the DD region. Subsequently, the DISC complex (Death-inducing signaling complex) is formed
[32][33][34]. This complex combines procaspases -8 and -10 and has autoproteolytic activation properties
[35]. Cleaved caspases -8 and -10 activate the implementing caspases and initiate changes in the cell structure leading to cell death
[32]. In addition, active caspases -8 and -10 activate BID (a pro-apoptotic BCL family protein), which leads to increased release of cytochrome C from mitochondria by its truncated form tBID (
Figure 3).
Figure 3. Intrinsic and extrinsic apoptotic pathway. THANK—TNF family receptor; LT—lymphotoxin; FasL—Fas Ligand; TRAIL—TNF-related apoptosis-inducing ligand; VEGI—Vascular Endothelial Growth Inhibitor; TNF—tumor necrosis factor; DISC—Death-inducing signaling complex; FADD—Fas-associated protein with death domain); TRADD—tumor necrosis factor receptor type 1-associated DEATH domain protein; p53—tumor protein p53; APAF-1—apoptotic protease activating factor-1; ROS—reactive oxygen species; Bax, Bak, Bid, Bim—proapoptotic BCL-family proteins; tBID—truncated BID.
An important apoptosis inducer is a p53 protein. This protein participates in the external and internal pathways of apoptosis. p53 interacts with BCL (B-cell lymphoma) proteins family contributing to the upregulation of mitochondrial channels and the cytochrome C efflux into the cytoplasm, activating the internal pathway of programmed cell death
[36][37]. It has been established that p53 also induces genes coding for death receptors and death ligands
[37].
3.2. The Intrinsic Apoptosis Pathway
This pathway is also called a mitochondrial pathway. It depends on energetic and metabolic processes in the cells and is induced by stress factors. These factors are oxidative stress, DNA damage, changes in cytoplasmic calcium ions concentration, and others. Furthermore, the production of reactive oxygen species (ROS) activates pro-apoptotic BCL- family proteins
[38]. As a result of these reactions, the mitochondrial membrane is leaking
[39], leading to the release of cytochrome C from mitochondria
[38]. Released cytochrome C binds with procaspase9 and apoptotic protease activating factor-1 (APAF-1), forming apoptosome complex. The complex activates the cascade of structural changes in the cell that contribute to cell death through active forms of executive caspases such as caspase-3, caspase-6, and caspase-7 (
Figure 3)
[40][41].
4. Functional Significance of PRODH/POX in Cell Metabolism
Proline oxidase (POX), also known as proline dehydrogenase (PRODH), is a mitochondrial flavin enzyme associated with the inner mitochondrial membrane. The enzyme catalyzes proline degradation by converting this amino acid to Δ1-pyrroline-5-carboxylic acid (P5C). During this reaction, electrons are transferred via flavin adenine dinucleotide (FAD) to cytochrome C in the respiratory chain, producing ATP molecules, facilitating survival. However, when electrons are transferred directly to oxygen, that happens in specific metabolic conditions, ROS are formed, inducing apoptosis or autophagy
[42][43][44][45].
Although the mechanism for switching from ATP to ROS production is not fully understood, it has been suggested that excessive rates of electron transport may contribute to ROS generation
[46]. The mechanism of this process is based on mitochondrial membrane potential driving ATP synthase and ATP production and the Kadenbach mechanism (occurring at high ATP/ADP radio) that involves binding of ATP to cytochrome c oxidase (CytOx) and inhibition of the enzyme. In stress situation, ATP-dependent inhibition is switched off and CytOx activity is determined by membrane potential leading to an increase in ROS production. Another mechanism depends on the quantity of electron transfer to the Heme aa3 of CytOx and, in case CytOx is inhibited by ATP, ROS production is decreased. Whether PRODH/POX-dependent ATP/ROS generation involves the same mechanism requires to be explored. However, it has been found that PRODH/POX binds to Coenzyme Q1 (coQ1) decreasing respiratory fitness that was counteracted by N-acetyl-cysteine, suggesting that the effect was mediated by PRODH/POX-dependent ROS formation
[47]. Of interest is also finding that PRODH/POX is inhibited by succinate alleviating PRODH/POX effects on respiratory fitness. It suggests that PRODH/POX-induced ATP or ROS formation is metabolic contextdependent.
5. Involvement of ER Agonists in PRODH/POX-Dependent Apoptosis
ERs regulate the expression of AMP kinase (AMPK), which stimulates the activity of PRODH/POX
[48][49].
The primary ligands for ER are estrogens, which represent a group of pleiotropic hormones. There are two dominant sources of estrogens in female physiology. In the pre-menopausal age, the ovaries are the principal producer of estrogens. In the post-menopausal age, when ovarian estrogen production declines, fat tissue becomes the main source. Adipocytes have a specific enzyme called aromatase, which converts testosterone to estrogen
[50]. ER ligands—estrone, estriol, estradiol, and 2-hydroxy estrone—play functional roles in the physiology of the central nervous system, bones, reproductive and cardiovascular system. However, they also play an important role in carcinogenesis, stimulating cancer cell growth. These hormones act on the cancer cells by targeting the steroid receptor complex to specific DNA sequences, activating specific gene transcription. Several studies have demonstrated this mechanism using tamoxifen, a selective estrogen receptor modulator that inhibits estrogen-dependent tumor growth
[51].
Estrogens regulate PRODH/POX-dependent functions at the level of ER, p53, substrate availability for PRODH/POX that is dependent on prolidase activity (proline supporting enzyme) and collagen biosynthesis (proline utilizing process), as well as HIF-1α. It seems that the most important player in determining pro-apoptotic/anti-apoptotic phenotype of cancer cells is the correlation between ERα, P53, and PRODH/POX. As pointed out in the above section, PRODH/POX is a P53-induced gene promoting apoptosis. However, ERα antagonizes P53-dependent apoptosis, promoting cell survival
[52][53][54]. Based on these data, it has been established the mechanism for ERα anti-apoptotic potential, suggesting the formation of ERα-P53 complex
[55]. Since ERβ was found to attenuate the complex formation, it was concluded that ERβ has pro-apoptotic activity
[55]. Whether pro-apoptotic activity of ERβ undergoes through PRODH/POX that has either pro-apoptotic or pro-survival potential requires further study.
6. Effects of ER Modulators on PRODH/POX-Dependent Apoptosis
Phytoestrogens are natural compounds that are ER modulators. They resemble estrogens in their structure. Phytoestrogen’s ability to binding ER induces an estrogenic response or an anti-estrogenic effect
[56]. This effect depends on the concentration of the compound and the type of target tissue. Isoflavones at low concentrations have an agonist effect, and at higher concentrations, they are antagonists. Due to this feature, phytoestrogens are called selective estrogen receptor modulators (SERMs)
[57]. Phytoestrogens exhibit a broad spectrum of anticancer activity. They inhibit proliferation, invasiveness and induce apoptosis of breast cancer cells. Furthermore, they modulate the activity of ROS-scavenging enzymes
[58][59]. For instance, genistein is a characteristic isoflavone found in soybean and is the most abundant natural ERβ modulator. It has an affinity for both ERα and ERβ. However, it has a ninefold preferential affinity for ERβ. By regulating ERβ expression, genistein exerts anticancer effects. Numerous in vitro and in vivo studies have shown that genistein decreases cancer cell proliferation by blocking the cell cycle in the G2/M phase. Induction of apoptosis is associated with the activation of caspase-9 and downregulation of cyclin B1
[60][61].