UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE MEDICINA DE BOTUCATU Jofer Andree Zamame Ramirez Autofagia em células tumorais: um mecanismo de carcinogênese e resistência aos quimioterápicos Botucatu 2017 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE MEDICINA DE BOTUCATU Autofagia em células tumorais: um mecanismo de carcinogênese e resistência aos quimioterápicos Jofer Andree Zamame Ramirez Dissertação apresentada à Faculdade de Medicina, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de Botucatu, como parte dos requisitos necessários para a obtenção do título de Mestre em Patologia. Orientador: Prof. Dr. Ramon Kaneno Botucatu 2017 Este trabalho foi desenvolvido no Departamento de Microbiologia e Imunologia do Instituto de Biociências da Universidade Estadual Paulista campus de Botucatu. Dedico este trabalho a minha família. Agradecimentos Primeiramente, gostaria de agradecer ao meu orientador Dr. Ramon Kaneno, por todas as lições, não só acadêmicas, mas também sobre a vida, por toda a sua paciência e apoio que recebi durante estes dois ótimos anos. Muito obrigado Professor! A meus pais Fernando Zamame e Sandra Ramirez, por todo o amor e apoio que me deram, apesar de que nos separam 4.500 km (+/- 500 km), estou orgulhoso de ser seu filho, Obrigado, papai e mamãe! A meus irmãos, embora longe senti seu carinho, agradecimentos para os risos e bons tempos! Obrigado Vladimir e Fernando! A minha prima Rachel Siccha (Kelly) Eu sempre serei grato; você foi a "catapulta" para vir aqui. Muito obrigado! A meu primo Ricardo Britske, como um guia (não remunerado) em tudo o que eu sempre tive dúvidas. Muito obrigado! Aos meus amigos do laboratório de Imunologia de Tumores. À Graziela Romagnoli pela sua paciência e por ser um modelo a seguir. À Carolina M.Gorgulho (Carú) pelas muitas horas de aulas que eu recebi. Ao Edson Comparetti, Sophia Sartori e Raquel Liszbinski por me ajudar quando mais precisava. À Bianca Falasco, Juliana Toscano e Nathalia Laszkiewicz, por me ensinarem que sempre posso melhorar. À Flávia Sardela, Angélica Amarral e Bruna Sanrromão, pela sua disposição. Obrigado por terem sido minha família nestes últimos dois anos! Aos meus amigos e colegas do Departamento de Microbiologia e Imunologia. Ao Reginaldo Keller (Regis), por sua ajuda e compreensão. Daniela R. Rodrigues e Ivy Rafacho, por todas as vezes em que me ajudaram. Mariana Romão, Mariana Matias, Priscila R. Nunes e Vanessa R. Ribeiro, pela bibliografia, sua disposição e os risos! À Livia Matsumoto, Ariane Rocha por terem sido minhas primeiras amigas, obrigado por toda a ajuda e informação! Elisa de Oliveira e Fernanda Lopes Conte, pela sua ajuda e sugestões. Aos professores do Departamento de Microbiologia e Imunologia. A professora Maria Terezinha Serrão para ser um guia e exemplo. A professora Alexandrina Sartori por ser a primeira professora que conheci e me ajudou a adaptar-me ao departamento. A professora Ângela Soares pelas sugestões. Ao professor Sílvio Luís de Oliveira, por esclarecer muitas dúvidas. Aos Professores Mauricio Sforcin, Ary Fernandez e Eduardo Bagagli por toda sua ajuda nestes dois anos! Aos funcionários do Departamento de Microbiologia e Imunologia. Aline Missio, Ana Cláudia Acerra e Luiz Severino Dos Santos (Lula), obrigado por toda sua ajuda, foi muito importante! Muito obrigado! A coordenadora do programa de pós-graduação em Patologia/FMB, Dra. Denise Fecchio por sua ajuda e incentivo para continuar o meu projeto de pesquisa. Obrigado por tudo! À secretária do programa de pós-graduação em Patologia/FMB Vania Soler pela sua gentileza e paciência! Muito obrigado! À CAPES pela bolsa concedida durante a realização do meu mestrado. Muito obrigado! “Aprender a leer es lo más importante que me ha pasado en la vida”. Mario Vargas Llosa 5 Índice 1. Introduction: Autophagy as a physiological process ...................................................... 8 2. The controversial role of autophagy in cancer ............................................................... 14 2.1. Autophagy as a tumor suppressor .................................................................................. 14 2.2. Autophagy as a tumor promoter ..................................................................................... 17 3. Autophagy and resistance to chemotherapy .................................................................... 20 4. Conclusion ......................................................................................................................... 25 References .............................................................................................................................. 25 Anexos ......................................................................................................................... 29 6 Autophagy in tumor cells: a mechanism of carcinogenesis and resistance to chemotherapeutic agents Revisão da literatura apresentada na forma de manuscrito, redigido de acordo com as normas para submissão à revista Cellular Oncology (Springer). Porcentagem de similaridade com textos da literatura=6.7% de acordo com a plataforma Academic Plagiarism. 