Attack cancer: Through autophagic modulations as suppressor or promoter

Abstract

Organelle integrity and maintenance of protein homeostasis and purpose is essential for fundamental equilibrium and survivability. Autophagy is the primary process which regulates the distribution of different cell loads to lysosomes for destruction and reuse. Extensive research illustrates the protective functions of autophagy against various diseases. Though in cancer, noticeably contrasting functions of autophagy have been evaluated in the prohibition of preliminary tumor evolution vs the continuance and, anabolic and catabolic variations of well-established and invasive tumors. Autophagy possesses numerous roles in tumor microenvironment (TME) establishment and associated immune cells function which is addressed in recent studies. Autophagic machinery which is employed in different autophagy-related pathways contributes to metastatic diseases and are distinct from classical autophagy. Therapeutic strategies based on the inhibition or induction of autophagy and related processes has helped in the designing of efficient anticancer drugs. According to the review, we evaluate and decipher the various purposes of autophagy and its association with autophagy mechanisms in course of tumor development, invasion and progression. We summarize the latest findings involving the role of these activities including tumor cells and TME and define further breakthrough in therapy aiming at autophagic activities in cancer.

Key Words: Autophagy; Tumor microenvironment; Autophagic machinery; Autophagy-related pathways; Autophagosomes

Core Tip: Autophagy plays a crucial role in the breakdown and recycling of cellular components. However, its role in cancer biology is complex and multifaceted. It can inhibit or help tumor growth depending on cancer type, stage, and situation. Autophagy helps cancer cells withstand environmental stress, such as lack of oxygen, nutrients, or chemotherapy. Recent studies have shown that autophagy signaling pathways are complicated and can be changed to treat it. Stopping autophagy may improve the sensitivity of cancer cells to chemotherapy. Increasing autophagy can kill cancer cells, especially apoptosis-resistant ones. Thus, autophagy targeting is a promising but difficult cancer treatment.

INTRODUCTION

Macroautophagy (herein mentioned as autophagy) is important equilibrium process, regulating destruction and reuse of cell’s materials[1]. The advantages of modulating autophagy in disorder management has gained attention, for example, in the destruction of protein mass contributive to neurodegenerative diseases. Autophagy roles seem to be more complicated and show dependency on stage, type of tumor and nearby microenvironment.

Out of the three variations of autophagy, macroautophagy is the best categorized pathway. The initiation starts with the double membrane structure formation from the cell, known as phagophore and then later it results in the expansion into autophagosome. Followed, by the engulfment of cell’s components into autophagosomes then its delivery to lysosomes for fusion and degradation. Microautophagy is considered to be the autonomous alteration in shape of membrane of lysosome upon direct engulfment of cytoplasmic components due to bulging or invagination[2]. The last of the three is chaperone-mediated autophagy (CMA), which involves a heat shock cognate 70 kDa protein that is cytosolic, and identifies substrate protein containing the particular pentapeptide KFERQ (Lys-Phe-Glu-Arg-Gln). Lysosome-associated membrane proteins (LAMPs) interact and direct lysosomal transport of cytosolic proteins/substrates for degradation and regulate CMA (Figure 1)[3,4].

Figure 1 Types of autophagy. Macroautophagy involves double membrane structure phagophore and then its expansion into the autophagosome. In microautophagy, change in shape of lysosome occurs. Chaperone-mediated autophagy, there is attachment of KFERQ to heat shock cognate 70 kDa and then followed by recognition via LAMP2 and finally degradation.

In autophagic machinery, a group of autophagy associated genes (ATG) production composes the emergence of a bilayered vesicle, referred to as the autophagosome, which condenses cell loads and combines to lysosomes, ultimately leading to destruction by the activity of lysosomal hydrolases[5]. The formation of autophagosome is initiated by Unc-51-like kinase (ULK) complex, which is composed of ULK1 and ULK2, FAK family-interacting protein (FIP200), ATG13 and ATG101. This group sends signals from signaling centers of nutritious and vitality sensing, involving mTORC1 signaling. VPS34, beclin1, ATG14, and VPS15 make up the autophagy-specific VPS34 complex I, which functions downstream of the ULK complex and catalyzes the production of PI3P on the autophagic layer. Autophagic machinery involving ATG16 L1-ATG5-ATG12 complex, ATG3 and ATG7 is persuaded by PI3P. The above-mentioned proteins enable the fatty acid fusion of the ATG8 family which includes microtubule-associated protein LC3 and gamma-aminobutyric acid receptor-associated protein (GABARAP) subfamilies that were essential for load enrolment and autophagosome maturation as well as other processes involved in this[6,7]. Autophagy can be highly selective in nutrient starved cells where autophagosomes are used to carry different cargoes for recycling critical nutritious substances including proteins or fats and in others can be non-selective as well. This selectivity is due to autophagic cargo receptors (ACRs). This system identifies loads that have been designated for destruction through dependent or independent ubiquitin mechanisms[8]. To supplementary complicate the process, works have demonstrated, role of ATG polypeptides in addition to autophagosome development, in so doing enhancing their functions and applications in diseases[9]. There are shreds of evidence of the existence of two pathways of lysosomal degradation processes with respect to autophagy that rarely need the involvement of ATG polypeptides. This encompasses autophagy and microautophagy mediated by chaperones, in that activity of chaperone and lysosomal layer introversion to capture cell’s matter facilitated through cargo delivery to the lysosome[1].

Autophagy is regarded as a double-edged sword in carcinoma, and works are contributive through the advancing knowledge of critical means via which autophagy impacts cancer succession[10]. The universally accepted fact is that autophagy represses tumor initiation, and also confirms that autophagy plays a role in recognized tumors by enhancing unchecked cell growth and increased metabolic activities, thereby priming the need for support from tumor maintenance aimed at autophagy. Autophagy exhibits crucial functions in tumor cells themselves (intrinsically) and in the nearby stroma (extrinsically), mutually having significances for tumor advancement and drug opposition. Tumor stage, specific oncogenic alterations and cell context possess effect on autophagy.

In this review, we converse the functions played by autophagy in cancer initiation, development and treatment. Also, the part of autophagic machinery is in maintenance of tumor microenvironment (TME) and works evaluating by what means, autophagy in the cells of stroma is able to affect tumor biology. There are increasing evidences that ATG proteins play an efficient role in autophagy and many different operations that are unassociated from the traditional process of autophagy, referred as “autophagy related” routeways. In present report, we explored the potential role of ATG polypeptides and their benefaction to malignant disease advancement. Tumor development can be addressed by targeting autophagy and their current therapeutic advances have been discussed.

