Inhibition of autolysosomes by repurposing drugs as a promising therapeutic strategy for the treatment of cancers

Abstract

Autophagy process coordinates lysosomal destruction of cytoplasmic materials including damaged organelles and proteins. The double-membranous ‘autophagosome’ selectively sequesters the cytosolic components, followed by ‘autolysosome’ formation for degradation. By recycling energy, autophagy preserves intracellular homeostasis. Autophagy also acts a crucial function in various cellular events including cell survival and death in response to stressed conditions. Thus, any dysfunction in autophagy causes a wide variety of pathologies including cancers. Multiple lines of evidence have decorated the importance of autophagy for the regulation of cancer development. Aberrant autophagy can govern healthy normal cells to endure the transformation of malignant cells. By comparison, cancer cells are apt to control autophagy to bloom it and to repel therapeutic challenges. Hence, the modulation of autolysosomes can assassinate malignant cells or sharpen them for therapy. Most current research is trying to manipulate autophagy appropriately for cancer therapies and a wide variety of medical objectives are turning forward to inhibit the acidification of lysosomes and autolysosomes with repurposing drugs. This review explores the lysosomotropic agents be utilized to treat cancers through their inhibiting roles in the acidification system and discuss the preclinical and clinical clues for hints the next generation of pharmacological agents for the treatment of cancers.

KEYWORDS:

• Autolysosome
• autophagy-lysosomal pathway
• lysosomal function
• drug repurposing
• chloroquine analogue
• cancer therapy

Introduction

Autophagy is a lysosome-oriented degrading process of potentially dented or redundant cytoplasmic entities for promoting nutrient recycling (Towers et al. 2020; Zada et al. 2021). Autophagy maintains cell homeostasis regarding several stressful states such as deprivation of nutrient, hypoxia or endoplasmic reticulum (ER) damage. Under basal level conditions, autophagy regulates metabolic and cellular homeostasis (Ceccariglia et al. 2020). This process is essential in all mammalian cells to generate building block molecules for supporting other cellular processes (Amaravadi et al. 2016; Yun and Lee 2018; Yazdani et al. 2019). This process also maintains cellular fitness by supplying energy and nutrients through both nonselective and selective degradation of macromolecules and cellular organelles in response to several stresses including hypoxia (He et al. 2018; Yu et al. 2018). Accumulating evidence has suggested that any dysfunctions in this intricate and securely regulated process such as inherited mutations in the highly conserved autophagy-related (ATG) genes have been concerned in a wide variety of physiological disorders and human diseases (Table 1).

Table 1. Dysfunctional autophagy is associated with different human diseases.

Cancers	Breast	Haplo insufficiency of Beclin 1 (Liang et al. 1999), mutation and deletion of EI24/PIG8 (Zhao et al. 2005)
	Ovarian	Haplo-insufficiency of Beclin 1 (Cai et al. 2014)
	Endometrial	ATG4 C, ULK4 and FIP200 along with MTOR-activating mutations (Devis-Jauregui et al. 2021)
	Prostate	Haplo-insufficiency of Beclin 1 (Levy et al. 2017)
	Brain	Mutation in PARK2 (Veeriah et al. 2010)
	Lung	Mutation in PARK2 (Xiong et al. 2015)
	Gastric/colorectal	Mutation in UVRAG and PARK2 (Kim et al. 2008; Poulogiannis et al. 2010; Park et al. 2014)
	Carcinoma (nasopharyngeal/ hepatocellular/ esophageal squamous cell carcinoma)	MicroRNA 106a-5p upregulation (Zhu et al. 2021), LncRNA NBR2 downregulation (Sheng et al. 2021); Genetic polymorphisms of ATG5 (Yang et al. 2017)
	Sarcoma	Overexpression of Bcl-2 and Bcl-XL; Mutation in p53 (Min et al. 2017)
Bone	Paget’s disease	Mutation in p62 (Usategui-Martin et al. 2020)
Lung	Asthma	Genetic polymorphisms of ATG5 (Martin et al. 2012)
	Cystic fibrosis	Mutation in CFTR (Groman et al. 2002); Sequestration of Beclin 1 in perinuclear aggregates (Luciani et al. 2011; Maiuri et al. 2019)
	Lymphangioleiomyomatosis	Mutations in TSC1/2 (Muzykewicz et al. 2009)
Heart/kidney	Ischemia/ reperfusion injury	Overexpression of Beclin 1 (Hamacher-Brady et al. 2006; Li et al. 2020)
Central nervous system	Alzheimer’s disease	Mutation in PSEN1 (Janssen et al. 2003; Tran and Reddy 2020)
	Parkinson’s disease	Mutations in PINK1 and PARK2 (Pickrell and Youle 2015; Barazzuol et al. 2020)
	Amyotrophic lateral sclerosis (ALS)	Mutation in p62; SOD1; OPTN; dynactin and dynein (Lee et al. 2015)
	SENDA	Mutation in WDR45 (WIPI4) (Mollereau and Walter 2019)
	Hungtington’s disease	Upregulation of mRNA expression of LC3A, ULK2, and LAMP2 and downregulation of PINK1, WDFY3 or ALFY, and FKBP1A (Martin et al. 2015)
	Hereditary spastic paraparesis	Mutations in TECPR2; SPG15 or KIF5A (Oz-Levi et al. 2012; Renvoise et al. 2014; Lopez et al. 2015)
Intestine	Crohn’s disease	T300A mutation in Atg16L1 (Ma et al. 2021); polymorphisms and mutations in NOD2 (Yamamoto and Ma 2009) and IRGM (Mehto et al. 2019)
	Ulcerative colitis	Downregulation of ATF4 and VDR (Shao et al. 2021); SNPs in REDD1/SMURF1 (Franke et al. 2010; Angelidou et al. 2018)
Polycystic kidney disease		Mutation in Pkd1/2 (Nowak and Edelstein 2020); BCL-2 deficiency (Veis et al. 1993)
Vacuolar myopathies in skeletal muscle		Accumulation of LC3-II (Margeta 2020), mutations in LAMP2, VMA21, and CLN3 (Weihl et al. 2015)
Liver	α1-Antitrypsin deficiency	Z mutation (SERPINA1) (Marciniak and Lomas 2010)
	NAFLD	Down-regulation of ATG7 (Khambu et al. 2018)
Immune diseases	Vici syndrome	Recessive mutation in EPG5 (Byrne et al. 2016)
	Lupus (SLE)	Polymorphisms in ATG5 and PRDM1 (Zhou et al. 2011)
	RA	Overexpression of Beclin 1, ATG5 and LC3 (Zhu et al. 2017)
Microbial infection	Mycobacterium tuberculosis	Polymorphism in IRGM (Xie et al. 2017)
	Mycobacterium leprae	Polymorphism in NOD2 (Leturiondo et al. 2020)
Metabolic Dysfunction	Type II diabetes	Loss of function of ATG7 and ER stress (Yao et al. 2021)
	Metabolic syndrome	Downregulation of ATG7 and ATG5; insulin sensitivity (Menikdiwela et al. 2020)
	Obesity (obesogenesis)	Deletion in ATG7, LAMP2 or TFEB (Zhang et al. 2018)
Aging		Overexpression of ATG5 in mice increases lifespan (Aman et al. 2021)
Lysosomal Disease	Danon disease	Mutation in LAMP2 (Lieberman et al. 2012; Majer et al. 2012)
	Pompe disease	Acid α-glucosidase (GAA) deficiency (Lieberman et al. 2012)
	Gaucher disease	Mutation in glucocerebrosidase (V394L) (Lieberman et al. 2012)

ATF4 (activating transcription factor 4), ATG16L1 (autophagy-related 16 like 1), CFTR (cystic fibrosis transmembrane conductance regulator), DRAM1 (DNA damage-regulated autophagy modulator protein 1), EPG5 (ectopic P-granules autophagy protein 5 homolog), FIP200 (RB1 inducible coiled-coil 1), FKBP1A (FK506-binding protein 1A), IRGM (immunity-related GTPase family M), KIF5A (kinesin heavy-chain), LncRNA NBR2 (long noncoding RNA neighbor of BRCA1 gene 2), NAFLD (Nonalcoholic fatty liver disease), NOD2 (nucleotide binding oligomerization domain containing 2), OPTN (optineurin), PAMPs (pathogen-associated molecular patterns), PARK2 (Parkin RBR E3 ubiquitin protein ligase), PINK1 (PTEN induced putative kinase 1), Pkd1/2 (polycystin 1/2), PSEN1 (presenilin 1), RA (rheumatoid arthritis), SENDA (Static encephalophagy of childhood with neurodegeneration in adulthood), SLE (systemic lupus erythematosus), SMURF1 (SMAD specific E3 ubiquitin protein ligase 1), SNPs (Single-nucleotide polymorphisms), SOD1 (superoxide disumutase 1), TSC1/2 (tuberous sclerosis complex 1 or 2), UVRAG (UV radiation resistance associated), VDR (vitamin D receptor), WDR45 (WD repeat domain 45).

Three major autophagic molecular features have been mechanistically described based on the target substrates involved in lysosomal degradation (Figure 1), namely macroautophagy (simply autophagy), chaperone-mediated autophagy (CMA) and microautophagy (Galluzzi et al. 2017). Autophagy occurs in multi-stages such as initiation, elongation, closure, docking and fusion with the endolysosomal system with the regulation of the ATG genes (Al-Bari 2020; Devis-Jauregui et al. 2021). Autophagy begins with an isolating membrane (IM) formation, previously familiar with phagophore. Although the origin(s) of IM in autophagy has long been debated, several membranous organelles such as mitochondria, ER, Golgi apparatus, or plasma membrane (PM) and their contact sites such as ER exit sites (ERES), ER–Golgi intermediate compartment (ERGIC) have been linked up the membrane donors for the nascent IM (Hurley and Young 2017; Al-Bari 2020). The IM is sequestrated in the cytoplasmic cargoes to form autophagosomes that then dock and fuse with the lysosomes for the generation of de novo structures called ‘autolysosomes’ (Al-Bari 2020). Finally, the degradation of autophagic cargo is triggered by the lysosomal hydrolytic niche (low pH with acidic hydrolases) (Noda and Inagaki 2015; Hurley and Young 2017; Al-Bari 2020; Devis-Jauregui et al. 2021) to split up into several nutritional constituents into the cytoplasm for energy production and cellular repairing (Lamb et al. 2013).

Figure 1. Different classes of autophagy process. The autophagy is classified as (A) macroautophagy where cytosolic constituents are submerged and delivered to the lysosome; (B) microautophagy where cytosolic wastage products are straightly submerged and co-opted by lysosome via redisposition of its membrane and (C) chaperone-mediated autophagy (CMA) where KFERQ motif holding cellular proteins documented by the cytosolic HSP70 chaperone and translocated to the lysosomal membrane via interacting with LAMP2 receptor leading to their degradation. Macroautophagy (simply autophagy) proceeds the isolation membrane (IM) formation. Cellular stresses culminate in AMPK stimulation and mTORC1 suppression that in turn makes the ULK1 complex dissociated with mTORC1 and initiates autophagy. Through a series of interchain responses, this IM confiscates its cargos into an autophagosome (AP, a double-membrane vesicle) that in turn kisses and fuses with the lysosome to make autolysosome (AL). The AL cargos are degraded by an acidic niche containing hydrolytic enzymes in lysosomal lumen. The degrading products are used for energy production or biosynthesis of macromolecules. TFEB coordinates the stress responses like starvation via lysosome and controls countless cellular processes including autophagy and lysosomal biogenesis (Al-Bari and Xu 2020).