7 Autophagy in tumor cells: a mechanism of carcinogenesis and resistance to chemotherapeutic agents Jofer Andree Zamame Ramirez 1,2, Graziela Gorete Romagnoli 2, Ramon Kaneno 2* 1 São Paulo State University – UNESP, Department of Pathology, School of Medicine of Botucatu, Botucatu, SP, Brazil 2 São Paulo State University – UNESP, Department of Microbiology and Immunology, Institute of Biosciences of Botucatu, Botucatu, SP, Brazil *Corresponding author: Ramon Kaneno: Departamento de Microbiologia e Imunologia, Instituto de Biociências de Botucatu, Universidade Estadual Paulista – UNESP, câmpus de Rubião Jr, 18618-970. Phone: +55 14 3880 0432; Fax +55 14 3815 3744; email:rskaneno@yahoo.com.br. Abstract Autophagy is a dynamic physiological macromolecular process, whereby intracellular substrates are exposed to lysosomes for degradation and recycling of damaged organelles, alleviating cellular stress conditions. Several studies have shown that autophagy plays a critical role in tumoral cell survival, performing a protective role by correcting carcinogenic damages. However, this physiological process can be subverted in some cells, leading to the promotion of carcinogenesis or allowing cell escape by increasing its resistance to chemotherapeutics. This review covers the basic mechanisms and genes involved in autophagy as well as the controversial findings on their role tumor cells; we also reviewed the processes by which drug resistance may be determined, for a better understanding of how autophagy works and how it can be handled as an antitumor therapeutic intervention. 8 1. Introduction: Autophagy as a physiological process The term autophagy was first used by deDuve in 1963(1), and was defined by Klionsky in 2003 as "self-eating" at subcellular level(2). Since then, many scientists have investigated the mechanisms that trigger this process. Current definition of autophagy is a conserved cellular degradation process, in which portions of cytosol and organelles are sequestered into a double-membrane vesicle, called autophagosome and fused with lysosome for breakdown and eventual recycling of resulting macromolecules(3). This process is required for the maintenance of cellular homeostasis and generation of amino acids to sustain viability during periods of nutrient privation, or under other kinds of metabolic stress like intracellular bacterial or viral infections, mutations or exposition to drugs(4-6). The autophagy process has three different forms: macroautophagy, microautophagy, and chaperone-mediated autophagy(3). Macroautophagy, which is often simply referred as autophagy, is the most characterized form and has been extensively investigated (4, 6, 7), being defined as the sequestration of a bulk of cytoplasm and organelles in a double- membrane vesicle called phagophore. This phagophore is originated from endoplasmatic reticulum and formed by expansion of autophagosome, and fuses with lysosome, forming autophagolysosome. Inside autophagolysosome, damaged organelles are degraded by lysosomal hydrolases, and finally, the resultant ATP and peptides are used to keep the cell viability (3, 8). In contrast, microautophagy is characterized by direct uptake of cytoplasmic substrates by invagination of lysosomal membrane, while chaperone-mediated autophagy occurs by shuttling soluble proteins into the lysosome, via lysosomal chaperone proteins such as hsc70(3, 8). Survival and homeostatic functions of autophagy have been evolutionarily conserved from yeast to mammalian. When cells have rich nutrient condition, autophagy is produced 9 at low levels (basal autophagy), providing tissues with a cleaning mechanism, to control intracellular quality through cytoplasmic rotation and removal of damaged or unnecessary organelles (9-12). However, when a cell is under different kinds of stress, such as nutrient deprivation, hypoxia, intracellular infections (13) and exposition to drugs(14), autophagy is rapidly induced in order to maintain the amino acid pool in the cytoplasm, and cell survives possibly through protein neo-synthesis, energy production and gluconeogenesis(12), that maintain cell ATP production. Autophagy involves a concerted action of highly conserved gene products and is controlled by two pathways involved in the regulation of cell growth and metabolism, the mTOR (mammalian target of rapamycin) and the AMPK (AMP-activated protein kinase) /UVRAG (UV irradiation resistance associated gene) signaling pathways. During normal situations (nutrient availability), mTOR complex I (mTORCI) inhibits autophagy since it phosphorylates Atg13, causing disaggregation of ULK1 complex (ULK1, Atg13 and FIP200), required to form autophagosome. However, when mTOR is inhibited by dephosphorylation (caused by cell starvation), ULK1 complex works and autophagy is induced (8). Conversely, AMPK, is activated during energy deprivation or hypoxia by increased conversion of ADP to ATP, and can inactivate mTORC1, and trigger autophagy (10, 11). In parallel, AMPK and UVRAG pathways activate the pro-autophagy kinase Vps34 (Class III PI3 Kinase) by phosphorylating and recruiting beclin-1 (proautophagy and tumor suppressor gene), which produces PI(3)P that is essential for to initiate autophagosomes. Autophagy triggering is illustrated in Figure 1. 10 Figure 1: Autophagy inhibition and activation routes. Nutrient availability allows mTOR to disaggregate ULK1 complex, releasing ULK1, Atg 13 and FIP200, and avoiding the formation of autophagosome. Nutrient starvation provokes mTOR dephosphorilation and reduces its activity. It keeps ULK1 complexed and able to induce autophagosome formation, triggering autophagy. In such a condition AMPK pathway is also triggered, increasing conversion of ADP to ATP, and inactivating mTORC1. AMPK also phosphorylates beclin-1 to induce PI(3)P and start autophagy. Autophagy starts with phagophore formation and there are two models to explain the biogenesis of phagophores. In the first, the maturation model, it derives from a preexisting endoplasmic reticulum (ER) membrane. This theory, created by Klionsky and Dunn, is supported by the observation of similar membrane thickness of these organelles. They used specific antibodies for ER and phagophores to demonstrate the similarity between these two membranes by immunoelectron microscopy(2). 