TUMOR DEVELOPMENT SUPPRESSION

Cell survival is promoted by autophagy which was found in the initial investigations of autophagy in yeast[11]. Formative studies reveal that autophagy was activated in response to nutrient deficient conditions inside cell through which dissipation of cellular elements for administrating nutriments is done and is highly conserved in higher eukaryotes[12]. Autophagy is considered highly adaptable and moderates various cell stresses including polypeptide and organelle injury and redox disproportion. It has become very much clear that autophagy not only functions in providing nutrients but also is a fundamental homeostatic mechanism inside cells that helps in cell unification, redox parity and proteostasis[1]. In line of above-mentioned purposes, it is well understanding that autophagy has parts that defends against cancer. In the given subdivisions, we concisely explain function of autophagy as a tumor repressor[13-17].

INDICATION OF AUTOPHAGY IN TUMOR DEVELOPMENT SUPPRESSION

The first evidence presents itself through the work of BECN1 gene that encipher BECN1 of the tumor suppressive role of autophagy. When examining cell lines for breast cancer and primary mammary tumors, it was established that they frequently exhibited a loss of BECN1 gene. Interestingly, mice that only have hemizygous of this gene are more susceptible to developing tumors[18-20]. Further works have raised doubts, indicating allelic lack of BECN1 can be due to relation of the BRCA1 tumor repressor on chromosome 17q21[21]. The importance of losing the BECN1 domain is still unclear. Studies report that genes involved in autophagy are often affected in early cancer development and that autophagy helps prevent tumor growth[22].

Autophagy modulates tumor establishment by both, tissue- and gene-specific manner. Works related to BECN1 gene in mice set up that in other tissues there were no effect but in lung, liver and lymphatic tissue the hemizygosity of Becn1 caused tumor formation[20,23]. Elimination of Atg7 solely excluding added genetic actions, leads to the establishment of tumor in liver only[24]. Initial events of liver tumor development are caused by the advent of hepatocyte-acquired progenitor cells that are formed due to the deprivation of autophagic machinery in liver and results in cycling of tissue demolition and regeneration[25]. Functions of autophagy are only indicative with other genetic lesions in additional tissues. Therefore, the question is being raised, whether autophagy has tumor suppressive activity or whether its elimination is consequent of microenvironment which is tumor-advancing. Various works stand for the head-on part of autophagy in tumor abolishment and it can be modulated by tumor suppressive pathways. Tumor protein 53 (p53) which is the main tumor oppressive vehicle has represented to regulate autophagic machinery in multitudinous methods. Cytoplasmic p53 suppresses autophagy in initial levels but once functional actively by cell stresses including DNA damage, its amount is increased tremendously leading to the activation of genes in autophagy advancement such as DRAM1 and PRKAB1[26,27].

The bond connecting p53 and autophagic machinery is complex, as works have shown, Atg7 can actually inhibit p53 activation, while CMA can lead to the degradation of mutated p53[28,29]. Studies have evaluated that cells sheltering inactivation of autophagic proteins during disease advancement are evidence of autophagic pathways as tumor repressors. In ovarian and breast tumors, allelic loss of BECN1 occurs. Thus, autophagy and tumor suppression in human cancer are not definitively correlated, and other studies have reported allelic absence or decreased BECN1 expression in various cancer types[30,31]. Latest studies have evaluated that various autophagic genes, or constituents that modulate ATG polypeptides, are alternated or deactivated to avoid tumor-repressive consequences of autophagic machinery as tumor advances. In gastrointestinal and liver malignancies, frameshift mutations have been found in a number of ATG genes, including ATG2B, ATG5, ATG9B, and ATG12. In addition, it has been discovered that expression of ATG5 and ATG7 is lessen in melanoma[32,33]. Furthermore, works on mouse prototypes showed that elimination of either the BCL2/adenovirus E1B 19 kDa BNIP3 or BCL2/adenovirus E1B 19 kDa BNIP3 L mitophagy receptors in reference of autophagy encouraged the advancement of breast and pancreatic cancer[34,35]. Impacts backing disturbance in autophagy should be necessarily studied to differentiate between effects arising from elimination of autophagy and also those affected by particular pathways.

PARTICULAR AUTOPHAGY IN TUMOR REPRESSION

There have been reported various types of autophagy in different disorders, including cancer which has been reviewed elsewhere[36-39]. Among these, two of the forms of autophagy have been selectively applicable to tumor repression, two of which have been intricated in releasing cell strain due to reactive oxygen species (ROS), that results in the destruction of DNA leading to modifications.

Mitophagy, involving exclusion of mitochondria was one of the prior embodiments of particular autophagy. The repair mechanisms for DNA of mitochondria and proteins are not so much complicated and less effective than those found in nucleus and cytoplasm. Nevertheless, the dependability of mitochondria remains intact during the process of autophagic destruction of damaged mitochondria and their subsequent replacement, before the initiation of new biosynthesis[40]. Cumulation of damaged mitochondria in biological system in which important autophagic genes are erased, opens to the gathering of ROS and DNA destruction, implicating essential roles of mitophagy in tumor suppression[41,42].

The other embodiment of particular autophagy which is associated with balancing ROS is pexophagy, through involvement of the excluded peroxisomes[43,44]. β-oxidation of fatty acids is essential in cancer and also that pexophagy performs an efficient character in balancing ROS[44], when comparing mitophagy with pexophagy the latter has been found to contribute less in cancer.

In addition, ACRs perform roles in particular autophagy. The foremost ACR evaluated was SQSTM 1 or p62. Further p62 has considerable characteristics in cancer which are mentioned beneath, involving induction of NF-κB and NRF2 routeways. Induction of one of the routeways is either tumor-promoting, or, at least, tumor-supporting. The major effect of autophagy is tumor repressive which is maintained by the adequate magnitude of p62 via autophagy-regulated destruction. This is better explained by work on liver cancer in mice, through which tumor formation is brought about when major autophagic genes are erased which is reciprocated by the deletion of p62 (Figure 2)[24].

Figure 2 Dual roles of autophagy. HCQ: Hydroxychloroquine.

PARTS IN TUMOR ADVANCEMENT

Early studies provide parts for autophagy in maintaining the equilibrium of advanced cancers which was supported by presence of LC3 puncta and lipid-attached LC3 (LC3-II), which is indicated by the accumulation of autophagosomes[45]. Further, the stable tissue-based data indicated the higher levels of autophagosomes, therefore they are not able to differentiate connecting progression of autophagy or disfigurement of autophagosome turnover. The loss of capability to investigate autophagic flux in tissue creates problem in evaluating autophagy in human cancer. Various preclinical works provided evidence that autophagy connects the development and metabolizing machinery of advanced tumors further downstream of induction of different carcinogenic genes and/or rendering inactive tumor repressors[41,46].

ONCOGENIC INDUCTION WHICH IS FOLLOWED BY AUTOPHAGY IN CANCER

Studies exploring genetically modified mouse models of cancer which have been operated by oncogenic Ras exposed an essentiality for functional autophagic routeways in tumor advancement. RAS genes are generally alternated in various cancers: 90% pancreatic ductal adenocarcinomas (PDAC) include alterations in KRAS genes[47]. The induced state, RAS activates tumor proliferation and viability and solely initiates tumor advancement. Further, these caused high demand on cell vitality and early predecessors needed, and, by self-digestion, autophagy resolves the restricted accessibility for extrinsic nutriments and hence assists and promotes tumor progression. Reports are indicative that these functions of autophagy create dependence in advancement of different RAS-driven cancers, and such tumors advances to fixed stage of autophagy. In few studies, autophagy exhibits tumor repressive effects in normal cells, however loss of autophagic machinery enhances the initial levels of tumor advancement, still in RAS-directed cancers, advancement to cancer was choked in lack of further genetic lesions[48-50].