In response to various stresses (intracellular or environmental), autophagy initiation is controlled chiefly by two important cellular pathways: mTOR (mechanistic target of rapamycin)-dependent and -independent pathways (Sabatini 2017; Al-Bari and Xu 2020). In response to amino acids or growth factors, mTOR pathway is induced which in turn phosphorylated and incorporated several ATG genes within mTOR complex and inhibited autophagy. For example, when mTORC1 is active in nutrient-rich states, associates with UNC51-like kinase 1 (ULK1) complex (not free from the ULK1 complex) through Raptor and phosphorylates ULK1 at Ser⁷⁵⁸ and Ser⁶³⁸, and ATG13 at Ser²⁵⁸ resulting inhibition of autophagy (Dossou and Basu 2019). Conversely, during nutrient deprivation, mTOR pathway is suppressed and initiated autophagy because inactive mTOR complex releases several autophagy. The major events of mTOR pathway-dependent regulation of autophagy are represented here. The initiation stage is controlled by AMPK (AMP-activated protein kinase), ULK1 and mTOR complex 1 (mTORC1). mTOR pathway is inhibited by AMPK that induces the autophagic process (Kim et al. 2011; Yun and Lee 2018). Here, mTORC1 acts as a chief inhibitor of autophagosome development by ULK1. Under glucose deprivation, the activated AMPK suppresses mTORC1, then phosphorylates ULK1 in a straight way and leads to initiate autophagy (Kim et al. 2011). Nucleation stage of the IM is interceded by the complex of Beclin-1-class III phosphatidylinositol 3-kinase (PI3 K) (class III PI3 K) that comprises Beclin-1, Vps34 (class III PI3 K), p150, ATG14L and the activating molecule in Beclin-1-regulated autophagy (AMBRA-1) (Mizushima et al. 2011). IM elongation stage turning to completed autophagosome is controlled by two ubiquitin-like protein complexes: ATG5–ATG12–ATG16L1 and the microtubule-associated proteins 1A/1B light chain 3B (called LC3-II). Several ATGs such as ATG7 and ATG10 are required peacekeepers of the process (Ichimura et al. 2000; Behrends et al. 2010). LC3-1, the key mammalian ortholog of ATG8 is transformed to LC3-II and tarnished after the fusion of autophagosomes with lysosomes. The final degradation stage is the production of nutrient materials (Al-Bari 2020).

At a glance of lysosomes

Lysosomes are housekeeping organelles that play a vital role in the conservation of intracellular homeostasis by degrading cellular and extracellular constituents (Ballabio 2016). Over the last 10 years, innovation in lysosome biology research has recognized an extensive role of lysosomes in the pathology of diseases as well as focused on targeting the lysosome in different diseases (e.g. cancers). The magnitude of lysosomes varies from 0.1 to 1.0 µm in mammalian cells, which is suggested to be lesser than autophagosome ranges (0.5–1.5 µm) (Burman and Ktistakis 2010; Mizushima and Komatsu 2011). Anatomically, the trafficking vesicles (Figure 2) may suggest as early endosomes (pH of 6.0–6.6), late endosomes (extra acidic pH of 5) or the highly acidic organelles lysosomes (pH of 4.5) (Mindell 2012; Nwadike et al. 2018; Chen et al. 2020). Therefore, the autolysosome, the fused form of lysosome with autophagosome would necessary to partake in a firmly practical adjustment to preserve optimal acidic pH for executing digestion of the intraluminal materials. The autolysosome retains lower pH than the lysosome based on the autophagosome. Lysosomes are surrounded by integral lipoprotein membranes called lysosome-associated membrane proteins 1 and 2 (LAMP1 and 2) (Saftig and Klumperman 2009) and contain an acidic interior with different soluble hydrolases (e.g. cathepsins) (Piao and Amaravadi 2016). LAMP1 and 2 are essential for lysosomal regeneration, maintenance lysosomal acidity as well as CMA (Saftig and Klumperman 2009). As a receptor of CMA, LAMP2 can be reallocated from the lumen to lysosomal membrane (Cuervo and Wong 2014). In addition, heat shock protein 70 (HSP70) can protect lysosomal membrane permeabilization (LMP) (Tang et al. 2017). Lysosomes contain at least 60 soluble hydrolases within the acidic niche (Saftig and Sandhoff 2013). Lysosomal acid hydrolases execute their best action at pH 4.5–5.0 within the acidic lysosome. These hydrolases are accountable for the degradation of waste materials and salvaging of essential nutrients to preserve cellular homeostasis (Saftig and Klumperman 2009; Stoka et al. 2016). The vacuolar type ATPase (v-ATPase), an ATP-driven hydrogen (H⁺) ion pump, uses the energy potential by hydrolyzing ATP into ADP to drive H⁺ ions into the intralysosomal lumen. As a result, ATP-driven proton (H⁺) pump maintains optimal acidic niche (pH of ∼4.5–5.0) comparatively to somewhat basic cytosol (pH 7.2) (Mindell 2012; Settembre et al. 2013) for proper activities of lysosomal hydrolases (Pamarthy et al. 2018). The acidic lysosomal pH is also crucial for the transport and maturation of lysosomes as well as autophagosomes (Al-Bari 2020). Moreover, the acidic lysosomal pH is also applied by transmembranous permeases to disseminate digestive products to the cytosol (Perera and Zoncu 2016).

Figure 2. The central position of lysosomes at the ALP. Activated lysosomes and autolysosomes are associated with terminal degradation stage through endocytic and autophagic pathways. Exogenous products are endocytosed by endosomes and autolysosomes act as an integral part of the autophagy for degradation. The membrane of lysosome plays as a podium for the gathering of mTORC1 complex, which plays a main role for nutrient sensing signals of lysosomes to control energy levels through downstream mediators including TFEB. Interior pHs of intracellular vesicles steadily decrease laterally from early endosomes to late endosomes to lysosomes, the mostly acidic organelles. Lysosomes create and preserve their pH homeostasis by applying v-ATPase carrier. The positive charged ions such as K⁺ can be dispatched by K⁺ ion channel or by exchanged by Cl⁻ influx carrier via ClC-7, a Cl⁻/H⁺ antiporter.

In the case of garbage disposal system in the cell, autophagosomes transport the intracellular components to the lysosomes, and endocytic and phagocytic systems transport exogenous factors to the lysosomes (Lopez-Hernandez et al. 2020). Here, the centrally acting lysosomes regulate autophagy in four main ways: supporting IM formation, lysophagy, autolysosomal degradation and autophagic lysosomal reformation (ALR). By interacting with special membrane contact sites of other organelles including ER, peroxisomes and mitochondria, lysosomes donate membranes for IM (Hurley and Young 2017; Simmen and Tagaya 2017; Al-Bari 2020). Selective autophagy controls the homeostasis of lysosomes when damaged lysosomes LMP are not repaired. The injured lysosomes are submerged by autophagosomes which in turn fuse with efficient lysosomes to eliminate them (Trivedi et al. 2020). The levels of lysosomes can be reestablished through ALR (autophagic lysosomal reformation) process (Chen and Yu 2018; Al-Bari 2020). The lysosomal homeostasis is then reestablished by its biogenesis.

Lysosomal repair and lysophagy

Under biological conditions, the lysosomal quality and quantity are constantly regulated by its homeostatic pathway poised between development and degradation (Maejima et al. 2013; Zhu et al. 2020). The integrity and optimum activity of lysosomes are very important for cell survival. Lysosomal membrane damage (LMD) disrupts cytosolic harmony causing the release of intralysosomal acidic components, which trigger a series of apoptotic pathways including ferroptosis (Zhu et al. 2020). Several signal stressors such as lysosomotropic drugs, pathogens, reactive oxygen species (ROS) and other factors may lose membrane integrity and inflict varying extent of LMP and LMD and these are potential hazards to cell fate (Zhu et al. 2020). For preserving lysosomal integrity and intracellular homeostatic balance, renewal of lysosomes is rapidly commenced in the LMD state, the process called lysosomal quality control (LQC) encompassing lysosomal repair, lysophagy and lysosomal regeneration (Maejima et al. 2013; Zhu et al. 2020). Indeed, the imbalance of lysosomal homeostasis is crucial for the development of several degenerative pathologies, including cancers, inflammatory diseases and infections (Zhu et al. 2020). Thus LQC makes a novel but potential target of lysosomal impairment and additional investigation for effective disease managements.

Interestingly, the LMD can be repaired and prevented the release of luminal hydrolases by the actions of HSP70 and ESCRT (endosomal sorting complex required for transport) machinery (Papadopoulos et al. 2020; Zhu et al. 2020). Earlier work appreciated the defensive activity of HSP70 for preserving lysosomal integrity. HSP70 binds to bis(monoacylglycerol)phosphate that leads to an increase in the activity of acid sphingomyelinase (ASM) to regulate optimum lipid configuration (Mahapatra et al. 2021) and prevent LMP (Papadopoulos et al. 2020). Recent studies showed that the ESCRT machinery comprising four complexes (ESCRT-0, -I, -II, -III) orchestrates for the recovery of LMD and cell survival through multiple mechanisms in a coordinated manner (Christ et al. 2017; Radulovic et al. 2018; Skowyra et al. 2018; Zhu et al. 2020). ESCRTs can renovate several tiny perforations in the lysosomal membrane at the initial stage of its injury (Skowyra et al. 2018). After LMP, the Ca²⁺ release from injured lysosomes into the cytosol enhances the lipid-binding capacity of ALIX (apoptosis-linked gene-2-interacting protein X), a constituent of the ESCRT machinery and activated the CHMP4B (multivesicular body protein 4B), a core factor of the ESCRT-III machinery for the regulation of the healing process. Moreover, TSG101 (tumor susceptibility gene 101 protein), a factor of the ESCRT-I machinery, is found to be vital in ALIX deficiency and helps the repairing machinery to the membranes of lysosomes (Papadopoulos et al. 2020; Zhu et al. 2020; Mahapatra et al. 2021). Although the exact mechanistic pathways by which ESCRT machinery heals the lysosomal membranes are ambiguous, they could be associated with promoting the development of filamentous helices and the tightening of pores in lipid bilayers on the membrane surface (Christ et al. 2017).

When an extensive injury of the lysosomal membrane occurs and the LMP cannot be repaired, selective autophagy of lysosomes, called lysophagy is initiated to clear the dented lysosomes (Serrano-Puebla and Boya 2016; Papadopoulos and Meyer 2017; Papadopoulos et al. 2020). Similar to other selective autophagy, in lysophagy the impaired lysosomes are marked with ubiquitin and ultimately fused with normal effective lysosomes through autolysosomal formation (Yao et al. 2021). Two ubiquitination pathways regulating lysophagy have been identified: cytosolic lectins such as galectins (Gals) and the ligase complex Skp1/CUL1/F-box (SCF) with F-box protein 27 (SCF^FBXO27) (Thurston et al. 2012; Maejima et al. 2013; Yoshida et al. 2017; Yao et al. 2021). The galactose-containing lectins, Gals are usually localized in the cytosols where galactose-moiety chains are anchored at the cell surface (Li et al. 2021). Gals are crucial for monitoring lysophagy because LMD and its elimination can be measured by observing the levels of Gals (Thurston et al. 2012; Maejima et al. 2013; Papadopoulos and Meyer 2017). Gals such as Gal-1, -3, -8, and -9 play as radars for various types of LMD by interacting with β-galactosides on the luminal side of its membrane (Chauhan et al. 2016; Zhu et al. 2020). For example, LMD permits the Gal-3 recruitment to its broken membrane (Paz et al. 2010; Li et al. 2021) and induces lysophagy by activating several autophagy regulators such as ULK1, Beclin 1 and ATG16L after binding with TRIM16 (tripartite domain containing protein 16) (Chauhan et al. 2016). Finally, Gal-3 is adapted by a K⁶³ ubiquitin chain and interacts with LC3 on the IM via p62, an autophagy receptor and finally forms an autolysosome (Chauhan et al. 2016). Likewise, Gal-8 and Gal-9 can also be engaged in dented lysosomes and promote lysophagy through various pathways (Thurston et al. 2012; Chauhan et al. 2016). For instance, Gal-8 specifically interacts with LC3 on the IM through CALCOCO2 (Ca²⁺-binding and coiled-coil domain-containing protein 2), an autophagy receptor, resulting in the degradation of dented lysosomes (Thurston et al. 2012; Zhu et al. 2020). For activation of lysophagy, SCF^FBXO27 is also engaged in impaired lysosomes and ubiquitinates LAMP2 by interacting with LC3 on the IM surface via p62 (Yoshida et al. 2017; Zhu et al. 2020).

