The Multifaceted Role of Autophagy in Endometrium Homeostasis and Disease

Abstract

Autophagy is a conserved fundamental cellular process with a primary function of catabolizing harmful or surplus cellular contents such as protein aggregates, dysfunctional/long-lived organelles, intracellular pathogens, and storage nutrients. An increasing body of evidence reveals that basal autophagy is essential for maintaining endometrial homeostasis and mediating endometrial-specific functions, including menstrual cycle, embryo implantation, and decidualization. However, perturbed levels of autophagy can lead to severe endometrial pathologies, including endometriosis, endometrial hyperplasia, endometrial cancer, adenomyosis, and leiomyoma. This review highlights the most recent findings on the activity, regulation, and function of autophagy in endometrium physiology and pathology. Understanding the mechanistic roles of autophagy in endometrium homeostasis and disease is key to developing novel therapeutic strategies for endometrium-related infertility and malignancies.

Introduction

Autophagy is an evolutionarily conserved catabolic process that plays an essential role in maintaining cellular homeostasis by facilitating lysosomal degradation and recycling of intracellular macromolecules and organelles. Depending on the type of cargo and route of delivery to lysosomes, autophagy can be classified into three categories: microautophagy (sequestration of cytoplasmic components directly into the lysosome, where acidic hydrolases mediate degradation), chaperone-mediated autophagy (selective degradation of unique motif-containing cargo proteins recognized and delivered to the lysosome by a chaperone complex), and macroautophagy (degradation of cytosolic material via sequestration into double-membrane vesicles called autophagosomes that subsequently fuse with lysosomes). This review focuses on macroautophagy (hereafter referred to as autophagy), which has been extensively studied for its role in endometrial physiology and pathophysiology [1–4]. In multicellular organisms, autophagy genes are intimately involved in regulating many fundamental cellular processes, such as regulation of cell death, cell proliferation, inflammation, and numerous innate and adaptive immune functions. Thus, perturbations in autophagy have been associated with aging and numerous diseases, including neurodegeneration, cancer, cardiovascular infection/inflammation, and autoimmune conditions such as inflammatory bowel disease [5]. The link between autophagy and disease pathogenesis is currently an intensive area of investigation complicated by the fact that autophagy can either be protective or harmful depending on the biological context. Recent studies revealed that autophagy also plays an indispensable role in the physiological and pathophysiological processes related to the endometrium, including cyclic menstruation, decidualization, implantation, and endometrium-related diseases such as endometrial hyperplasia, endometrial cancer, and endometriosis [6–11]. Autophagy as a basic biological activity is closely related to cell proliferation and apoptosis, which ubiquitously occur in the dynamic refreshment of the endometrium during each menstrual cycle. Thus, studying the role of autophagy in the endometrium may provide us with a better understanding of its physiology and offer clues on how to treat endometrium-related diseases or infertility. This article reviews and summarizes the most recent knowledge of autophagy in the physiology and pathology of the endometrium.

Molecular Mechanism of Autophagy

The process of autophagy involves six major steps: induction, nucleation, elongation, closure/maturation, fusion, and degradation (Fig. 1). Several distinct complexes composed of evolutionarily conserved autophagy-related proteins (ATGs) cooperate with membrane trafficking components to drive each step of autophagy (Table 1). The induction stage for the formation of autophagosome is initiated by the activation of ULK1 (also known as ATG1) complex, comprised of ULK1, ULK2, ATG13, FIP200/RB1CC1, and ATG101. Autophagosome formation is regulated by the mammalian target of rapamycin (mTOR), which acts as a nutrient sensor to directly inhibit the induction of autophagy by suppressing the kinase activity of the ULK1 complex under nutrient-rich conditions. Furthermore, nucleation of autophagosome formation requires generation of phosphatidylinositol 3-phosphate (PtdIns3P) by the PtdIns3K complex, consisting of PI3K catalytic subunit type 3 (VPS34), PI3K regulatory subunit 4 (VPS15), Beclin 1, Beclin 1-associated autophagy-related key regulator (ATG14L), and activating molecule in Beclin 1-regulated autophagy protein 1 (AMBRA1) (Fig. 1).

Fig. 1.

Autophagy pathway and its components. Autophagy is a multistage process involving six major steps: initiation/nucleation, elongation, closure/maturation, fusion, and degradation. Upon induction, the ULK1 complex comprises of serine/threonine protein kinases ULK1 and ULK2, ATG13, FIP200/RB1CC1, and ATG101-phosphorylates multiple substrates, including components of the phosphoinositide 3-kinase (PI3K) III nucleation complex to promote phagophore nucleation. The PI3K III complex comprises of VPS34, VPS15, beclin 1, ATG14L, and AMBRA1 protein. For the elongation step, expansion of phagophore required two ubiquitin-like conjugation systems, ATG12-ATG5-ATG16L and LC3 conjugation system. The conversion of cytosolic LC3-I into membrane-bound LC3-II is indicative of autophagy induction and autophagosome formation. Once completed, the autophagosome then fuses with a lysosome to form an autolysosome, in which the sequestered cytoplasmic material and cargos are degraded by lysosomal hydrolases into amino acids and macromolecules and eventually released for recycling into the cells

Table 1.

