Autophagy: A Targeting Tool for Prevention and Control of Breast Carcinoma and Role of Phytoconstituents

One of the most universal cancers, malignant neoplasm of breast claims the lives of over a million people globally and has a high death rate. The most prevalent and dangerous form of malignancy that affects women is breast cancer. The majority of breast cancer patients have the same features, such as responding to hormone treatment and expressing the estrogen (ER) and progesterone (PR) receptors. [1] Survival rates have grown as a result of hormonal treatment, chemical therapy, and radiation therapy. Still, there has been a rise in the incidence of breast cancer. For these patients, therefore, the creation of innovative therapeutic approaches is essential. According to recent research, a very small group of cells called breast cancer stem cells (CSCs) may be able to self-renew and develop into the full tumor. [2] In multicellular organisms, cell death is a complicated event that may happen in both pathological and normal circumstances. Necrosis, autophagy, and apoptosis are three typical methods of cell death. Necrosis is widely acknowledged to be a type of unprogrammed cell death that is unrelated to the caspase family. Two mechanisms of programmed cell death include autophagy and apoptosis. Dr. Yoshinori Ohsumi received the 2016 Noble Prize for Medicine or Physiology in recognition of his research on the principles behind autophagy. During nutritional shortage, the conserved catabolic process autophagy can support cell homeostasis. [3] Literally, autophagy means "self-eating." Christian de Duve first used the term "autophagy," which means "eating of self" in Greek, more than 40 years ago. The concept was primarily based on the observation of mitochondria and other intracellular structures degrading in lysosomes of rat liver perfused with the pancreatic hormone glucagon. [4] A cell kills outdated or damaged cellular components through a multifaceted, intricate process that allows the cell to recycle these components later on to suit its metabolic demands. [5] Autophagy is a self-digestive mechanism that guarantees misfolded proteins and organelles that are damaged are broken down by lysosomes. [6] The primary catabolic process inside cell that breaks down and recycles long-lived proteins and organelles is called autophagy. Autophagy is a system that has been persisted throughout evolution for the deterioration of organelles, ribosomes, and macromolecules. All eukaryotes have a highly conserved mechanism for cellular recycling and breakdown called autophagy. [7] (fig. (1))

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 Fig. (1). Overview of the autophagy process [8]

1.1 Outline of the autophagy mechanism [9]

Signals that initiate the autophagic process (initiation) usually come from a variety of stressful situations, including protein aggregation, hunger, hypoxia, oxidative stress, and stress on the endoplasmic reticulum (ER). The Unc-51-like kinase 1 (ULK1) complex, which is made up of ULK1, autophagy-related protein 13 (ATG13), RB1-inducible coiled-coil protein 1 (FIP200), and ATG101, is the common target of these signaling pathways. This complex then causes the nucleation of the phagophore by phosphorylating elements of the class III PI3K (PI3KC3) complex I, which is made up of class III PI3K, vacuolar protein sorting 34 (VPS34), Beclin 1, ATG14, activating molecule in Beclin 1-regulated autophagy protein 1 (AMBRA1), and general vesicular transport factor (p115). This, in turn, activates the production of phosphatidylinositol-3-phosphate (PI3P) at an ER structure known as the omegasome. Then, through association with their PI3P-binding domains, PI3P attracts the PI3P effector proteins zinc-finger FYVE domain-containing protein 1 (DFCP1) and WD repeat domain phosphoinositide-interacting proteins (WIPIs; here, WIPI2) to the omegasome. It has been demonstrated recently that WIPI2 directly binds ATG16L1, thereby attracting the ATG12~ATG5–ATG16L1 complex, which in turn strengthens the ATG3-mediated conjugation of ATG8 family proteins (ATG8s), such as γ-aminobutyric acid receptor-associated proteins (GABARAPs) and microtubule-associated protein light chain 3 (LC3) proteins, to membrane-resident phosphatidylethanolamine (PE). This results in the formation of the membrane-bound, lipidated forms; for instance, in this conjugation reaction, LC3-I is transformed into LC3-II, which is the distinctive signature of autophagic membranes. ATG8s are necessary for the elongation and closing of the phagophore membrane in addition to drawing in other autophagic machinery components that include an LC3-interacting region (LIR). Furthermore, LC3 plays a crucial role in selective autophagy by facilitating the sequestration of cargo that has been selectively labeled into autophagosomes through cargo receptors that include LIR. The Golgi complex, mitochondria, recycling endosomes, plasma membrane, and other cellular membranes all donate membrane material to help the autophagosomal membrane elongate (part of these lipid bilayers are delivered by ATG9-containing vesicles, nonetheless, it is currently uncertain where the remaining lipid bilayer came from). The autophagosome is a double-layered vesicle that forms when the autophagosomal membrane seals. As it grows, it loses its ATG proteins and eventually unites with the lysosome. The autophagic cargo is broken down by acidic hydrolases in the lysosome, releasing the nutrients that were saved and allowing the cell to use them again in the cytoplasm. Ub, ubiquitin. (Fig.2)

1.2 Autophagy mechanism

When autophagy is induced, ATGs are attracted to a particular subcellular location known as the phagophore assembly site (PAS), and an isolation membrane that forms the phagophore -a cup-shaped structure- is nucleated (Fig. 1). The phagophore expands into a sphere that encircles a section of the cytosol due to the gradual elongation of the curved isolation membrane. Eventually, the isolating membrane closes to form an autophagosome, a double-membraned vesicle that traps the cytosolic materials that has been consumed as autophagic cargo. The autophagosome's outer membrane merges with the lysosomal membrane to produce an autolysosome once the majority of ATGs have been cleared and transported to the lysosome by microtubules. A single-membrane autophagic body is released into the lysosomal lumen as a result of this fusion, and the autolysosomal hydrolytic milieu then breaks down the autophagic body and its contents. [10], [11], [12]. [9] The highly controlled process of autophagy entails many phases. Autophagy-related (Atg) proteins are usually considered crucial for the control of autophagy; these proteins were first identified in yeast [13]. The process of autophagy induction involves the recruitment of certain regulators to the phagophore assembly site (PAS), where membrane structures are nucleated to create the phagophore, a cup-shaped structure. The phagophore enlarges to absorb cargos for degradation before closing into an autophagosome, a double-membrane vesicle with an enclosed interior. Autolysosomes are single-membrane, degradative vesicles that are produced when autophagosomes are transported and combine with lysosomes in subsequent stages [9] [12]. [14] (Fig. (2))