Two important signaling pathways can induce the biogenesis of new lysosomal components. One pathway is the ESCRT machinery-dependent restoration of dented lysosomes. The second pathway is initiated by the detachment of mTORC1 from the dented lysosomes (Jia et al. 2018; Papadopoulos et al. 2020). Since mTOR usually preserves an inactive form of the TFEB (transcription factor EB) by phosphorylation, mTORC1detachment causes TFEB dephosphorylation and nuclear translocation leading to lysosomal generation by TFEB interacting with CLEAR (coordinated lysosomal expression and regulation) (Settembre et al. 2013; Papadopoulos et al. 2020).

Lysosomal involvement in cancer

Lysosomal action and dysfunction have been shown to act vital roles in cancers; however, the mechanisms in which lysosomes impart to develop cancer and metastasis are still being explored. Quickly dividing cells like cancers are extremely reliant on efficient lysosomes to maintain homeostasis under stress conditions in the TME (Ballabio 2016; Ballabio and Bonifacino 2020). In parallel, dramatic variations in volume, composition and subcellular localization of lysosomes occur during the conversion of normal pre-malignant cells to tumorigenesis and progression (Kirkegaard and Jaattela 2009; Kallunki et al. 2013). Many cancer cells use alternative metabolic pathways that can increase ROS turning to dysregulation of lysosomes and cause LMP (Aits and Jaattela 2013). Moreover, in large arrays of cancers, lysosomal cathepsins are strongly expressed and delocalized during cancer progression via LMP (Kallunki et al. 2013). Mislocalization of these cathepsins to the extracellular matrix (ECM) stimulates tumor cell survival, angiogenesis and invasion (Gocheva et al. 2006). In addition, secretory lysosomal glycosidases such as β-N-acetylglucosaminidase (β-NAG) enable ECM degradation (Ramessur et al. 2010). Sphingosine kinase (SK) is up-regulated (Le Scolan et al. 2005) and ASM is downregulated (Petersen et al. 2013) in different types of cancer cells. These variations also affect the structural integrity of the lysosomal membrane turning to LMP. Lysosomes also act an essential function in drug resistance in cancer by confiscating weakly basic chemotherapies within the cells as well as an exocytotic mechanism (Zhitomirsky and Assaraf 2016). Thus cancer cells become resistant to chemotherapy and the importance of lysosomes as a therapeutic approach for cancer treatment.

It is growing evidence that the dysregulation of homeostatic dynamics in tumor cells by modifications of metabolic pathways and pH gradient reversal is a key factor for succeeding cancer development, invasion and metastases (Fais et al. 2007; Taylor et al. 2015). Cancer cells cannot endure extreme proton (H⁺) in their niches without appropriate removal. These cells exploit numerous routes to expel H⁺ ions, causing basic intracellular and acidic extracellular TME, commonly called as pH gradient reversal. In the pH gradient reversal, acidic extracellular pH (pHe) changes from normal healthy cells (∼7.4) to cancer cells (6.5–7.0) whereas preserving neutral-to-basic intracellular pH (pHi) of normal healthy cells (∼7.2) or additional basicity in it as high as value 7.6 in tumor lesions (De Milito et al. 2010). The acidic tumor pHe initiates cell survival and multiplications, tends to chemoresistance and provokes metastatic budding (Luciani et al. 2004; Rofstad et al. 2006) whereas the preservation of basic pHi bears resistance to cytotoxicity (Coss et al. 2003). Moreover, the acidic tumor pHe stimulates immune escape of tumor cells and efficient proteolytic degradation of ECM by invasive tumor cells (Brown and Murray 2015; Huber et al. 2017). The possible mechanisms conveying pH gradient reversal comprise the upregulation of multiple PM-oriented ion pumps including Na⁺/H⁺ exchanger 1 (NHE1), v-ATPase as well as carbonic anhydrases recognized as proteins for net acid bulge (White et al. 2017). Moreover, v-ATPase expels H⁺ ions from the cytoplasm into intracellular organelles including lysosomes which act as main intracellular H⁺ ion stores (Maxson and Grinstein 2014; Oot et al. 2017). The activity of v-ATPase is boosted in tumor cells (Sennoune et al. 2004) and knockdown of the c subunit of v-ATPase diminishes cell proliferation of hepatocellular carcinoma (HCC) and sensitizes breast cancers to chemotherapies (You et al. 2009) suggesting that dysfunctional pH gradient in cancers is well adjusted by several ion transporters (De Milito et al. 2010). Moreover, v-ATPase acts as a master mediator of the transcription factor, E2F1-mediated lysosomal trafficking and mTORC1 stimulation in malignancy (Meo-Evoli et al. 2015). Recent study suggests that STAT3 (signal transducer and activator of transcription-3) confines to the lysosomal membrane and associates with coiled-coil domain in v-ATPase. STAT3 augments lysosomal acidity by activating v-ATPase. Moreover, STAT3 transports between nuclei and lysosomes on the basis of cytosolic pH, shifting its actions as a v-ATPase activator. This action is important for preserving basic cytosol in normal state and once cancers challenge acidifying stress (Liu et al. 2018). Thus the pH gradient reversal is emergently associated with as a widespread trademark of cancer detected in malicious tumors (Stock and Pedersen 2017; White et al. 2017). This differential pH gradient concept becomes a potential exploitable avenue for the treatment of cancer by exploiting features of TME to destroy cancer cells.

Autophagy-lysosomal pathway and its regulation

The autophagy-lysosomal pathway (ALP) regulates a variety of signaling pathways via autophagy and endocytosis to preserve cellular homeostasis (Aits and Jaattela 2013; Saftig and Sandhoff 2013; Kroemer and Galluzzi 2017). In recent years, the insight of ALP has changed that the pathway participates in many other cellular processes (Ballabio 2016; Ballabio and Bonifacino 2020) such as PM restoration, exosome release, cell migration, and tumor metastasis (Figure 2). However, defects of ALP have been shown to trigger different types of sporadic lysosomal storage disorders (LSDs) (Parenti et al. 2015; Platt et al. 2018). In addition, the role of dysregulated ALP has been widely studied in current years in several common human diseases including cancers (Parenti et al. 2015; Platt et al. 2018; Chang and Dong 2020). Thus the ALP is an important validated target of therapeutic interventions with importance on their significance for cancer therapy.

Regulation of autophagy-lysosomal pathway

The ALP is an essential pathway for regulating intracellular homeostasis. Predictably, the ALP is co-ordinately regulated in many pathways to ensure the clearance efficacy of excessive and damaged proteins and organelles. The well-known controller of autophagy is the mTORC complex (Figure 3) and based on the structural differences, this complex is subdivided into mTORC1 and mTORC2. mTORC1 replies to numerous input signals including nutrition status, growth factor levels and cellular energy demands. Amino acid-mediated cellular sensing occurs within the lumens of lysosomes, while other amino acids are sensed by cytosolic proteins (Jewell et al. 2013). Specifically, amino acid sensors govern the localization and activation of mTORC1. For example, glutamine (Gln), leucine (Leu), and arginine (Arg) are sensed by solute carrier family 38 member 9 (SLC38A9) and influence mTORC1 stimulation via mechanisms connecting to lysosomal v-ATPase activity and the complex ‘Ragulator’. It has been found that the localization of mTORC1 to the membrane of lysosome is activated by amino acid-mediated enrolment of GTP-binding protein RHEB and Ras-related GTP binding protein A (Rag GTPase). The v-ATPase on the lysosomal membrane is also tangled to amino acid sensing and connects with Rag GTPase and Ragulator (Al-Bari and Xu 2020). Amino acids stimulate RAGs with Ragulator facilitated the activation of RAGs followed by interacting with mTORC1. Thus the above mechanistic pathways are called the LYNUS (lysosome nutrient-sensing) machinery (Figure 1).

Figure 3. Control of ALP by mTORC1-dependent and independent signal transductions. Amino acid (AA) signal, growth factors like insulin and cellular ATP regulate autophagy via mTORC1 mTORC1-dependent and independent pathways. Insulin stimulates both mTORC1 and mTORC2 via the traditional PI3K-AKT-TSC1/2 (tuberous sclerosis complex 1/2)-RHEB pathway. In Ras-MAPK (ERK) signal, ERK and RSK suppress TSC2 to induce mTORC1. Low nutrition status induces AMPK and TSC complex, suppressing RHEB and mTORC1. Carriage of AAs controls mTORC1 action via AA transporters like SLC3A2 in cell membrane as well as SLC38A9 in lysosomal membrane. The RAGULATOR complex ties up the RAGs to the surface of lysosome. V-ATPase is essential for the induction of mTORC1 on lysosomal surface. RAG A/B is controlled by the GATOR1 complex and GATOR2. KICSTOR complex acts as an essential action in tethering GATOR1 to the lysosomal surface, thus suppressing the activity of RAG A/B and mTORC1. Lysosomal membrane-oriented arginine (Arg) transporter, SLC38A9 binds with the RAGs and RAGULATOR to stimulate mTORC1. DNA damage inhibits mTORC1 activity by activating p53 in an AMPK- and TSC1/2-conditional way. Activated mTORC1 controls TFEB intracellular position via its phosphorylation (at Ser²¹¹) and subsequent confiscation with 14-3-3 protein. mTORC2 regulates autophagy via FOXO3 pathway. Phosphorylated FOXO3 interacts with 14-3-3 protein in cytoplasm and prevents induction of gene transcription. mTOR-independent cell signaling controlling autophagy comprises cAMP-Epac-PLCϵ-IP3, Ca²⁺-calpains–G_sα and inositol (Ins) signaling pathways. Intracytosolic Ca²⁺ status can be amplified by multiple Ca²⁺ channels including the TRP channel. An upsurge in cytosolic Ca²⁺ levels stimulates calpains and then Gsα/AC/cAMP/Epac/Rap2B/PLCϵ signal which regulates autophagy by endorsing IP3 via its receptor, IP3R activation. IP3 binds to IP3 receptor (IP3R) on ER to release stored Ca²⁺ that impairs AP maturation by blocking AP-lysosome fusion. Ca²⁺ efflux through lysosomal NAADP triggered TRP channel can be extra augmented by Ca²⁺ secretion from IP3R causing activation of the CaMKKβ–AMPK signal and AP formation. However, Ca²⁺ secretion from lysosomes can influence lysosomal pH and repeal fusion of lysosomes with APs. On the other hand, an upsurge Ca²⁺ concentration in cytosol next stimulates CAMKK-β, tracked by AMPK-mediated autophagy induction. In normal cases, phosphorylated TFEB by mTORC1 is sequestered with 14-3-3 protein in cytoplasm. Under fasting, lysosomal mTORC1 is inactive form and upsurge Ca²⁺ is secreted from lysosome via TRPML1 channel. Then Ca²⁺ stimulates CaN (calcineurin) that interacts and dephosphorylates TFEB. This form of TFEB is incapable to interact with 14-3-3 protein and easily moves to the nucleus (Al-Bari and Xu 2020).

Transcriptional regulation of autophagy-lysosomal pathway

Several stressful environments such as starvation, hypoxia and chemotherapies stimulate autophagy in cancer cells via transcriptional regulation. In tumor cells, autophagy usually supports cell growth in hypoxic sections (Singh et al. 2018). HIF-1α (hypoxia-inducible transcription factor 1α) transcribes several genes that endorse angiogenesis as well as reduce mitochondrial biogenesis and respiration (Wang et al. 2017). As a master controller, TFEB regulates the harmonization of several steps in ALP such as autophagosome, autolysosome and lysosomal biogenesis (Al-Bari 2020; Al-Bari and Xu 2020). TFEB also interacts with a promoter region for the CLEAR network element that regulates several processes such as autophagy, biogenesis of lysosomes and PM repair (Palmieri et al. 2011).

