Autophagy in the physiological endometrium and cancer

Figures & data

Figure 1. Schematic representation of the autophagy process. Autophagy is a multi-stage process that comprises several steps: initiation, expansion, closure, and fusion with the endolysosomal system. The process initiates with the progressive sequestration of a portion of the cytoplasm that is enclosed within a double-membrane (a phagophore) to finally form an autophagosome. The initiation involves the transmission of the autophagy signal, which is triggered by multiple conditions, such as nutrient starvation, and canalized through specific cell sensors such as AMP-activated protein kinase (AMPK), HIF1A (hypoxia-inducible factor alpha) and mechanistic target of rapamycin kinase complex 1 (MTORC1) to the membrane of origin at which the nucleation takes place. As a consequence, there is a recruitment of the key initiation complexes. The ULK1 (unc-51 like autophagy activating kinase 1) complex plays a central role in the initiation stage, and it is the target of these signaling pathways and, when activated, triggers the nucleation of the phagophore by phosphorylating components of the class III phosphatidylinositol 3-kinase (PtdIns3k) complex I. Then, WIPI2 (WD repeat domain, phosphoinositide interacting 2), and ZFYVE1 (zinc finger FYVE domain containing 1) are recruited to the omegasome. WIPI2 binds and is activated by phosphatidylinositol 3-phosphate (PtdIns3P). Once activated, WIPI2 recruits at phagophore assembly sites the ATG12–ATG5-ATG16L1 complex that enhances the ATG3-mediated conjugation of LC3 (microtubule-associated protein light chain 3) to phosphatidylethanolamine. LC3-I is converted into its lipidated form, LC3-II. Membrane material for the nucleation of the phagophore and the elongation of the autophagic membrane arises from the ER, mitochondria, recycling endosomes, Golgi apparatus, and plasma membrane from which ATG9-containing vesicles provide portions of lipid bilayers. Finally, the expansion continues until the complete formation and closure of the autophagosome. The outer membrane of the autophagosome subsequently fuses with the lysosome, giving rise to the autolysosome. After lysosomal digestion, the sequestered cytoplasmic material is degraded into amino acids and macromolecules, transported across the lysosomal membrane to the cytosol, and finally recycled for anabolic processes

Box 1. Definitions

Autophagy: An intracellular catalytic process that recycles damaged cellular organelles and other constituents by delivering them into the lysosomes. Chaperone-mediated autophagy: a form of autophagy characterized by the delivery and degradation of cellular constituents by a chaperone-dependent selective process. Microautophagy: a form of autophagy whereby cellular cargo is degraded within lysosomes by direct lysosomal/vacuolar engulfment. Macroautophagy: a form of autophagy characterized by the formation of double-membrane vesicles that will fuse with the lysosomes to degrade organelles and other cellular components.

Figure 2. Illustration of endometrial layers and tissue components dynamic changes during the course of the different phases of the menstrual cycle. The endometrium, composed of a single layer of columnar epithelial cells plus stroma, can be subdivided into two layers: the functional, which faces the uterine cavity, and the basal layer in direct contact with the myometrium. Both layers undergo structural changes during the menstrual cycle. Menstruation constitutes the first phase of the menstrual cycle, and it is characterized by the complete desquamation of the functional layer of the endometrium and bleeding (menses). This stage precedes the proliferative phase, characterized by the increase in the synthesis and secretion of estrogens as the ovarian follicles mature (ovarian follicular phase). The estrogens will promote the regeneration of the functional layer from the basal layer of the endometrium. By day 14, a peak in LH secretion triggers a decrease in estrogen production, ovulation, and the formation of the corpus luteum (luteal ovarian phase), which represents the primary source of progesterone. Progesterone will prepare the endometrium for future implantation by fostering global endometrial thickening characterized by increased spiral artery length and coiling, uterine secretions, and reduced smooth muscle cell contractility. In the absence of pregnancy, the demise of the corpus luteum will cause a dramatic drop in the progesterone and estrogen levels, and the initiation of the menstruation starts again

Box 2. Definitions

Implantation: a coordinated process by which the blastocyst orientates, attaches, invades, and finally embeds itself into the maternal endometrium. Decidualization: the differentiation process that undergoes endometrial fibroblast-like mesenchymal stromal cells to epithelioid-like cells in order to accommodate pregnancy. Menopause: a condition that develops at the end of lifelong menstrual cycles characterized by an insufficient stimulation of the endometrium in response to a primary ovarian failure and a dramatic decrease of circulating estrogen. The endometrium might become thinner as a result of low estrogen levels and is described as being atrophic.