11 The second, called assembly model, is commonly observed in yeast cells and states that phagophore is assembled de novo from lipids in the cytoplasm to form a pre- autophagosomal structure (PAS)(15, 16), that eventually form phagophore and autophagosome membrane. Use of a green-fluorescent protein (GFP) labeled anti-Aut7 (related with LC3) allowed to show that PAS plays a crucial role just before or during the formation of autophagosome in yeasts. Anyway, independent of the proposed models, autophagy can be summarized in four steps (12, 16, 17) as shown in figure 2.  Nucleation: It is the initial formation of autophagosomes and requires the beclin-1 complex (beclin-1, VPS34, Atg14L, VPS15). This complex is formed and regulated by UV irradiation resistance associated gene (UVRAG), produces PI(3)P by phosphorylating phosphatidylinositol (PI) of the hydroxyl group at position 3 of the inositol ring. Accumulation of PI(3)P generates a platform to recruit effector proteins. PI3P-binding proteins (Atg2-Atg18) are required to form the isolation membrane for sequestering autophagic substrates.  Elongation: Refers to the closure of isolation membrane to form autophagosomes. This process requires conjugation of Atg proteins (“Autophagy proteins”) that causes sequestration of cytoplasmic constituents. Meanwhile, PI(3)P recruits Atg12-Atg5-Atg16 complex (Atg5 complex), that conjugate LC3 (LC3-I/Atg8) to phosphatidylethanolamine (PE) to form LC3-II. This protein increases stability of autophagosome elongation by setting microtubule-associated proteins, like bricks building a wall.  Maturation (Fusion): Molecular mechanism underlying autophagosome maturation is largely unknown, but according to some theories this process is mediated by TECPR1 (Tectonin Beta-Propeller Repeat Containing 1) from lysosome, binds to Atg12-Atg5 complex and PI(3)P to promote autophagosome- 12 lysosome fusion and stability. Alternatively, autophagosomes can fuse with endosomal vesicles, such as endosomes and multivesicular bodies, to form amphisomes, which ultimately fuse with lysosomes.  Degradation: After fusion with lysosomes, sequestered materials are hydrolyzed and inner membrane of autophagosomes and cargoes are degraded by lysosomal enzymes. Breakdown products are released back into the cytosol for further recycling. 13 Figure 2: Autophagy steps. After autophagy induction, UVRAG stimulates recruitment of beclin-1 complex, which includes beclin-1, VPS34, Atg14L, and VPS15, In order to form an autophagosome, elongate it and close the isolation membrane, two protein conjugation systems are required, the Atg12–Atg5–Atg16 complex (Atg5 complex) and the Atg8/LC3– phosphatidylethanolamine (PE) Finally, closed autophagosomes fuse with lysosome, which enzymes degrade autophagosome contents. 14 2. The controversial role of autophagy in cancer Autophagy and autophagic defects have been implicated in different diseases, including neurodegeneration, myopathy, Crohn's disease and cancer. However, the role of autophagy in carcinogenesis is controversial, since there are both evidence that it suppresses tumor transformation, and that it can promote or facilitate survival and adaptation of tumor cells. Thus, autophagy appears to play a protective role in the early steps of tumor development, regulating oncogenic genes and molecules, while established cancer appears to be benefited by autophagy. Therefore, in this review, we will first present the evidence of a protective role of autophagy in the early phases of carcinogenesis and then, those found as pro-carcinogenic events in the later steps of this process. 2.1. Autophagy as a tumor suppressor One of the most important connections between autophagy and tumor suppression is the regulation of reactive oxygen species (ROS). The increase of ROS production induces nitration and deamination reactions on DNA bases, accelerating mutagenesis, increasing the activation of oncogenes, and thus stimulating carcinogenesis(18, 19). Mitochondria are considered the main source of intracellular ROS and its production increases as these organelles age or become damaged. In this context, autophagy helps to avoid damage through selective degradation of defective mitochondria (mitophagy). Consequently, in early steps of cancer, inhibition of autophagy facilitates genomic instability by activating oncogenes and inducing genotoxic effects as observed in autophagy-defective cells(20). Thus, selective removal of potentially damaged mitochondria reduces excessive ROS production and thereby limits tumor-promoting effects. Accordingly, inhibition of 15 autophagy leads to accumulation of defective mitochondria, with consequent cell transformation(21). The gene p53 is called “guardian of the cellular genome”, because his product (protein p53) inhibit cell cycle progression. Normal activity of p53 induces generation of the protein p21 that complex to cell division stimulating protein cdk2 (22). Fail of p53, do not block cell cycle, allowing the development of transformed cells. P53 can be activated by variety of stresses and subsequently works as a transcription factor to orchestrate several biological outputs, such as transient cell cycle arrest(22), apoptosis (22), cell senescence (22), metabolism (23), and regulation of autophagy(24). Being a suppressor gene for oncogenesis, defects or suppression of p53 break the genomic stability and facilitate cancer development. However, during autophagy, part