Under fed conditions, TFEB is provisionally engaged to lysosomes via binding to active RAG GTPase, a constituent of the mTORC1-RAG GTPase-regulator complex at the lysosomal surface (Martina et al. 2012; Settembre et al. 2012; Bahrami et al. 2020). This complex promotes TFEB phosphorylation at both serines, S²¹¹ via mTOR and Ser¹⁴² via extracellular signal-regulated kinase 2 (ERK2). This event provokes TFEB dislocation from the lysosomes and attaches it with YWHA, a 14-3-3 family protein that can preserve TFEB in the cytoplasm (Martina et al. 2012; Settembre et al. 2012). However, stressful situations such as caloric restriction or lysosomal stress cause inactivation of RAG GTPases as well as mTORC1 and prevent TFEB phosphorylation (Martina et al. 2012; Settembre et al. 2012). Consequently, dephosphorylated TFEB accumulates in cytosol, separates it from YWHA binding and translocates it in perinucleus (Figure 3). Moreover, TFEB also auto-controls its expression via starvation-activated autoregulatory loop (Settembre et al. 2013). Pharmacological inhibition of mTOR can turn to TFEB dephosphorylation and relocation to the nucleus (Settembre et al. 2012). In the nucleus, TFEB can activate CLEAR network gene expressions (Palmieri et al. 2011). These genes encode for several proteins that coordinate the translocation and actions of lysosomal enzymes contributing to the autophagic degradation (Palmieri et al. 2011). Several pathways can be regulated by the CLEAR network genes such as peroxisome proliferator-activated receptor gamma (PPARγ) co-activator-1 (PGC1), which is associated with mitochondrial biogenesis and the AMPK signaling pathway (Palmieri et al. 2011). Moreover, the lysosomal Ca²⁺ channel, mucolipin-1 (Emanuele 2014; Medina et al. 2015) (TRPML1, gene MCOLN1) can regulate TFEB (Figure 3). Thus TFEB positively controls several genes that are keys for cellular degradation and clearance through ALP (Palmieri et al. 2011). Under the fasting state, Ca²⁺ is dispersed from lysosomes through the TRPML1 channel, a TFEB transcriptional target (Medina et al. 2015), and a Ca²⁺ microdomain is twisted adjacent to lysosome (Emanuele 2014; Bahrami et al. 2020). This restricted raise in Ca²⁺ stimulates calcineurin which promotes TFEB dephosphorylation and translocation in the nucleus (Figure 3) resulting transcription of its targeting genes (Medina et al. 2015).

Forkhead box O (FOXO) family member proteins, FOXO1 and FOXO3, act as important stimulators of ALP in case of starvation (Mammucari et al. 2007) by controlling the expressions of ATG proteins (Morel et al. 2017). Since mTORC1 acts as the main sensor of nutrient deprivation and growth factor signaling pathways, ALP can also be controlled by mTORC2 via mTORC2-(protein kinase B) AKT-FOXO3 pathway (Lamming et al. 2012). Here, mTORC2 causes AKT phosphorylation (at Ser⁴⁷³) that in turn results in FOXO3 phosphorylation (at Thr³²). As a result, cytosolic FOXO3 is retained in the cytosol via 14-3-3 interaction (Tzivion et al. 2011) and therefore transcriptionally suppresses the autophagy pathway (Figure 3). In the starvation environment, stimulated FOXO3 helps the transcription of genes that induce autophagy (Mammucari et al. 2007).

Autophagy-lysosomal pathway in cancer: the ‘double-edged sword’ role

In cancers, several signaling molecules associated with autophagy have been discovered to be both overexpressed and deregulated indicating the evidence that autophagy acts ambiguous dual roles in cancer progress (Yun and Lee 2018; Yazdani et al. 2019). Autophagy functions as either tumor inhibitor or inducer based on the type, stage, and TME context (Figure 4). While autophagy restricts cancer progress in the initial stages, it can assist a protumoral action in advanced stages, inducing cell growth and metastasis (Yun and Lee 2018). Additionally, autophagy in stem cells maintains its exclusive properties such as cell differentiation and self-renewal (Chang 2020). Although autophagy in normal healthy cells usually happens at low expression levels and many well-known malicious cells have high expressions of basal autophagy even in fed states (White 2015). Moreover, increasing evidence suggests that autophagy alters response to selective types of anticancer chemotherapy and thus represents a resistant mechanism to chemotherapy in many malignancies (Sui et al. 2013). Moreover, the autophagy regulation imparts to the expressions of tumor suppressive proteins or oncogenes. Tumor suppressive proteins are down-regulated by mTOR signaling pathway ensuing an initiation of autophagy and inhibition of cancer commencement (Avalos et al. 2014). On the other hand, oncogenes can be stimulated by mTOR pathway resulting in an inhibition of autophagy and promotion of cancer development (Yun and Lee 2018). While these dual intricate pathways lead autophagy an exciting target for anticancer therapies, an advanced understanding of autophagic roles in diverse stages of cancer development and precise cellular and extra-cellular context should be reserved for attention to connect autophagy in cancers.

Figure 4. The Janus-faced contradictory actions of autophagy in cancer cells. Autophagy acts as a Janus-faced play or the Yin-Yang faced function on cancer development. In healthy cells, it functions as a tumor inhibitory mechanism by ensuring optimal energy supply, preserving genomic integrity, promoting the degradation of damaged organelle and cellular oncogenes, and endorsing first-line immunity against microbial infection. In advanced tumors, however, autophagy actions can be reinstated. Autophagy functions as an oncogenic phenomenon by endorsing cancer development via declining stresses, the preservation of cancer stemness potential and metastasis; executing cells resistant to anoikis; helping the persistence of cancer cells in a dormant state and supporting the senescent cell state.

Tumor-suppressive role of autophagy-lysosomal pathway in pre-malignant cells

Fascinatingly, a complicated relation between autophagy and cancer is recognized when Beclin 1, an important promoter for IM formation is indicated to inhibit breast cancer development (Liang et al. 1999). The Beclin 1 deficiency results in autophagy inhibition and upsurges in growth in cancer cells, suggesting that Beclin 1 plays as a tumor inhibitor (Liang et al. 1999; Qu et al. 2003). Moreover, Beclin 1 depletion is found in various human prostate, breast and ovarian cancers (Qu et al. 2003) and the down-regulated Beclin 1 levels in various cancers like HCC (Cai et al. 2014; Zhang et al. 2019). However, the overexpressed Beclin 1 inhibits cell proliferation of colon cancer (Zhang et al. 2019) and cervical cancer (Sun et al. 2010). Likewise, UVRAG (UV radiation resistance-associated gene) and Bif-1 (Bax interacting factor-1) associated with Beclin 1 complex act as tumor inhibitors and certainly control autophagy (Morselli et al. 2009; He et al. 2015). More considerable data for tumor-inhibiting role of autophagy is the mouse liver adenoma progression in ATG5 and ATG7 deficiencies (Takamura et al. 2011). Thus the following multiple mechanisms proposed to explain the tumor-preventing role of autophagy in pre-malignant cells.

1. In early tumorigenesis, autophagy suppresses the initiation and expansion by eliminating the damaged proteins, dysfunctional or damaged organelles like mitochondria (called mitophagy) and the transforming active pathogens, all of which can induce genotoxic oxidative stress by producing ROS, chromatin fragments containing damaged DNA, genomic instability such as micronuclei, endogenous retrotransposons through aberrant mutations and ultimately cancer initiation and progression (Guo et al. 2014; Bartsch et al. 2017).

2. In stress conditions such as ER stress or starvation, tumor cells having malfunctioning and/or deficiencies in autophagy favorably accumulate damaged macromolecules (e.g. p62, an adaptor protein, ER chaperones) and injured organelles (particularly mitochondria) (Mathew et al. 2007; Pecoraro et al. 2020), subsequently inducing ROS and genomic damage and chromatin instability (Wang et al. 2016; Yun and Lee 2018). These events result in the upregulation of the new autophagy cycle and the promotion of tumor progression. However, the correct autophagy favors DNA repair by preventing nuclear p62 accumulation and coordinates mitotic fidelity (Wang et al. 2016).

3. Autophagy controls the pre-malignant cell necrosis that limits to provoke a chronic inflammatory response, which can further become a strong oncogenic driver through cytokine production (Coussens et al. 2013). Autophagy destroys pro-inflammatory objects like injured mitochondria that can produce ROS abundantly as well as deliver mitochondrial (mt)DNA and type I interferon (IFN) signaling components (Zitvogel et al. 2012).

4. Efficient autophagy may enable oncogene-induced senescence (OIS) (Perez-Mancera et al. 2014; Dou et al. 2015) that provokes cell cycle arrest permanently and suppresses the cancer cell proliferation (Garcia-Prat et al. 2016).

5. Autophagy-mediated apoptosis in cancers does not succumb to cell death which is caspase independent (Rybstein et al. 2018).

Thus integral responses of autophagy in pre-malicious cells offer vigorous protection to the cellular homeostasis of healthy tissues.

Tumor-promoting action of autophagy-lysosomal pathway in malignant cells

Cancer cells require plentiful nutrients and oxygen supply during development and invasion to retain their rapid proliferation. An increased autophagic activity of cancer cells is thought to relate to their survival in hostile TME (Morselli et al. 2009). Activated autophagy in tumor cells based on stress conditions, thus, it makes a pro-survival action of cancer cells (White 2015). Autophagy in neoplastic cells influences tumor development in an intricate way linking both basic and extrinsic mechanisms of cancer cells. The following ways where autophagy is stolen the normal cellular homeostasis for developing tumor cells.

1. The efficient autophagy helps the detached tumor cells from ECM to promote cell survival, circumvent apoptotic cell death and preserve latency in a detached and antagonistic niche including hypoxia and acidic TME until they form new colonies (Qureshi-Baig et al. 2020).

2. Autophagy also plays a part in tumor metastasis. Cancer cells are capable to involve in metastasis by invading and colonizing new tissues and organs. In advanced metastatic stages, autophagy performs a pro-metastatic action through the enhancement of cell viability and colony formation in inferior sites (Kenific et al. 2010; Sosa et al. 2014). For undertaking metastasis, cancer cells must be detached from ECM (Vanharanta and Massague 2013) and autophagy allows ECM-detached cells to circumvent anoikis (programmed cell death upon detachment) (Avivar-Valderas et al. 2013).

3. Autophagy affects the TME that comprises numerous factors such as hypoxia, cytokines chemokines and other inflammatory factors. In the case of established tumors, autophagy helps tumor cells supply energy and metabolic products for continuous proliferation and protect cytotoxicity under hostile TME (Li et al. 2017; Daskalaki et al. 2018). Under therapeutic stressful conditions, robustly induced autophagy may help the cancer cells to endure, adapt and proliferate rapidly for the promotion of tumor development (Rybstein et al. 2018).

4. RAS protein-mutated cancer cells preserve an elevated level of basal autophagy. For example, H-RAS/K-RAS mutation causes elevated autophagy which in turn augments oncogenesis and progression of several fatal cancers including colon cancers (Karnoub and Weinberg 2008; Goel et al. 2015).

5. Autophagy is activated during cellular senescence. Although limited proliferation, senescent cancer cells can still support detachment by inducing TME depending on activated autophagy. Interestingly, autophagy facilitates oncogene RAS-tempted senescence and suppression of autophagy postpones senescence in cancer cells (Young et al. 2009).

6. Inflammatory cytokines are highly expressed in TME suggesting that inflammatory signals participate in tumorigenesis (Franklin et al. 2014). Inflammation induces elevated ROS levels in cancer cells, and immune cells including macrophages which release cytokines such as IL-6 (interleukin-6), IL-10 and TNF-α (tumor necrosis factor α) into TME. Autophagy regulates these inflammatory cytokines by secreting DAMPs (degradation of damage-associated molecular patterns) such as HMGB1 (high mobility group box 1) and (mt)DNA (Pan et al. 2014).

7. In cancer biology, the EMT (epithelial–mesenchymal transition) induces metastasis ensuing in an absence of cell–cell adhesion, increase of cell motility and invasiveness (Thiery et al. 2009). EMT-induced cancer cells have elevated levels of autophagy for cell survival under several stress conditions (Cai et al. 2015). For example, autophagy in HCC has been found to accelerate metastasis by overexpressing EMT (Li et al. 2013).

8. Stimulation of protective autophagy in cancer cells is the main challenge in antineoplastic therapy because of the development of chemoresistance (Chen et al. 2019).