The first stages of a double-membrane vesicle include vesicle nucleation, which is the membrane's isolation, vesicle elongation, and vesicle completeness [15]. During autophagy, an isolating double-membrane vesicle of nonlysosomal origin that is sealed sequesters the cytosolic materials that need to be broken down, forming an autophagic vacuole or autophagosome. The enzymes needed for the breakdown of sequestered components are produced by the fusion of lysosomes and autophagosomes [16]. The endoplasmic reticulum forms the first phagophores, which encircle and pack organelles to create autophagosomes [17]. Autophagy has been recognized as a multifunctional mechanism that is triggered by several stimuli including as toxins, chemotherapeutic drugs, viral infections, microenvironmental stress, and hypoxia-induced intracellular damage. The advancement of mutations in cancer cells that provide resistance to apoptosis suggests that autophagy may potentially play a role in cell death. Potential targets for innovative methods to programmed cell death (PCD) include nonapoptotic forms [18]. [7] A paradigm for breaking down the autophagic process into distinct phases has been created by analyses of yeast Atg mutants. Among them are: [17]

As shown, the two procedures may be broken down into a number of distinct phases. The insets display the proteins that are involved in each stage. With one notable exception -the cytoplasm to vacuole targeting (Cvt) route employs components necessary for the packaging of particular cargo- the majority of the molecular machinery is shared by the two pathways. Furthermore, number of variables that interact with Atg1 are exclusive to a single pathway. The names of proteins that have been revealed to have functional orthologs in higher eukaryotes are highlighted and displayed in rectangular boxes. The following are the main stages of autophagy: [17]

1. Induction Regulation

The autophagy activation and the transition between autophagy and the Cvt pathway are regulated by Tor kinase and its downstream impactors. Hyperphosphorylation of Atg13 results in reduced affinity for Atg1 kinase, which is either directly or indirectly caused by Tor. Alterations in the activity of Atg1 kinase might lead to a transition between the two pathways.

2. Cargo Packaging

Certain autophagic pathways, such as the Cvt pathway and peroxisome degradation, need cargo detection and packing. Precursor aminopeptidase I (Ape1) dodecamers develop into massive complexes that engage the receptor Atg19 during the Cvt pathway. The cargo then interacts with the ubiquitin-like protein Atg8 through a subsequent interaction with Atg11, enabling inclusion into the sequestering vesicle that forms from the preautophagosomal structure (PAS).

3. Nucleation Of Vesicles

The central mechanism of two different lipid kinase complexes includes the yeast PtdIns 3-kinase, Vps34. Complex I produces PtdIns(3)phosphate at the PAS and is necessary for the Cvt and autophagy processes.

4. Expanding Vesicles

Autophagy-related pathways require two ubiquitin-like (Ubl) proteins. Following two posttranslational processing processes, Atg8 is recruited to the PAS membrane and conjugated to phosphatidylethanolamine (PE). Similar to the E1 ubiquitin activating enzyme, the Atg7 protein is essential for the activation of Atg8, the second Ubl protein, and Atg12. Atg12's C-terminal glycine is connected to Atg5's internal lysine. The development of a multimeric complex is facilitated by the oligomerization of Atg16, which is bound by the Atg12-Atg5 conjugate. While Atg3 and Atg10 are analogues of E2 ubiquitin conjugating enzymes, Atg4 is a cysteine protease.

5. Retrieval

The sole structural protein that is known to be attached to the finished autophagosome or Cvt vesicle is Atg8. While the integral membrane protein Atg9 is eliminated via a particular retrieval mechanism involving Atg2 and Atg18, other peripheral membrane components involved in vesicle production most likely cycle on and off the membrane.

6. Fusion and Docking

All routes that end in the vacuole have the same components needed for the fusion of the autophagosome or Cvt vesicle with the vacuole. It has just been demonstrated that two elements, Ccz1 and Mon1, function during the tethering or docking step, which brings the vesicles close to the vacuole before fusion.

7. Breakdown of Vesicles

Although many subcellular trafficking pathways terminate at the vacuole, known about the turnover of membranes in this compartment. A putative lipase called Atg15 is needed for the intralumenal breakdown of Cvt and autophagic bodies. It takes the multivesicular body pathway to reach the vacuole.

2. Types of autophagy

Three primary types of autophagy are distinguished in mammalian cells: chaperone-mediated autophagy, microautophagy, and macroautophagy. [19] [20]  Specifically, the lysosomal degradation mechanism macroautophagy is an adaptive response to several stressors, including cellular damage caused by chemicals, infection, hypoxia, restriction of nutrients, and growth factor depletion. [21] In this situation, autophagy may provide cytoprotection by either providing nutrition and energy during fasting and other stressful situations, or by specifically removing potentially harmful components like misfolded proteins or damaged mitochondria. This process is known as "selective autophagy." [9] In addition to its cytoprotective function, autophagy has been suggested as a mechanism for death of cells due to the observation of autophagic characteristics in dying cells; this phenomenon is known as autophagic cell death (ACD). [22] Consequently, a wide range of illnesses, including as viral disorders, cancer, and neurodegeneration, are influenced by autophagic activity. [23] [24] Cellular proteins and organelles go through a catabolic process called autophagy, whereby they are taken up by autophagosomes, broken down in lysosomes, and then recycled to maintain cellular metabolism. About 16–20 highly conserved autophagy-related genes (ATG) govern autophagy in mammals by regulating the development of autophagosomes and their fusion with lysosomes. The four stages of this process—initiation, nucleation, maturation, and degradation—involve the varied actions of ATG proteins at various stages. [25] When ATGs proteins are recruited to a certain subcellular region known as the phagophore assembly site (PAS), the initiation and nucleation proteins aid in the genesis of the autophagic vesicle membrane. The phagophore expands into a double-membraned vesicle known as the autophagosome with gradual elongation of the curved isolation membrane, trapping the absorbed cytosolic material as autophagic cargo. After the autophagosome and lysosomal membrane combine to produce an autolysosome, the autophagic body and its contents are degraded. [20]  [26]