of the p53 molecules is degraded, resulting in its underexpression and tumor growth facilitation(25). Increasing evidence indicate that p53 and AMPK/mTOR pathway are the most important mediators of the senescence response (26, 27). Thus, p53 can function as a suppressor of cell senescence by blocking the mTOR pathway (26). Under serum starvation, an increased autophagic flow was observed in HCT-116 p53+/+ but not in HCT-116 p53-/- cells, suggesting that p53 promotes autophagy. Then, p53-dependent autophagy protects cancer cells from starvation-induced death, while inhibition of autophagy (by the treatment with autophagy inhibitors) results in a high rate of cell death. Also, PALB2 (Partner and localizer of BRCA2) knockout mice develop breast adenocarcinoma when p53 is mutated and partial inhibition of autophagy by monoallelic loss of beclin-1 increases apoptosis and delays tumor growth in a p53 dependent way (28, 29). 16 Autophagy also permits degradation of protein aggregates. Defects in the autophagic process is associated with accumulation of protein aggregates and autophagy substrate p62/SQSTM1. Protein p62 is a selective autophagy substrate that accumulates when autophagy is reduced. This protein contains three regions called PB1 (Phox and Bem1p) domain that permits protein oligomerization, UBA (ubiquitin-associated domain) required for binding to polyubiquitinated proteins, and LIR (LC3-interacting region) necessary for association with LC3. Interestingly, p62 levels are commonly elevated in breast and prostatic tumors, suggest an association between reduced autophagy and carcinogenesis (30, 31). Another possible tumor suppressor role of autophagy was shown in studies of beclin-1 (BECN1 gene). Overexpression of BECN1 promotes autophagy in cancer cells and inhibits tumorigenesis in a murine model(33). This gene plays an important role in autophagy, regulating and binding to BCL2 (gene of B-cell lymphoma 2), UVRAG (UV Radiation Resistance Associated), Atg14 (Autophagy-related gene 14) and VMP1 (Vacuole Membrane Protein 1), which are important genes for autophagosome formation. BECN1 resides on chromosome 17q21, commonly deleted in breast, ovarian, and prostate cancers(34). BECN1 deletion can be monoallelic, as happens in breast cancer cell lines (35). Furthermore, BECN1 +/- mice suffer from a high incidence of tumors, including mammary gland neoplasia, lymphoma, lung adenocarcinoma, and hepatocellular carcinoma (36, 37), suggesting that BECN1 may be a haploinsufficient tumor suppressor. Analysis by RT-qPCR showed expression of beclin-1 is upregulated in mamospheres of breast cancer cell lines starved. Knocking out beclin-1 gene in adult mice causes avoidance of autophagy and increases the incidence of lymphomas and carcinomas, indicating that autophagy can work to eliminate tumorigenic defects. This same scenario also occurs in ovarian and prostate cancer(38). 17 Mutations of UVRAG are also found in human colon cancer cells. This protein recruits beclin-1 increasing autophagy events, while mutations interfere with its tumor-suppressing functions and enhance transformation of colorectal cancer cells, as happens with many human colon cancer cell lines (HCT15, HCT116, KM12, LIM2405, LS180, RKO and SW48)(17, 39, 40). 2.2. Autophagy as a tumor promoter Besides the above-mentioned evidence that autophagy can protect cells from malignant transformation, several studies point out this phenomenon as a way to escape from antitumor response. Tumor cells usually have high proliferation rates, which translate into higher bioenergetic and biosynthetic requirements than non-transformed cells. These requirements can be satisfied by increasing autophagy as a mechanism to obtain both ATP and metabolic intermediates(32). Importantly, for tumor cells, in which oncogene Ras is active, high levels of basal autophagy and dependence on this mechanism for survival are observed (34, 41). Then, autophagy is thought to promote tumor cell survival by increasing tolerance to stress and providing a pathway to get nutrients to supply their energetic requirements (32). Small GTPases of Ras family are involved in signaling pathways important for proliferation, cell survival, and metabolism. Ras-activating mutations are present in 33% of all human cancers(42), being linked to the development of some of the most lethal cancers, including those of the lung, colon, and pancreas (34). Activation of Ras oncogene is initiated by cell surface receptors, that induces RasGEFs (guanine-nucleotide exchange factors) to exchange GDP by GTP on Ras. Once activated, Ras stimulates diverse downstream effectors leading to the initiation of an array of cell signaling networks, including AMPK/UVRAG autophagy pathway(43). Since Ras works as a positive regulator 18 of the mTOR pathway, it would be expected this gene would negatively regulate autophagy. However, Ras is also involved in the regulation of a vast number of other signaling pathways, and its implication in autophagy regulation is multifaceted(42, 43). In vitro studies showed that several cell lines with Ras-activating mutations exhibit high levels of basal autophagy and marked autophagy-dependent survival under conditions of nutrient deprivation. Accordingly, silencing these genes involved in autophagy promotes the accumulation of dysfunctional mitochondria, low oxygen consumption, and decreased cell growth(20). In summary, current evidence points towards autophagy as a mechanism that ensures adequate mitochondrial metabolism in Ras+ cancers by supplying mitochondrial intermediates via degradation of macromolecules under both basal and starvation