Autophagy-related genes with their specific roles in autophagy

Stage of autophagy	Autophagy-related gene	Autophagy-related function
Induction/nucleation	ATG1	Kinase required for vesicle formation and the cytoplasm-to-vacuole targeting (Cvt) pathway
	ATG13	Autophagosome formation; autophagy factor and target of TOR kinase signaling pathway
	RB1CC1/FIP200	Autophagosome formation
	C12orf44/ATG 101	Induction of autophagy
	PIK3C3/VPS34	Generates PI3P; complexes with BECN1
	PIK3R4/pl50	Part of PtdLns3K complex; involved in the nucleation of the phagophore
	BECN1	Autophagy promoting gene; part of PtdIns3K complex; complexes with UVRAG to regulate PIK3C3
	UVRAG	Interacts with PtdIns3K complex
Elongation	ATG5, ATG12, ATG16L1	Autophagic vesicle formation; forms complex that associates with phagophore membrane
	Atg8/LC3	Autophagosome maturation/expansion gene; adaptor protein for selective autophagy
	ATG9	Forms Cvt and autophagic vesicles; cycles between PAS and peripheral sites
Closure/maturation	LC3B-II	Selects cargo for degradation; receptor for phagophore
	p62/SQSTM1	Adaptor molecules on target cargo; interacts with LC3B-II
Fusion & degradation	VTI1B	Fusion of autophagosomes with endosome

Following nucleation, the membrane begins to expand. At this stage, it is called a phagophore, which is the primary double-membrane sequestering compartment. In the elongation stage, the expansion of the phagophore involves two ubiquitin-like conjugation systems, the ATG12–ATG5–ATG16L system and microtubule-associated protein light chain 3 (LC3)/ATG8 conjugation system. Autophagy conjugate 12–5 (ATG12–ATG5) is activated by the enzymes ATG7 and ATG10. The ATG12–ATG5 complex conjugates with ATG16 to expand the autophagosome membrane. The precursor of LC3 is processed by the protease ATG4 to generate the LC3-I form. Then, LC3-I is conjugated with phosphatidylethanolamine (PE) by the ATG7 and ATG3 enzymes to generate the LC3-II form, which serves as an autophagosome marker. The closure/maturation stage involves LC3B-II, which acts as a “receptor” on the phagophore, and interacts with “adaptor” molecules on the target (e.g., protein aggregates, mitochondria) to promote their selective uptake and degradation [12]. Lastly, the fusion and degradation stages first require transport of the autophagosomes to lysosomes within the cell (Fig. 1). Cytoskeleton components and cytoskeleton-related motor proteins mediate the movement of autophagosomes to lysosomes for tethering. Several proteins including UVRAG, Rubicon, presenillin-1, Valosin-containing protein, and syntaxin-5 SNARE complex proteins have been identified to help autophagosomes fusion to lysosomes [13]. Ultimately, the degradation products, including macromolecules, are then exported to the cytosol for re-utilization by the cell.

Autophagy in Endometrium Homeostasis

The endometrium is a complex dynamic tissue that undergoes sequential phases of proliferation and differentiation to support embryonic implantation during the conceptive cycle and subsequent shedding and regeneration during the non-conceptive cycle [14]. Different lines of evidence have defined the role of autophagy in maintaining endometrium homeostasis and establishment of successful pregnancy, as detailed below.

Menstrual Cycle

The human menstrual cycle consists of three sequential phases: the proliferative, secretory, and menstrual phases. Each phase is marked by physiological changes controlled by circulating levels of estrogen and progesterone secreted from the ovary. The proliferative phase is dominated by estrogen, leading to extensive glandular and luminal proliferation of the endometrium. The secretory phase is dominated by progesterone, causing inhibition of estrogen-induced proliferation and resulting in a shift from epithelial cell proliferation to differentiation [15]. In the absence of pregnancy, the abrupt fall in the levels of circulating estrogen and progesterone leads to the shedding of the endometrial layer, which marks the menstrual phase. The association of autophagy and cycling human endometrium has been demonstrated by Choi et al. (2012), where they showed the regulation of endometrial cell autophagy by ovarian steroids during the proliferative and secretory phases of the menstrual cycle. Specifically, they detected increased levels of caspase-3 and lipidated MAP1LC3A/LC3A or LC3A-II with the progression of the menstrual cycle, reaching its peak during the late secretory phase. Also, the immuno-localization for LC3A-II was higher in the glandular epithelium compared to stromal cells during the late secretory phase [6]. Earlier reports observed increased apoptosis in the glandular epithelium of the late secretory phase and menstruating endometrium compared to the proliferative phase [16–18], suggesting a potential connection between autophagy and apoptosis in the cyclic remodeling of the human endometrium. In animal models, an increased number of autophagosomes was observed in the cytoplasm of uterine epithelial cells (not stromal cells) in ovariectomized rats compared with the sham-operated rats. Similarly, the level of autophagy was higher in postmenopausal human uterine epithelial cells compared to premenopausal uterine epithelial cells indicating the onset of autophagy upon estrogen deprivation [19]. This is consistent with a significant increase in the expression of LC3 in the endometrial Ishikawa cell line upon withdrawal of progesterone or estrogen, which mimics the changes in the menstrual phase [6]. In conclusion, these findings suggest that basal levels of autophagy are maintained in the cycling endometrium and are regulated by ovarian steroid hormone levels (Fig. 2).