2.1 Microautophagy

The mechanism known as "microautophagy" allows cytoplasmic components to enter the lysosome by invaginating or deforming the lysosomal membrane. [27]  In microautophagy, cargo is directly taken up by invaginating the lysosomal membrane. [20] Microautophagy is a kind of autophagy in which, in yeast and plants, the vacuole directly absorbs cytoplasmic entities intended for breakdown by membrane invagination. . [28], [29] A comparable process uses late endosomes in cells from humans and Drosophila melanogaster. This mechanism is called "endosomal microautophagy" and it also happens in yeast cells. [30], [31], [32] Numerous substrates, including peroxisomes (a process known as "micropexophagy," which was historically the first form of yeast microautophagy to be described) [28], parts of the nucleus [33], damaged mitochondria [34], and lipid droplets [35], have been engaged in the degradation of yeast through microautophagy. Microautophagy mediates the anthocyanin breakdown process in plants [36]. Last but not least, endosomal microautophagy breaks down cytosolic proteins either universally or specifically (i.e., only proteins that have an HSPA8-recognized KFERQ-like motif) [30], [31], [32]. It's interesting to note that some proteins absorbed by multivesicular bodies through direct membrane invagination may escape destruction and instead be released into the extracellular environment within exosomes. [37]. [5]

2.2 Chaperone-mediated autophagy

Cytosolic proteins are directly transported to the lysosome for destruction is a element of CMA [38]. Translocation of individual unfolded proteins occurs directly through the lysosomal membrane by chaperone-mediated autophagy (CMA). This process is highly specific; all CMA substrates share a pentapeptide targeting motif that is biochemically related to KFERQ, unlike macroautophagy and microautophagy, which may both nonspecifically engulf bulk cytoplasm. [39].  [20] The unique characteristic of CMA is that substrates are delivered to lysosomes without the need for vesicles or membrane invaginations because a protein-translocation complex at the membrane of the lysosome allows substrates to access the lysosomal lumen [38]. CMA does not break down organelles, other macromolecules such lipids, nucleic acids, or proteins that are essential to membranes [40] [41] [42]. It exclusively breaks down soluble proteins that have a KFERQ-like motif bound to HSPA8 [39]. Major regulatory functions in various pathophysiological scenarios, including metabolic regulation [43] [44], genome integrity preservation [45], aging [46], [47], [48], T-cell activation [49], neurodegeneration [50], and oncogenesis [51] are attributed to CMA, based on the numerous cytosolic proteins on which it has been demonstrated to operate. Furthermore, the cytosolic proteome's linear sequencing analysis indicates that around 30% of its constituents may be broken down by CMA [39]. [52] CMA substrates unfold and dissociate from chaperones [42], and are translocated into the lysosomal lumen via oligomeric LAMP2A complexes stabilized by a cytosolic pool of glial fibrillary acidic protein [53] and a lysosomal pool of heat shock protein 90 alpha family class A member 1 (HSP90AA1; also known as HSP90) [54]. [55]. [56]. [57]. [57]. [58]. [32]. [58]. [5]

2.3 Macroautophagy

In order to sequester and transfer cargo to the lysosome, macroautophagy depends on the de novo production of cytosolic double-membrane vesicles, or autophagosomes. As previously mentioned, one of the ways that macroautophagy differs from microautophagy and CMA is that the first site of sequestration takes place outside of the lysosome's limiting membrane and is accomplished by the creation of cytosolic vesicles that carry the cargo to this organelle. The morphological characteristic of autophagosomes, which sequester cargo, distinguishes macroautophagy from other intracellular vesicle-mediated trafficking processes. Specifically, autophagosomes form by expansion rather than membrane budding, meaning that they do not bud from an already-existing organelle [59]. Instead of starting at a single PAS, autophagosome formation in mammalian systems occurs at many locations throughout the cytoplasm [60], [61]. According to a number of research, animals may use omegasomes, endoplasmic reticulum (ER)-associated structures, as starting sites [62] [63]. After commencement, the membrane starts to swell. It is known as a phagophore at this point, and it is the main compartment with a double membrane for sequestering fluid [64]. The membrane eventually bends to produce a spherical autophagosome as the phagophore grows. Following its formation, the autophagosome must transport its contents to either the functionally similar vacuole found in yeast and plants or the lysosome found in humans. The autophagosome's outer membrane will merge with the lysosomal/vascular membrane upon reaching its destination. However, in mammals, lysosome-autophagosome fusion does not result in the production of autophagic bodies [65]. In mammalian cells, an autolysosome is the result of the merger of an autophagosome with lysosome [59]. The inner membrane of the autophagosome and its contents degrades when exposed to the acidic lumen and resident hydrolases of the lysosome/vacuole. The autophagic cargo is then exported back into the cytoplasm by lysosomal permeases, where it can be utilized by the cell for energy production or other biosynthetic processes [66]. [20]

2.4 Selective Autophagy of Organelles

By preserving both organelle integrity and quantity in the face of changing conditions and pressures, selective autophagy of organelles plays a critical function in the preservation of cellular homeostasis. Organellophagy, a process of clearing organelles, is distinct from bulk breakdown, which happens during starvation-induced autophagy, for example, since it involves the particular sequester of cellular components. There are many different kinds of organellophagy covered in this review, they all are initiated by a signal that sets off subsequent events that produce degradation cues for a particular target, molecules that identify the target as cargo to be broken down, and autophagy-related elements that sequester and remove the cargo. There is evidence that autophagy will able to digest a number of organelles, including mitochondria, peroxisomes, lysosomes, the ER, nucleus, and chloroplasts in different taxa. [67] The autophagolysosome breaks down cargo, such as organelles, during selective autophagy. Ligands like ubiquitin are frequently used to identify cargo, allowing it to interact with receptor proteins and the autophagosomal membrane. By attaching to Atg proteins like Atg8/LC3 or interacting with adaptor proteins, this interaction takes place. The autophagosome forms after the cargo reaches the phagophore. The cargo can subsequently be broken down by the autophagolysosome, which is formed when it combines with the lysosome. [67]

2.4.1 Mitophagy

The selective elimination of excess or damaged mitochondria by macro- or micro-autophagy is known as mitophagy.