Table 1. Targeting autophagy in endometrial cancer

References	Model	Drug combination	Effects on autophagy and related molecules	Role of autophagy
Fukuda T et al., Gynecol Oncol 2015 [71]	In vitro: Ishikawa, KLE, and AN3CA TP53 mutated EC cell lines, as well as the HEC-6, HEC-150, HEC-108 cells	CQ	Increased accumulation of LC3-II and SQSTM1	Cell proliferation, cytoprotective effect (cell survival)
	In vitro: Established Ishikawa cisplatin-resistant cell line	Cisplatin	Increased expression of LC3-II, slightly decreased of SQSTM1, and accumulation of autophagosomes
		Cisplatin + CQ	NA
Sun M et al., Biotechnol Lett 2017 [72]	In vitro: Ishikawa EC cell line and its cisplatin-resistant sub-clone	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	Cell survival
		Cisplatin + siRNA HOTAIR	Reduced GFP fluorescence in contrast to control	Apoptosis, cell death
Lin Q et al., Oncol Lett 2017 [73]	In vitro: Ishikawa EC cell line	Cisplatin	Increased number of autophagosomes and LC3 expression, reduced expression of p-Akt, p-MTOR and PIK3R1	Not assessed
		Cisplatin + IGF1	Decreased number of autophagosomes and LC3 expression compared to cisplatin alone
Kao C et al., Cell Death Dis 2014 [80]	In vitro: Ishikawa EC cell line	Bortezomib	Accumulation of SQSTM1, LC3B expression, inhibition of cathepsin B, and stimulation of p-ERK	Cell survival
Umasankar et al., Int J Mol Sci 2018 [82]	In vitro: Ishikawa EC cell line	MHY2256	Increased number of acidic vesicular organelles and levels of LC3-II and ATG5	Potential cell death
	In vivo: Ishikawa EC cells were injected subcutaneously. MHY2256 (5 mg/kg) was injected intraperitoneally (twice per week)		NA
Zhou W-J et al., Neoplasia 2018 [83]	In vitro: Ishikawa, RL95-2 and KLE endometrial cancer cells. IL27-overexpressed EC cells and control were treated ± rapamycin and/or cisplatin for 48 h and were co-cultured with NK cells. NK cells were collected and co-cultured with fresh EC cells	Rapamycin	Increased mRNA expression of BECN1, LC3B, and MTOR in Ishikawa cells. SQSTM1 mRNA levels were decreased only in IL27 overexpressed-Ishikawa cells	Promotion of NK cells cytotoxicity
	In vivo: Nude mice were inoculated subcutaneously with Ishikawa EC cells (IL27-overexpressing cells on the right or negative control on the left). Rapamycin ± cisplatin or PBS was intraperitoneally injected for 3 weeks after xenotransplantation. Human NK cells from peripheral blood were transferred to Ishikawa-xenografted nude mice every 7 days for the first 2 weeks	Rapamycin+NK	Increased number of autophagic vacuoles and autolysosomes
		Cisplatin+NK
		Rapamycin+Cisplatin+NK	Higher rate of autophagosomes and autolysosomes in comparison with the administered treatments
Felip I et al., Gynecol Oncol 2019 [87]	In vitro: Ishikawa, AN3CA, HEC-1A, ARK-1 and ARK-2 EC cell lines	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))	Potential cell death mechanism
	In vitro: Established 3D cultures from a PTEN knock-out mice model, with the tamoxifen-induced CRE recombinase (Cre:ER^+/−) under the estrogen promoter		Increased LC3-II levels
	In vivo: HEC1-A subcutaneous model	ABTL0812	Increased TRIB3 levels and LC3-II conversion
	PDX models (endometrioid and serous)	ABTL0812 + Carboplatin + Paclitaxel	NA
Liu S et al., Int J Oncol [88]	In vitro: Hec-1A, JEC, Ishikawa and AN3CA EC cell lines. The highest IC50 values were found in HEC-1A cells and JEC cells, respectively, and were identified as “paclitaxel-resistant cells”	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	Cell survival
		Paclitaxel + CQ	Accumulation of LC3-II and impaired SQSTM1 degradation
		Paclitaxel + NAC (N-acetyl-cysteine)	Decreased LC3 conversion in HEC-1A and JEC cells, and increased SQSTM1 expression
Ran X et al., Int J Clin Exp Pathol [89]	In vitro: Ishikawa and RL95-2 EC cells. Paclitaxel-resistant cells were also generated	Paclitaxel	Accumulation of LC3 puncta, high LC3-II:LC3-I ratio and BECN1 expression	Cell survival
		Paclitaxel + MIR218 mimics	Decreased BECN1 expression, high level of LC3-I to LC3-II conversion and decreased accumulation of LC3B puncta
Wang H et al., Oncol Lett 2016 [90]	In vitro: Ishikawa and HEC-1A EC cell lines	Everolimus (RAD001)	Inhibition of MTOR and RPS6KB1/p70S6 kinase phosphorylation, increased autophagosome formation and LC3-II expression level	Cell death, apoptosis
		Paclitaxel + everolimus	NA