Absence of factors that limit tumor development not only advances cancer but is also operated by the induction of carcinogenic factors like RAS. The tumor repressor genes can be induced by carcinogenic agents likewise, RAS49, and they have been considered in reference of autophagy in tumor advancement. There are two principal tumor repressor genetic codes in malignancy which are p53 and Pten. Reports in mice have illustrated loss of one of these genetic codes that can ease the blockage of tumor advancement in lack of autophagic machinery, however this does not contribute to completely grown cancers[48,50-53]. Development of pancreatic cancer seems to be dependent on p53 levels, when complete lack of p53 takes place, it advances tumor[48], on the other hand, hemizygous deletion or mutant p53 did not[50]. Further in lung cancer loss of p53 alone or in synergistic manner with mutant KRAS permits tumor development which differs from the mutation of KRAS alone, but only causes benign tumors that has numerous non-functional mitochondria[53]. Absence of tumor repressor does not always evade autophagy dependency. Lung tumors of mouse models have low ability to adapt to nutriments and energy starvation when absence of AMPK activator and tumor repressor LKB1 takes place. Following this lack, it was evident that few tumors depend on autophagic machinery to sustain lipid and amino acid reservoirs, and lack of pair of LKB1 and ATG7 was synthetically lethal[54]. The above all precedents prove that functionality of autophagic machinery in cancer is contingent on type of carcinogenic abrasions that is responsible for transformation. Additional studies are essential in other tumor categories and in further models to explain where and when autophagy causes inhibition or promotion in tumor advancement. These evaluations are the key to earmark the autophagic pathways therapeutically in various cancer categories.

TUMOR AND AUTOPHAGY METABOLIZING MACHINERY

Usual part of autophagy in tumor continuance and normal advancement is to release cell strain and thus sustain equilibrium and cell survivability[11,12]. This equilibrium function spans from providing nutriments during starvation which is in less vascularized regions of advancing tumors, to maintain ROS levels, which if not controlled may contribute to cell death.

One major dissimilarity among tumor and normal tissues is in their metabolizing machinery. Tumors alter metabolism to be efficiently anabolic, including the transition from oxidative phosphorylation to glycolysis and then moving onto glycolytic intermediates into biosynthetic routeways such as the pentose phosphate routeway[55]. Though there is less need for the production of adenosine triphosphate (ATP), mitochondrial integrity is still required, and autophagy conserves it, as is indicated that the absence of autophagy opens on to buildup of dysfunctional mitochondria in KRAS-driven mutagenesis[53]. When Atg7 is erased in BRAFV600E-driven lung cancer leads to the deprivation of glutamine, that is much needed for mitochondrial respiration and viability of cells of the tumor operated by BRAFV600E[56]. These impact the overall inhibition on autophagy which may result into the metabolic and redox adaptations that supports metastasis. Mammary cancer cells exhibiting dysfunctional mitophagy possess metastatic stability[34]. These changes are raised from the buildup of impaired mitochondria in mitophagy-deprived tumor cells, leading to higher ROS and subsequently a switch from oxidative to glycolytic metabolizing machinery, which is supposed to favor principal tumor progression along with metastasis.

The buildup of ACR p62 in breast cancer cells that lack autophagy averts the proteasomal destruction of the important glycolysis regulator, PFKFB3. This leads to increased proliferation and development of latent metastatic tumor cells[57]. Increased ROS levels, in autophagy-deprived cells are moderated by activation consequent to the accumulation of p62, of NRF2-mediated antioxidant transcriptional applications[58]. In various cancer models NRF2 activation has been indicative of the promotion of metastasis[59,60]. By these results, it can be shown that autophagy deprivation leads to NRF2-driven antioxidant processes and glycolytic metabolism, that results in induction of metabolic functions that enable the dissemination of tumor cells.

AUTOPHAGY “THE DOUBLE-EDGED SWORD”

The part of autophagy in contributing to oncogenesis which is pioneer factor of impermanence in cancer patients is very much debatable. Several early works are evident that autophagy induces various biological pathways critical for metastasis which includes invasion and migration[61-63], regulation of epithelial-mesenchymal transition (EMT)[64,65], opposition to anoikis[66], adjustment to nutriment deficiency and anoxia[64], and survivance in distant tissue niche[46]. Inhibition of autophagic machinery is now a plausible therapeutic strategy to avert metastasis related diseases and in other late reiterative disease in several cancers[46]. Preclinical evaluation utilizing mouse models illustrates decreased metastasis on lack or inhibition of autophagic machinery. Hepatocellular carcinoma in vivo model demonstrated that autophagic machinery induces anoikis opposition and metastasizing dissemination[65]. Thus, it can be made clear that autophagy provides benefits to tumor cells which lack adhesion to extracellular matrix as they circulate to peripheral organs[63]. Further, reports utilizing polyoma middle T oncogene-driven (PyMT) mammary tumor illustrated that lack of Fip200, a crucial modulator of autophagic machinery activation, ultimately led to decreased primary tumor growth and an associated scaling down in metastasis to lung[62]. Furthermore, this early work did not observe particular consequences of autophagy on pioneer tumor vs metastasis[67].

Recent studies using multiple models illustrate that autophagy restricts major rate-limiting steps in metastasis. Several cancers, including prostate, breast and melanoma, have been demonstrated to circulate tumor that are quiescent, and clinically not detectable, in metastasizing tissue for long period. Then the cells go through proliferative growth, leading to macro-metastatic abrasions that aggregates in demise of the patient. The mechanism of circulating tumor into deadly metastasis is labeled ‘metastatic colonization’ which is contemplated to be the major rate-limiting step in metastatic advancement[66,68]. Reports have illustrated, key roles of autophagic routeways in regulating appearance from dormancy and to greater extent, particularly in decreasing metastatic seeding and protuberance. For example, relocated D2. OR mammary metastasizing cells possess quiescent characteristic and breaks down to advance into activated metastasis in syngeneic hosts[69]. Knockdown of Atg3 in this type of cells forces it to cut out quiescence, leading to expanding metastasizing cells with high stemness characteristics, signifying that autophagic blockade provides birth to combative subgroups in vivo[57]. Likewise, in models of latent breast cancer brought on by doxorubicin therapy, consistent suppression of autophagy through Atg5 knockdown led to both the release from dormancy and the early recurrence of metastasis compared to autophagy-competent control cells[70]. Through this work, it is worth noting that autophagy-deprived metastasis possessed increase occurrence of expanding polyploid-like cells, indicating absence of autophagic machinery that induces genomic unpredictability; though, it remains not certain by which mechanism does autophagic machinery protects tumor cells from genomic unpredictability or whether these events put up to metastatic recurrence in this representation.