Numerous factors, including hypoxia, uncouplers, and Reactive Oxygen Species (ROS), which harm mitochondria, can initiate mitophagy. As the mitophagy receptor in yeast, Atg 32 interacts with Atg 8 on the autophagosomes inner membrane and binds the adaptor protein Atg11. Mammals: Pink1 phosphorylates several targets, such as ubiquitin (Ub), and then attracts Parkin, which then ubiquitinates proteins on the mitochondrial surface to intensify the signal. Cargo receptor proteins can then identify these ubiquitinated proteins, causing the mitochondria to form autophagosomes for eventual destruction. Moreover, LC3 can be recognized by mitochondrial surface receptor proteins, which would help in phagophore recruitment. Through interactions with LC3 that are dependent on the LIR motif, AMBRA1 also localizes to damaged mitochondria, facilitating both classical PARKIN-dependent and -independent mitochondrial clearance. Furthermore, TBK1 phosphorylates autophagy receptors to establish a mitophagy signal amplification loop. [67]

2.4.2 Pexophagy

A macroautophagic reaction that targets peroxisomes preferentially is called pexophagy. Drugs that multiply peroxisomes or changes in nutritional circumstances can both cause proteophagy. As the autophagy receptor in yeast, Atg30 (P. pastoris) or Atg36 (S. cerevisiae) binds peroxisomes by their association with one or more peroxin and attracts autophagic machinery through their connection with Atg proteins. Atg11 can interact with both Atg30 and Atg36 since Atg36 is phosphorylated by Hrr25. It has been established that NBR1 or p62 have a role in pexophagy in animals. NBR1 interacts with phospholipids and ubiquitin by binding to peroxisomes via its JUBA domain. In order to bind ubiquitinated proteins, p62 supports and collaborates with NBR1. In response to ROS, ATM kinase phosphorylates PEX5, which causes PEX5 to become ubiquitinated. Afterward, PEX5 is identified by p62, which directs pexophagy into peroxisomes. Pex2 ubiquitinates both PMP70 and Pex5, and PMP70, also been linked to pexophagy. [67]

2.4.3 Nucleophagy

An autophagic reaction that targets nucleus at specific regions is known as nucleophagy.  The removal of nuclear contents by autophagy by means of the cargo receptor Atg39 or piecemeal micronucleophagy (PMN) is known as nucleophagy. In yeast, PMN is caused by the joining of the nucleus and the vacuole generated by the two essential proteins, Vac8 and Nvi1. A nucleus portion buds off at these junctions, releasing a vesicle into the vacuole where vacuolar hydrolases break it down. A large part of the basic autophagy machinery is used in this process. The nucleophagy receptor Atg 39 localizes to the nuclear envelope and perinuclear ER of yeast, where it promotes the destruction of nuclear components. The mechanism behind nucleophagy in mammals is less well understood. [67]

2.4.4 Reticulophagy

The selective autophagic breakdown of specific ER regions are known as reticulophagy. Certain writers claim that reticulophagy, also known as ER-phagy. Triggers that can induce ER-phagy include ER stress, TCPOBOP withdrawal, and nutritional stress. Atg39 and Atg40, which localize to different ER subdomains and interact with Atg8, promote ER-phagy in yeast. The functional equivalent of Atg40 in mammals is FAM134B. Moreover, p62 and BNIP3/Nix have been connected to ER-phagy. [67]

2.4.5 Ribophagy

A particular kind of autophagy that targets ribosomes is called ribophagy.

2.4.6 Aggrephagy

Aggrephagy is an autophagic reaction that is particular to aggregates of proteins.
2.4.7 Lipophagy

The specific autophagic breakdown of neutral lipid droplets is known as lipophagy.

2.4.8 Bacterial xenophagy

The elimination of cytoplasmic bacteria—that is, bacteria that break out from the phagosomal compartment during phagocytosis—and damaged phagosomes carrying bacteria is known as bacterial xenophagy.

2.4.9 Viral xenophagy

A macroautophagic reaction to fully formed cytoplasmic virions or their constituent parts is known as viral xenophagy, sometimes known as virophagy.

2.4.10 Proteaphagy

The word "proteaphagy" was developed to describe the macroautophagic reactions unique to dormant proteasomes.

2.4.11 Lysophagy

The particular macroautophagic elimination of damaged lysosomes in mammalian cells is known as lysophagy. [5]  Numerous triggers that cause lysosome destruction can also stimulate lysophagy. Galectin-3 and LC3 are drawn to the wounded lysosome following lysosomal damage. Since p62 co-localizes with ubiquitinated lysosomal membranes, it is likely that p62 recruitment follows ubiquitination in this process. The precise processes governing lysophagy control are yet unknown, though.  [67]

3. The Core Pathway of Mammalian Autophagy

At least five molecular elements are involved in the basic route of mammalian autophagy (Fig. 1), which starts with the creation of an isolating membrane, also known as a phagophore. These components include:

1. The complex known as Atg1/unc-51-like kinase (ULK)
2. Two transmembrane proteins, Atg9 and vacuole membrane protein 1 (VMP1)
3. Two ubiquitin-like protein conjugation systems, Atg12 and Atg8/LC3)
4. The Beclin 1/class III phosphatidylinositol 3-kinase (PI3K) complex
5. Proteins that facilitate fusion between autophagosomes and lysosomes [68]

Cellular stress signals directly regulate a few of these essential autophagy pathway elements. [21]

4. Regulation of autophagy

To adjust to shifting environmental conditions, eukaryotes have evolved signaling networks that regulate translation, transcription, and protein modification. Cells must preserve basic and necessary functions in order to conserve energy and nutrients during times of scarcity. A network of conventional and distinct signaling cascades tightly regulates autophagy, a significant cellular catabolic activity. Thus, it is crucial to consider both cues coming from inside the cell and signaling that is started in reaction to outside alterations (Figs 1 and 2). It is well known that the major regulator of cell homeostasis, mTOR in humans, or Ser/Thr kinase TOR (target of rapamycin) in yeast, is responsible for controlling autophagy [7]. Two different protein complexes, TORC1 or TORC2, include TOR [8,9]. While both TOR complexes control cellular metabolism, only TORC1 has a direct connection to autophagy regulation. [10]