Fig. 2.

Cyclic changes of ovarian steroid hormones, endometrium, and autophagy level during the different phases of endometrial cycle in normal endometrium

Implantation and Decidualization

Implantation is a complex developmental process involving a reciprocal dialogue between the blastocyst and receptive uterus and is essential for embryonic development within the uterus to achieve successful pregnancy. The synchronized development of the embryo to the active stage of the blastocyst, differentiation of the uterus to the receptive state, and crosstalk between the blastocyst and uterine luminal epithelium are essential to the implantation process. For successful implantation to occur, the endometrium must undergo decidualization, a hormone-driven process in which endometrial stromal cells (ESCs) proliferate and differentiate into decidual cells [20]. In mice, this process is triggered by the attachment and implantation of the blastocyst, whereas in humans, this differentiation occurs in the luteal phase of each menstrual cycle independent of embryo implantation [20]. Successful decidualization is essential to establish and support a healthy pregnancy.

Mice lacking key genes that are important in decidualization have shown reduced fertility and pregnancy complications, such as preterm birth. Recent evidence from both in vitro and in vivo studies suggest that induction of autophagy favors decidualization, as indicated by the increased autophagic flux during the decidualization of an immortalized cell line of human endometrial stromal cells (t-HESC) [21] and increased levels of autophagy markers such as p-ULK1 in the stimulated uterine horn of mice compared to unstimulated horn [22]. Knockdown of the autophagy genes ATG5 and ATG7 impaired decidualization in t-HESC cells, which indicates the involvement of autophagy in human endometrial decidualization [21]. More recently, studies by Oestrich et al. (2020) using conditional knockout (cKO) of the autophagy genes ATG16L as well as FIP200 in the mouse uterus provided direct evidence for their role in mediating endometrial decidualization [7, 8]. Human endometrial cells lacking ATG16L or FIP200 autophagy genes in the uterus showed failed decidualization [7, 8]. Moreover, FIP200 cKO mice showed aberrant progesterone signaling in stromal cells with sustained uterine epithelial proliferation and impaired decidualization, indicating that basal autophagy is crucial for progesterone-mediated stromal cell proliferation [8].

Impaired decidualization has also been observed in diet-induced obese mice [22]. Rhee et al. demonstrated that a high level of palmitic acid, a common metabolite observed in obese individuals due to an imbalance of energy metabolism, can inhibit autophagy and cause failure of mice as well as human endometrial cell decidualization [22]. This might be one of the mechanisms that lead to poor reproductive outcomes and early pregnancy loss in obese women, thus creating a potential link between autophagy and obesity in the context of reproductive fitness. Conversely, folate acid has been reported to induce the autophagy of the endometrium, promoting the decidualization process [23]. Autophagy modulators may therefore provide potential therapeutic alternatives for the treatment of obesity-associated subfertility.

Autophagy in Endometrium-Related Pathologies

Endometriosis

Endometriosis is an estrogen-dependent disorder characterized by the presence and growth of endometrial-like tissue in locations outside the uterine cavity, including the ovaries, pelvic peritoneum, and recto-vaginal septum [24–26]. Currently affecting approximately 10% of women of reproductive age and 20–50% of infertile women, endometriosis is the third leading cause of gynecologic hospitalization each year in the USA. Moreover, endometriosis is hard to diagnose at early stage, and the treatment strategies are very limited implicating for the need of better understanding of underlying molecular pathogenesis involved therein. Recent findings have highlighted a potential connection between autophagy and pathogenesis of endometriosis. Until now, both opposing kinds of concepts have been proposed for the induction or suppression of autophagy in the progression of endometriosis [9, 27]. Decreased autophagy, along with increased mTOR activity, has been reported in postmenopausal ovarian endometriosis [27]. While Allavena et al. (2015) have reported a significant upregulation of autophagy in ovarian endometriomas compared with the eutopic endometrium of patients with endometriosis or healthy women [28], Choi et al. reported differential induction of autophagy by mTOR activity in normal and endometriotic tissue [9]. In this study, the authors demonstrated induction of autophagy and apoptosis by mTOR inhibition during the progression of the menstrual cycle in the eutopic endometrium. However, the levels of autophagy, mTOR activity, and apoptosis were constant throughout the menstrual cycle in ectopic endometriotic tissues, suggesting that a constant level of autophagy is maintained by the refrainment of mTOR activity in ectopic endometriotic tissues and is related to decreased apoptosis [9]. The same authors also demonstrated that dienogest-mediated mTOR inhibition induces autophagy leading to apoptosis of endometriotic cells. These findings suggest a possibility for new therapeutic agents targeting the mTOR pathway or other autophagy-related molecular pathways to induce autophagy and treat endometriosis [29].