When working on Fip200 in[66], PyMT cells genetically lacking either Atg12 or Atg5 demonstrated pioneer tumor development when orthotopically transplanted into mammary glands[71].

Though, on editing of principal tumors, autophagy-deficient tumors exhibited high spontaneous metastasis recurrence when related to autophagy-competent complements. Follow-up studies illustrated a particular restrictive genetic deletion of Atg5 or Atg12 in tumors following circulation to lungs, leading to an increase proliferative subpopulation possessing the ability of enhanced metastatic protuberance[71]. In metastasizing replica, constructed on 4T1 mammary tumor cells, almost identical outcomes were found when Atg12 was knockdown[71]. In contrast, increasing autophagic machinery through genetic decrease of rubicon (Rubcn), a negative autophagy regulator, was capable of inhibiting macro-metastatic protuberance. Obstruction of the autophagic machinery increases the amount of tumor subpopulations of cells with basal epithelial differentiation, as indicated by overexpression of transcription factor p63 and keratin type I cytoskeletal 14[71]. These studies conclude that autophagy works in a stage-specified repression of metastasis.

The exact processes through which autophagy inhibition promotes metastatic seeding and elongation remain a critical area for investigation. Reduced turnover of ACRs, through which there is mediation of selected autophagy and function as multidomain signaling focal point are being scrutinize in recent years. The ACRs, mainly p62, activates oncogenic advancement and resistance to therapeutics in autophagy-deficient cells through various incompatible selected pathways[10,72]. The key purpose of p62 as signaling molecule is its potential to induce pro-oncogenic NF-κB signaling that has been associated with high prime tumor development in the background of autophagic deprivation[62,73]. Whether p62 associated induction of NF-κB routeways promotes metastasis is still unclear. Additionally, p62 has showed to repress the destruction of transcription factor TWIST1, the principal modulator of EMT. It has been shown that upregulation of p62 induces mesenchymal differentiation and enables metastatic tumor development in vivo[74]. Mammary cancer models of mouse possessing dysfunctional autophagy resulted in the buildup of NBR1, leading in advancement of invasive subpopulations of tumors displaying pro-metastatic ground level differentiation[71]. Functional work provides that high expression of NBR1 are essential and sustainable for pulmonary metastasizing colonization and the procurement of the ground level differentiation characteristics[71]. ACRs p62 and NBR1 in autophagy-deprived cells play role as the major activators of metastasizing traits.

KEY SWITCH FACTORS OF AUTOPHAGY THAT INFLUENCE ITS PROMOTION OR SUPPRESSION

Another concept that is related with autophagy is that whether inhibition of autophagic machinery does have a therapeutic impact and this is termed as “Autophagic Switch”. In this review, the alterations associated with autophagy have been highlighted. In early or pre-malignant cells there is requirement for the degradation of dysfunctional organelles to maintain homeostasis. Therefore, autophagy is suppressed. This bounds the DNA damage, genomic instability, and inflammation-conditions that drive tumor initiation. The studies have found that loss of BECN1 in early stages of tumor formation leads to activation of autophagy. At later stages of malignancies where it is well established, autophagy plays a potent role in minimizing the metabolic stress, hypoxia, and nutrient deprivation, especially in poorly vascularized regions. Autophagy provides cancer cells the ability to recycle components to generate ATP and building blocks, enabling them to survive harsh conditions and resist therapy[75].

In addition, RAS and BRAF are the key oncogenic switching factors of autophagy that regulates its promotion or suppression depending upon the stages of tumor and nutrient availability. Active KRAS suppresses autophagy when nutrients are abundant. While, in KRAS mutant cancers there is upregulation of autophagy because of the hypoxic TME to sustain mitochondrial metabolism. When therapy-induced stress is caused in tumor cells oncogenic BRAFV600E can increase autophagy via ERK activation. BRAFV600E tumors often activate ER stress pathways (PERK/eIF2α) and JNK signaling, leading to enhanced autophagy[76]. In normal cells, wild-type BRAF signaling through ERK can activate mTOR, suppressing autophagy under growth-promoting conditions.

The role of p53 in regulating autophagy is dependent on its localization in the cell. In early stages of tumor, when nutrient availability is high, p53 acts as suppressor of autophagy as its localization is in the cytoplasm via inhibiting AMPK pathway. When p53 is localized in the nucleus under nutrient poor conditions, autophagy is accelerated to meet the energy demands of the cancer cells. PTEN primarily promotes autophagy through mTOR inhibition, acting as a tumor suppressor. LKB1 is a master upstream activator of AMPK and in turn, promotes autophagy. LKB1 phosphorylates and activates AMPK under conditions of low energy (high AMP/ATP ratio). Activated AMPK inhibits mTORC1 (via TSC2 and Raptor phosphorylation) and directly activates ULK1, initiating autophagy. The role of TME in promoting autophagy is represented in Figure 3.

Figure 3 Induction of autophagy dependent on tumor microenvironment. TME: Tumor microenvironment; ROS: Reactive oxygen species; CAF: Cancer-associated fibroblast.

ROLES IN THE ESTABLISHMENT OF TME

While much research on the role of autophagic machinery in cancer has focused on the hereditary absence of ATG genes in tumors, a significant aspect of studying autophagic modifiers in vivo is understanding how these mediators regulate autophagy not only in the tumor cells themselves, but also in local and remote stromal cells within host. Research on organismal models has commenced to demonstrate the effects of systemic hereditary autophagy suppression in different carrier. Atg 7 deletion in mice leads to the promotion of tumor growth[77]. This loss of autophagy in the model shows the noticeable high regression of KRAS operated tumors linked to autophagic inhibition in tumor[77,78]. Additional after-effects on tumor regression happened frequently than the deadly metabolic and neurological deteriorations which led to Atg7 lack in adult mice. The outcomes are indicative of the existence of systemic inhibition of autophagy as anti-cancerous therapy. Most of the mice surrendered to neurodegenerative disease, indicate that toxicity of inhibitors of autophagy could be compensated by evolving mediators that are unable to bypass blood-brain barrier[77]. Additionally, in model of systemic autophagic inhibition attained through the appearance of a dominant-negative Atg4b mutant, acute inhibition of autophagy in Kras-operated pancreatic tumors led to tumor regression, indicating that both tumor cell and host autophagy giving rise to tumorigenesis[79].