(A) Essential autophagy regulators are amino acids. When mTORC1 levels are high, they are directed towards the lysosomal membrane, where Rheb activates it and phosphorylates the subunits of the Ulk1 complex, inhibiting autophagy. (B) Insulin inhibits autophagy by activating mTOR through the PI3KC1/Akt/TSC pathway when insulin binds to its receptor (IR). After FoxO transcription factors are blocked by Akt, the expression of proteins associated to autophagy is suppressed. Hexokinase-II, a mTOR activator, is inhibited by glucose 6-phosphate, which prevents autophagy. (C) When EGFR is activated by its ligand, autophagy is inhibited either directly through the phosphorylation of Beclin1 or indirectly through the phosphorylation of STAT3, which releases eIF2a and causes the production of autophagy-related proteins. (D) When LPS binds to TLR4, it activates, attracting adaptor proteins to the plasma membrane. Consequently, Beclin1 undergoes Lys63-linked ubiquitination as a result of the recruitment of TRAF6, which enables Beclin1 to bind PI3KC3 and trigger autophagy. P62 expression is upregulated when Nrf2 is activated. [10] (Fig. (5))

Autophagy is regulated by internal signals at several intracellular regions and at various levels. Through AMPK, mTORC1 activity is controlled at the lysosome and peroxisome. By phosphorylating raptor and activating the TSC1/2 complex, which both result in the suppression of mTORC1, active AMPK indirectly suppresses autophagy. Phosphorylation of Ulk1, Vps34, and Beclin1 directly inhibits autophagy. ROS molecules upregulate the expression of autophagy-related proteins and trigger autophagy at the mitochondria, ER, and plasma membrane. The intracellular stores of Ca2+ in the mitochondria and the ER mediate Ca2+ signaling. It is yet unclear how NO induces AMPK, which regulates autophagy. Mitophagy is regulated by NO via cGMP. [10] (Fig. (6))

5. Diverse Biological Functions of Autophagy Genes Contribute to Their Roles in the Regulation of Mammalian Disease [25] (Fig. (7))

6. Cancer and the function of autophagy

Numerous human diseases, including microbial infections, inflammatory and immunological disorders, pulmonary diseases, heart and cardiovascular disorders, kidney diseases, metabolic diseases, and neurodegenerative disorders (Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis) have been studied in relation to autophagy. Tumor or cancer is more autophagy reliant than these illnesses and can sometimes result in "autophagy addiction" [8]. Autophagy appears to have a variety of functions in the genesis of cancer. While some research support autophagy's tumor-promoting functions, others demonstrate that it suppresses tumors [69]. The intricate relationship between autophagy and cancer biology might be attributed to several factors such as stressor kinds, tumor types, and stage. Therefore, developing successful anticancer treatment options requires a knowledge of the functions that autophagy plays under various situations. [14] Autophagy has two functions in cancer: cell survival that encourages the growth of pre-existing tumors or tumor suppressor by limiting the accumulation of destroyed proteins and organelles [70] [71]. Selective autophagy essential in cellular quality control, lowering the generation of reactive oxygen species (ROS), preventing chronic tissue damage and inflammation, and promoting genome stability by blocking the accumulation of oncogenic p62/SQSTM 1 protein aggregates and damaged organelles like mitochondria [72][73]. In this sense, ROS and the DNA damage response were inhibited in autophagy-defective cells by p62/SQSTM 1 knockdown [74]. Mutations in autophagy, such as heterozygous knockdown of Beclin1 and ATG7 in mice, have been linked to a greater risk of metabolic stress, genomic damage, and cancer [67]. On the other hand, overstimulating autophagy as a result of overexpressing Beclin1 can prevent the growth of tumors [75]. Additional data suggests that autophagy mostly contributes to stress tolerance in cancer cells, which keeps tumor cells alive [76][77]. It appears that cancer cells trigger autophagy to maintain their elevated frequency of multiplication, which demands a lot of ATP. Hypoxia and food restriction are examples of cytotoxic and metabolic stressors that can trigger autophagy, which recycles cellular components to support cancer cell survival [76][78][79]. Autophagy levels are increased in several cancers. Specifically, Beclin1 was shown to be elevated in cancers of the colon, stomach, liver, breast, and cervical regions [26], indicating that increased autophagy may contribute to carcinogenesis. Autophagy plays a crucial function in cancer, and inhibiting it in tumor cells to boost the effectiveness of anti-cancer medications [80][81]. According to other research, cancer cells with mutations in H-RAS or K-RAS can undergo high levels of autophagy [82][83]. In these cells, the reduction of vital autophagy proteins prevents the cells from growing, suggesting that autophagy is a crucial survival mechanism in these malignancies. Autophagy plays a vital role in the development of cancer metastasis. This correlation was observed in metastases from melanoma, glioblastoma, hepatocellular carcinoma, and breast cancer. Furthermore, autophagy appears to be important for distant colonies to survive while facing a variety of environmental stresses, including as food shortage, hypoxia, and separation from the extracellular matrix (ECM) [26].  Autophagy involved in tumor suppression and malignant cell death in addition to its well-documented function in carcinogenesis. Because autophagosomes and autolysosomes have been seen to gather in the dying cells of cytoplasm without triggering apoptosis, the induction of ACD was postulated as cell death mechanism [84]. Long-term stress leads to increased autophagic activation, which may result in an excessive turnover of proteins and organelles that exceeds the cell's capacity and lead to ACD [85]. A kind of ACD known as "autosis" has been observed in starving dying cells that exhibit autophagy-related morphological features rather than apoptosis or necrosis; autophagy inhibition may be able to stop this cell death [86][87]. In this particular context, the observation that apoptosis is stimulated in cancer models indicates a relationship between autophagy and apoptosis when autophagy is inhibited. Otherwise, other types of cell death, including ACD, could happen in preclinical animals with impaired apoptosis. [26] In cancer, autophagy exhibits a dualistic role: it may either promote carcinogenesis or inhibit the start of tumors [88]. Autophagy can effectively prevent the onset of tumour in some situations, while deficiencies in Atg can lead to tumor development through mechanisms such chromosomal instability, chronic inflammation, and the genomic damage response [89][90][91]. Autophagy loss lead to p62 accumulation in a autophagy related 5 (Atg5) deletion model of mouse, which aided in the tumor development [92]. Additional research revealed that autophagy's involvement in the formation of cancer is reliant on the p53 status, and that autophagy suppression activates p53 independently of p53-independent processes that restrict tumor growth [93]. Furthermore, Human Epidermal Growth Factor Receptor 2 (HER2) amplification inhibits basal autophagy, and elevated autophagy guards against HER2-mediated carcinogenesis [94]. As a result, autophagy suppresses tumors and its suppression increases the risk of malignant transformation in healthy cells. On the other hand, autophagy is often thought of as a more widely applicable survival strategy that cancer cells may consciously utilize in response to different cellular stress circumstances. Up-regulated basal autophagy promotes carcinogenesis in retrovirus-associated DNA sequence (RAS)-driven cancer cells, whereas inhibiting key autophagy proteins Atg5 or Atg7 inhibits cell development [82]. Elevated autophagy, however, is a reactive survival mechanism with a good pancreatic cancer prototype, whereas autophagy suppression leads to tumor regression [95]. According to a recent study, autophagy is essential for tumor maintenance through host and tumor cell-intrinsic processes, and in pancreatic ductal adenocarcinoma, autophagy inhibition results in tumor suppression [96]. In certain situations, pharmacological or genetic autophagy suppression may enhance anti-cancer treatments. As a result, autophagy's tumor suppression keeps healthy cells from turning into neoplastic precursors. However, mild autophagy only promotes cell survival in malignant cells when they are subjected to different cytotoxic stresses. [8] (Fig. (8)

