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Introduction (What is autophagy and questions function?)
Death of cells is associated with three different processes: apoptosis, autophagy and necrosis1. The term autophagy mean literally eating of self. There are 3 types of autophagy: chaperone-mediated autophagy, microautophagy,and macroautophagy. This report will focus on macroautophagy (referred from now as autophagy). Autophagy is the catabolic process in which a cell digest its cytoplasmic contents. Autophagy recycles energy and nutrients when stress and starvation occur2. When cells are in starvation, autophagy increases and degrades proteins and organelles. As a results of this, cells can obtain macromolecules, such as fatty acids, nucleotides and amino acids which would be unavailable when autophag y did not occurred. This results suggest that autophagy has an protective role in cells, especially when cells are in nutrient deprivation4. Autophagy is regulated by genes called autophagy regulators (atg). In research it was found that ATG plays an important role in the initiation and progression of cancer. Activation of autophagy in cancer occurs in response to stress, such as hypoxia, starvation and unfolded protein response (endoplasmatic reticulum (ER) stress2. Autophagy plays a major role as a survival mechanism in this response to stress. Studies showed that autophagy has an important homeostasis role in the balance between synthesis, degradation and recycling of components8.
Normally, autophagy function in clearing protein aggregates and damaged organelles. When autophagy fails, due to multiple causes, multiple pathological conditions can occur like cancer, bacterial and virus infections and neurodegenerative diseases.3. The role of autophagy in cancer leads till discussion. The protective role of autophagy as mentioned above means that when tumor cells are in starvation as a results of limited angiogenesis, autophagy helps the tumor cells to survive. In contrast, there is evidence that autophagy has an anticancer role. The autophagy gene beclin1 (atg 6 in yeast) is monoallelically lost in human tumorss in the breast and ovarian2. The tumors suppressor genes p53 and PTEN, which are often mutated in tumorss, induces autophagy5,6, which suggest that autophagy serves as anticancer role. The mechanism of how autophagy serves an anticancer role is unclear, but recent studies suggest that autophagy limits tumors cell growth, reduces mutagenesis and other damages caused by reactive oxygen species (ROS)7. The above mentioned data suggest that autophagy can stimulate and prevent cancer, depending on the context. In this report, the regulation and mechanism autophagy will be mentioned. Further, the role of autophagy in cancer will be analysed, and possible therapies of cancer in relation to autophagy will be discussed.
Normal Molecular mechanism autophagy. (LC3,phagosome, lysosome etc.)
Autophagy is a lysosomal pathway involved in the turn over of organelles and long living proteins9. Autophagy starts with a phagophore, which is probably derived from a lipide bilayer made by the endoplasmatic reticulum (ER) and the trans-golgi system and endosomes10. This phagophore expends to take up cell compartments, such as protein aggregates, ribosomes and organelles. In this phagophore the cell compartments are taken up in a double membramed autophagosome11. Then the autophagosome with the uptaken cell compartments matures cause the autophagosome fusions with the lysosome. This process promotes the degradation of autophagosomal
contents by lysosomal acid proteases. Products of degradation, such as permeases and transporters export amino acids, are released back into the cytoplasma, where these products can be used for building macro molecules and for metabolism. This mechanism shows that autophagy recycles cellular compartments, which also creates ATP and has a protective function in cells by removing damaged cell compartments11.
In yeast, the phagophore membrane formation is formed around the pre-autophagosomal structure (PAS), but no evidence is found that this happens also in mammels12. Probably the PAS in mammals is directly made in the ER in transaction with the trans-golgi and late endosomes13. For phagosome formation in yeast, activity of the atg1 kinase in complex with the Atg13 and Atg17 is required, possible for regulating the protein atg9 that may act by promoting lipid recruitment to the expanding phagophore12. This process is regulated by the energy sensing TOR, which phosphorylates Atg13. This phosphorylation prevents that Atg13 interacts with Atg1, which leads to rendering initiation of autophagy14. ULK-1 and ULK-2 are mammalian homologues of the Atg1 in yeast. It is unknown which of these two is the analogue in promoting autophagy in mammals15. The step of autophagosome formation in mammals requires further in investigation.
The role of Class 3 PI-3 Kinases (PI3K), notably vesicular protein sorting 34 (vps34) and his binding partner beclin-1 (atg6) in phagophore formation and autophagy is well understood in mammals. Vps34 is involved in membrane processed in the cell, but is only involved in autophagy when it is complexed to beclin-116. The interaction of beclin-1 with vsp34 increased level of PI3K, but how this is regulated when cells are in starvation is not clear yet.