9. Autophagy supports natural tumor progression by protecting the structure and function of cancer stem cells (CSC) that participate in oncogenesis, resistance to chemotherapy and invasion (Liu et al. 2017). Numerous studies have examined the way to preserve the stemness in these cells and found that autophagy acts an essential role to regulate the homeostasis of CSCs (Pan et al. 2013; Sharif et al. 2017).

To summarize, these complex paradoxical dual functions of autophagy have directed to debate over whether or how autophagy modulation would be endeavored in effective cancer treatment. Thus these Janus-faced contradictory phenomena lead the autophagy as a challenging but auspicious target for anticancer therapies (Rubinsztein et al. 2012; Levy et al. 2017; Towers et al. 2020). Here, in this review implication in inhibition of autophagy by repurposing drugs may be a gorgeous novel approach to protect and treat cancers.

Drug repurposing in cancer treatment

Despite incredible sources and strategies being investigated for cancer treatment, cancer is one of the most principal reasons of morbidity worldwide (Turanli et al. 2018). Over the last decades, the de novo development and commercialization of a new drug is a complicated, time-consuming, expensive and often failure-prone process (Yella et al. 2018) despite the advances in genomics, bioinformatics, life sciences and biotechnology as well as enhanced knowledge of human diseases. Research expenses by the NIH (National Institutes of Health) USA and investments in pharmaceutical companies have steadily increased, while the proportionate number of novel anticancer drug approvals has stagnated (Pammolli et al. 2011; Scannell et al. 2012; DiMasi et al. 2016). Moreover, the major presently available conventional antitumor chemotherapeutic agents are extremely costly, minimal expansion in survival rate and accompanying with several toxic effects that considerably diminish the lifespan of the patients (Langedijk et al. 2015). Thus scientists and clinicians have approved several approaches to decrease the production cost and time associated with cancer drug discovery. One such approach is to assess the reputable non-cancer drugs that have already been permitted for non-cancer diseases, the approach is called drug repurposing.

Drug repurposing (also known as drug repositioning/ recycling/ redirecting/ retasking/ reprofiling, therapeutic/ indication switching) has increased substantial consideration over the last 5 years (Nosengo 2016) because of providing a rapid opportunity to convert the new therapeutic strategies into clinical trials. This approach frequently encompasses a novel formulation, route of administration or dosage form of the drug and a diverse drug target comparative to the original pharmacological indication (Urquhart 2018). The main merit of this approach is the availability of biopharmaceutical and toxicokinetic data of drugs and thus reducing the need for additional studies to investigate clinical trials (Waring et al. 2015) and can substantially reduce drug discovery cost and time with established human safety (Baker et al. 2018). Besides, drug repurposing may uncover new cellular pathways associated with a particular disease like carcinogenesis or reveal new molecular targets for therapy (Galluzzi et al. 2017). Thus drug repurposing is receiving increased interest from governments, pharmaceutical companies, nongovernmental agencies and academic researchers.

Potential drugs targeting the autolysosomes in cancers

Autolysosome, at the center stage of autophagy, emerges as an optimistic goal for the progress of innovative mediators against a wide variety of diseases including cancers (Galluzzi et al. 2017). Although several known compounds inhibit autophagy at different stages, regrettably real selective pharmacological suppressors of autolysosomes are not yet existing for application in cancer patients (Galluzzi et al. 2017). However, there are five groups of agents that can be classified that can hit the autolysosomes as well as lysosome in cancer (Table 2). Most of the agents are still being examined in the preclinical trials excluding the antimalarial drug chloroquine (CQ) and its analogue hydroxychloroquine (HCQ) which are the clinically permitted autophagy inhibitor widely applied in many clinical trials in combination with several anticancer therapies. Although CQ analogues have been verified as impartial therapies, a more applied and rational strategy is to practice in their combined therapy with conventional cancer therapies. The combination anticancer drugs having synergistic effect without increasing toxicity have been reported using tumor cell lines, animal models (Table 3), and cancer patients (Table 4). There are some foundations sustaining to the approaches:

1. It is practicable that autophagy does not show a real or steady response to cancer therapies.

2. In general, cancer cells have an elevated level of autophagy for cell survival and adaptation under stress conditions. Autophagy in cancer cells circumvents parent autophagy inhibition (Towers and Thorburn 2019).

3. Many cancer therapies are known to stimulate autophagy and the induced autophagy converses capability of avoiding the attacks from chemotherapies, thus subsequent in chemoresistance.

4. Autophagy inhibitors as monotherapies alone execute a limited capacity for tumor development and unpredictable results from clinical trials.

5. Autophagy deficiencies are connected to many human diseases (Table 1) and autophagy suppression in tumor cells is more expected to be relatively harmful to normal cellular homeostasis because autophagy routinely eliminates the damaged or dysregulated proteins.

Table 2. Selected examples of ALP inhibitors with therapeutic benefits.

Agent	Derivative	Target of actions	Stage of autophagy	Indications	Remarks	Affected cancer cell type tested
Lysosomal lumen alkalizers: chloroquine analogues
Chloroquine	Aminoquinolines	Impaired lysosomal acidity	Fusion and degradation	Permitted for malaria	Nonselective suppression of ALP	Different cell types (McAfee et al. 2012; Selvakumaran et al. 2013; Levy et al. 2017; Rebecca et al. 2017)
Hydroxy-chloroquine	Aminoquinolines	Impaired lysosomal acidity	Fusion and degradation	Permitted for malaria, SLE and RA	Nonselective suppression of ALP	Different cell types (McAfee et al. 2012; Rangwala et al. 2014; Rebecca et al. 2017)
Quinacrine	9-aminoacridine	Impaired lysosomal acidity	Degradation	Putative not classic female sterility	Nonselective suppression of ALP	Different cell types (Goodall et al. 2014)
Mefloquine	Quinoline	Impaired lysosomal acidity	Degradation	Permitted for Malaria	Nonselective suppression of ALP	Different cell types (Sharma et al. 2012; Goodall et al. 2014)
Quinine	Quinoline	Impaired lysosomal acidity	Degradation	Permitted for malaria	Nonselective suppression of ALP	Different cell types (Liu et al. 2016)
Spautin-1	MBCQ	Suppression of USP10 & USP13	Nucleation	Preclinical cancers	Exception of CQ analogue	Different cell types (Liu et al. 2011)
Lys05	Aminoquinolines	Impaired lysosomal acidity	Fusion and degradation	Preclinical, cancer	Nonselective suppression of ALP	Different cell types (Amaravadi and Winkler 2012; McAfee et al. 2012)
ARN16090	ARN5187 analogue	Impaired lysosomal acidity	Fusion and degradation	Preclinical cancer	Suppression of NR1D2/REV-ERBβ	Colon cancer HCT116 (De Mei et al. 2015; Torrente et al. 2015)
SAR405	–	Inhibition of Vps34	Nucleation	Preclinical cancer	Kinase selectivity profile	Renal tumor cells (Pasquier 2015)
EAD1	Substituted triazole	accumulation of LC3-II	Nucleation	Experimental	More potent than CQ and HCQ	Human lung cancer cells (Nordstrom et al. 2015; Sironi et al. 2019)
Verteporfin	Benzoporphyrin	Suppression of LC3 lipidation	Elongation	Permitted as PDT	Exception of CQ analogue	Different cell types (Lu et al. 2009; Donohue et al. 2011)
VATG-027	1,2,3,4 tetrahydroacridine	Impaired lysosomal acidity	Fusion	Preclinical trials	Potent suppression than CQ	Mutant BRAF melanomas (Goodall et al. 2014)
Clioquinol ionophore	8-hydroxyquinoline	Impaired lysosomal acidity	Degradation	Permitted urinary infections	Autophagy initiation by interruption of mTOR signaling	Different cell types (Cao et al. 2014)
Nitazoxanide	2-AN-5NB	Impaired lysosomal acidity	Degradation	Antiprotozoal drug	Broad-spectrum anti-infection agent	Bladder cancer cells (Wang et al. 2018)
Carvedilol	methoxyphenoxy- 2-propanol	Impaired lysosomal acidity	Degradation	Synthetic antihypertensive	Nonselective β-adrenoceptor blocker	Different cell types (Meng et al. 2018)
ML-9	Naphthalene sulphonamide	Impaired lysosomal acidity	Diffusion and degradation	Inhibitor of Akt	Nonspecific inhibition of kinase	Prostate cancer cells (Kondratskyi et al. 2014)
L-asparagine	Nonessential amino acid	Inhibition of efflux pathway of lysosome	Fusion	Preclinical	Increase catalytic activity of L-asparaginase	Breast cancer cells (Knott et al. 2018)
Azithromycin	Macrolide antibiotic	Prevention of autophagosomal acidification	Fusion and degradation	Chronic infection	Failure to kill intracellular mycobacteria	Myeloma cells (Renna et al. 2011; Moriya et al. 2013)
Clarithromycin	Macrolide antibiotic	Prevention of autophagosomal acidification	Fusion and degradation	Infection	Failure to kill intracellular mycobacteria	Myeloma cells (Nakamura et al. 2010; Moriya et al. 2013)
Ciprofloxacin and norfloxacin	Quinolone Antibiotics	LMP induction	Diffusion and degradation	Infection	Caspase-independent cell death with MMP	Cervical carcinoma (Groth-Pedersen and Jaattela 2013)
Nanomycin and siomycin A	Quinone antibiotics	LMP induction Cathepsin D	Diffusion and degradation	Infection	Selectively inhibits DNMT3B	Colon carcinoma (Groth-Pedersen and Jaattela 2013)
Liensinine	Isoquinoline alkaloid	Impairment of RAB7 recruitment to lysosomes	Fusion and degradation	Preclinical	Unaffected lysosomal pH	Breast cancer cells (Zhou et al. 2015)
Matrine	Quinolizidine alkaloid	Impaired endosomal/lysosomal acidification	Diffusion and degradation	Preclinical	Induction of autophagy	Different cell types (Wang et al. 2013; Zhang et al. 2020)
MHY1485	Morpholino-triazine	Impaired lysosomal acidity	fusion	In vitro studies	mTOR inducer and autophagy inhibitor	Experimentally used (Yang et al. 2017)
Vacuolar H^+-ATPase inhibitors
Bafilomycin A1	Antibiotic	v-ATPase suppression	Fusion and degradation	Clinical trial	Global v-ATPase suppressor	Experimentally used (Li et al. 2016)
Doxorubicin Adriamycin	Anthracycline antibiotic	v-ATPase inhibition	Fusion and degradation	Permitted for leukemia	DNA-damaging agent	MEFs (Aits and Jaattela 2013; Li et al. 2016)
Manzamine A	Alkaloid	v-ATPase suppression	Fusion and degradation	Preclinical	Global v-ATPase suppressor	Cervical cancers (Kallifatidis et al. 2013; Karan et al. 2020)
Cleistanthin-A	Glycoside	v-ATPase suppression	Fusion and degradation	In vitro works	Global v-ATPase suppressor	Different cell types (Zhao et al. 2015; Liu et al. 2020)
Archazolid	Macrocyclic lactone	v-ATPase suppression	Fusion and degradation	In vitro works	Decrease of cathepsin B activity	Mammary epithelial cells (Kubisch et al. 2014; Merk et al. 2017)
E64d	Fungal metabolite	Protease suppression	Incomplete proteolysis	Not recorded for Leishmania	Nonspecific suppressor	Experimentally used in MCF-7 cells (Schurigt et al. 2010)
Lysosomal hydrolase inhibitors
Omeprazole	Pyramidal racemate	V-ATPase inhibitors	V-ATPase inhibitors, cathepsin B/L	Acid reflux, ulcers	Risk of gastric cancer for long-term use	Leukemia, lymphoma (Groth-Pedersen and Jaattela 2013; Cheung and Leung 2019)
Elaiophylin	Macrodiolide antibiotic	Abolition of maturation of cathepsin B and D.	Degradation	Antimicrobial and antihelminthic actions	Raise of autophagosome accrual	Ovarian cancer cells (Zhao et al. 2015)
Pepstatin A	Hexapeptide metabolite	Inhibition of cathepsin D	Partial lysosomal proteolysis	Not recorded	Reversible nonspecific suppressor	Experimentally used (Maynadier et al. 2013)
Vildagliptin	Cyanopyrrolidine	Cathepsin K Inhibitor	Partial Lysosomal proteolysis	Not recorded	Selective DPP-4 inhibitor	Colorectal cancers, lung cancer cells (Jang et al. 2015; Davidson and Vander Heiden 2017; Jang et al. 2019)
Acid sphingomyelinase (ASM) modulators
Siramesine	Sigma (σ) receptor agonist	ASM modulator	Modulation of LMP	treatment of anxiety	Lysosomotropic agent	Breast carcinoma (Groth-Pedersen and Jaattela 2013; Petersen et al. 2013)
HPS70 inhibitors
PES	Pifithrin-μ	ASM modulator	Modulation of LMP	HPS70 inhibitor	Lysosomotropic agent	NSCLC cells (Granato et al. 2013; Zhou et al. 2017)
Tetrandrine	bisbenzylisoquinoline alkaloid	Inhibition of lysosomal acidification	Diffusion and degradation	Preclinical	Activation of autophagy in HCC	Different cell types (Qiu et al. 2014; Wong et al. 2017)
Thapsigargin	Sesquiterpene lactone	Inhibitor of SERCA; elevation of cytoplasmic Ca^2+ levels	Nucleation, Fusion and degradation	Experimental agent	Induction of autophagy caused by ER stress	Different cell types (Ganley et al. 2011)
Paclitaxel (taxol)	Taxane	Inhibition of Vps34 and autophagosome trafficking	Initiation, degradation	Approved for breast, ovarian and lung cancers	Inhibition of autophagy reverses taxol resistance	Different cell types (Wang et al. 2020)
Geldanamycin	Natural product	Inhibition of cathepsin D	Initiation, degradation	Hsp90	Sustaining proliferative Signalling	Different cell types (Davidson and Vander Heiden 2017)