Autophagy affects several facets of the development of cancer. Elevated autophagic activity is thought to provide cytoprotective properties and inhibit the development of cancer. However, autophagy is frequently enhanced in early tumors (after successful initiation) to combat challenges brought on by both low nutrition availability (starvation) and rapid development (such as protein stress). The spread of cancer is linked to several cellular mechanisms that interact with the autophagy system. Since multiple transcription factors that promote epithelial-mesenchymal transition (EMT) are downregulated in an autophagy-dependent way, it appears that the activation of autophagy during EMT inhibits EMT. On the other hand, autophagy is upregulated in many malignancies, which promotes anoikis resistance, a step necessary for cancer cells to acquire the ability to migrate and disseminate, albeit the underlying processes remain unknown. Both autophagy (focal adhesion turnover) and autophagic degradation of the actin dynamics regulator transforming protein RHOA can restrict or stimulate cell migration. On the one hand, autophagy is induced by cancer therapy and plays a role in resistance development. Conversely, autophagy has been shown to be antitumorigenic and has been shown to be necessary for immunogenic cancer cell death. (Fig. (8)) There are two distinct mechanisms in which autophagy modulates cell migration. Firstly, autophagy ubiquitination-dependently breaks down active (GTP-bound) RHOA and the RHOA guanine nucleotide exchange factor (GEF) H1 by recognizing them through autophagy receptor sequestosome 1 (p62). Actin dynamics are impacted, which prevents cell migration and other cellular functions that are dependent on RHOA. It's interesting to note that anoikis resistance is facilitated by autophagic RHOA degradation. Interestingly, it has been demonstrated that RHOA inhibits signaling upstream of mTORC1, perhaps inducing a negative feedback loop via increasing autophagy. However, by breaking down a number of focal adhesion components, autophagy facilitates the disintegration of focal adhesions and promotes enhanced cell motility. Extracellular matrix, also known as Ub ubiquitin, mTORC1, mTOR complex 1, and focal adhesion kinase, FAK. (Fig. (9))  [9]

6.1 Autophagy Modulation to Improve Cancer Treatment

The most cutting-edge method of inhibiting autophagy for cancer treatment is lysosome dysfunction caused by hydroxychloroquine (HCQ). HCQ inhibits autophagic flow and cargo degradation and decreases tumor development in a number of preclinical models (60). Although HCQ is now being tested in clinical studies to see whether it may be used to treat cancer, it is currently used to prevent malaria in people. HCQ is associated with positive clinical outcomes (60–67). If administered at a high enough dose, HCQ can control autophagy in individuals and is well tolerated even in combination therapy. A portion of patients have responded to HCQ, and some have experienced stable illness, despite the tiny sample sizes making it impossible to determine efficacy. It is unknown if HCQ will be effective if it has enough strength to prevent autophagy in human cancers. This has led to the creation of HCQ compounds that are more active, including Lys05 (68). A further benefit of targeting lysosomes is that it prevents both extracellular protein scavenging by macropinocytosis and intracellular protein scavenging by autophagy (69). For K-ras-driven pancreatic cancer, macropinocytosis and lysosomal degradation of albumin (and maybe other extracellular proteins) are crucial survival mechanisms (69, 70). First attempts to specifically target the autophagy mechanism have concentrated on blocking the enzymes required for autophagosomes. The production of the lipid phosphatidylinositol 3-phosphate, which is necessary for autophagosome membrane development, depends on the class III PI3K Vps34. Specific inhibitors of Vps34 that can both accelerate the mortality and decrease the proliferation of cancerous cells (3, 71, 72). The autophagy-initiating kinases ULK1 and 2 are another possible target, and recently created inhibitors prevent autophagy in vitro (73, 74). Additional targetable enzymes are ATG4b, which processes ATG8/LC3, which is necessary for its autophagosome membrane attachment, and ATG7, an E1-like enzyme necessary for the conjugation stages and the creation of autophagososmes.  It is genetically proven that ATG7 deletion inhibits carcinogenesis (5), and one may anticipate that an inhibitor would have the similar effect. It is necessary to conduct more research to determine whether an inhibitor might be beneficial since overexpression of a dominant-negative ATG4b can either enhance toxicity or promote tolerance to cytotoxic treatments (75). [91]

6.2 Autophagy: A promising target for cancer treatment

Autophagy is a sophisticated, tightly controlled process. The development and spread of cancer are facilitated by the dysregulation of autophagy. Because autophagy plays a role in cancer, treating it with this method can be beneficial. From autophagy induction and autophagosome formation to lysosomal breakdown, druggable targets have been found throughout the autophagic process. Autophagic medicines targeting autophagosome-autolysosome fusion and disintegration, particularly HCQ, the most often studied class of medications in clinical trials. Modulating autophagy to cure cancer is also effectively achieved by medications that operate on the upstream signalling cascades governing autophagy, in addition to focusing on the ULK1 complex's initiation of autophagy and its completion, the core autophagy machinery. A few of these signalling modulators, such as mTOR, AMPK, and PI3K inhibitors, are undergoing clinical trials [97]. [14] Autophagy is often seen in tumor cells as a response to therapy, which may be cytotoxic or cytoprotective. Beclin 1 mutations or allelic loss are commonly observed in prostate, ovarian, and breast cancers. Beclin 1 established the initial link between Atg and cancer. According to certain theories, autophagy is vital for cancer's ability to chemo-resistant certain treatment options that usually cause an apoptotic reaction. [7] (Fig. (10))