AUTOPHAGY CHAINS HOST TUMOR METABOLIC COOPERATION

Tumors are not separate; rather, they are associated with the host's stromal and immune cells and grow alongside them. Autophagy plays a part in the metabolic rate of tumors. The production of the non-essential amino acid alanine in transplantation models of PDAC is mostly dependent on the autophagic process in pancreatic stellate cells, a vital component of the tumor stroma. Pancreatic tumor cells then use this alanine to help them grow and survive in various microenvironments[80]. Distant site tumor gets support of autophagy from another organ. The established anabolic state associated with tumor advancement creates increased need for arginine, a non-essential amino acid, all tumor cells are dependent on this amino acid[81]. The arginine-degrading enzyme arginase is released into the bloodstream by the liver after autophagy is completely eliminated from the body or liver. This causes a drop in the levels of arginine circulating in the body, which in turn prevents the development of a main tumor in the lung[78]. This is necessary in tumors with diminished arginine-succinate synthase activity, which is required for de novo production of arginine[82]. This renders tumors auxotrophic for arginine, rendering them effective targets for the inhibition of autophagy in the liver.

These experiments additionally implicated utilizing the model of autophagic inhibition attained through the inductive expression of a negative dominating mutant Atg4b. This model showed tumor regression which results in entire body autophagic inhibition in colonized Kras driven pancreatic tumors[79]. This study provides evidence that both host and tumor cell autophagic machinery contribute to tumor advancement. Work on Drosophila RasV12; Scribble deficiency (scrib)^−/− tumor model illustrated that these tumors arise autonomously and systemically, not within a cell that operates autophagic machinery throughout host tissues[83,84]. Autophagy in stromal cells of the host triggers the activation of aggressive protuberance and invasion of RasV12; scrib^−/− tumor throughout the fly. In a similar manner, the studies on mice have shown that the absence of mature systemic autophagy has a more pronounced impact on inhibiting tumor development and proliferation in flies with RasV12; scrib^−/− tumors, compared to the absence of autophagy solely in tumor compartment[83]. The inhibition of systemic autophagy through Atg5 knockdown for transient period of time has been illustrated to repress the intake of glucose and lactate into KrasG12D/⁺; p53^−/− lung tumors in mice, that ultimately leads to dysfunctional tumor growth, explaining how stromal cells undergoing autophagy may broadened the host-tumor metabolite transfer[85].

These studies provide evidence of the fact that autophagy provides all the essential needs for the proliferating tumor cells to maintain core metabolic functions. The results also revealed that systemic focusing on autophagy could have unintended adverse effects in healthy cells like neurons, and that autophagic suppression in the host increases responsiveness to tumor therapies when compared to tumor cell-specific targeting of autophagy.

SUPPORT OF AUTOPHAGY TO CANCER-LINKED FIBROBLASTS

Autophagy of stromal cells has been demonstrated to be essential in tumorigenesis, particularly in regulating protein secretion. Recent research has illuminated the involvement of stromal autophagy in cancer-associated fibroblasts (CAFs), which are fibroblasts found in solid tumors. These CAFs play a crucial role in regulating tumor cell proliferation and behavior through a variety of mechanisms[86]. Proteases, extracellular matrix constituents, inflammatory cytokines, and different growth and angiogenic agents are all secreted by CAFs. Elevated autophagic levels in fibroblasts are linked to unfavorable patient outcomes in head and neck cancer[81]. Consequently, suppressing the process of autophagy of fibroblast was associated with a decrease in tumor progression in laboratory co-culture models. This was due to a reduction in the release of various pro-tumorigenic substances, such as interleukin (IL)-6, IL-8, and basic fibroblast growth factor[87].

A major secretory event needed for desmoplastic stromal response is autophagy in CAFs. Tumor desmoplasia is referred to fibrotic and inflaming microenvironment related with low prognosis in various human solid tumors. Cytological studies have demonstrated that desmoplasia is characterized by the stimulation of fibroblasts and the buildup of type I collagen, resulting in increased tissue rigidity and inflammation[88]. It has been shown that autophagy in pancreatic stellate cells, which cause the desmoplastic fibrotic stroma present in PDACs, increases the release from CAFs of inflammatory cytokines as well as extracellular matrix components[89]. Other studies offer valuable insights into the role of fibroblast autophagy in activating the desmoplastic response. These studies have examined mammary tumor models, both autochthonous and orthotopic transplant, that are driven by the PyMT oncogene. It has been found that the absence of autophagy in CAFs effectively reduces tumor growth and improves the survival of the host carrying the tumor[90]. Furthermore, certain abnormalities in procollagen proteostasis that lead to fibroblasts' lack of autophagy cause defective secretion of type I collagen in both in vitro and in vivo settings[90,91]. Atomic force microscopy investigations have demonstrated that a reduction in the concentration of type I collagen in the stroma derived from fibroblasts lacking autophagy leads to decreased tissue stiffness. This decrease in tissue stiffness serves as a biophysical trigger for the development of cancer[92]. In addition, the lack of autophagy in fibroblasts has an impact on the secretion of type I collagen and tissue stiffness. This finally supports the function of autophagy in fibroblasts in regulating several secretory events involved in tumor desmoplastic response by causing a decrease in the secretion of pro-inflammatory cytokines and neo-angiogenesis factors[90]. The previously mentioned investigations underscore the pivotal function of stromal autophagy in the advancement of primary tumors and exhibit noteworthy methods that may augment the possible therapeutic advantages of autophagy inhibition in every facet of cancer for anticancer therapy.

SECRETORY AUTOPHAGY

These reports illustrate, efficient role of autophagy in host stroma controlling extracellular secretion. The central autophagy has been observed to be executed in both conventional and unconventional secretory routeways in response to its involvement in lysosome destruction. The majority of the mechanical work conducted to gain insight into autophagy-dependent secretion has been devoted to the non-traditional secretion of proteins that are deficient in the N-terminal signal peptide. This process is collectively referred to as secretory autophagy[93,94]. There exist alternative methods that allow proteins to circumvent the Golgi network and reach the plasma membrane directly for secretion outside the cell, in contrast to proteins that follow the standard endoplasmic reticulum-Golgi pathway. The first evidence of ATG proteins was found in the non-traditional yeast Acb1 secretion[95,96]. In mammals, a number of targets for secretory autophagy have been found. These targets include cathepsins, insulin-degrading enzymes, the integral membrane protein cystic fibrosis transmembrane conductance regulator, the high mobility group protein B1, and IL-1β and IL-18[94]. Mechanical perceptions have been generated as a result of the evaluation of IL-1β, a critical moderator of the inflammatory response, among these objectives. A study that was informative illustrated that active IL-1β is combined into autophagosomes, but it is next transferred for secretion, to the plasma membrane as opposed to degradation through fusion with lysosomes[97]. Subsequent reports determined that IL-1β is spatially combined within the outer and interior membranes of double-membrane autophagosome intermediaries[98]. According to recent research, the vesicular structure may resemble the Golgi and endoplasmic reticulum's intermediate apartments, and IL-1β is delivered to this apartment by the protein channel TMED10[99]. Inflammasome stimulation leads to uncontrolled IL-1β release across gasdermin D pores in the plasma membrane. This suggests that autophagic-bypassing pathways are the major method of IL-1β secretion in physiologic situations[100,101]. IL-1β has a significant impact on the TME, influencing various functions such as inflammation and angiogenesis, which contribute to tumor progression and metastasis[102]. Future research should focus on how secretory autophagy and gasdermin D affect IL-1β secretion levels.

