Autophagy: mediator of cellular homeostasis and cell survival

Introduction

Autophagy operates as a highly integrated cellular homeostasis maintenance process. Because of its centralized role as a housekeeping and cellular stress monitoring system, its operative effects within any cell type vary on a case-by-case basis. A nice example of this concept is seen when looking at autophagy’s relationship with cancer. Depending on the nature of a cancer cell-type or developmental stage of a cancer, autophagy can act either as a cancer promoter or suppressor. Christian de Duve, a Nobel Prize-winning biochemist was first credited with coming up with the name autophagy [1]. The term “Autophagy” derives from the Greek root-words auto (self) eating (phagy) due to its intracellular housekeeping actions. Like apoptosis, autophagy is a genetically controlled/evolutionarily conserved programmed cell death process. Autophagy is most commonly known for its cell survival actions; enabling cell survival during nutrient deprivation conditions. In a broader sense, autophagy serves the critical role of helping cells weather extended periods of a wide range of physiological stress conditions. Some of the more well-known stressors include nutrient deprivation, metabolism related stressors such as ROS production, hypoxia, and anti-cancer treatment protocols.

Autophagosome Assembly Overview

Autophagy is reliant upon the assembly of an intracellular membrane enclosure structure through which expendable damaged or aging intracellular components can be gathered up for autolysosome processing. Currently, three different forms of autophagy have been identified and differentiated by the means in which the captured cytosol content is presented to the lysosome [2]. These three forms have been classified as: 1) macroautophagy, 2) microautophagy, and 3) chaperone-mediated autophagy. Macroautophagy, the most abundant and well-studied form of autophagy, is usually referred to simply as autophagy in the published literature. Autophagosome assembly involves a multi-step assembly process (Figure 1). It begins with the creation of an isolation membrane or autophagophore. Autophagophores are nucleated at an endoplasmic reticulum (ER) -emanating membrane domain enriched for the lipid phosphatidylinositol 3-phosphate (PI3P), known as the omegasome [3, 4]. Complete encirclement of this structure with captured cytosolic content leads to the formation of an autophagosome. This lipid bilayer enclosed structure can merge (fuse) with an enzyme laden lysosome forming an autolysosome. Incorporation of lysosome-origin hydrolases facilitates the breakdown of the autolysosomal cargo, completing the component recycling process [5].

Figure 1

Figure 1. Assembly of membrane intermediates leading to autolysosome formation during autophagy. 

Selective and non-selective cytosolic component targeting

There are two types of autophagy: ongoing basal level autophagy and inducible autophagy [6]. Basal level autophagy is a constitutive housekeeping process for removal of outdated or defective cellular or organelle components. Inducible autophagy acts as the cell’s response to various types of stressors. Some of these stress-factors include nutrient or cell energy deficiencies, oxidative stress, hypoxia, or from a cancer treatment perspective, cytotoxic stress emanating from chemotherapy. Inducible types of autophagy can be further broken down into selective and non-selective autophagic targeting [7]. In the case of non-selective autophagy, the main purpose is harvesting a generic spectrum of intracellular cytoplasm and organelle component content for the purpose of sustaining metabolic activities to include energy production. In contrast, selective forms of autophagy target specific intracellular organelle or membrane-protein structure. Autophagy action using this more precise selective process provides the cell with a means by which to limit organelle removal to organelles that are damaged beyond normal function [8]. Although the targeting action involves some form of ubiquitination process, the actual signaling stimulus seems to vary with the organelle involved [8]. Ubiquitin tagging of protein targets allows them to be identified and bound by autophagy receptors with the aid of LC3/Atg8 for incorporation into autophagosomes [8, 9]. Autophagic flux is a general term used to define the rate of autophagosome-mediated intracellular degradation activity in response to or associated with a particular physiological situation [10].