DNMT (DNA methyltransferases), DPP-4 (dipeptidyl peptidase-4), LMP/MMP (lysosomal/ mitochondrial membrane permeabilization), MBCQ (4- [[3,4-(Methylenedioxy) benzyl] amino]-6-chloroquinazoline), MEFs (mouse embryonic fibroblasts), NAC (N-acetyl-L-cysteine), NSCLC (non-small cell lung cancer), PES (2-phenylethyenesulfonamide).

Table 3. Selected examples of therapeutic benefits from ALP inhibition in animal models.

Agent	Host	Tumor type	Therapy	Comments
In vivo efficacy studies of CQ analogue as monotherapy
CQ	nu/nu mice	U373-EGFRwt (H)		ITR to epithelial tumor (Jutten et al. 2013; Galluzzi et al. 2017; Verbaanderd et al. 2017; Mainz and Rosenfeldt 2018)
	Athymic BALB/c nu/nu mice	CD133^+ and CD133^- cells (H)	–	Sensitive to LCSCs in TME
	Nude mice	HepG2-GFP (H)	–	Sensitive to liver cancer
	NOD-SCID mice	SKMel23 cells (H)	–	Selectively promotes apoptosis in melanoma cells
	nu/nu mice	B16-F10 cells (M)	–	ITR to melanoma model (Maes et al. 2014)
	Nude nu/nu mice	MCF7 and MDAMB231 cells	–	Potential response to TNBC
	NMRI nu/nu mice	HCT116 and HT29 cells	–	Limitations in efficacy of CQ in TME (Pellegrini et al. 2014)
CQ (Lys05)	nu/nu mice	1205Lu cells, C8161 cells, HT29 cells (H)	–	Standalone therapeutic effect in melanoma and CRC (McAfee et al. 2012)
HCQ	nu/nu mice	Pancreatic cancer (H)	–	ITR to pancreatic cancer
In vivo efficacy studies of autophagy inhibitor as combination therapy
CQ	Athymic nu/nu mice (4-6 wk)	U87MG glioma cells (H)	TMZ	ITR to glioblastoma
	C.B.17 SCID mice (8-10 wk)	HT29 colon carcinoma cells (H)	Bevacizumab, oxaliplatin	ITR to treatment in CRC (Selvakumaran et al. 2013)
	Swiss nu/nu mice (6 wk)	MDA-MB-231 breast cells (H)	CyP and/or doxorubicin	ITR to breast cancers
	NOD/SCID mice	HT29 cells (H) PROM1/CD133^+ CCSC	PDT	ITR to CRC, restoration of sensitivity to PDT
	BALB/c nude mice (5-6wk)	FaDu hypopharyngeal cells (H)	Cisplatin	ITR to HSCC model
	Wistar rats (8 wk)	C6 brain cells	TMZ (+curcumin)	ITR to brain tumor
	BALB/c nu/nu mice (4-6 wk)	EC109/CDDP oesophageal cells (H)	Cisplatin	ITR of cisplatin
	BALB/c mice (8 wk)	SGC7901 gastric cancer cells (H)	Cisplatin	Chemosensitivity to cisplatin
	NMRI nude mice (8 wk)	MDA-MB-231 and MCF-7 breast cancer cells	Rapamycin	Tumor growth reduction
	BALB/c nu/nu and BALB/c mice (4-6 wk)	CaR-1, HT-29, colon26 colon cancer cells	Temsirolimus (TEM)	Suppressed tumor growth and triggered enhanced apoptosis
	BALB/c nude mice (6 wk)	PC-9/wt and PC-9/gefB4 lung cancers	Gefitinib	Suppressed tumor growth
	BALB/c nude mice	HCC-827 lung cancers	Gefitinib (+ Akt inhibitor)	Significantly inhibited tumor growth
	Swiss albino mice	Ehrlich ascites carcinoma cells	Sunitinib	Significantly induced apoptosis and reduced tumor growth
	CD-1 nude mice (6-7 wk)	H3122CR-1 lung cancer cells	Crizotinib	Sensitization of drug resistant lung cancer cells to crizotinib
	NOD-SCID mice (6 wk)	Karpas-299 lymphoma cells	Crizotinib	Significantly suppressed tumor growth and increased apoptosis
	BALB/c nude mice (6-8 wk)	U251 glioblastoma cells	Vandetanib	Increased apoptosis and reduced tumor growth
	Athymic nude mice (4-5 wk)	JIMT-1 breast cancer cells	Trastuzumab	Significantly suppressed tumor growth
	New Zealand White rabbits	VX2 liver tumors	TACE	Significantly reduced tumor growth
HCQ	Rag2^−/− mice	MDA-MB-231 cells (H)	Epirubicin	Enhanced response to breast cancer model
	Nude NCr Nu-M mice (6 wk)	UACC903 melanoma cells	Temsirolimus	Promotes apoptosis and tumor suppression
	Athymic nude mice (5-6 wk)	H358 or H460 human NSCLC cells	Erlotinib	Significant sensitization to erlotinib therapy
	Athymic BALB/c nude mice (5-6 wk)	SPC-A1 lung cancer cells (H)	Crizotinib	Increased apoptosis and reduced tumor growth

5-FU (5-fluorouracil), ATM (ataxia telangiectasia mutated), CCSC (colorectal cancer stem cells), CQ (chloroquine), CRC (colorectal carcinoma), EGFR (epidermal growth factor receptor), H (human), HCC (hepatocellular carcinoma), HCQ (hydroxy-chloroquine), HDIL-2 (high-dose interleukin-2), HSCC (hypopharyngeal squamous cell carcinoma), ITR (improved therapeutic response), LCSCs (liver cancer stem cells), M (mouse), NMU (N-methyl-N-nitrosourea), NOD (non-obese diabetic), PDAC (pancreatic ductal adenocarcinoma), PDT (photodynamic therapy), SCID (severe combined immunodeficiency mice), TACE (transcatheter arterial chemoembolisation), TME (tumor microenvironment), TMZ (Temozolomide), TNBC (triple-negative breast cancers), wk (week-old), wt (wild-type).

Table 4. Ongoing and completed clinical trials with ALP inhibitors.

Treatment strategy
Inhibitor	Other agents	Disease	Trial phase	Identifier	Sponsor
CQ	–	SCLC	1/T	NCT01575782	Maastricht Radiation Oncology
	–	SCLC	1/T	NCT00969306	Maastricht Radiation Oncology
	TMZ	Gliosarcoma	1/NR	NCT04397679	Barbara Ann Karmanos Cancer Institute
	Trametinib	Metastatic N-RAS Melanoma	R	NCT03979651	Hospices Civils de Lyon
HCQ	Radiation	COVID-19, cancer	2/R	NCT04381988	Memorial Sloan Kettering Cancer Center
	Trametinib	Biliary cancer	2/NR	NCT04566133	National Cancer Institute (NCI)
	Ulixertinib	GI neoplasms	1/R	NCT04145297	University of Utah
	Trametinib	Pancreatic cancer	1/R	NCT03825289	University of Utah
	Letrozole, Palbociclib	Breast cancer AJCC v8	1/2/R	NCT03774472	M.D. Anderson Cancer Center
	Azithromycin	Cancer, COVID 19	2/R	NCT04341207	Gustave Roussy, Cancer Campus, Grand Paris
	LY3214996	Pancreatic cancer	2/R	NCT04386057	Kimberly Perez
	Cobimetinib, Atezolizumab	GI cancer	1/2/R	NCT04214418	Columbia University
	Binimetinib	Metastatic pancreatic adenocarcinoma	1/R	NCT04132505	M.D. Anderson Cancer Center
	Everolimus	Breast cancer stage IIB	2/R	NCT03032406	University of Pennsylvania
	Binimetinib	NSCLC	2/NR	NCT04735068	University of Pennsylvania
	Abemaciclib	Breast cancer	2/NR	NCT04523857	University of Pennsylvania
	Sorafenib	Hepatocellular Cancer	2/R	NCT03037437	The University of Texas Health Science Center at San Antonio
	–	Prostate cancer recurrent	2/R	NCT04011410	Andrew C. James, MD
	SUBA-itraconazole	Prostate cancer	1/2/R	NCT03513211	St Vincent's Hospital, Sydney
	TMZ	Glioblastoma	2/3/R	NCT03008148	Johnpro Biotech, Inc.
	CPI-613	Myelodysplastic syndromes	1/2/NR	NCT03929211	Wake Forest University Health Sciences
	Trametinib	Metastatic N-RAS melanoma	R	NCT03979651	Hospices Civils de Lyon
	Dabrafenib, Trametinib	Glioma	1/2/R	NCT04201457	Pediatric Brain Tumor Consortium
	Nivolumab, Ipilimumab	Melanoma	1/2/R	NCT04464759	Ravi Amaravadi, MD
	Docetaxel, Gemcitabine	Recurrent Osteosarcoma	1/2/R	NCT03598595	M.D. Anderson Cancer Center
	Carfilzomib, Dex	Multiple Myeloma	1/R	NCT04163107	Norwegian University of Science and Technology
	CPI-613	Sarcoma, Clear Cell	1/2/R	NCT04593758	Rafael Pharmaceuticals Inc.
	ABC294640	Cholangiocarcinoma	2/R	NCT03377179	RedHill Biopharma Limited
	–	2019 Novel Coronavirus	1/2/NR	NCT04731051	King Hussein Cancer Center
	Radiation	COVID-19, Cancer	2/R	NCT04381988	Memorial Sloan Kettering Cancer Center
	Trametinib	Biliary Cancer	2/NR	NCT04566133	National Cancer Institute (NCI)
Verteporfin	SpectraCure P18 System	Recurrent Prostate Cancer	1/R	NCT03067051	SpectraCure AB
Doxo-rubicin	Cisplatin	Ovarian cancer	1/R	NCT02475772	Ruhr University of Bochum
	Trastuzumab	Breast cancer	1/R	NCT02562378	Roche Pharma AG
	–	Cutaneous T cell lymphoma	1/R	NCT02192021	University of Pittsburgh
	Ixazomib, gemcitabine	Bladder cancer	1 /2/R	NCT02420847	M.D. Anderson Cancer Center
	–	Glioblastoma	2/R	NCT02758366	Meyer Children's Hospital
	–	Liver cancer	1/R	NCT02753881	Yale University
	Pembrolizumab	Sarcoma	1/2/R	NCT02888665	Fred Hutchinson Cancer Center
Clarithro-mycin	Lansoprazole, amoxicillin, metronidazole	Gastric diffuse large B-cell lymphoma	2/R	NCT02388581	National Health Research Institutes, Taiwan
	Selinexor, Dex, lenalidomide	Multiple myeloma	1/2/R	NCT02343042	Karyopharm Therapeutics Inc
	Antimicrobial agents	Neoplasms	2/R	NCT02366884	Dr. Frank Arguello Cancer Clinic
	Lenalidomide, Dex	Multiple myeloma	3/R	NCT02575144	PETHEMA Foundation
	Lenalidomide, Dex	Multiple myeloma	3/R	NCT04287660	The First Affiliated Hospital of Soochow University
	Triple therapy	Gastric cancer	4/R	NCT02047994	International Agency for Research on Cancer
	Daratumumab, Pomalidomide, Dex	Multiple myeloma	2/NR	NCT04302324	Weill Medical College of Cornell University
	Lenalidomide, Dex	Multiple myeloma in Relapse	2/R	NCT04063189	The First Hospital of Jilin University
Cipro-floxacin	Metronidazole, neomycin	Rectal cancer	2/NR	NCT02682485	University of Chicago
	Etoposide	Leukemia	1/2/R	NCT02773732	University of Florida
	Bile Acid Binder	Colon adenocarcinoma	R	NCT04003181	University of Aarhus
	–	Pancreatic ductal adenocarcinoma	1/R	NCT04523987	National University Hospital, Singapore
	Antibiotic	Acute lymphoblastic leukaemia	NR	NCT04678869	University College, London
Paclitaxel	Enzalutamide + carboplatin	Endometrial cancer	2/R	NCT02684227	M.D. Anderson Cancer Center
	fruquintinib	Advanced gastric cancer	3/R	NCT03223376	Sun Yat-sen University
	PU-H71	Metastatic breast cancer	1/R	NCT03166085	Memorial Sloan Kettering Cancer Center
	Gemcitabine	Small cell lung cancer	2/R	NCT02769832	Celgene Corporation
	Gemcitabine	Pancreatic adenocarcinoma	4/R	NCT03401827	Seoul National University Hospital
	Gemcitabine	Adenocarcinoma pancreas	4/ R	NCT02812992	AIO-Studien-GmbH

