Co‐targeting of lysosome and mitophagy in cancer stem cells with chloroquine analogues and antibiotics

Abstract The catabolic autophagy eliminates cytoplasmic components and organelles via lysosomes. Non‐selective bulk autophagy and selective autophagy (mitophagy) are linked in intracellular homeostasis both normal and cancer cells. Autophagy has complex and paradoxical dual role in cancers; it can play either tumour suppressor or tumour promoter depending on the tumour type, stage, microenvironment and genetic context. Cancer stem cells (CSCs) cause tumour recurrence and promote resistant to therapy for driving poor clinical consequences. Thus, new healing strategies are urgently needed to annihilate and eradicate CSCs. As chloroquine (CQ) analogues show positive clinical outcome in several clinical trials either standalone or combination with several chemotherapies. Moreover, CQ analogues are known to eliminate CSCs via altering DNA methylation. However, several obstacles such as higher concentrations and dose‐dependent toxicity are noticeable in the treatment of cancers. As tumour cells predominantly rely on mitochondrial actions, mitochondrial targeting FDA‐approved antibiotics are reported to effectively eradicate CSCs alone or combination with chemotherapy. However, antibiotics cause metabolic glycolytic shift in cancer cells for survival and repopulation. This review will provide a sketch of the inhibiting roles of current chloroquine analogues and antibiotic combination in CSC autophagy process and discuss the possibility that pre‐clinical and clinical potential therapeutic strategy for anticancer therapy.

for eliminating very quickly propagating cells, the failure rate of conventional therapies is likely associated with a relatively rare slowly proliferating culture of cancer cells stay in tumour, called cancer stem cells (CSCs). 5,6 CSCs have been exhibited to be resistant to traditional chemotherapy as well as radiation. 7 Residual memory CSCs disappeared after clinical treatment are suggested responsible for the re-survival of tumours and for their progressive metastasis. 8 It has also been suggested that the most metabolic active CSCs have heightened biogenetic rate of mitochondria as compared to normal cell correspondents. 9,10 Thus, a great attempt has been concerned to the new drug development that is capable to correctly target biogenesis of mitochondria-associated CSCs.
The conventional drug discovery and development process are an indeed challenging field in terms of rising and unsustainable costs, and time-exhausting tasks, with a high frequency of failure rate. 11 Thus, pharmaceutical companies have decided to decrease annual investment regarding classical drug discovery 12 and healthcare systems have faced the substantial challenge in their survival for commercial sustainability inflamed by paying of prescription drugs. 13 In this context, drug repurposing (new therapeutic uses or indications are found for existing drugs) appears as a new platform for the pharmaceutical industries, patients and healthcare payers. 14,15 Moreover, drug repositioning (also called drug repurposing) approach may conquer many tremendous obstacles involved in new drugs discovery because of having established pharmacokinetics, pharmacodynamics and toxicity profiles, approval by several regulatory agencies FDA (US) and EMEA (Europe), and these recognitions accelerate the assessment of the agents in clinical trials. 16,17 Furthermore, drug repurposing may discover novel molecular regulatory pathways involved in cancer regrowth or admit new molecular targets for cancer therapy. 18 It has been exemplified that repurposing drugs chloroquine (CQ) analogues and antibiotics are known to accelerate the therapeutic capacity of chemotherapy by eliminating CSC traits of invasive progression in tumours. 19,20 Thus, repurposing drugs play an important role to eradicate CSC-mediated tumorigenesis.

| ROLE OF BULK AUTOPHAGY IN C AN CER
Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved, cellular homeostatic process that facilitates nutrient recycling via lysosomal degradation of potentially harmful cytoplasmic entities. 21,22 It has been widely established that the bipolar nature of autophagy exists in cancers. 23,24 Autophagy act as either tumour suppressor or tumour promoter depending on tumour type, stage of tumour development, tumour microenvironment and genetic context. 25,26 Although autophagy limits cancer development in the early stages of tumorigenesis, it can also have a pro-tumoral role in more advanced cancers, promoting primary tumour growth and metastatic spread. 25 Under normal conditions, cells utilize basal levels of autophagy to aid in the maintenance of biological function, homeostasis, quality control of cell contents and elimination of old proteins and damaged organelles. 27 Additionally, autophagy in stem cells is related to the maintenance of their unique properties, including differentiation and self-renewal. 28,29 However, many established malignant cells have high levels of basal autophagy even in fed conditions. 30,31 In contrast, autophagy in normal cells generally occurs at low levels and is only up-regulated in response to stressful conditions such as starvation. Moreover, some anticancer drugs can regulate autophagy. Therefore, autophagy-regulated chemotherapy can be involved in cancer-cell survival or death. 32 Additionally, the regulation of autophagy contributes to the expression of tumour suppressor proteins or oncogenes. Tumour suppressor factors are negatively regulated by mechanistic target of rapamycin (mTOR) resulting in the induction of autophagy and suppression of the cancer initiation. 33 In contrast, oncogenes may be activated by mTOR, class I PI3K (phosphoinositide 3-kinases) and AKT (also known as protein kinase B), resulting in the suppression of autophagy and enhancement of cancer formation. 25,34 Although these dual-complex mechanisms make autophagy a challenging target for anticancer therapeutics, a better understanding of the autophagic roles in different stages of tumorigenesis, specific cellular and extracellular context and the crosstalk between autophagy and apoptosis should all be taken into consideration to better harness autophagy in cancer treatment. 35