conditions(41). The role of autophagy has also been studied in other different contexts independent of Ras. For instance, in a model of breast cancer, the inhibition of autophagy by FIP200 deletion suppresses mammary tumor initiation and progression. Deletion of FIP200 results in multiple autophagy defects including accumulation of ubiquitinated protein aggregates and p62/SQSTM1, deficient LC3 conversion, and increased number of mitochondria with abnormal morphology in tumor cells(44). Authors observed the FIP200 ablation increase the number of mitochondria with abnormal morphology in mammary tumor cells and significantly reduces their proliferation (45). Thus, it would be interesting to determine whether FIP200 contribute to dependence on autophagy for cell proliferation. Other genes involved in autophagy are LC3 genes or microtubule-associated protein 1 light chain 3 (MAP1LC3A and MAP1LC3B). Authors observed that 65 out of 67 samples obtained from patients with breast carcinoma have significantly higher expression of MAP1LC3A/B protein as compared with normal tissues(45). It indicates that expression of LC3 is closely associated with development of breast cancer. 19 Immunohistochemical analysis of ULK1 expression in two independent cohorts of nasopharyngeal carcinoma (NPC) indicates a correlation of high expression of this protein with resistance to therapy, whereas patients with good therapeutic response express lower levels(46). It suggests that high ULK1 expression is closely associated with an aggressive clinical feature of NPC patients. Since ULK1, one of three main genes required to form phagophore, is regulated by mTOR complex, increased levels of this molecule mean higher levels of autophagy and the consequent facilitation of tumor cell grow. Therefore autophagy can work both for tumor suppression (Beclin-1, UVRAG, ULK1 and p53) and for oncogenesis (FIP200, LC3, p62 and Ras) depending on the level of alteration they present, and the imbalance towards one of these hands, as illustrated in figure 3. 20 Figure 3: Autophagic imbalance. Proposed genes by which autophagy may suppress or promote tumorigenesis. Genes Beclin-1, UVRAG, ULK1 and p53, expressed in early stages of cancer, trigger autophagy as a process to fix all damages caused by stress and thus prevent tumor growth. But, in later stages, changes occurring in tumor suppressor genes provide the balance for the oncogenesis side. Genes FIP200, LC3 (Atg5), Ras and p62, when are overexpressed or mutated, behave like oncogenes, promoting oncogenesis. As illustrate in the image, genes that stimulate tumor growth are more prevalent and more expressed in final stages of tumorigenesis. 3. Autophagy and resistance to chemotherapy Growing knowledge on the mechanisms involved in tumor formation and metastatic progression has not been directly translated into more effective treatment and cure of cancer. Reasons for failure of presently available anticancer treatment are mainly related to 21 cellular heterogeneity of tumors and rise of inherent or therapy-induced resistance of tumor cells to the therapeutic agents (47). The inability of conventional treatments to completely eradicate all invasive tumor cells is the major cause of treatment failure, allowing tumor recurrence or relapse. It has been proposed that small subsets of cancer cells, called cancer stem cells (CSCs) are responsible for cancer genesis, tumor growth, recurrence, and development of drug resistance(29). CSCs have been identified as immortal tumor initiating cells that can self-renew and have pluripotent capacity(48), and was found in several kinds of solid tumors(49). In recent years, CSCs have gained intense interest as key tumor-initiating cells that may also play an integral role in recurrence following chemotherapy. Then, a number of mechanisms of chemoresistance have been identified in CSCs (36, 50, 51), including autophagy. Gong et al.(52) observed that muted beclin-1 is crucial for maintenance of CSC and tumor development in athymic mice, this study highlighting the role of autophagic pathway for CSC maintenance and consequently for tumor survival and growth. Serum-deprived mesenchymal stem cells (SD-MSCs) support MCF-7 tumor growth. Tumor injected with SD-MSCs exhibited higher cellularity, decreased apoptosis, and differentiation of tumoral cells. Muted Beclin-1 staining indicated autophagic areas surrounded by actively proliferating tumoral cells. In addition, in vitro studies demonstrated that SD-MSCs survive using autophagy and secrete paracrine factors that support tumor cells following nutrient/serum deprivation(51). Several studies show that inhibition of autophagy turns cancer cells sensitive to a broad spectrum of therapies (53-55). For example, LC3 expression positively correlates with expression of aldehyde dehydrogenase 1 (ALDH1) in pancreatic cancer tissues. ALDH1 is a CSC marker and a high co-expression of LC3/ALDH1 can be associated with 22 both poor overall survival and progression-free disease (53). In pancreatic cancer cell lines, higher LC3-II expression was observed in the sphere-forming cells than in the bulk cells. Blockade of autophagy by silencing ATG5, ATG7, and BECN1 or administration of chloroquine (CQ) markedly reduces the CSC population, ALDH1 activity, sphere formation, and resistance to gemcitabine in vitro and in vivo(54). The combination of temsirolimus, an mTOR inhibitor (clinically used in renal cancer and melanoma) with hydroxychloroquine (HCQ), drug that increases the pH of the lysosome, disrupting its functions and blocking its union with the autophagosome, augments cell death in preclinical