ANT (active, not recruiting), C (completed), Dex (dexamethasone), IELSG (International Extranodal Lymphoma Study Group), NCI (National Cancer Institute), NR (not yet recruiting), NSCLC (Non-Small Cell Lung Cancer), R (recruitment), S (suspended), SCLC (small cell lung cancer), T (terminated), TMZ (temozolomide).

Lysosomal lumen alkalizers: chloroquine analogues

Most of the effective drug repurposing method has chiefly been by chance discovery or clinical reflection rather than systematic, hypothesis-oriented results. One of the serendipitous discoveries for drug repurposing achievement histories connects to the (re)use of CQ analogues (Al-Bari 2015). These analogues are still chosen for malaria treatment as they are cheap and effective agents with a low toxicity profile in patients (Al-Bari 2015; Zhang et al. 2015). Although as an anti-cancer drug, CQ analogues particularly HCQ alone have been shown to suppress cell growth and proliferation, these analogues have been widely used in preclinical and clinical trials with auspicious outcomes (Galluzzi et al. 2017). CQ analogues in combination with antitumor agents impair clonogenic cancer cell growth and proliferation, improve tumor milieu and increase cell death, especially under metabolic stresses (Galluzzi et al. 2017). CQ analogues preferentially accumulate in and/or damage lysosomal or autolysosomal acidification system, thus abrogating lysosomal enzymatic activities (Piao and Amaravadi 2016). It has been found that CQ analogues are more potential sensitizer in cancer treatment mainly in cases of chemotherapy resistance (Pascolo 2016; Morel et al. 2017). In tumor treatment, CQ analogue is frequently applied in blending with chemotherapies and radiation since it is revealed to improve the cancer cell killing efficiency. These analogues have coactive actions with selective chemotherapies applied to treat several cancers such as melanoma, multiple myeloma, glioma and advanced/refractory solid tumors without causing extra adverse effects (Morel et al. 2017). CQ has also been found to enhance the therapeutic efficiency of antitumor drugs that cause autophagy induction (Torgersen et al. 2013). For example, CQ analogue causes an extra 30–40% decrease in cell survival rate treated with histone deacetylase inhibitors like valproic acid or vorinostat in acute myeloid leukemia (AML) cells (Torgersen et al. 2013). Interestingly, many weak basic drugs for cancer treatment gather in lysosomes without causing inhibition of autophagy (Piao and Amaravadi 2016). One clarification, HCQ can dislocate other mutual therapies from the lysosome to cytosolic niche by enhancing their cytosolic availability (Piao and Amaravadi 2016). In line with this notion, CQ analogues in combination with clinically approved chemotherapies have been revealed to suppress cancer cell growth in autophagy reliant on ways (Maes et al. 2014; Eng et al. 2016). These analogues offer significant protection against both the cancer cells and the TMEs especially when combined with standard anticancer therapies.

Current chloroquine analogues for autophagy inhibitions

The basic chemistry of CQ analogue comprises a heterocyclic fraction (7chloro-quinoline) standing on an alkaline side chain at carbon-4 position (see S. Figure 1a). Two H⁺ bearing sites on the side chain are accountable for its mildly alkaline property with pK's of 8.3 and 10.2 correspondingly (see S. Figure 1b) which keeps it soluble in the abdomen (Al-Bari 2015). Basically, HCQ varies from CQ by presenting a hydroxyl (OH^–) group (see S. Figure 1a) at the terminal of the side chain. This structural alteration of HCQ causes a reduction the potential for retinal toxicity and making HCQ more satisfactory safety profile to CQ (Al-Bari 2015). Since retinal toxicity, a main adverse effect of CQ and incapable to arrive in tumor cells effectively due to protonation in the acidic ECM in TME of many tumors (Pellegrini et al. 2014; Morel et al. 2017), many scientists have discovered more new CQ analogues and or CQ-based hybrid compounds in challenges to progress anticancer action. Currently, several CQ analogues target the different stages of autophagy as well as lysosome in cancers including Lys05 (Amaravadi and Winkler 2012; McAfee et al. 2012), Spautin-1 (Liu et al. 2011; Shao et al. 2014), SAR405 (Ronan et al. 2014; Pasquier 2015), quinacrine, mefloquine (Wang et al. 2015), verteporfin (Liang et al. 2020; Mae et al. 2020), clioquinol (Khan et al. 2020; Mizutani et al. 2021), nitroxoline (Mitrovic and Kos 2019; Xu et al. 2020), EAD1 (a triazole-functionalized CQ derivative) (Nordstrom et al. 2015), and VR23 (a 4-piperazinylquinoline scaffold) (Piao and Amaravadi 2016). A ligand of REV-ERBβ (reverse of ERBα-β variant protein, a member of the REV-ERB-A family of transcription factor), ARN5187 inhibits both REV-ERB-facilitated transcriptional control and autophagy. Strangely, while ARN5187 and CQ impart the same lysosomotropic power and inhibitory action on autophagy, ARN5187 is suggestively higher cytotoxicity in BT-474 cells (De Mei et al. 2015). Moreover, VATG-027 (two acridine ring derivative of CQ) and VATG-032 (tetrahydroacridine ring derivative) have been identified to inhibit autophagy up to 150-fold than CQ by inhibiting acidification of lysosomes and weakening turnover of arriving vesicles like autophagosomes (Goodall et al. 2014). Vacquinol-1 (NSC13316, a quinolone derivative) stimulates cell death in glioblastoma multiforme. NSC23925 (a synthetic analogue to vacquinol-1) has been reported to reverse P-glycoprotein-mediated efflux of several cancer drugs in multidrug-resistant cancer cell lines (Sander et al. 2017). Complex derivatives such as ferroquine (ferrocene-4-aminoquinoline), platinum (II)-CQ complex, polymeric CQ (copolymerization of N-(2-hydroxypropyl) methacrylamide with methacryloylated HCQ), gold(I)-CQ complexes [Au (CQ) (PPh3)PF6] (de Souza Pereira et al. 2021) have also been found to inhibit ALP. Most of these analogues are still being examined in the preclinical trials excepting CQ and HCQ.

Chloroquine analogues: just act as autophagy inhibitors?

In ongoing clinical trials for cancer therapies, HCQ is frequently applied in blending with chemotherapies, radiation and immunotherapy. CQ or HCQ treatment can suppress autophagy-oriented cell survival rate and potentiate anticancer action of chemotherapies. But, current evidences suggest that CQ and its majority analogues mediated suppression of the final degradation stage of autophagy is not the only mechanistic way in case of anticancer activity; CQ analogue can also influence other mechanistic pathways such as LMP. Alike HCQ also stimulates mitochondrial membrane potential (MMP) and the proapoptotic Bcl-2 family members Bax/Bak-dependent MMP to activate caspase stimulation (Seitz et al. 2013). Maycotte (Maycotte et al. 2012) also described that CQ can hypersensitize breast cancer cells to anticancer drug independent of autophagy suppression. Besides autophagy-independent anti-tumor actions, CQ analogues may have other ways such as dropping nutrient scavenging actions, anti-inflammatory properties to sensitize chemotherapies (Maycotte et al. 2012; Maes et al. 2014; Eng et al. 2016). Thus the achievement of clinical trials using CQ/HCQ in combination with antitumor drugs may not only the suppressive effects CQ/HCQ on autophagy activated by chemotherapies, but also the other mechanisms that inhibit it. Therefore, a comprehensive idea of the cellular pathways of CQ/HCQ would be suggested in the ongoing clinical trials wherever CQ/HCQ are applied as suppressors of autophagy.

Caveats against efficacies of chloroquine analogues in autophagy inhibitions

Chloroquine analogues like HCQ are presently clinically accepted autophagy suppressors for cancer treatment. There are many human clinical trials discovering suppression of autophagy as a therapeutic approach applied CQ analogues because of their extended track of safety record in human patients (Table 4). However, there are many highly debatable caveats as well as enquiries lingering as the analogues represent the best effective drugs for suppressing autophagy. For example,

1. These analogues can be necessitated in highly concentrated (even μM) solutions to obtain sufficient autophagy suppression that is contradictorily attainable in human patients than traditionally applied for malaria and rheumatoid arthritis (RA) (Pascolo 2016). Hence, HCQ combined with chemotherapy and/or radiation has been found to cause low efficacy in preliminary trials. Additionally, high HCQ doses applied in clinical trials yield a substantial interpatient variability of autophagy suppression. Since autophagy activation and dependency are exclusive to cancer cells, hypothetically extended autophagy suppression can offer a therapeutic index to preferentially influence cancer cells. However, the sustained period of HCQ dose for a tumor patient will inevitably influence nontransformed normal healthy cells as well.

2. CQ analogue fails to inhibit autophagy in acidic ECM (pH 6.5) in tumor for a decrease in cellular absorption of the agents (Pellegrini et al. 2014). Although CQ analogues exert their inhibitory effects on autophagy in one of the mechanisms, the analogues are possible harmful to tumor cells by fetching additional targets. Because CQ analogues cause an amplified apoptosis index, tumor relapse, and suspension in cancer reappearance in patients.

3. Furthermore, the biological halflives of CQ analogues are calculated as practically lengthy periods (e.g. over 3 weeks for HCQ) which can govern chronic adverse effects such as retinopathy and indigestion (Al-Bari 2015). Thus many clinical trials have shown dose-limiting side effects like neutropenia, thrombocytopenia, and sepsis when HCQ is used in combination with chemotherapies (Al-Bari 2015).

4. CQ analogues can have autophagy-independent anticancer actions via extra mechanisms such as suppressing nutrient scavenging activities, anti-inflammatory properties and can sensitize to other chemotherapies (Maycotte et al. 2012; Eng et al. 2016). Moreover, CQ is capable to sensitize cancer cells to chemotherapies once the earlier stages of autophagy were blocked (Maes et al. 2014).