Lists of main topics
• Autophagy is an emerging potential therapeutic target for multiple disorders including multiple malignant tumours. Autophagy has both suppression role in tumour initiation and promotion action in tumour progression, and this controversy role of autophagy has led to dilemma over whether or how targeting of autophagy therapeutically should be undertaken for efficient treatment of cancers.
• Chloroquine analogues are established autophagy inhibitors from malaria treatment. When lysosomotropic action of chloroquine analogues is elucidated, these drugs have become popular for autophagy suppressors. Interestingly, an intricate link between autophagy and cancer is established when Beclin 1 (BECN1), an essential autophagy gene, is found to suppress breast tumorigenesis. 36,37 In several cancer-cell lines and mice models, the loss of BECN1 results in an inhibition of autophagy and an upsurge in cell proliferation. 36,38,39 In addition, the BECN1 gene is monoallellicaly deleted in 40%-75% of breast, ovarian and prostate cancers. 36,40,41 It is also found that the overexpression of Beclin 1 can inhibit the growth of colon cancer cells, 42 nasopharyngeal carcinoma 43 and CaSki cervical cancer cells. 44 Due to the genomic close proximity of the BRCA1 (breast cancer 1, early-onset gene) and the BECN1 gene at the 17q21 chromosome, it was assumed that BECN1 deletions are rather a passenger event. 45 Tumour suppressor gene deletions require additional modulators to form cancer.
In human breast and ovarian cancers, BECN1 is often co-deleted with BRCA1. This led to the hypothesis that BECN1 loss is a passenger event and is only deleted due to its proximity to BRCA1. 45,46 BRCA1 is frequently mutated in familial cases of breast and ovarian cancer, being relatively rare in sporadic cancers, and it is a classical tumour suppressor, as only one copy is sufficient to maintain its function. By contrast, the loss of just one allele of BECN1 is sufficient to induce tumorigenesis, 38,39 and therefore, it is suggested as a haploinsufficient tumour suppressor. Furthermore, two survival analyses on the TCGA (Cancer Genome Atlas Project) and METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) data set showed that a worse survival probability was associated with the lower BECN1 but not with the BRCA1 mRNA expression in all breast cancer types, 47 indicating that in sporadic breast cancers, BECN1 is a driver rather than a passenger event.
Autophagy also maintains cancer-cell re-survival during metabolic stressful conditions, and these mediate resistance to therapies such as chemotherapies or radiation. 48,49 Thus, induction of autophagy in cancer cells is associated with stress tolerance mechanism when these cells are experienced to nutrient starvation, hypoxic conditions or anticancer therapies. [50][51][52] In well-established tumours, the stress-induced autophagy allows tumour cell regrowth which in turn expedite tumour cell advancement and negotiate resistance to anticancer therapies. 53 As a result, inhibiting pro-survival (cytoprotective) autophagy in cancer cells has been shown to augment the effectiveness of anticancer therapy by promoting apoptotic cell death. 54,55 Although these dual-complex mechanisms make autophagy a challenging target for anticancer therapeutics, a better understanding of the autophagic roles in stages of tumorigenesis, specific cellular and extracellular context and the crosstalk between autophagy and apoptosis should all be taken into consideration to better harness autophagy in cancer treatment. Although autophagy modulation has promised as an emerging therapeutic strategy for certain cancer types, major challenges remain unclear. For examples, higher chemotherapy doses may cause toxic side effects and it is contradiction whether autophagy-modulating agents may significantly affect the tumour cells. Furthermore, there is doubt existence about an actual tissue-derived autophagy measurement, especially inaccessible in solid tumours. 18,56 Therefore, a better intervention of chemotherapeutic combination is required for modulation of inherent autophagy properties. Thus, treatment strategies of cancers that modulate autophagy both inducing and inhibiting concomitantly emphasize a better understanding for improved therapeutic outcome.