Regulation of autophagy

Over the past decade, much has been learned about the intricate mechanisms by which autophagy is regulated. mTOR (mechanistic target of rapamycin), an evolutionarily conserved serine/threonine kinase, acts as a central regulator of cellular anabolic and catabolic processes. These include monitoring signals emanating from both intracellular and extracellular locations [11]. Named for its sensitivity to the anti-cancer agent, rapamycin, this nutrient-sensing kinase, is central to controlling the autophagy initiation process. mTOR exists as two different biologically active forms: mTORC1 and mTORC2 [12]. Each form represents a distinct multiprotein complex having different regulatory activities [13]. mTORC1 is involved in the direction of cellular proliferation whereas the less clearly understood mTORC2 seems to have cell survival and cytoskeletal organization directives [14]. Using the often-used nutrient depletion response example, during nutrient depletion conditions, mTORC1 activity is inhibited. mTORC1 inhibition leads to unc-51-like kinase 1 (ULK) dephosphorylation and subsequent activation [15]. ULK1 activation serves as a positive regulator of autophagosome formation [9]. mTORC1 is regulated by AMP-activated protein kinase (AMPK). Inhibition of mTORC1 with increased AMPK activity leads to an increase in the autophagic process [16]. In times of nutrient abundance, mTORC1 freely binds to and phosphorylates ULK1, interfering with its ability to bind with AMP-activated protein kinase (AMPK), leading to autophagy inhibition.

Defective autophagy-associated diseases

Autophagy’s central role in regulating and maintaining cellular homeostasis portends its involvement in the control of a broad range of mission-critical cellular functions. Logic would then dictate that any performance defect within autophagy’s wide-ranging activity functions would manifest itself across a broad range of human diseases states [17]. Thus, an initial belief that autophagy’s main function was to act as a nutrient deficit mediating cell-survival mechanism during nutrient deprivation cycles was grossly incomplete. Because of our interest in limiting our discussion to the general mechanisms propelling the autophagic processes, along with the role autophagy plays in cancer treatment, we limited our autophagy-defect pathologies to the more prominent autophagy-origin diseases. Some of these include neurodegenerative diseases, heart disease, autoimmune/inflammatory driven pathologies (inflammatory bowel diseases, Crohn’s disease, diabetes, obesity, lupus), and cancer [17, 18].

Autophagy as a cancer agonist/antagonist

Autophagy as a cancer antagonist  

When reviewing current autophagy publications, much attention has been given to its role in controlling the onset and expansion of human cancer. How much of an active role does autophagy play in disease onset and progression? As briefly stated in the introduction, autophagy plays dual and conflicting roles as both an advanced-stage-cancer survival promoter as well as an early-stage-cancer formation suppressor. As an early cancer cell genesis suppressor, autophagy’s basal level housekeeping function proactively prevents the creation of mutagenic intracellular environments favorable to new cancer cell genesis. Autophagosomes effect the removal and hydrolysis of damaged mitochondria, misfolded proteins, and protein-aggregates which if left untended can lead to harmful ROS production [19]. ROS-associated DNA damage, leading to mutation driven genetic instability, is fertile ground for cancer cell genesis. Autophagy’s housekeeping activities efficiently remove potentially harmful cytosolic debris in a multistep process (Figure 1) [6, 20].

Autophagy as a cancer agonist

Acting as a pro-cancer survival mediator, stressor induced autophagic-flux amplification serves to increase per-cell numbers of autophagasome bodies. The endgame is assuring cancer cell survival during nutrient limiting or environmentally harsh (hypoxic, ROS containing) conditions.  Upregulating autophagy activity also provides the necessary cellular component “fuel” to produce additional energy. Additional energy is needed to support accelerated cell growth rates occurring within most tumor expansion scenarios. Furthermore, it is common knowledge that autophagy can come to the rescue of established cancers undergoing chemotherapy treatment. Upregulated autophagy activity, as a direct result of ongoing chemotherapy exposure, brings about a multiple drug resistance (MDR) phenotype within target cancer populations [21]. Stress-induced autophagy, whether it arises from nutrient deprivation, routine chemotherapeutic treatments, or other introduced stressor elements, can lead to cancer tumor cell dormancy. Unfortunately, with cancer cell dormancy comes the possibility of resumed tumor cell growth later once the stressor factor has been removed from the microenvironment of the cells [22].

Targeting autophagy as an additional tool for cancer treatment

Targeting autophagy inhibition as a strategy for improving chemotherapy treatment outcomes arose from well-documented evidence implicating an active/functional autophagy system with greatly enhanced survivability of all stages of cancer. As stated in the prior section above, autophagy is known to be responsible for MDR following extensive chemotherapy procedures. By its very nature as a genetically conserved cell survival mechanism, it provides an opportunity for improved cancer cell viability within harsh environmental conditions (nutrient deficiency, hypoxic conditions) known to occur within the microenvironment of solid tumor cell expansion. Enabling metastatic tumor cells to survive in new and potentially hostile microenvironments gives autophagy a central role in tumor invasion and metastasis [23]. Autophagy also provides opportunities for cancer cell transformation into a dormancy state. Cancer cell dormancy opens the door for future resurrection and metastasis once chemotherapy treatments have ceased. Unfortunately, a false sense of security arising from treatment-associated cancer cell/tumor reduction can give the impression that the cancer battle has been won. Too often, however, dormant stage cancer cell populations surge back with a renewed vengeance once the chemo-treatments have concluded! So, suffice to say that there is ample justification for seeking out a pharmaceutical autophagy inhibitor(s) solution to be used as a standalone treatment or in tandem with chemotherapeutic agents.