5. CQ analogue has been applied as lysosomal suppressors but is incapable to suppress lysosomal localized mTOR pathway, thus it also has autophagy-independent effects as anti-cancer agents (Maycotte et al. 2012; Eng et al. 2016; Towers and Thorburn 2017). For example, CQ has known to bind DNA for many years (Kwakye-Berko and Meshnick 1989). However, Ravi Amaravadi and Jeffrey Winkler groups find out dimeric quinacrines as effective antitumor molecules, which simultaneously suppress mTOR signaling and autophagy (Rebecca et al. 2017). The quinacrine linker DQ661 inhibits PPT1 (palmitoyl-protein thioesterase 1) as a molecular goal in vitro and suppresses tumor growth of several mouse models including colorectal cancer, and thus can be securely united with chemotherapy (Rebecca et al. 2017).

6. Although pharmacokinetic data are well known, it is practically very undefined as to whether CQ/HCQ can exactly attain concentrations in the cancer cells that will efficiently suppress autophagy; in reality, it is persuaded to claim that the purpose that CQ/HCQ has confirmed to be comparatively safe to patients because the analogue is unable to truly inhibit autophagy at concentrations that are attained in standard clinical treatments.

7. Another important question with applying CQ/HCQ for cancer treatment is related to their immunosuppressive actions (Taherian et al. 2013). It is generally known that the stimulation of anticancer immunity is critical for extended therapeutic efficiency in patients influenced by major malignancies (Fridman et al. 2017). In this situation, the systemic management of CQ/HCQ may be harmful as it would approve the fleeing of cancer cells from immunosurveillance (Kroemer et al. 2015) and incapable to amend response rate and total longevity (Rojas-Puentes et al. 2013). Thus CQ analogue in combination with standard therapeutic schedules may consult for temporary assistances that are not duplicated by a real expansion in patient longevity. Further study would be necessary to explore more active and tolerable CQ analogues as selective inhibitors, as well as delineates the timing, interval and dose intensity that turns to maximal therapeutic actions during tumor treatment (Chude and Amaravadi 2017; Zeh et al. 2020).

Other drug repurposing lysosomal lumen alkalizers include antidepressant drugs such as nortriptyline, imipramine, clomipramine, desipramine, and siramesine. These agents have revealed efficacy in chronic lymphocytic leukemia (CLL), colon cancer and breast cancer. Antihistaminic agents like loratadine and terfenadine have also shown effectiveness by stimulating apoptosis in lung and breast cancer cells. Stilbenoid antioxidants including pterostilbene and antidepressant drugs such as thioridazine, chlorpromazine, and aripiprazole have also shown therapeutic efficiency in breast cancer cells (Levy et al. 2017). The particulars of these drugs are concisely discussed in Table 2.

V-ATPase inhibitors

Vacuolar H⁺-ATPase (v-ATPase), a proton pump, generates H⁺ ions by hydrolysis of ATP into the lysosomal lumen, thus making the lysosomes acidic. Suppression of v-ATPase can be attained by the action of several microbial compounds such as bafilomycin A1, cleistanthin A, archazolid, and manzamine A (De Milito et al. 2010). Although bafilomycin A1 is suggested to be the classical inhibitor of v-ATPase, its high toxicity profile has limited to use in clinical intervention for blocking autophagy in vivo (Piao and Amaravadi 2016). Archazolid, another v-ATPase inhibitor has shown the capability to decrease the action of the cathepsin B protease (Kubisch et al. 2014). The v-ATPase inhibitors manzamine A (Kallifatidis et al. 2013) and cleistanthin-A have also shown cytotoxic actions on several cancer cells (Zhao et al. 2015). The anti-melanoma effect of esomeprazole, a clinically approved proton pump inhibitor (PPI), is allied to inhibit the homeostatic mechanism of cancer cells that help their viability by eliminating H⁺ and acidic metabolites. Besides, esomeprazole stimulates autophagy-mediated apoptosis in gastric cancers (De Milito et al. 2007).

Acid sphingomyelinase modulators

Multidrug resistance is the prime obstacle to successful cancer treatment. Acid sphingomyelinase (ASM), a hydrolase within the lysosome cleaves sphingomyelin to ceramide which assists as a substrate for the production of other members of sphingolipids including S1P (sphingosine 1-phosphate) in response to cellular stresses (Petersen et al. 2013; Savic and Schuchman 2013). ASM action is important for lysosomal membrane integrity and revival of tumor cells as well as for resistance phenotypes of multidrug. Cancer cells usually exhibit lower ASM activity than in normal healthy cells, resulting in elevated sphingomyelin levels. Thus cancer cells display high level of vulnerability to ASM treatment because blocking ASM expression outcomes in higher sphingomyelin levels which restricts the normal action of lysosomal membrane. Several ASM modulators including cationic amphiphilic agents such as antidepressants (chlorpromazine, siramesine) (Petersen et al. 2013), CQ and antiarrhythmic drugs (amiodarone) decrease ASM action by relocating ASM from the lysosomal membrane resulting in LMP and eventual tumor cell death (Saftig and Sandhoff 2013). Moreover, siramesine and clomipramine also effectively inhibit autophagic flux by neutralization of the lysosomal pH effectively (Petersen et al. 2013).

Heat shock protein inhibitors

As chaperone molecules, HSP70 can protect the lysosome from permeabilization and maintain cellular protein homeostasis (Penke et al. 2018). Cancer cells in different types of origins upregulate HSPs in particular HSP70 (Granato et al. 2013) and HSP90 to cope with their basal stress that promotes cell metastasis deliberating a survival benefit (Mjahed et al. 2012). By selectively interacting with bis monoacylglycero phosphate in the lysosomal lumen, the nanomachine HSP70 stimulates ASM and supports maintaining the lysosome membrane integrity (Petersen et al. 2013). Elevated expressions of HSP90 and HSP70 in cancer cells appear to associate with quicker disease development and are frequently involved in amplified chemotherapy resistance and poor patient diagnosis (Kumar et al. 2016). Targeting HSP70 thereby inactivating ASM in cancer cells would upsurge LMP resulting in cell death (Saftig and Sandhoff 2013; Howe et al. 2014). For instance, PES (2-phenylethyenesulfonamide, also called pifithrin-μ), a specific suppressor of stress-inducible HSP70 suppresses lysosomal action and autophagy and exerts prominent anticancer effects by promoting tumor cell death (Granato et al. 2013; Howe et al. 2014). HSP90 chemical inhibitors such as 17-AAG or 17-DMAG are currently developed and 17-AAG in combination with chemotherapies is presently undergoing phase I/II trials for solid and hematologic malignancies (Workman 2020).

Therapeutic targeting of cathepsins in cancer

Currently, lysosomal proteases like cathepsins have grown substantial curiosity in the branch of oncology (Fuchs et al. 2020; Holzen et al. 2020). Under physiological conditions, lysosomal cathepsins maintain tissue homeostasis (Pu et al. 2016). However, any alterations in cathepsin expression (usually upregulation) or localization (such as in cytoplasm, nucleus, mitochondria, or ECM architecture) have been associated with cancers (Pogorzelska et al. 2018). For example, overexpression of cathepsins B/S has been prominently compromised the development of solid tumors (Fuchs et al. 2020). Also, cathepsins are involved in response to antitumor drug within TME and they can play important actions in the progression of resistance to the chemotherapies (Rudzinska et al. 2019). Numerous tactics have been explored to inhibit tumor-associated cathepsin action, including chemical inhibitors and antibodies (in cancer immunotherapy). Although several cathepsin inhibitors have been explored for cancer treatment (Kos et al. 2014), there are presently not available cathepsin inhibitors for clinical trials in cancer. However, the main effort of research on the progression of cathepsin suppressors is on osteoporosis (Drake et al. 2017) and arthritic diseases (Wei et al. 2020). Since cathepsins display ideal prognostic markers these can be investigated for manipulating improved drug delivery tactics (Wilkinson et al. 2015; Yan et al. 2017). For example, cathepsins B/L are suggested as biomarkers for cancer identification where their expressions are frequently inversely connected with patient responses (Zhang et al. 2014).

Other potential autophagy-lysosomal pathway targeting

The transcription factor TFEB regulates the ALP suggesting that the several TFEB-controlled genes may comprise novel targets for drug discovery in cancer treatment. Current data have also suggested that ALP can firmly control the iron, heme (Dong et al. 2015), and calcium (Medina et al. 2015) metabolisms. Thus targeting transcription factors and nonprotein residues of the lysosome including Ca²⁺, Fe²⁺, and heme components can offer an additional therapeutic approach for aiming the lysosome in cancer.

Opinions against inhibition of autophagy for cancer treatment

Numerous studies have proposed that the suppression of autophagy is not practically ideal clue an immunologic standpoint for cancer treatment as it would suppress anticancer T-cell functions (Rao et al. 2014). The argument behindhand, autophagy in dead cancer cells is necessary for immunological clearance, which assists in effective appreciation and stimulation of the immunity for efficient anticancer immune response (Michaud et al. 2011). Autophagy induces antigen cross-presentation that offers extra latent way where suppression of autophagy could hinder a vigorous anticancer immune reply (Levy et al. 2017; Li et al. 2017). In patients, suppression of autophagy is not selectively aimed at cancer cells, hence global autophagy inhibition may cause potential toxicity. For example, the global ATG7^-/- adult mice are died due to lethal neuronal toxicity, interruption of glucose homeostasis, and amplified vulnerability to infection (Karsli-Uzunbas et al. 2014; Levy et al. 2017). Still, in the provision of positive clue, the extended use of HCQ for the treatment of rheumatic disorders like RA and the ailment of several cancer patients with CQ for prolonged times without producing toxicity indicates that an extended schedule strategy with autophagy suppressors is practicable (Levy et al. 2014).

Executive summary and perspective

Nutrient sensing property of the lysosome in ALP has provided a great influence on cellular physiological processes. Autolysosomes regulate metabolic homeostasis in cancer cells through the digestion of their cellular content. The evolving actions of autolysosomes in cancer cells may include many complex phenomena like nutrient sensing, signal transduction, apoptosis, and immunological clearance. Besides, its action as an inhibiting or promoting mechanism in cancer persists in dispute. Presently knowledge about autolysosomes and ALP is still inadequate, and several open questions are so far to be clarified.

Open questions

1. Autophagy acts the Yin-Yang faced functions in cancer cells which is dependent on the types and stages of cancer development. How ALP can trigger the cancer pathology and/or the carcinogenesis keeps to be elucidated.

2. In the early stage, autolysosomes display cancer oppressive action while in metastatic stage, these endorse cell survival by supplying nutrients under metabolic stress conditions. Whether and how autolysosomes have a role in the apoptotic pathway remain uncertain.

3. Stimulation of ALP obeys the ‘survival of the fittest’ rule for cancer cells. Dented proteins and organelles are eliminated by ALP which provides reserved energy and cancer cells use this energy for persistence against anticancer drugs. Thus understanding the exact changes of ALP may lead to new therapeutic strategies for a wide variety of diseases like cancers.

4. Moreover, the precise mechanistic pathways of lysosomal trafficking and function need to be clarified. Effectively analysis of the dynamic events occurred within the ALP vesicles due to diverse stresses needs to be clarified.

5. Since TFEB orchestrates as a master regulator of the ALP, transcriptional regulation of ALP activity and the precise roles of the transcribed genes associated with ALP biogenesis are still being explained.

To answer these questions, scientists should explore the genome editing roles of autolysosomes and lysosomes within the TME. Genomic and metagenomic investigation by applying high throughput screening techniques such as transcriptomics, proteomics and metabolomics to examine patient-derived cells may result in an advanced knowledge of lysosome-mediated cancer treatment.

Authors’ contributions

MAAB conceived, designed, edited, revised, and proof-read the manuscript.

Acknowledgements

The author does not take any allowance from known funding agencies in the public–private sectors, pharmaceutical companies, or not-for-profit organizations.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing does not apply to this article as no new data were created or analyzed in this study.