| Targeting lysosome in autophagy by chloroquine analogues
Chloroquine (CQ) analogues such as hydroxychloroquine (HCQ), quinacrine (QN), mefloquine (MQ), Lys05, verteporfin, clioquinol SAR405, spautin-1 (specific and potent aut phagy inhibitor 1), ARN5187, VATG (Van Andel-T-Gen)-027 and VATG-032 and its other derivatives are well-known repurposing success stories because these analogues are effective, inexpensive, well-tolerated in humans. 57,58 CQ analogues, for example HCQ, MQ and verteporfin, are FDA-approved agents generally applied for the treatment of malaria, systemic lupus erythematosus, rheumatoid arthritis and photodynamic therapy, but their potentials as anticancer agents have currently appeared. 57 As lysosomotropic agents, CQ analogues efficiently deacidify lysosomal lumens by changing permeability of lysosomal membrane potential (LMP). 59 Accumulating lines of evidence suggest that CQ analogues favourably induce apoptosis and necrosis in cancer cells such as breast cancer, colon cancer, glioma and glioblastoma compared with normal cells either in standalone or in combinations with chemotherapy. 53,60 In the context, it has been found that CQ analogues have direct actions on diverse kinds of cancers that influence chemotherapeutic actions, for example inhibition of both multidrug resistance pump and autophagy, intercalation in DNA and improving the penetration of chemotherapies in cancer cells or solid tumour tissues. 61,62 In these cases, the lysosome-deacidifying property of CQ analogues seems the most vital parameter for improving efficacy and specificity for cancer therapies.
CQ analogues also sensitize triple-negative breast cancer (TNBC) cells, categorized by a plenty of chemotherapy-resistant breast cancer stem cells (CSCs) as well as chemotherapy-resistant pancreatic CSCs to where CQ analogues efficiently prevent autophagy. [62][63][64] Thus, CQ analogues need to be more discovered in the scientific background as their victory may benefit to further quickly progress the poor diagnosis of patients with TNBC or pancreatic cancer.
Interestingly, recent evidence suggests that HCQ in combination treatment with mTOR inhibitors such as temsirolimus significantly suppresses tumour growth in vitro and in vivo. 65,66 Here the period of treatment and acceptable dose of HCQ differentially affect medical profits (best outcome achieved with 1200 mg HCQ twice daily).
Another clinical trial (phase 1 study) HCQ (600 mg) in blending with temozolomide (TMZ) indicates suppression of autophagy in humans.
However, an increased dose of HCQ is indispensable for noticeable clinical outcome. 67 Moreover, CQ also potentiates the cytotoxic effect of TMZ by inhibiting mitophagy in glioma cells. 68  and (d) finally, CQ-associated chemo-sensitization to chemotherapy seems to be an autophagy-independent occurrence. 72 These data strongly support a necessity to investigate better therapeutic strategy with specific molecular mechanism in modulating of autophagy in cancers. Further research will be required to identify and develop for additional effective and acceptable CQ analogues as autophagy suppressors, as well as outline the prime dose and dose interval that leads to highest the therapeutic activity during cancer therapy.
However, the successful drug repositioning approach has primarily been by serendipitous discovery or clinical observation, such as the rich history and serendipitous indications for chloroquine 59 (Table 1 and Figure 1). Thus, scientists from the repurposing drugs in oncology (ReDO) project highlighted the potentiality of CQ analogues for cancer treatment by acting on both the cancer cellular level and the tumour niches and suggested that these analogues could propose important clinical advantages for cancer patients, particularly in combination with conventional anticancer treatments.