Consistent with these findings, Mei and colleagues also found unaltered levels of autophagy in the ectopic endometrium of endometriosis patients during the menstrual cycle. However, there were reduced levels of Beclin 1 in ectopic secretory phase endometrial stromal cells compared to eutopic secretory phase endometrial stromal cells [30]. Other promising observations come from the Ruiz et al. study, in which authors established a mouse model of endometriosis and analyzed mRNA levels of autophagy markers [31]. The authors observed a decrease in mRNA levels of at least 13 autophagic markers in the uterine horns of endometriosis-induced mice compared to controls, indicating a possible mechanism for survival of endometriotic lesions by maintaining a suppressed level of autophagy [31]. Similarly, another study found reduced levels of Beclin 1 at both the mRNA and protein levels in ectopic and eutopic endometriotic endometrium, further confirming the suppressed status of autophagy in endometriosis. The same study also demonstrated a negative correlation between Beclin 1 protein expression and serum cancer antigen 125 (CA-125; a glycoprotein biomarker for endometriosis) [32].

Borahay et al. (2015) reported that Mullerian-inhibiting substance (MIS) can inhibit the cell growth and induces autophagy, as well as apoptosis, in ectopic endometrial cell lines indicating that MIS may have a potential as a novel approach for medical treatment of endometriosis [33]. Hydroxychloroquine, an autophagic flux inhibitor, was also found to reduce the in vitro survival capacity of human endometriotic and endometrial cells [31]. In two recent studies by Jamali et al. and Yin et al. (2020), it has been shown that treatment with quercetin or metformin alone or in combination caused decrease levels of autophagy markers and mTOR expression in a rat model of endometriosis [34, 35]. Another combinatorial drug treatment that included MK2206 and chloroquine effectively inhibited autophagy in endometriosis and prevented the recurrence of the disease [36]. These studies show a potential for therapeutic strategies targeting autophagy in the treatment of endometriosis that should be further explored. Despite these observations, the role of autophagy in endometriosis is still obscure. To gain a better understanding, additional studies are required to cement the role of autophagy in endometriosis.

Endometrial Hyperplasia

Endometrial hyperplasia (EH) is a common pelvic gynecological condition affecting women of all age groups, with the majority of cases representing with abnormal uterine bleeding. Most common gynecological diagnosis for EH is the abnormal growth of endometrial glands with or without atypia [37]. EH is a significant risk factor for the development or even co-existence of endometrial cancer with prolonged unopposed estrogenic stimulation and may also occur because of chronic disorders such as obesity, diabetes, polycystic ovarian syndrome, and delayed childbearing [38]. Endometrial hyperplasia condition is commonly managed with progestin therapy with surveillance or hysterectomy. Moreover, therapeutic options for EH patients who wish to preserve fertility are challenging and require nonsurgical management. Therefore, future studies should focus on understanding the underlying molecular mechanism involved in pathogenesis of endometrial hyperplasia and devise new treatment strategies.

The role of autophagy in endometrial hyperplasia has not been explored in depth. Currently, there are only two studies that have attempted to define the role of autophagy in endometrial hyperplasia. One study by Feng et al. demonstrated an Nrf2-dependent upregulation of an autophagy adaptor, SQSTM1, that promoted endometrial hyperplasia [10]. Using a tamoxifen-induced endometrial hyperplasia rat model, the authors observed an increase in both Nrf2 and SQSTM1 expression. Treatment with Nrf2 inhibitor, brusatol, attenuated expression of both Nrf2 and SQSTM1, suggesting that targeting the Nrf2-SQSTM1 signaling cascade could prevent tamoxifen-induced endometrial hyperplasia [10]. However, studying whether inhibition of SQSTM1 or more autophagic markers could reverse the tamoxifen-induced effect would have provided more clear evidence for the direct involvement of autophagy in EH. Another study by Wang et al. [39] showed that treatment with metformin and sorafenib alleviated endometrial hyperplasia in polycystic ovary syndrome (PCOS) by promoting apoptosis and regulating autophagy. In this study, the authors showed that endometrial and myometrial cells treated with metformin or sorafenib could upregulate LC3-II and downregulate expression of p62/SQSTM1 with metformin inducing mammalian target of rapamycin complex 1 (mTORC1) and sorafenib inducing mTORC2 expression. These findings suggest that treatment with metformin and sorafenib could decrease the risk of EH in PCOS by promoting autophagy through inhibition of mTORC1 and mTORC2, respectively [39]. However, due to the limited research, the role of autophagy in EH pathogenesis is yet to be clearly defined. Future research is warranted to explore the precise expression patterns of autophagy in EH and underlying mechanisms to develop new therapeutic agents to treat the disease.