Pharmaceutical development of autophagy-inhibiting agents is still in its early stages. Currently, the monomeric aminoquinoline analogs, chloroquine (CQ) and hydroxychloroquine (HCQ) are undergoing Phase II/III clinical trials for their benefit in cancer therapy (Figure 2) [24].

Figure 2a

 

Figure 2b

Figure 2. Molecular structures of Chloroquine (left) and Hydroxychloroquine (right). Diagram sourced from Wikipedia.

Prior to their use as autophagy inhibitors, CQ and HCQ were used as anti-malaria and anti-inflammatory mediating agents [25]. Presently, CQ and HCQ show evidence of enhancing effectiveness of chemotherapy treatment. Autophagy inhibition makes tumor cells more sensitive to chemotherapy agents. CQ and HCQ demonstrate autophagic-flux blocking properties [26]. When we state that HCQ and CQ block autophagic flux processes, we imply that they inhibit autophagic degradation processes. Consensus opinion concludes that they inhibit autophagy by interfering with lysosomal fusion with sequestered cargo within autophagosomal bodies [27]. A major criticism of the CQ and HCQ autophagy inhibitors is their lack of autophagy suppression potency [28]. To remedy CQ potency issues, dimeric analogs of CQ were synthesized leading to more potent autophagy inhibitor molecules, Lys01 and Lys05 (Figure 3) [29].

Figure 4

 

Figure 3. Molecular structure evolution of chloroquine analogs leading to synthesis of a dimeric chloroquine form, Lys05 with enhanced autophagy inhibition properties. Diagram sourced from Quentin McAfee et al. (2012) Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency. PNAS 109 (21) 8253-8256.

Lys05, a water-soluble version of Lys01 was synthesized to enable in vivo studies[29]. This group concluded that the dimerized CQ analog, Lys05, provided greater autophagy inhibition properties as well as single-agent antitumor activity [29]. A more recent study found that a slightly different analog of the Lys05, DC661, also exhibited improved autophagy inhibition potency relative to the monomeric aminoquinoline prototype inhibitor constructs (Figure 4) [28].

Figure 5

Figure 4. Molecular structure of a more recent dimeric CQ analog, DC661. This dimeric CQ analog demonstrates improved penetration of acidic environment of lysosome and blockage of autophagy. Diagram sourced from Vito W. Rebecca et al. (2019). PPT promotes tumor growth and is the molecular target of chloroquine derivatives in cancer. Cancer Discovery; 9, 220-229.

For the sake of transparency, multiple other small molecule driven autophagy inhibitors are being evaluated for their autophagy inhibition and anti-cancer efficacy properties. Suffice to say, because of autophagy’s strategic role in the expansion and resilience of cancer, great emphasis will continue to be placed on advancing this clinical cancer research agenda.

Summary

Early impressions of autophagy’s role as a cell survival mechanism during nutrient deficiency has evolved over the past fifteen-plus years, subsequently assigning autophagy a central role in a wide range of physiological activities. This includes significant roles in developmental processes, including organ development. Autophagy continues to be recognized for its important role as a centralized cellular stress response mechanism. By providing cells with a means of extending available intracellular resources by recycling non-essential or outdated cellular components, acquisition of an operational autophagy process played a critical in the evolution of higher life forms. Recycling intracellular components enables the continued production of energy supplies without which life cannot exist.

Unfortunately, a negative consequence of autophagic processes lies in their actions to promote the growth of human cancer. In addition to providing cancer cells with a means of surviving nutrient deficiencies, autophagy also provides a means by which fast growing tumor cells, in need of additional energy resources, can sustain their rapid growth rates. In addition, the acquisition of MDR phenotypes in cancer cells can also be attributed to autophagy. Together these autophagy-associated attributes make the battle against human cancers a much more challenging undertaking.