Takahashi A et al., Cancer Cell Int 2014 [97]	In vitro: Ishikawa EC cell line	Metformin	Increased acidic vesicular organelles formation, LC3-II:LC3-I ratio and SQSTM1 degradation	Cell death, apoptosis
Kanda R et al., BMC Cancer 2018 [99]	In vitro: Ishikawa EC cell line	Liraglutide	Induced LC3 expression, p-AMPKα and decreased SQSTM1 protein levels	Potential cell death mechanism
		Liraglutide + AICAR	Enhanced autophagosome accumulation
Zhou WJ et al., Cell Commun Signal 2019 [100]	In vitro: Ishikawa and KLE EC cell lines	CB-839	Increased BECN1 and LC3B expression, decreased SQSTM1 levels	Potential cell death mechanism
			Enhanced autophagosome accumulation
	In vivo: Subcutaneous injection of Ishikawa cells, and implant estrogen pellets (60 d time release, at 0.72 mg β-estradiol/pellet). When the tumor volume reaches approximately 100–150 mm3, the mice were treated orally twice a day with the vehicle or the 200 mg/kg CB-839 solution.		Autophagosome and autolysosome accumulation
Gu C-J et al., J Transl Res 2017 [101]	In vitro: Ishikawa and RL95-2 EC cell lines	PPD and/or Metformin	Increased BECN1 expression, LC3B-II:LC3B-I ratio and decreased SQSTM1 protein levels	Potential cell death mechanism
	In vivo: Ishikawa cells were inoculated subcutaneously into nude mice. PPD and/or metformin were injected intraperitoneally once a day after xentransplantation		NA
Zhuo et al., Arch Gynecol Obstet 2016 [105]	In vitro: Ishikawa EC cell line and Ishikawa progesterone-resistant cells	Metformin	Upregulation of LC3, p-AMPK and BECN1 protein levels	Potential cell death mechanism
Liu H et al., Onco Targets 2017 [106]	In vitro: Ishikawa and progestin-resistant Ishikawa EC cells, by exposing Ishikawa to long-term MPA treatment	MPA	Increased protein expression of KIAA1324/EIG121, BECN1, SQSTM1, LC3B, ATG3, and ATG5 in parental Ishikawa cells and unchanged in progestin-resistant cells	Anti-proliferative effect
			Decreased p-AKT-1 in parental cells, while increased p-AKT-1, KRAS, p-MTOR and decreased PTEN expression observed in progestin-resistant cells
		Everolimus (RAD001)	Inhibition of MTOR phosphorylation, and increased expression of LC3B, ATG3, and ATG5
Eritja N et al., Autophagy 2017 [127]	In vitro: 2D model HEC-1A, Ishikawa, RL95-2, and KLE EC cell lines	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	Cell survival, cytoprotective effect
		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	Cell survival, cytoprotective effect
		Sorafenib + CQ	LC3B-II puncta was further enhanced and the decrease in SQSTM1 levels was blocked
	In vitro: 3D organotypic Ishikawa cells	Sorafenib	Increased LC3-II expression, autophagosome formation and decreased SQSTM1
	In vivo: Subcutaneous model: HEC-1A cells were injected in immunodeficient SCID hr/hr mice. Mice were treated with an intraperitoneal injection of CQ and/or sorafenib administered by oral gavage.	Sorafenib + CQ	Decreased SQSTM1 and increased LC3B protein levels compared with sorafenib alone
	In vivo: Retro-orbital metastasis model: EGFP-luciferase MFE-296 cells injected retro-orbitally into the sinus of immunodeficient SCID females.	Sorafenib and/or CQ	NA
	In vivo: PDOX model: endometrial orthoxenografts were developed in nude mice after ortothopic implantation of primary human fresh surgical specimens. Mice were randomly allocated into the treatment groups: i) placebo; ii) sorafenib; iii) CQ; and iv) sorafenib + CQ.
Conza et al., J Cell Physiol [129]	In vitro: Ishikawa, HEC-1B, and AN3CA EC cell lines	SI113 (SGK1 inhibitor)	Increased LC3B-II and BECN1 expression	Cell death
Bao X–X et al., Chin Med J 2012 [136]	In vitro: HEC-1A EC cell line	Nifepidine	Increased autophagic dots, increased LC3 and BECN1 expression, and decreased RPS6KB1 levels	Cell survival
Fukuda T et al., Oncol Lett 2016 [137]	In vitro: Ishikawa EC cell line	Resveratrol	Increased LC3-II expression and autophagosome accumulation	Cell survival
Wu C-H et al., Oncotarget 2016 [142]	In vitro: Ishikawa and HEC-1A EC cell line	Isoliquiritigenin	Presence of autophagosomes, increased LC3-II levels and SQSTM1 expression	Cell survival
	In vivo: HEC-1A subcutaneous xenografts		NA

*NA: data not available

Figure 3. Illustration of the principal autophagy-related function into the human endometrium. Autophagy has a crucial role in both normal and pathological endometrium and has been linked to endometrial cancer development

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