Endometrial Cancer

Endometrial cancer (EC) was the second most common and the fourth leading cause of gynecological cancer deaths worldwide in 2018 [40]. According to the International Agency for Research on Cancer, the incidence rate of EC has increased rapidly since 2018 and is estimated to increase by more than 50% worldwide by 2040 [41]. The epidemiological risk factors associated with EC are diverse and include estrogen use, obesity, diabetes, smoking, null parity, early menarche, and late menopause. EC patients often present with low-grade, estrogen-driven, type 1 early-stage disease and have a favorable prognosis. However, those with advanced-stage, hormone-independent, type 2 metastatic disease have few treatment options and poor survival outcomes. Currently, major treatment strategies include endocrine therapy, chemotherapy, immunotherapy, radiotherapy, and hysterectomy. The clinical effectiveness of these strategies is variable among patients, implying that novel molecular therapies that target specific cellular processes associated with cell survival, such as autophagy, could be used to improve the success rates of current EC therapies.

The role of autophagy in EC is complicated and controversial because it is both pro-survival and pro-apoptotic. It has been reported that conventional antitumor therapies, including hormones, chemotherapy, and ionizing radiation, may activate autophagy in tumors as a pro-survival response, thus contributing to treatment resistance [42]. However, progressive autophagy can be cytotoxic and can result in apoptosis of tumor cells [42]. Thus, the opposing functions of autophagy during carcinogenesis make it difficult to predict which modulation strategies will result in successful treatments. In recent years, diverse approaches have been employed to delineate the precise role of autophagy in EC. An interesting study by Lebovitz et al. (2015) defined the mutational landscape of autophagy genes in endometrial cancer. Two hundred eleven autophagy-associated genes for tumor-related alterations were screened from the Cancer Genome Atlas, leading to the identification of mTOR activating mutations as well as somatic mutations in four autophagy core genes, including RB1CC1/FIP200, WDR45, ULK4, and ATG7. Moreover, type I endometrioid tumors with inactive PTEN showed over-activation of mTOR genes, indicating that autophagy is less active in type I tumors. In contrast, type II serous tumors exhibited a significant increase of the autophagy inducer CDKN2A [43], suggesting that more aggressive type II tumors might exploit autophagy as a source of energy or nutrients to promote tumorigenesis [11]. More recently, a study by Zhang et al. (2020) examined the differential expression of 37 autophagy-related genes (ARGs) in endometrial cancer compared to normal endometrium. Among the identified differentially expressed ARGs, the authors validated the induced expression of CDKN2A, PTK6, ERBB2, and BIRC5 and predicted their potential as independent predictive biomarkers and therapeutic targets for endometrial cancer [44]. A study by Sivridis et al. [45] also reported induced autophagy levels in high-grade endometrial tumors. In a series of 360 endometrial carcinomas, LC3A reactivity was recognized in three basic patterns: diffuse cytoplasmic, cytoplasmic/juxta-nuclear, and “stone-like structures” (SLS). Contrary to the diffuse and the juxta-nuclear staining that shows basal levels of autophagy and low malignant potential, SLS positive staining was associated with highly aggressive tumors, representing exaggerated autophagy levels underscoring a potential role for autophagy in tumor aggressiveness [45]. Thus, induced autophagy levels may reflect an aggressive neoplastic cell population with a high invasion and metastasis ability. This could be considered an important parameter to evaluate endometrial tumor aggressiveness, though more in-depth studies are warranted in this direction.

Aberrant expression of other autophagy core components has been shown in EC. For instance, increased Beclin 1 expression has been observed in endometrial adenocarcinomas and is associated with poor prognostic factors, such as high tumor grade, myometrial invasion, and low estimated survival [46]. A positive correlation between HIF1α and Beclin 1 expression was also found, suggesting the tumor-promoting activities of Beclin 1 under hypoxic microenvironments [46]. In contrast, another study reported a decrease in Beclin 1 and PTEN expression throughout the neoplastic transformation from endometrial hyperplasia to carcinoma [47]. However, in this study, the expression of Beclin 1 was correlated only with cell differentiation and histological type, but not with the myometrial infiltration or the pathologic stage of the tumor, which may explain the conflicting report.

Many researchers have demonstrated aberrant activation of autophagy pathways and investigated the therapeutic potential of anti-cancer drugs to modulate autophagy in EC. Several natural compounds exhibited promising effectiveness against EC, including itraconazole [48], isoliquiritigenin (ISL) [49], resveratrol [50], ABTL0812 (fatty acid-derived molecule), Fucus vesiculosus (brown seaweed) extracts [51], and triterpenoids echinocystic acid and its glycosides [52]. These compounds can induce autophagy and have been proposed as attractive therapeutic agents to treat aggressive endometrial cancers. Autophagy has also been associated with drug resistance in some EC treatment therapies as explained above. Both cisplatin and paclitaxel are the first-line chemotherapeutic drugs used for endometrial cancer and have been reported to induce autophagy in endometrial cancer cells [53]. Intriguingly, autophagy inhibition by chloroquine has been used as an effective tool to increase the sensitivity of both cisplatin and paclitaxel-resistant EC cells towards these drugs [53, 54]. Sensitivity of endometrial cancer cells to cisplatin and paclitaxel was also improved by knocking down ATG5, ATG7, or Beclin 1, emphasizing that autophagy suppression could be an effective and potent strategy to improve cisplatin or paclitaxel treatment outcomes in EC.