models. A phase I dose-escalation study evaluated the effects of the maximum tolerated dose (MTD=200 mg/day), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of the combination of these two drugs. Authors studied 27 patients with advanced solid malignancies and observed by positron emission tomography (PET) that such a combination produced metabolic stress in tumors of those patients that experienced clinical benefit. Pharmacodynamic evidence of autophagy inhibition was observed in tumor biopsies of patients treated with daily HCQ. This study indicates that combination of HCQ with temsirolimus is safe and tolerable, modulates autophagy in patients, and has significant antitumor activity(55). Moreover, active clinical trials are ongoing to test the clinical efficacy of autophagy inhibitors (mainly CQ/HCQ) as cancer therapeutics(56-60). At least 14 clinical trials using CQ/HCQ as adjuvant in cancer therapy are currently ongoing as summarized in Table 1. In a single blind phase I trial (NCT01480154) it was observed that combination of HCQ at maximum tolerated dose with AKT-inhibitor downregulated autophagy and extend survival among patients with advanced solid tumors(56). 23 A phase II trial (NTC01026844) was proposed to use Erlotinib, a epidermal growth factor receptor inhibitor, commonly used for treatement of lung cancer, in conjunction with HCQ at different concentrations for the treatment of lung cancer. The study began in 2009 and was completed in 2017, but of little response against drugs, authors resolves finished and discarding what was found(57). Another phase II study (NTC01842594) used Sirolimus (Rapamicyn), an mTOR inhibitor (1mg) along with HCQ (200mg) against soft tissue sarcoma of 10 patients, with moderate toxicity (nausea, diarrhea and skin rash) being found in 30% of patients(58). Moderate toxicity was also observed in 52% of patients with untreated B-CLL lymphoma, in this phase II study (NTC00771056) (59). Other studies enrolled in table 1 are still in the "ongoing" phase, recruiting patients or in phase of pre-publication analysis. It is possible that some of them did not achieved the hypothesized clinical results, making it difficult to be published. For instance a phase I/II trial (NCT01206530) was launched in 2010 to test the efficacy of combined therapy of HCQ with fluorouracil, leucovorin, calcium, oxaliplatin and bevacizumab to treat colorectal cancer (CRC)(60);but until now results are not publically available. Nevertheless, none of these trials has reached Phase III, implying that inhibiting autophagy remains an incompletely know approach, requiring efforts understand the complexity of events that such approaches can trigger. Table 1: Currently approved clinical trials using adjuvant CQ/HCQ as cancer therapies Trial ID Start Year Phase of Trial Cancer Type Dose of Adjuvant Therapeutic agents NCT01480154 2011 I Advanced solid tumors, melanoma, prostate or HCQ MTD Akt inhibitor MK2206 24 kidney cancer NTC01026844 2009 II Non-Small Cell Lung cancer HCQ (400-600-800- 1000mg/day) Erlotinib NTC01842594 2012 II Soft tissue sarcoma HCQ (200mg/day) Sirolimus NTC00771056 2008 II B-Cell Chronic Lymphocytic Leukemia HCQ (400mg/day) --- NCT02316340 2015 II Refractory metastatic colorectal cancer HCQ (600 mg/day) clinical efficacy with progression free survival Vorinostat NCT01978184 2013 II Pancreatic cancer HCQ (1200 mg/day) antitumor Efficacy Gemcitabine, abraxane NCT01649947 2011 II Recurrent non- small cell lung cancer CQ (200 mg BID two times in day) response to therapy Bevacizumab, carboplatin, paclitaxel NCT01494155 2011 II Pancreatic cancer HCQ (400 mg BID) progression-free survival Proton beam radiation therapy and capecitabine NCT01828476 2013 II Metastatic castrate refractory prostate cancer Response with/without HCQ treatment Navitoclax and abiraterone acetate NCT01006369 2009 II Metastatic colorectal cancer HCQ (200 mg/day) partial response and overall survival XELOX- bevacizumab NCT02013778 2013 I, II Liver cancer and hepatocellular carcinoma HCQ MTD Transarterial chemoemboliz ation (TACE) NCT01550367 2012 I, II Metastatic renal cell carcinoma HCQ (600–1200 mg/day) safety and toxicity IL-2 (aldesleukin) 25 Source: National Cancer Institute (USA) 4. Conclusion Although the controversy about the action of autophagy as tumor promoter or tumor suppressor, there is evidence that it inhibition helps to kill tumor cells mostly dependent on the kind of tumor and the therapeutic agents to be associated with. Therefore, we need a better understanding of the functional relevance of autophagy in the tumor microenvironment and enhance the ongoing dialogue between the laboratory and clinical researches in order to provide a new focus on therapeutic strategy to prevent resistance and increase the effects of cancer therapies. References 1. de Duve C. Lysosomes revisited. European Journal of Biochemistry. 1983;137(3):391-7. doi: 10.1111/j.1432-1033.1983.tb07841.x. 2. Klionsky DJ, Cregg JM, Dunn Jr WA, Emr SD, Sakai Y, Sandoval IV, et al. A Unified Nomenclature for Yeast Autophagy-Related Genes. Developmental Cell. 2003;5(4):539-45. doi: http://dx.doi.org/10.1016/S1534-5807(03)00296-X. 3. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature. 2008;451(7182):1069-75. doi: 10.1038/nature06639. PubMed PMID: PMC2670399. 4. Cuervo AM. Autophagy: In sickness and in health. Trends in Cell Biology. 2004;14(2):70-7. 5. Ciechanover A. Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat Rev Mol Cell Biol. 2005;6(1):79-87. 