7. Autophagy: Targeting in Breast Cancer

7.1 Breast Cancer Treatment and Autophagy

Endocrine resistance and endocrine therapy. Endocrine treatment is provided as an aromatase inhibitor (AI) such as letrozole or exemestane, or as an antiestrogen (AE) such as tamoxifen (TAM) or fulvestrant (FAS; Faslodex; ICI 182,780). It is a possibly more effective and less toxic medication for the curement of hormone-dependent breast tumors. For more than 30 years, antiestrogens -TAM in particular- have been the "gold standard" of first-line endocrine treatment [7]. With this medication, more than 15 million patient per years of clinical experience [98]. Furthermore, TAM is the only medication effective in treating invasive breast cancer in both postmenopausal and premenopausal women. Regretfully, many women with ER+ illness are still not cured by endocrine therapy today. It's unknown exactly how estrogen withdrawal (or AI treatment) or AE treatment causes breast cancer cells to die. For instance, even in the case of p53 mutation, breast cancer cells react to AEs and to the removal of estrogen. Even while one of the outcomes of apoptosis is cell death, these treatment responses may be explained by earlier processes that were sparked by autophagy signals [99]. The stimulation of this process in response to hormone treatment has been linked to autophagy. According to recent research, endocrine treatment alters the quantity of autophagosomes, boosts LC3 protein cleavage, and lowers p62 expression. In line with previous findings, PCD II is linked to the endocrine therapy's growth-inhibiting effects on breast cancer cells. [7]

7.2 Autophagy: Bortezomib Treatment against Breast Cancer

Three important endoplasmic reticulum resident transmembrane proteins -pERK, IRE1, and ATF6- may acts to accumulate and aggregate misfolded proteins in the ER lumen due to the 26S proteasome's inhibition. This could activate an unfolded protein response (UPR). A member of the protein kinase family called activated protein pERK phosphorylates the cytosolic Eukaryotic Translation Initiation Factor eIF2a subunit. This reduces the amount of protein synthesized globally and causes activating transcription factor 4 (ATF4) and other selected mRNAs to be translated preferentially. According to several publications, the eIF2a/pERK pathway and endoplasmic reticulum stress are strong inducers of macroautophagy, which is beneficial for cell survival. According to a recent study using the MCF7 cell line, LC3B protein and mRNA levels dramatically rose in a dose- and time-dependent way while receiving boretinomib therapy. Bortezomib-treated cells' increased autophagy was reliant on ATF4's overexpression of LC3B. Furthermore, RNA interference (RNAi) targeting pERK, ATF4, or LC3B transfected MCF7 cells increases their sensitivity to boratezamib treatment. Additionally, following 48 and 72 hours of treatment, there was an enormous rise in the staining of dead cells for both Annexin V and propidium iodide in cases where LC3B or ATF4 were lost. From a therapeutic perspective, targeting autophagy would be an appealing option to improve breast cancer's response to boretinomib and make it more susceptible to environmental stress, which is typically present in solid tumors. [7]

7.3 Autophagy: Trastuzumab Treatment against Breast Cancer

The first immunotherapeutic medication to successfully treat breast carcinomas overexpressing the HER2 (erbB-2) oncogene was trastuzumab (Tzb and Herceptin). According to recent research, LC3-II expression increases in Tzb-resistant HER2-positive breast cancer cells (SKBR3 cell line) when compared to Tzb-naïve SKBR3 parental cells. This suggests that increased autophagy is a corollary of Tzb-conditioned cells acquired Tzb autoresistance. Additionally, treatment with 3-methyladenime (3-MA), a pharmacological inhibitor of autophagy, inhibited the development of preautophagosomal structure, which significantly decreased the vitality of Tzb-resistant HER2-positive BC cells, but not Tzb-naïve SKBR3 parental cells. Further proof that autophagy is essential for Tzb-insensitive high-rates of cell proliferation in Tzb-refractory cells was obtained by blocking LC3-dependent autophagosome formation using the strong and very sequence-specific technique of RNA interference (RNAi). This test demonstrated the severe fragility of TzbR cells while avoiding any off-target adverse consequences that can complicate the interpretation of data obtained with autophagy inhibitors. All of these results proved unequivocally that basal autophagy hyperactivation is vital for the survival of Tzbrefractory TzbR cells when they are reexposed to Tzb. Therefore, in patients who show no signs of improvement to trastuzumab treatment, the combination of Tzb and autophagy inhibitors may be a potential approach. [7]

7.4 Role of Autophagy in the Enhancement of the Inhibitory Effect of Breast Cancer Treatments

Though it shares 54-59% homology with Ras proto-oncogenes, ARHI encodes a small GTP-binding protein that is part of the Ras/Rap superfamily and serves as a tumor suppressor gene in ovarian and breast carcinoma. ARHI is expressed in typical epithelial cells in breast, but it is dramatically downregulated in over 70% of breast carcinoma. Tumor development from in-situ to invasive carcinoma associated with loss of ARHI expression. A cytotoxic medication called paclitaxel induces apoptosis and G2/M cell-cycle arrest, which can stop the development of cancer cells. The HDAC inhibitor TSA promotes autophagy and can activate a number of tumor suppressor genes. According to recent research, ARHI causes breast cancer cells to undergo autophagy. A number of LC3 punctates increased in SKBR3 and MDA-MB231 cells, expressed in small quantities of endogenous ARHI transfected with ARHI. This observation indicates that LC3 membrane-bound form accumulated on autophagic vesicles. Additionally, TSA administration increases autophagy; however, the impacts of TSA were inhibited by siRNAARHI transfection, indicating that ARHI is necessary for the activation of autophagy. [7]