Exposure to excess estrogen unopposed by progesterone signaling has been well documented as a risk factor for EC. Interestingly, a group of investigators elucidated the hormonal-based regulation of autophagy in EC. A recent report by Zhou et al. demonstrated that high levels of estrogen can inhibit autophagy and induce endometrial growth by promoting glutamine metabolism. In 90% of early-stage, low-grade, hormone receptor-positive EC cases, higher expression of the progesterone receptor has been shown. This can be treated with progestin, a conservative endocrine treatment. However, about 30% of these patients are resistant to progestin treatment. Liu H. et al. reported that inhibition of autophagy through over-activation of the PI3K/AKT/mTOR pathway can promote progestin resistance in EC [55]. The authors suggested that pharmacological inhibition of the mTOR pathway using RAD001 or mTOR silencing could re-sensitize progestin-resistant cells to progestin by promoting autophagy. This suggests that mTOR is a potential target to treat the progestin-resistant EC. In another report, metformin was shown to overcome the progestin resistance in EC by promoting progesterone receptor expression, simultaneously inhibiting the mTOR pathway, and inducing autophagy [56, 57]. Similarly, metformin treatment impaired the growth of endometrial cancer cells by inducing cell cycle arrest and concomitant autophagy and apoptosis [58]. These findings demonstrate the potential use of metformin in the treatment of progesterone-resistant EC through the modulation of autophagy.

Adenomyosis

Uterine adenomyosis is a common benign condition characterized by the presence of irregular ectopic nests of endometrial glands and stroma within the myometrium [59–61]. Adenomyosis is commonly associated with menorrhagia, dysmenorrhea, chronic pelvic pain, and dyspareunia and can even interfere with embryo implantation [62, 63]. Nearly 10 to 66% of women are diagnosed at the time of hysterectomy; however, most women are diagnosed at late stages of disease due to a lack of disease bio-markers [64]. Severely symptomatic women not responding to pharmacological therapy often undergo invasive surgical intervention (hysterectomy) [63]. A recent study by Yoo et al. (2020) suggested that adenomyosis is an ovarian steroid hormone-dependent disorder resulting from high estrogen levels unopposed by progesterone, similar to endometriosis, endometrial hyperplasia, and endometrial cancer [63]. However, the precise etiology, pathophysiology, and the best methods of treating adenomyosis are not known.

Studies on the role of autophagy in adenomyosis are limited and controversial. A report by Yuefang et al. (2010) showed decreased beclin-1 expression in eutopic endometrium of women with adenomyosis, indicating reduced levels of autophagy. Conversely, a recent study by Zhou et al. (2021) highlighted that autophagy is activated in eutopic and ectopic endometrium from adenomyosis patients through decreased Indian hedgehog signaling, implying that induced autophagy may promote aberrant survival of endometrial cells at ectopic sites (65).

Other studies have shown that eutopic and ectopic endometria of women with adenomyosis develop a certain degree of progesterone resistance [65–67]. Moreover, increased expression of the estrogen receptor (ER-β) along with reduced expression of progesterone receptor (PR) has been identified in endometrium from women with adenomyosis [67, 68]. Thus, it is plausible that unopposed estrogen signaling, like other endometrial pathologies, may suppress autophagy in adenomyosis. However, further studies are required to confirm steroid regulation of autophagy in adenomyosis.

Uterine Fibroids/Leiomyoma

Uterine fibroids (UF) or leiomyoma is benign gynecological tumor that arise from the smooth muscle compartment of the uterus (myometrium) [69]. UFs are characterized by the excessive proliferation of muscle cells and fibroblasts, along with abnormal deposition of fibrous extracellular matrix [70]. It is estimated that up to 80% of women by the age of 50 are diagnosed with UF, and this condition is a common cause of heavy menstrual bleeding, pain, infertility, and pregnancy complications. Among other factors impacting the development of UF, estrogen is known as key regulator of their proliferative growth.