6. Shoji-Kawata S, Levine B. Autophagy, antiviral immunity, and viral countermeasures. Biochimica et biophysica acta. 2009;1793(9):1478-84. doi: 10.1016/j.bbamcr.2009.02.008. PubMed PMID: PMC2739265. NCT01206530 2010 I, II Colorectal cancer HCQ (600–800 mg/day) Fluorouracil leucovorin calcium oxaliplatin, bevacizumab NCT01510119 2011 I, II Kidney cancer HCQ MTD rate of progression in 6 months Rad001(everol imus) http://dx.doi.org/10.1016/S1534-5807(03)00296-X 26 7. Mehrpour M, Esclatine A, Beau I, Codogno P. Overview of macroautophagy regulation in mammalian cells. Cell Res. 2010;20(7):748-62. 8. Eskelinen E-L, Saftig P. Autophagy: A lysosomal degradation pathway with a central role in health and disease. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2009;1793(4):664-73. doi: http://dx.doi.org/10.1016/j.bbamcr.2008.07.014. 9. Maycotte P, Thorburn A. Autophagy and cancer therapy. Cancer Biology & Therapy. 2011;11(2):127-37. doi: 10.4161/cbt.11.2.14627. PubMed PMID: PMC3047083. 10. Janku F, McConkey DJ, Hong DS, Kurzrock R. Autophagy as a target for anticancer therapy. Nat Rev Clin Oncol. 2011;8(9):528-39. doi: http://www.nature.com/nrclinonc/journal/v8/n9/suppinfo/nrclinonc.2011.71_S1.html. 11. Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in cancer. Nat Rev Cancer. 2007;7(12):961-7. 12. Alva AS, Gultekin SH, Baehrecke EH. Autophagy in human tumors: cell survival or death? Cell Death Differ. 2004;11(9):1046-8. 13. He C, Klionsky DJ. Regulation Mechanisms and Signaling Pathways of Autophagy. Annual review of genetics. 2009;43:67-93. doi: 10.1146/annurev-genet-102808-114910. PubMed PMID: PMC2831538. 14. Rubinsztein DC, Codogno P, Levine B. Autophagy modulation as a potential therapeutic target for diverse diseases. Nature reviews Drug discovery. 2012;11(9):709-30. doi: 10.1038/nrd3802. PubMed PMID: PMC3518431. 15. Takeshi Noda KS, Yoshinori Ohsumi. Yeast autophagosomes: de novo formation of a membrane structure. TRENDS in Cell Biology 2002;12(5):231-5. 16. Reggiori F, Klionsky DJ. Autophagosomes: biogenesis from scratch? Current Opinion in Cell Biology. 2005;17(4):415-22. doi: http://dx.doi.org/10.1016/j.ceb.2005.06.007. 17. Virgin HW, Levine B. Autophagy genes in immunity. Nat Immunol. 2009;10(5):461-70. 18. Filomeni G, De Zio D, Cecconi F. Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell Death and Differentiation. 2015;22(3):377-88. doi: 10.1038/cdd.2014.150. PubMed PMID: PMC4326572. 19. Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochemical Journal. 1996;313(Pt 1):17-29. PubMed PMID: PMC1216878. 20. Gomes LC, Di Benedetto G, Scorrano L. During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Nature cell biology. 2011;13(5):589-98. doi: 10.1038/ncb2220. PubMed PMID: PMC3088644. 21. Ding W-X, Yin X-M. Mitophagy: mechanisms, pathophysiological roles, and analysis. Biological chemistry. 2012;393(7):547-64. doi: 10.1515/hsz-2012-0119. PubMed PMID: PMC3630798. 22. Li T, Kon N, Jiang L, Tan M, Ludwig T, Zhao Y, et al. Tumor suppression in the absence of p53-mediated cell cycle arrest, apoptosis, and senescence. Cell. 2012;149(6):1269-83. doi: 10.1016/j.cell.2012.04.026. PubMed PMID: PMC3688046. 23. Berkers Celia R, Maddocks Oliver D, Cheung Eric C, Mor I, Vousden Karen H. Metabolic Regulation by p53 Family Members. Cell Metabolism. 2013;18(5):617-33. doi: 10.1016/j.cmet.2013.06.019. PubMed PMID: PMC3824073. 24. Tasdemir E, Maiuri MC, Galluzzi L, Vitale I, Djavaheri-Mergny M, D'Amelio M, et al. Regulation of autophagy by cytoplasmic p53. Nat Cell Biol. 2008;10(6):676-87. doi: http://www.nature.com/ncb/journal/v10/n6/suppinfo/ncb1730_S1.html. 25. Tom Strachan AR. Cancer Genetics, in Human Molecular Genetics1999. 26. Astle MV, Hannan KM, Ng PY, Lee RS, George AJ, Hsu AK, et al. AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for targeting mTOR during malignancy. Oncogene. 2012;31(15):1949-62. doi: http://www.nature.com/onc/journal/v31/n15/suppinfo/onc2011394s1.html. http://dx.doi.org/10.1016/j.bbamcr.2008.07.014 http://www.nature.com/nrclinonc/journal/v8/n9/suppinfo/nrclinonc.2011.71_S1.html http://dx.doi.org/10.1016/j.ceb.2005.06.007 http://www.nature.com/ncb/journal/v10/n6/suppinfo/ncb1730_S1.html http://www.nature.com/onc/journal/v31/n15/suppinfo/onc2011394s1.html 27 27. Tasdemir E, Maiuri MC, Morselli E, Criollo A, D'Amelio M, Djavaheri-Mergny M, et al. A dual role of p53 in the control of autophagy. Autophagy. 2008;4(6):810-4. doi: 10.4161/auto.6486. 28. Huo Y, Cai H, Teplova I, Bowman-Colin C, Chen G, Price S, et al. Autophagy opposes p53- mediated tumor barrier to facilitate tumorigenesis in a model of PALB2-associated hereditary breast cancer. Cancer discovery. 2013;3(8):894-907. doi: 10.1158/2159-8290.CD-13-0011. PubMed PMID: PMC3740014. 29. Tripathi R, Ash D, Shaha C. Beclin-1–p53 interaction is crucial for cell fate determination in embryonal carcinoma cells. Journal of Cellular and Molecular Medicine. 2014;18(11):2275-86. doi: 10.1111/jcmm.12386. PubMed PMID: PMC4224560. 30. Bjørkøy G, Lamark T, Pankiv S, Øvervatn A, Brech A, Johansen T. Chapter 12 Monitoring Autophagic Degradation of p62/SQSTM1. Methods in Enzymology. Volume 452: Academic Press; 2009. p. 181-97. 31. Inami Y, Waguri S, Sakamoto A, Kouno T, Nakada K, Hino O, et al. Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. The Journal of Cell Biology. 2011;193(2):275- 84. doi: 10.1083/jcb.201102031. PubMed PMID: PMC3080263. 32. Mathew R, Karp C, Beaudoin B, Vuong N, Chen G, Chen H-Y, et al. Autophagy Suppresses Tumorigenesis Through Elimination of p62. Cell. 2009;137(6):1062-75. doi: 10.1016/j.cell.2009.03.048. PubMed PMID: PMC2802318. 33. Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 1999;402(6762):672-6. 