At present, research is being conducted to investigate the alternative secretion of proteins through small extracellular vesicles (EVs), which are referred to as exosomes, using moderators of autophagy. It has been shown that the ATG8 conjugation machinery promotes LC3-dependent EV loading and secretion (LDELS), a process that allows a range of RNA binding proteins to be payload into EVs[103]. The activation of neural sphingomyelinase, which is associated to the formation of multivesicular bodies during EV biogenesis, is dependent on LC3 in LDELS[104]. The specific functions of LDELS in cancer are still not fully understood, but it is important to mention that the GABARAPL1 protein of the ATG8 family helps the loading of cargo and the generation of pro-angiogenic EVs in hypoxic tumor cells[104]. LDELS asserts that ATG8 family members play a role in the release of extracellular DNA and histones in a manner that is not dependent on EVs. However, the precise role of ATG proteins in this mechanism remains unknown[105]. Current work has showed that there is existence of another secretory autophagy routeway which gets activated upon the inhibition of lysosome during the administration of the medication hydroxychloroquine (HCQ) that inhibits autophagy through anticancer therapy by increasing the lysosomal pH. Various autonomous studies have illustrated lysosome inhibition by drugs enhances EV-mediated secretion of lipidated LC3 and autophagosomal substrates through secretory routes[106-108]. Specifically, lysosome blockage triggers secretion of ACRs into the extracellular milieu, such as p62, which are released as EV-associated nanoparticles as part of the EV and particles (EVPs) segment of EVs[106]. This secretory autophagic machinery, which occurs during the duration of lysosome inhibition (SALI), necessitates the presence of numerous ATG proteins to progress through the formation of autophagosomes, as well as RAB27A, which enables the departure of vesicles from cells. Furthermore, in vivo diagnosis of ACRs emitted through secretory autophagy during lysosome inhibition (SALI) is achieved through the isolation of EVPs from blood plasma after receiving HCQ therapy. The autophagy-dependent EVP secretome has the potential to function as a biomarker in the treatment of cancer[109]. Autophagy modulators and endolysosomal acidification were connected in this study to the regulation of unconventional expulsion by EV and EVPs. The intracellular communication between tumor, stromal, and immune cells in the TME is facilitated by EVPs, as evidenced by the maintenance of pre-metastatic niches that facilitate metastatic progression[110]. A one-sided question that is left unanswered is how autophagy regulates particular EVP loads which affects cancer advancement and therapeutic response.

In spite of various genetic evidence providing an important role of ATG proteins in regulating growth factors and cytokines being secreted in different cancers, our withstanding of how autophagic machinery mediates conventional secretion remains unclear. Autophagy plays a part in promoting the extracellular discharge of IL-6, IL-8, and related inflammatory mediators that stimulate tumor growth, according to research on cancer fibroblasts[87,89,90]. Till date, there is no certainty that does autophagic pathways have any direct mechanism in facilitating extracellular release of pro-tumorigenic factors. However, the need of the hour is to know the actual mechanism of secretion reliant on autophagy, not just in cancer but also in different metastatic diseases.

AUTOPHAGY AND TUMOR IMMUNITY

Functions of autophagy such as degradation and trafficking, including destruction and presentation of extrinsic antigens on major histocompatibility complex (MHC-II), as well as the cross-presentation of antigens on MHC-I have been described[111,112]. Emerging attentiveness in identifying the part of tumor linked immunity in tumor progression and anticancer therapy specifically immune checkpoint blockade therapy, numerous studies have evaluated these immunomodulatory functions of autophagic machinery. The authors of a study on PDAC have detailed an unexpected function of autophagy in evading of immune attacks. They have directed MHC-I in cancer cells for autophagic degradation through selective mechanisms, including NBR1[113]. This mechanism must be appropriately regulated, as the absence of MHC-I will lead to an immune attack by natural killer cells (NK). The renewal of MHC-I was the result of autophagic obstruction, which is reversible due to the immune evasion observed in PDAC. This led to the synergistic elicitation of immune checkpoint blockade therapy[113].

Genome-wide screening work showed that autophagic machinery plays an evident role in regulating host immune responses that modulate tumor maturation[114]. Furthermore, it was shown that autophagy promotes regulatory T cells in the liver, which in turn decreases anticancer T cell responses. Reduced antigen processing and presentation is associated with autophagy triggered by loss of LKB1, which facilitates immune checkpoint blockade therapy[115,116]. This contradicts other results that indicate a beneficial effect of autophagy on antigen presentation. As previously emphasized, autophagy facilitates the breakdown of cargo to generate antigens, which are then displayed on the cell surface for detection by immune cells[117,118]. Autophagy inhibition by directing ULK1 re-established antigen presentation, and synergized with blockade of PD1[116]. Autophagy not only plays role in antigen presentation but also in immune trafficking through altering the expression levels of chemokine and cytokine in TME. Early studies provided for example, that the high immune trafficking in response to inhibition of autophagy was seen upon FIP200 deletion in PyMT mammary tumors, which resulted to elicited making of chemokine CXCL9 and CXCL10, chemokines that activate, appointment of antitumor CD8⁺ cytotoxic T cells into tumors[66]. Genetic or pharmacological loss of autophagic machinery in B16-F10 melanoma cells leads to an upregulation and secretion of CCL5, which elicits NK infiltration into tumors[119]. Cytotoxic T cells and NK are crucial for immune checkpoint blockade and antitumor immunity. However, the impact of tumor cell autophagy on infiltration and the role concerning these cytotoxic immune subsets remain under intensive investigation.

ATG PROTEINS IN ALTERNATE PATHWAYS

Several ATG proteins, including autophagy, play essential roles in different cellular pathways (Table 1)[9]. The genetic regulation of ATG modulators impacts both canonical destructive autophagy, non-canonical autophagy and other related processes. Canonical autophagy is the standard autophagy process that operates inside the cell mostly, following all the required steps starting with ULK1 activation, then the utilization of PI3K-Beclin1 complex, both ATG conjugation systems (ATG12-ATG5-ATG16 L1 and LC3 lipidation), and forming a double-membrane structure autophagosome that fuses with a lysosome to recycle cell components. On the other hand, non-canonical autophagy is an alternative to the classical canonical autophagy as it skips or changes certain processes. It might not need sometimes ULK1 or the PI3K-Beclin1 complex, or only use LC3 without the ATG12 conjugation system, and often works on single-membrane structures like phagosomes or endosomes instead of double-membrane autophagosomes. Mentioned below we take into account, present scenario and how these processes are beneficial for cancer.

Table 1 Functions of autophagic proteins.