8. Cytotoxic autophagy is induced in cancer cells by phytochemicals.

When used to treat cancer patients, phytochemicals are thought to be safe and helpful at extending their lives. Numerous herbal substances activate ACD; as a result, they may be promising options for malignancy curement. Phytochemicals that can trigger cytotoxic autophagy in variety of malignancies are listed below. Plubagin, (quinonoid) extracted from Plumbago zeylanica L. (root), suppresses cell growth by cells to undergo autophagic cytotoxicity (ACD). Its anti-cancer effect may be suppressed by the Atg inhibitor bafilomycin [100]. A triterpenoid saponin called saikosaponin-d (Ssd) causes cervical and breast cancerous cells to undergo cytotoxic autophagy. When Atg inhibitor 3-MA is added, Ssd-induced ACD is suppressed [101]. Natural berberine (Coptidis rhizome) stimulates Atg in HCC cells; co-administration of 3-MA results in a decreased cell survival rate, indicating a reduction in the quantity of berberine-mediated ACD. [102]. When an Atg inhibitor is added, a natural polyphenol from the culinary herb carnosol causes apoptosis and ACD in breast cancerous cells, and the viability of the cells rises [103]. Bufalin (soluble digoxin-like substance) isolated from the skin of toads, is necessary for promoting ACD in human HCC. Additionally, 3-MA-induced autophagy suppression results in reduction of apoptotic ratio, indicates bufalin-induced Atg may encourage apoptosis [104]. Co-admistration with CQ diminishes the anti-cancer action of licorin A (Myristica fragrans) increase autophagy and death of lung cancerous cells [105]. Glycyrrhiza uralensis includes a potent flavonoid called isoliquiritigenin, which stimulates autophagy, prevents the development of OC cells, and reduces cytotoxicity when 3-MA is present [106]. [8]

8.1 Phytochemicals triggers cytoprotective autophagy in cancer cells

Certain phytochemicals have the ability to cause ACD, whilst other phytochemicals can cause tumour cells to undergo protective autophagy. When paired with the Atg inhibitors 3-MA and CQ, triptolide -a diterpene triepoxide derived from Tripterygium wilfordii Hook F.- causes protective autophagy in cells of prostate cancer, hence promoting triptolide-induced prostate tumor expansion suppression [107]. Autophagy is a favored mode of cell death in cases where apoptosis is compromised [108]. Breast malignant cells can accumulate autophagosomes when Eriocalyxin B (EriB), an ent-kaurane diterpenoid (Isodon eriocalyx var. laxiflora), is exposed to them. Co-administation with Atg inhibitors or knockdown of the Atg gene Atg5 promotes cell death, suggesting that EriB-induced Atg is an adaptive mechanism for cell survival [109]. Autophagy inhibitors can increase the harmfulness to cells of isobavachalcone, a naturally occurring chalcone found in Psoralea corylifolia L. seeds, which causes autophagy and death in Multiple Myeloma Cells [110]. A new lipid-soluble chemical called β-Elemene was isolated from Curcuma zedoaria. β-Elemene-induced Atg protects against malignant cell death since it increases Atg in breast cancer and that autophagy suppression with CQ lowers cell viability [111][112][113]. The active ingredient in ginseng, called ginsenoside F2, is known to trigger cytoprotective autophagy. When CQ is added to ginsenoside F2-induced death of cell in breast cancer, this process is amplified [114]. Natural substance Toxicarioside O (Antiaris toxicaria), induces Atg in colorectal cancerous cells. Toxicarioside-induced apoptotic death of cell is enhanced when paired with CQ [115]. Rabdosia rubescens contains a substance called oridonin, which increases Atg in prostate cancerous cells and decreases cell survival with 3-MA [116]. Isodeoxyelephantopin (ESI), a new sesquiterpene lactone derived from Elephantopus scaber L., stimulates protective autophagy and its anti-cancer impact on lung cancer cells is enhanced when 3-MA is pretreated [117]. Natural compounds such as oleanolic acid cause Atg in breast cancerous cells. When oleanolic acid and 3-MA are combined, the survival of the cells is decreased, suggesting that the autophagy generated by oleanolic acid functions as a defense mechanism against the anti-tumor effect of 3-MA [118]. Lung cancer cells undergo protective autophagy when exposed to curcumin E, a naturally occurring triterpenoid that is extensively dispersed in the kingdom of plants; autophagy suppression promotes apoptosis [119]. [8]

8.2 Autophagy inhibition via phytochemicals

Autophagy is a highly conserved cellular degradation pathway that plays a crucial role in maintaining homeostasis by degrading and recycling damaged organelles, misfolded proteins, and other cytoplasmic components. However, in cancer, autophagy plays a dual role, acting both as a tumor suppressor and as a survival mechanism under stress conditions. In breast cancer, autophagy often promotes tumor survival by allowing cancer cells to adapt to metabolic stress, chemotherapy, and hypoxia, leading to treatment resistance. Therefore, inhibiting autophagy has emerged as a promising strategy to enhance the effectiveness of cancer therapies. Phytochemicals, derived from natural plant sources, have been identified as potential autophagy inhibitors and modulators in various cancer types, including breast cancer.  [26] [120] [121] Phytochemicals might enhance anti-tumor therapy techniques by inhibiting Atg, it may represent a unique therapeutic approach for the curement of cancer. Mundulea sericea is the source of the retinoid deguelin, which inhibits Atg in human pancreatic cancerous cells and makes them more susceptible to doxorubicin-induced cytotoxicity [122]. Compound 20(S)-Ginsenoside Rg3, increases the susceptibility of HCC cells to doxorubicin, hence inhibiting autophagy [123]. Oblongifolin C is a caged xanthone that is isolated from Garcinia yunnanensis hu. It is a new autophagic flux inhibitor that boosts the anti-tumor effectiveness of food restriction linked with autophagy suppression in different cell lines [124]. Astragaloside II, inhibits autophagy by interfering with lysosomal activity, which speeds up cisplatin-induced apoptosis and increases cell death [125]. The catechin in green tea, epigallocatechin-3-O-gallate, suppress autophagic flux caused by doxorubicin, hence augmenting its anti-cancer properties [126] (Tables 1, 2, 3 & 4 and Fig. (11)). [8]

Table 1: Herbal Compounds Triggers Cytotoxic Autophagy In Breast Cancer  [8]

Table 2: Herbal Compounds Triggers Cytoprotective Autophagy In Breast Cancer [8]

Table 3. Natural and Synthetic Compounds as Modulators of Autophagy [14]

Table 4: Key Phytochemicals Involved in Autophagy

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Fig. (11) Key Phytochemicals Involved in Autophagy