As in other neoplasms, autophagy is a key contributor to pathogenesis of leiomyoma [71, 72]. Dysregulated autophagy, including decreased expression of ATG4D, has been shown to promote the growth of uterine fibroids in humans [72]. Deregulation of ATG4 is frequent in tumors of the female reproductive system such as ovarian, uterine, and breast cancers. A recent study by Barbara et al. (2019) reported modulation of autophagy in leiomyoma cells with ulipristal acetate (UPA), a novel selective progesterone receptor [71]. UPA is currently under phase-II clinical trials and is the only drug approved by the European Medicines Agency for short-term treatment of uterine fibroids prior to hysterectomy and, more recently, for intermittent treatment of moderate to severe symptoms of uterine fibroids in adult women of reproductive age [73]. However, the precise mechanism by which UPA ameliorates its effect on uterine fibroids is not fully defined yet. In Barbara et al. (2019) study, treatment with UPA was shown to selectively induce expression of autophagy markers, including LC3-II protein, LC3-II/LC3-I ratio, p62/SQSTM1, and ATG4D, in leiomyoma cells, indicating its therapeutic potential in treating leiomyoma by modulating autophagy [71].

High expression of fibroblast activation protein (FAP) is closely associated with the occurrence of UF [74]. A study by Luo et al. (2014) demonstrated that estrogen promotes the uterine fibroblasts proliferation by inducing FAP, extracellular matrix components (fibronectin, laminin, collagen I) expression, and release of growth factors (TGFβ and IGF-1) [75]. Moreover, silencing FAP expression significantly decreased the growth promotional effects of estrogen on tumor-associated fibroblasts suggesting that FAP plays an important role in estrogen-mediated fibroblast activation. Additionally, decreased FAP expression has also been known to reduce expression of phosphorylated Akt, indicating that FAP may function as an upstream regulator of PI3K/Akt signaling in the initiation and regulation of autophagy. Thus, increased FAP expression in the presence of estrogen and induction of the PI3K/Akt pathway could be a possible mechanism for autophagy suppression in uterine fibroids, though further studies for more detailed understanding are required.

Overall, based on the findings described above, it can be concluded that unopposed estrogen levels, as reported in different endometrium-related diseases, might inhibit autophagy as a survival mechanism to promote cell growth at ectopic sites (Fig. 3).

Fig. 3.

Estrogen-mediated regulation of autophagy in different endometrial pathologies

Potential Therapeutic Interventions of Autophagy in Endometrial Diseases

In recent years, several efforts have been made to determine the therapeutic benefits of autophagy modulation for endometrium-related diseases, including endometrial cancer, endometrial hyperplasia, endometriosis, and leiomyoma. The major research findings involving the use of potential therapeutic compounds to treat endometrial diseases, including the mechanism of action and autophagy modulation, are summarized in Table 2.

Table 2.