Stages	Complex	Key proteins	Functions
Activation of autophagy	ULK1	ULK1	Phosphorylates Beclin1 and plays role in the initiation of autophagy
		ATG13	Localizes PAS to ULK1
		FIP200	Earliest protein to reach PAS, acts as a scaffold for downstream ATG protein assembly at the PAS
		ATG101	Interact with Atg13 and forms the ULK-ATG13-ATG101-FIP200 complex
Assembly and formation of autophagosome	Class III PI3K complex I	VPS34	Catalytic PI (3) kinase
		VPS15	VPS 34 regulatory subunit
		Beclin1	Central regulator, shows interaction with major proteins including ATG14 L, UVRAG, Rubicon, Bcl 2
		ATG14 L	Interacting protein (with Beclin1)
	ATG9A Trafficking System	ATG9A	Recruits LC3 and WIPI1/2
		WIPI1/2	Involved in forming ATG9A trafficking system
		ATG2A	Interacting protein (with WIPI1/2)
	ATG12 ubiquitin-like conjugation system	ATG12	Ubiquitin like protein
		ATG7	E1 Enzyme
		ATG10	E2 Enzyme
		ATG5	Interacting protein (with ATG12)
		ATG16 L1	Provides stability to ATG12-ATG5 complex
	LC3 ubiquitin-like conjugation system	LC3A/B/C	Microtubule associated protein
		ATG7	E1 Enzyme
		ATG3	E2 Enzyme
		ATG4A/B/C/D	Enzyme that efficiently cleaves LC3 precursors and LC3-I/PE
Autophagosome formation and cargo sequestration, enabling subsequent lysosomal degradation and recycling of autophagosomal contents	ATG12 ubiquitin-like conjugation system+	Same as above	Autophagosome membrane elongation and closure
	LC3 ubiquitin-like conjugation system

ATG: Autophagy associated gene.

LC3-ASSOCIATED MECHANISMS IN TUMOR DEVELOPMENTS

The observation of a small number of phagocytic vesicles containing LC3 protein provided evidence for an unconventional function of ATG proteins beyond their role in autophagosome production[120]. Subsequent investigations have furthered the understanding of LC3-associated phagocytosis (LAP) by identifying LAP-like LC3 complexes on endosomes[121], LC3-associated endocytosis (LANDO)[122], and LDELS[103]. The combination of ATG8 proteins on single membranes, which is currently referred to as conjugation of ATG8 to single membranes (CASM)[123], demonstrates shared characteristics among these mechanisms. The requirement for specific ATG complexes results in the differentiation of CASM processes[124].

The activation of a distinctive VPS34 complex, which includes VPS34, UVRAG, BECN1, and VPS15, and Rubcn, an inhibitor of autophagosome formation, is required for the LC3 combination through LAP[125]. LAP induces lysosomal appointment to phagocytic vacuoles and degradation of phagocytosed material, which represses pro-inflammatory signals by facilitating the complete removal of phagocytosed substrates. The lack of RUBCN in myeloid cells inhibited LAP, which subsequently activated interferon type I signaling in tumor-linked macrophages, resulting in T-cell repression of tumor progression[126]. Rubcn upregulation requires LAP but not canonical autophagy[127-129]. This phenomenon is observed in various malignancies, such as stomach, liver, and breast cancer, and is associated with poor prognosis in patients[130]. The subject that remains unresolved is whether LAP possesses tumor activating properties in various types and stages of cancer. LAP-facilitated repression of immune cells may have contrasting effects during tumor induction and sustainment, similar to canonical autophagy. LC3 Lipidation has been observed in many cellular compartments, in addition to the LAMPs. This process is referred to as LAP-like LC3 Lipidation, and its significance in cancer is expected to become apparent in the near future. Lysosomotropic drugs, such as higher dosages of HCQ and ionophores, can stimulate LAP-like LC3 Lipidation[121]. HCQ functions as an autophagy inhibitor during anticancer treatment. Therefore, it would be beneficial to assess the impact of LAP-like LC3 Lipidation on the antitumor effectiveness of HCQ. The entosis mechanism has developed to induce LC3 Lipidation on the entotic vacuole that surrounds the absorbed cell, similar to LAP. The process of LC3 Lipidation, in turn, increases the death and lysosomal breakdown of phagocytosed cells and produces chemicals that promote the growth of the host cell[131]. Therefore, entosis has pro-tumorigenic role by enhancing the evolution of tumor and killing the neighboring normal cells, thus providing other function of LC3-associated processes during tumor growth[132].

Non-degradative functions can also be played by LC3- associated mechanisms. Recycling of surface receptors on the cell was regulated by LANDO, and their inhibition in myeloid cells stops the recycling of receptors involved in intake of Aβ amyloid, including CD36, toll like receptor 4 and triggering receptor expressed on myeloid cells 2 (TREM2)[122]. When LANDO is inhibited, there is an increase of Aβ amyloids outside the cells and an inflammatory reaction in the brains of mice. Recent studies have demonstrated that the increased expression of TREM2 is linked to unfavorable cancer prognosis[120]. It remains to be determined whether the response to the immune therapy and the growth of the tumor are influenced by the recycling of TREM2 or other receptors facilitated by LANDO.

ROLE OF AUTOPHAGIC MEMBRANES AS SIGNALING PLATFORMS

There are pieces of evidence that suggest that tissue and tumor cells when taken from autophagy-deprived mice possess decreased carcinogenic signaling for example, AKT-PI3K and MAPK-ERK signaling routeways[77,133,134]. This is the consequence of the tumor-activating functions of autophagy, which have been emphasized in this Review. However, the primary interactions between autophagic machinery mediators and growth factor signaling have also been investigated. Autophagic proteins, LC3B, can co-localize with the receptor (RTK) MET and phosphorylated ERK during hepatocyte growth factor induction. The LC3 Lipidation apparatus is required for the optimal promotion of MET and downstream signaling (Figure 4)[123,135]. The ULK1 complex, which includes ATG13, is essential for the promotion of MET. This underscores the fact that autophagic proteins are associated with signaling centers, which are referred to as autophagy-related endomembranes. These endomembranes are distinct from the canonical autophagosomes[135]. Likewise, the phosphorylated ERK colocalizes with LC3 and the ATG16 L1-ATG5-ATG12 complex, but not with ULK1 or VPS34, and epithelial growth factor (EGF)-induced ERK signaling appears to be dependent on principal ATG mediators, including ATG5 and ATG7[134]. All this results into the promotion of few RTKs, autophagy-related membranes which can be utilized for effective signaling. It is still unclear that whether signaling hubs are present on double or single membranes within cells and more analysis is required to differentiate their nature.

Figure 4 Crosstalk between the canonical autophagy machinery and pro-tumorigenic signaling pathways.

ATG mediators can alter growth factor-facilitated signaling through supplementary pathways. Epidermal growth factor receptor (EGFR) signaling can be modifiable through the autophagy-mediated degradation of a specific set of early endosomes that are inflamed and labeled with galectin-8[127]. When the autophagic machinery is lost, EGFR can accumulate on early endosomes, leading to the destruction of their endocytic recycling and impairing signaling. For instance, members of the ATG8 family, such as LC3C, quickly bind to MET, resulting in autophagic degradation and thereby negatively regulating MET signaling[128]. All of these findings offer proof that there is a complicated interaction between the autophagic machinery and carcinogenic signaling pathways. This warrants further investigation to carefully analyze its role in tumor development and/or later stages of cancer progression.