34. Liu J, Xia H, Kim M, Xu L, Li Y, Zhang L, et al. Beclin1 Controls the Levels of p53 by Regulating the Deubiquitination Activity of USP10 and USP13. Cell. 2011;147(1):223-34. doi: 10.1016/j.cell.2011.08.037. PubMed PMID: PMC3441147. 35. Tang MKS, Kwong A, Tam K-F, Cheung ANY, Ngan HYS, Xia W, et al. BRCA1 deficiency induces protective autophagy to mitigate stress and provides a mechanism for BRCA1 haploinsufficiency in tumorigenesis. Cancer Letters.346(1):139-47. doi: 10.1016/j.canlet.2013.12.026. 36. Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. The Journal of Clinical Investigation. 2003;112(12):1809-20. doi: 10.1172/JCI20039. 37. Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(25):15077-82. doi: 10.1073/pnas.2436255100. PubMed PMID: PMC299911. 38. Fu L-l, Cheng Y, Liu B. Beclin-1: autophagic regulator and therapeutic target in cancer. Int J Biochem Cell Biol. 2013;45(5):921-4. doi: 10.1016/j.biocel.2013.02.007. PubMed PMID: 23420005. 39. White E. The role for autophagy in cancer. The Journal of Clinical Investigation. 2015;125(1):42-6. doi: 10.1172/JCI73941. 40. He S, Zhao Z, Yang Y, O'Connell D, Zhang X, Oh S, et al. Truncating mutation in the autophagy gene UVRAG confers oncogenic properties and chemosensitivity in colorectal cancers. Nature Communications. 2015;6:7839. doi: 10.1038/ncomms8839. 41. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nature reviews Cancer. 2011;11(11):761-74. doi: 10.1038/nrc3106. PubMed PMID: PMC3632399. 42. Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 2003;3(1):11-22. 43. Ge J, Chen Z, Huang J, Chen J, Yuan W, Deng Z, et al. Upregulation of Autophagy-Related Gene-5 (ATG-5) Is Associated with Chemoresistance in Human Gastric Cancer. PLOS ONE. 2014;9(10):e110293. doi: 10.1371/journal.pone.0110293. 28 44. Wei H, Wei S, Gan B, Peng X, Zou W, Guan J-L. Suppression of autophagy by FIP200 deletion inhibits mammary tumorigenesis. Genes & Development. 2011;25(14):1510-27. doi: 10.1101/gad.2051011. PubMed PMID: PMC3143941. 45. Othman EQG, Kaur G, Mutee AF, Tengku Muhammad TS, Tan ML. Immunohistochemical expression of MAP1LC3A and MAP1LC3B protein in breast carcinoma tissues. Journal of Clinical Laboratory Analysis. 2009;23(4):249-58. doi: 10.1002/jcla.20309. 46. Yun M, Bai H-Y, Zhang J-X, Rong J, Weng H-W, Zheng Z-S, et al. ULK1: A Promising Biomarker in Predicting Poor Prognosis and Therapeutic Response in Human Nasopharygeal Carcinoma. PLOS ONE. 2015;10(2):e0117375. doi: 10.1371/journal.pone.0117375. 47. Cancer multidrug resistance. Nat Biotech. 2000;18:18-20. 48. Abdullah LN, Chow EK-H. Mechanisms of chemoresistance in cancer stem cells. Clinical and Translational Medicine. 2013;2:3-. doi: 10.1186/2001-1326-2-3. PubMed PMID: PMC3565873. 49. Guan J-L, Simon AK, Prescott M, Menendez JA, Liu F, Wang F, et al. Autophagy in stem cells. Autophagy. 2013;9(6):830-49. doi: 10.4161/auto.24132. PubMed PMID: PMC3672294. 50. Kantara C, O’Connell M, Sarkar S, Moya S, Ullrich R, Singh P. Curcumin Promotes Autophagic Survival of a Sub-Set of Colon Cancer Stem Cells, which are Ablated by DCLK1-siRNA. Cancer research. 2014;74(9):2487-98. doi: 10.1158/0008-5472.CAN-13-3536. PubMed PMID: PMC4013529. 51. Sanchez CG, Penfornis P, Oskowitz AZ, Boonjindasup AG, Cai DZ, Dhule SS, et al. Activation of autophagy in mesenchymal stem cells provides tumor stromal support. Carcinogenesis. 2011;32(7):964-72. doi: 10.1093/carcin/bgr029. PubMed PMID: PMC3128555. 52. Gong C, Bauvy C, Tonelli G, Yue W, Deloménie C, Nicolas V, et al. Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like/progenitor cells. Oncogene. 2013;32(18):2261-72. doi: 10.1038/onc.2012.252. PubMed PMID: PMC3679409. 53. Fujii S, Mitsunaga S, Yamazaki M, Hasebe T, Ishii G, Kojima M, et al. Autophagy is activated in pancreatic cancer cells and correlates with poor patient outcome. Cancer Science. 2008;99(9):1813-9. doi: 10.1111/j.1349-7006.2008.00893.x. 54. Yang M-C, Wang H-C, Hou Y-C, Tung H-L, Chiu T-J, Shan Y-S. Blockade of autophagy reduces pancreatic cancer stem cell activity and potentiates the tumoricidal effect of gemcitabine. Molecular Cancer. 2015;14:179. doi: 10.1186/s12943-015-0449-3. PubMed PMID: PMC4603764. 55. Rangwala R, Chang YC, Hu J, Algazy KM, Evans TL, Fecher LA, et al. Combined MTOR and autophagy inhibition: Phase I trial of hydroxychloroquine and temsirolimus in patients with advanced solid tumors and melanoma. Autophagy. 2014;10(8):1391-402. doi: 10.4161/auto.29119. PubMed PMID: PMC4203516. 56. Institute NC. Akt Inhibitor MK2206 and Hydroxychloroquine in Treating Patients With Advanced Solid Tumors, Melanoma, Prostate or Kidney Cancer. ClinicalTrials.gov [Internet]: Bethesda (MD): National Library of Medicine (US). ; 2011 [cited 2017 Mar 31]. https://clinicaltrials.gov/ct2/show/NCT01480154]. 57. Hospital. MG. Hydroxychloroquine With or Without Erlotinib in Advanced Non-small Cell Lung Cancer. ClinicalTrials.gov [Internet]: Bethesda (MD): National Library of Medicine (US). 2009 [cited 2017 Mar 31]. https://clinicaltrials.gov/ct2/show/NCT01026844]. 58. Hospital SKWH-SM. A Phase II Trial of Combined Hydroxychloroquine and Sirolimus in Soft Tissue Sarcoma ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2012 [cited 2017 Mar 31]. https://clinicaltrials.gov/ct2/show/NCT01842594]. 59. Health N. Hydroxychloroquine in Untreated B-CLL Patients ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2008 [cited 2017 Mar 31]. https://clinicaltrials.gov/ct2/show/NCT00771056]. 60. Pennsylvania ACCotUo. FOLFOX/Bevacizumab/Hydroxychloroquine (HCQ) in Colorectal Cancer ClinicalTrials.gov [Internet]. : Bethesda (MD): National Library of Medicine (US). ; 2010 [cited 2017 Mar 31]. https://clinicaltrials.gov/ct2/show/NCT01206530]. 29 Anexos