Targeting autophagy in endometrial-related diseases

Disease	Drugs	Effect on autophagy	References
Endometrial cancer	Liraglutide + AICAR CB-839	Enhanced autophagosome accumulation Increased BECN1 and LC3B expression, decreased SQSTM1 levels	[76]
	Liraglutide	Induced LC3 expression, p-AMPKα, and decreased SQSTM1 protein levels	[77]
	ABTL0812 (fatty acid-derived small molecule)	Reduced phosphorylation levels of AKT and RPS6KB1, increased levels of LC3-II and autophagic flux (mRFP-GFP tandem fluorescent-tagged LC3B construct (tfLC3)) Increased TRIB3 levels and LC3-II conversion	[78]
	Rapamycin	Increased mRNA expression of BECN1, LC3B, and mTOR in Ishikawa cells. SQSTM1 mRNA levels were decreased only in IL27 overexpressed-Ishikawa cells	[79]
	Rapamycin + NK	Increased number of autophagic vacuoles and autoly so somes	[79]
	Cisplatin + NK Rapamycin + cisplatin + NK	Higher rate of autophagosomes and autolysosomes in comparison with the administered treatments	[79]
	MHY2256	Increased number of acidic vesicular organelles and levels of LC3-II and ATG5	[80]
	Cisplatin + siRNA HOTAIR cisplatin	Reduced GFP fluorescence in contrast to control Increased number of autophagosomes and LC3 expression, reduced expression of p-Akt, p-mTOR and PIK3R1	[81]
	Cisplatin + IGF1	Decreased number of autophagosomes and LC3 expression compared to cisplatin alone	[81]
	Cisplatin	Reduced GFP fluorescence in contrast to normal Ishikawa cells by using a dual-color DsRed-LC3-GFP reporter to study the increased autophagy, based on GFP-tagged LC3 reporter introducing two readouts for autophagy activity (GFP is separated from the C-terminus of LC3 by a recognition site for the autophagic protease, ATG), and autophagosomes formation	[82]
	PPD and/or metformin	Increased BECN1 expression, LC3B-II/LC3B-I ratio, and decreased SQSTM1 protein levels	[83]
	MPA	Increased protein expression of KIAA1324/EIG121, BECN1, SQSTM1, LC3B, ATG3, and ATG5 in parental Ishikawa cells and unchanged in progestin-resistant cells Decreased p-AKT-1 in parental cells, while increased p-AKT-1, KRAS, and p-MTOR and decreased PTEN expression observed in progestin-resistant cells	[55]
	Sorafenib	Increased LC3B-II and increased LC3B-II puncta. Not altered SQSTM1 mRNA levels but enhanced proteolysis. Increased autophagic flux monitored by using a chimeric mRFP-GFP tandem fluorescent-tagged LC3B construct, as well as increased phagophore and autophagosome formation Increased LC3B-II and increased LC3B-II puncta. Not altered SQSTM1 mRNA levels but enhanced proteolysis. Increased autophagic flux monitored by using a chimeric mRFP-GFP tandem fluorescent-tagged LC3B construct, as well as increased phagophore and autophagosome formation	[84]
	Sorafenib + CQ	LC3B-II puncta was further enhanced, and the decrease in SQSTM1 levels was blocked	[84]
	SI113 (SGKl inhibitor)	Increased LC3B-II and BECN1 expression	[85]
	Everolimus (RAD001)	Inhibition of mTOR and RPS6KB1/p70S6 kinase phosphorylation, increased autophagosome formation, and LC3-II expression level	[86]
	Resveratrol	Increased LC3-II expression and autophagosome accumulation	[50]
	Isoliquiritigenin	Presence of autophagosomes, increased LC3-II levels, and SQSTM1 expression	[49]
	Paclitaxel	Increased LC3-II/LC3-I ratio, LC3 punctate dots in the cytosol, decreased SQSTM1, and increased BECN1 levels in HEC-1A and JEC cells	[77]
	Paclitaxel + CQ	Accumulation of LC3-II and impaired SQSTM1 degradation	[77]
	Paclitaxel + NAC (N-acetylcysteine)	Decreased LC3 conversion in HEC-1A and JEC cells and increased SQSTM1 expression	[77]
	Paclitaxel	Accumulation of LC3 puncta, high LC3-II/LC3-I ratio and BECN1 expression	[87]
	Paclitaxel + M1R218 mimics	Decreased BECN1 expression, high level of LC3-I to LC3-II conversion, and decreased accumulation of LC3B puncta	[87]
	CQ	Increased accumulation of LC3-H and SQSTM1	[53]
	Cisplatin	Increased expression of LC3-II, slightly decreased of SQSTM1, and accumulation of autophagosomes	[53]
	Metformin	Increased acidic vesicular organelles formation, LC3-II/LC3-I ratio and SQSTM1 degradation	[58]
	Metformin	Inhibits mTOR activation and induces autophagy	[55, 56]
	Bortezomib	Accumulation of SQSTM1, LC3B expression, inhibition of cathepsin B, and stimulation of p-ERK	[88]
	Nifedipine	Increased autophagic dots, increased LC3 and BECN1 expression, and decreased RPS6KB1 levels	[89]
Endometrial hyperplasia	Metformin and sorafenib	Enhanced LC3-II expression and decreased level of mTORC1 and P62	[39]
Endometriosis leiomyoma	Mullerian-inhibiting substance	Induced autophagy with increased LC3-II and Beclin-1 expression	[33]
	Retinoic acid	Induces autophagy	[90]
	Metformin, quercetin, or metformin + quercetin	Decrease autophagy
	MK2206 + chloroquine	Decrease autophagy
	ulipristal acetate	Induces autophagy, Increase LC3-II protein, LC3-II/LC3-I ratio, and p62/SQSTMl protein expression	[71]

Conclusion and Future Perspectives

Autophagy has diverse cellular functions in the endometrium. Substantial evidence supports the important role of autophagy in maintaining endometrial physiology and pathology (Fig. 4). From the aforementioned findings, we can now conclude that aberrant levels of hormones can promote endometrium-related pathologies by modulating autophagy in the endometrium. Despite recent progress in understanding the role of autophagy in endometrium homeostasis, several questions remain unaddressed. The mechanism by which ovarian steroid hormones regulate autophagy in the endometrium has not yet been elucidated. Both epithelial and stromal cells in the uterus employ unique molecular signaling pathways to carry out uterine specific events, such as implantation and decidualization. Therefore, it will be important to determine if autophagy in epithelial or stromal cells is required to promote these events. In addition to the many questions surrounding endometrial physiology, there are numerous unknown facts about endometrial pathology. Autophagy acts as a double-edged sword in endometrium-related pathologies with a very limiting knowledge regarding the mechanisms underlying its multifaceted role. Specifically, there are very few studies on the role of autophagy in adenomyosis, leiomyoma, and menorrhagia. To this end, future studies using uterine-specific conditional knockout mice for important autophagy genes may provide detailed mechanistic insights into the role of autophagy in the endometrium. The development of methods to monitor and modulate autophagy will be vital for advances in basic research and the development of therapeutic applications.

Fig. 4.

Illustration of the role of autophagy in endometrial homeostasis and disease

Abbreviations

ATG

Autophagy-related gene

BECN1

Beclin 1

CMA

Chaperone-mediated autophagy

LC3B-II

Microtubule-associated proteins 1A/1B light chain 3B

mTORC

Mechanistic target of rapamycin complex

ULK

unc-51-like kinase

UVRAG

UV resistance-associated gene

HESCs

Human endometrial stromal cells

EC

Endometrial cancer