AUTOPHAGY-INDEPENDENT ROLES OF ATG PROTEINS IN TUMOR PROGRESSION

It is important to note the major impact of non-autophagy-related functions of ATG proteins on tumor growth when considering the regulation of autophagy in cancer. For instance, the suppression of VPS34 lipid kinase activity or the elimination of its binding partner BECN1, which are commonly used to block autophagy. VPS34 is essential for generating PI3P on many cellular membranes, such as endosomes[129]. Therefore, the observed morphological alterations resulting from the repression of VPS34 can be attributed to the inhibition of autophagy, endocytosis, or both processes.

As stated above, autophagic proteins have registered functions in secretion of EVs[136]. This can happen through either autophagy-dependent or autophagy-independent processes. It has been observed that the formation of a non-canonical conjugate involving both ATG13 and ATG3 is not necessarily necessary for LC3 Lipidation. ATG12-ATG3 can interact with Alix, needed for transport complex, to regulate the movement of late endosomes and the release of EVs[137]. ATG5 and ATG16 L1 are essential for the release of EV through a process called lipidation-independent recruitment of LC3, which leads to the reduction of acidity in multivesicular bodies[138]. In this concept, the release of EVs is believed to have role that triggers the migration and spread of breast cancer cells. This suggests that ATG proteins have a role in cancer that is not related to autophagy.

The working of TBK1, which is a key modulator of innate immune response and autophagic payload binding can be repressed by FIP200.

This control on the activity of TBK1 by FIP200 occurs through autophagic dependent and autophagic independent functions of FIP200[139]. A preliminary study indicated that FIP200 and autophagy have a role in the advancement of mammary gland tumors via regulating cancer growth and the infiltration of T cells[68]. The latest research has demonstrated that FIP200 has a crucial role in promoting tumor growth, which is not dependent on its autophagy-related functions[140]. The authors have demonstrated that the autophagy-independent functions of FIP200 repress antitumor immune responses that are capable of modulating TBK1 activity, despite the fact that the autophagy-dependent functions of FIP200 are necessary for tumor development and metastasis. This was achieved by presenting an autophagy-deprived mutant of FIP200[140].

Numerous autophagy-unrelated activities have been described to ATG proteins with diverse applications in immunological reactions, membrane trafficking pathways, cytotoxic demise and p53 modulation[9]. How these roles can affect tumor growth is still unclear and needs to be resolved for future investigations.

CURRENT GAPS IN THE KNOWLEDGE

In some instances, autophagy is limited or has no effect, may be because our understanding of its function in cancer tumorigenesis is still limited. To address this, various questions regarding the same need answers: How is autophagy modulated differently in numerous cancers? Will it be better to target it in early or late stages of the disease for better efficacy? How do we design effective treatments that use autophagy in a more specific manner? Do these drugs targeting autophagy possess higher selectivity with lesser side-effects which includes the recent ones like CQ and HCQ? Gaining a better knowledge of the molecular mechanisms of autophagy at various stages of cancer will be critical in answering these issues and developing effective control measures, ultimately enhancing cancer treatment.

FUTURE DIRECTIONS IN THE KNOWLEDGE

Future implementation for controlling cancer via autophagy regulation should emphasize on developing stage- and context- specific strategies, as autophagy plays dual role both in tumor promotion and suppression depending on the type and stage of cancer. Identification of potent biomarkers to quantify autophagy activity and flux in patients will be necessary for administering treatment decisions. There is requirement to move beyond general regulators like CQ and HCQ toward more specific agents that precisely target particular autophagy components, including VPS34, ULK1, or LC3 conjugation systems, thereby lowering side effects including increased toxicity and resistance. Combination of autophagic modulators with chemo-, radio- or immunotherapy provides potential to surpass drug resistance and exploit lethal interactions while directing TME and immune system could further elicit therapeutic outcomes. Different drug delivery methods like nanoparticles or antibody drug-conjugates, may provide specific tumor targeting to minimize systemic side-effects. Tumor genomics and profiling of metabolism can help identify cancers that are dependent on autophagy, making them the key candidates for such therapies.

Improving preclinical models, that include patient derived spheroids and immune-competent systems, along with standardized assays for clinical trials, will be efficient for translating these methods into effective therapies. Thus, understanding the non-canonical autophagy pathways, working on long term safety concerns, and addressing potential resistance mechanisms will be crucial for sustainable therapeutic advantages. Finally, combining computational biology and system approaches could enable better prediction of response of patients and the personalization of combining regimens, would further ultimately lead to more particular and efficient cancer control through autophagy regulation.

CONCLUSION

In the last 15 to 20 years, reports defining functions of the role of autophagy in cancer and its potential as a therapeutic target has gained momentum. From a scientific perspective, it is essential to thoroughly examine the effects of depriving specific ATG genes, such as ATG5 or ATG7, in mice. This investigation aims to determine whether the resulting cancer morphology is influenced by autophagy or if it involves other processes, such as those associated with CASM. Furthermore, regarding the role of autophagy in tumor suppression and tumor growth, it has been temporarily emphasized that autophagy exhibits both beneficial and detrimental effects in cancer. Thus, there is a need for a clearer understanding of the fact that how tumors get control of the growth-repressive impacts of autophagy for the survival and maintenance of recognized tumors. We require effective and efficient models that allow us to promote or inhibit autophagic machinery in diverse tissues and at varying stages of tumor progression. It is very much clear that we need more potent drugs that can direct autophagy. These formulations can be designed to specifically affect several aspects of autophagy: (1) Regulating the turnover rate of autophagy by controlling lysosome activity; (2) Initiating autophagy by targeting specific mediators like VPS34 or ULK1; or (3) Inducing excessive autophagy activation. Identifying techniques to control autophagy in cancer is crucial in order to prevent unwanted side effects associated with suppressing autophagy, such as recurrent metastasis or dementia. To address this, it is necessary to delineate the roles of autophagy in different types of malignancies and at different phases, such as primary tumor vs advanced metastasis. Additionally, it is important to ascertain the reliance of other tumors on autophagy.

Currently, the majority of solutions rely on analyzing the steady-state levels of autophagosomes or LC3-II. However, this approach is inadequate for distinguishing between autophagosome maturation arrest and the activation of the autophagic machinery. Although significant advancements have been achieved in understanding the impact of autophagy on cancer, it is only after resolving these issues that we can effectively utilize current and future innovative approaches to suppress autophagy for the benefit of cancer patients. Moreover, as previously mentioned, these breakthroughs have the potential to be used in conjunction with traditional chemotherapy, incorporating new medicines that induce autophagic reliance in tumors or tumor-supporting stroma, or methods to engage the immune response against cancers.