| ROLE OF MITOPHAGY IN C AN CER
Mitophagy (mitochondrial autophagy) is the selective identification, degradation and removal of spoiled mitochondria at the autophagolysosome. 73 Mitophagy definitely varies from non-selective bulk autophagy due to its selectivity and regulation of the autophagic cargo. 74 Mitochondrial autophagy is co-ordinately related to cellular F I G U R E 1 An overview of mammalian autophagy process. Starvation, growth factor deprivation, low energy and hypoxia are wellestablished autophagy (specifically, macroautophagy) inducers. These culminate in mTORC1 inhibition and AMPK (5' AMP-activated protein kinase) activation, which, in turn, positively regulate the UNC51-like kinase 1 (ULK1) complex through a series of phosphorylation events. Induction of the ULK1 complex subsequently activates the class III PI3K complex, which leads to PI3P (phosphatidylinositol 3-phosphate) synthesis in isolation membranes (IMs) and initiates autophagy. Numerous molecular events are subsequently activated in the autophagy pathway, including initiation, nucleation, elongation, autophagosome maturation and cargo degradation. The IMs appear to have several sources, such as the ER membrane, Golgi apparatus and trans-Golgi network, plasma membrane, endosomal compartment and mitochondria. The two ubiquitin-like conjugation systems AuTophaGy-related 12 (ATG12)-ATG5-ATG16L1 complex and LC3 (microtubule-associated proteins 1A/1B light chain 3B)-II participate after their activation in the expansion of the double membrane and the closure of the isolation membrane. Once it is completed, the structure is called an autophagosome. After elongation and closure, the newly formed autophagosome may fuse with a late endosome to form an amphisome, or it may fuse directly with a lysosome to form an autolysosome, allowing the degradation of autophagic substrates. Once the cargos are degraded, the product macromolecules are exported to the cytosol to be recycled by the cell for ATP production and biosynthesis homeostasis that responds to extracellular deviations (eg stress, energy, nutrients). On the one hand, autophagosome formation occurs at the junction of mitochondria with endoplasmic reticulum upon the stimulation of autophagy initiation. In this process, mitochondria participate from the outer mitochondrial membrane lipids to nascent isolation membrane of autophagosomes. 75,76 On the other hand, autophagy donates mitochondria maintenance by regulation of mitochondrial integrity, which may also be related to regulatory higher living processes. 77 Mitophagy is triggered by stresses, DNA damage, inflammation, etc, and is an important mechanism for quality control of cellular bioenergetics and homeostasis by preserving mitochondrial integrity and actions. 78 Any imperfections in mitophagy lead to mitochondrial dysregulation that changes metabolic pathways and alters cell fate which in turn initiates the incidence and aetiology of diseases, including cancer. 79 During initiation of tumour, mitochondria perform a main role in supplying nutrients essential for boosted cell propagation and angiogenesis. 74 In addition, mitochondria contribute several events of cancers such as apoptosis resistance, oncogene-associated transformation, reprogramming of metabolism, translation of protein, stemness of cancer, malicious repopulation and drug resistance. [84][85][86] These solid foundation and proof-of-concept results strongly support the fact that mitochondria act as a fundamental metabolic centre vital for tumorigenesis. Thus, mitophagy mechanisms such as bioenergetics, biogenesis and cellular transductions of tumorigenesis have drawn the great attention for designing superb anticancer therapeutics. inhibits CSCs by impairing mitochondrial bioenergetic performance. 96 Atovaquone performs as an oxidative phosphorylation (OXPHOS)

| Targeting mitophagy by antibiotics
inhibitor and significantly inhibits sphere formation in breast and colorectal CSCs without affecting normal fibroblasts. 97 Pyrvinium pamoate, an anti-parasitic agent, behaves as an OXPHOS inhibitor aiming mitochondrial complex II and competently stops mammosphere production. 98 Doxycycline binds preferentially to the small subunit 28S ribosomes in mitochondria and erythromycin metabolites or chloramphenicol specifically fix to the mitochondrial ribosome large subunit 39S, thereby blocking biogenesis of mitochondria and thereby preventing protein translation as well as sufficient reduction in mammosphere production and bonafide CSC markers. 98 Thus, it is interesting that FDA-approved antibiotic-mediated mitochondria targeting may contribute to eradicate cancer cells particularly CSCs and the anticancer efficacies of the antibiotics (Table 2).

| TARG E TING LYSOSOME IN AUTOPHAGY AND MITOPHAGY BY CHLOROQUINE ANALOG UE AND ANTIB I OTI C S
It has been found that CQ analogues at low concentration suppress bone resorptive activity of osteoclasts without affecting bone-forming cells, 99 and subtherapeutic antibiotic treatment (STAT) causes an increase in bone mineral density. 100 Thus, combination of chloroquine analogues and mitochondrial-targeted agents in subtherapeutic level (at low concentration) would be more therapeutic potentials against CSC-related cancers and revolutionize the cancer research field without affecting normal cells.

| CON CLUS I ON S AND FUTURE PER S PEC TIVE
According to the vast evidence on in vitro and mammalian/ animal models, it is expected to find positive impacts of the combination

TA B L E 3 (Continued)
together with a diversity of chemotherapeutic drugs in cancer treatments (Table 3). It is predicted that such type of combinatory autophagy inhibitors with understanding of the molecular regulatory mechanism of the autophagy will direct to certain revolution in the treatment of multiple human diseases including cancer in the near future.

ACK N OWLED G EM ENTS
The authors thank Professor Dr Paul A Townsend, University of Manchester, for technical support of this manuscript.

CO N FLI C T O F I NTE R E S T
The author declares that there are no known competing financial interests or personal relationship that could have appeared to influence the work reported in this paper.

AUTH O R CO NTR I B UTI O N
Md. Abdul Alim Al-Bari: Writing-review & editing (equal).