References

  1. De Duve, C. and R. Wattiaux, Functions of lysosomes. Annu Rev Physiol, 1966. 28: p. 435-92.
  2. Mizushima, N., Autophagy: process and function. Genes Dev, 2007. 21(22): p. 2861-73.
  3. Axe, E.L., et al., Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol, 2008. 182(4): p. 685-701.
  4. Tooze, S.A. and T. Yoshimori, The origin of the autophagosomal membrane. Nat Cell Biol, 2010. 12(9): p. 831-5.
  5. Korolchuk, V.I., et al., Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates. Mol Cell, 2009. 33(4): p. 517-27.
  6. Lane, L.V.M.a.J.D., The autophagosome: current understanding of formation and maturation. Research and Reports in Biochemistry, 2015. 5: p. 39-58.
  7. Rabinowitz, J.D. and E. White, Autophagy and metabolism. Science, 2010. 330(6009): p. 1344-8.
  8. Anding, A.L. and E.H. Baehrecke, Cleaning House: Selective Autophagy of Organelles. Dev Cell, 2017. 41(1): p. 10-22.
  9. Kuma, A. and N. Mizushima, Physiological role of autophagy as an intracellular recycling system: with an emphasis on nutrient metabolism. Semin Cell Dev Biol, 2010. 21(7): p. 683-90.
  10. Loos, B., A. du Toit, and J.H. Hofmeyr, Defining and measuring autophagosome flux-concept and reality. Autophagy, 2014. 10(11): p. 2087-96.
  11. Laplante, M. and D.M. Sabatini, mTOR signaling at a glance. J Cell Sci, 2009. 122(Pt 20): p. 3589-94.
  12. Dibble, C.C. and B.D. Manning, Signal integration by mTORC1 coordinates nutrient input with biosynthetic output. Nat Cell Biol, 2013. 15(6): p. 555-64.
  13. Foster, K.G. and D.C. Fingar, Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. J Biol Chem, 2010. 285(19): p. 14071-7.
  14. Cork, G.K., J. Thompson, and C. Slawson, Real Talk: The Inter-play Between the mTOR, AMPK, and Hexosamine Biosynthetic Pathways in Cell Signaling. Front Endocrinol (Lausanne), 2018. 9: p. 522.
  15. Torii, S., et al., Identification of PPM1D as an essential Ulk1 phosphatase for genotoxic stress-induced autophagy. EMBO Rep, 2016. 17(11): p. 1552-1564.
  16. Kim, J., et al., AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol, 2011. 13(2): p. 132-41.
  17. Jiang, P. and N. Mizushima, Autophagy and human diseases. Cell Res, 2014. 24(1): p. 69-79.
  18. Xie, W. and J. Zhou, Aberrant regulation of autophagy in mammalian diseases. Biol Lett, 2018. 14(1).
  19. Mathew, R., et al., Autophagy suppresses tumorigenesis through elimination of p62. Cell, 2009. 137(6): p. 1062-75.
  20. Apel, A., et al., Autophagy-A double-edged sword in oncology. Int J Cancer, 2009. 125(5): p. 991-5.
  21. Bishop, E. and T.D. Bradshaw, Autophagy modulation: a prudent approach in cancer treatment? Cancer Chemother Pharmacol, 2018. 82(6): p. 913-922.
  22. Lu, Z., et al., The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells. J Clin Invest, 2008. 118(12): p. 3917-29.
  23. Kenific, C.M., A. Thorburn, and J. Debnath, Autophagy and metastasis: another double-edged sword. Curr Opin Cell Biol, 2010. 22(2): p. 241-5.
  24. Shi, T.T., et al., Research progress of hydroxychloroquine and autophagy inhibitors on cancer. Cancer Chemother Pharmacol, 2017. 79(2): p. 287-294.
  25. Haladyj, E., et al., Antimalarials – are they effective and safe in rheumatic diseases? Reumatologia, 2018. 56(3): p. 164-173.
  26. Xu, R., et al., The clinical value of using chloroquine or hydroxychloroquine as autophagy inhibitors in the treatment of cancers: A systematic review and meta-analysis. Medicine (Baltimore), 2018. 97(46): p. e12912.
  27. Mauthe, M., et al., Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy, 2018. 14(8): p. 1435-1455.
  28. Rebecca, V.W., et al., PPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer. Cancer Discov, 2019. 9(2): p. 220-229.
  29. McAfee, Q., et al., Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency. Proc Natl Acad Sci U S A, 2012. 109(21): p. 8253-8.