Two ubiquitin like systems play a role in promoting autophagy: the Atg5-Atg12 conjugation and the microtubule associated protein light chain 3 (LC3) processing step. In the Atg5-Atg12 conjugation step, the Atg12 is activated by Atg7 and is transferred to Atg10. Comjugated Atg5-Atg12 complexed forms pairs with Atg16L to form a complex that associated with the extending phagpophore17. When the autophagosome is formed, the complex dissociated from the membrane, which makes the conjugated Atg5-Atg12 a poor marker of autophagy. Genome wide association studies linkes a mutation in the in Atg16L to Crohnâ€™s disease18. The other ubiquitin like system involved in the formation of the autophagosome is the processing of microtubuleassociated protein LC3B, which is the mammalian homologue Atg8. LC3B is expressed as a cytosolic protein, that is cleaved by Atg4 to generate LC3B-1. Activated LC3B-1 is transferred to Atg3 and then LC3B-2 is generated. Dependent of the Atg5-Atg12, recruitment and intergration of LC3B-2 happens. LC3B-2 is found on the external and internal surfaces of the autophagosome, and plays a role in hemifusion of membranes and selecting cell compartments for dergradation19. During autophagy, the synthesis and processing of LC3 is increased, which makes it a good marker for levels of autophagy. The role of LC3-related moclecules in not clear. It is thought that that differences in protein interactions may determine which cell compartments are selected for uptake by the autophagosome20.
Uptake of cell compartments by autophagosomes was thought as a random process, but evidence is growing that the membrane of phagophore can interact with protein aggregates and organelles. LC3B-2 may act as a receptor at the phagophore, which interacts with cell compartments that are a target for autophagy. The molecule p62/SQSTM1 is am multifunctional molecules that promotes turnover of poly-ubiquitinated protein aggregates. Mutation of p62/SQSTM1 is linked to Pagetâ€™s disease21. In yeast, Uth1p and Atg31 promotes selective uptake of mitochondria, also known as mitophagy22.
The autolysosome is formed when the autophagosome fuses with the lysome11. In the autolysosome cell compartments are degradated by lysosomal proteases of engulfed molecules. This degradation leads to smaal molecules, mostly amino acids, and these are transported back into the cytosol for protein synthesis and maintenance of cellular functions under starvation canditions24.
Figure1. Mechanism of autophagy. (a) Capture of random or selective targets for degradation, (b) completion of the autophagosome, (c) fusion of the autophagosome with the lysosome (d) proteolic degradation by lysosomal proteases of engulfed molecules40
Major pathways and molecules autophagy
There are different pathways that leads to autophagy. The central player in these pathways is the target of rapamycin (TOR) kinase. TOR kinase plays a major role sensensing ATP and amino acids, and it cam integrate hormonal stimuli wit the PI3K/PKB pathway. The TOR compex 1 (TORC1) is inhibited by rapamycin. When TORC1 is inactivated, autophagy is stimulated in the presents of nutrients, which suggest that TOR down regulates autophagy25. It is thought that mTORC1 is phosphorylated when nutrients are available26. In presence of amino acids, hVps34 is stimulated, which results in mTOR activation and autophagy inhibition27. The Ras/cAMP dependent protein kinase a (PKA) pathway plays a major role in glucose sensing in mammals. When nutrients are available, small GTPases Ras1 and Ras2 are activated and this leads to elevation of cAMP generation. The cAMP binds to Bcy1 and this inhibits PKA28. Activation of the Ras/PKA pathway suppresses autophagy. TSC is also a regulator in the autophagy pathway. TSC has a GTPase activating protein function, which hydrolyzes GTP, which regulates Rheb negatively, and inhibits mTORC1 activity54.
Hormones also may play a important role in the regulation of autophagy. The hormones glucagon and ecdysone (in Drosophila) inhibits TOR by downregulating PI3K, which results in an increase of autophagy. The hormone insuline has the opposite effect, and has an inhibitory effect on autophagy29. As said, amino acids are the final product of autophagy. Amino acids acts as a negative feedback for autophagy. The amino acids TOR signalling pathway can be influenced by the AMP dependent protein kinase (AMPK). The presence of ATP in also inhibits autophagy30. If ATP is low in cells, the concentration of AMP will increase. AMPK is activated when the ratio of ATP/AMP is different then in normal situations. Autophagy can be suppressed by AMP due to activation of AMPK. It is known that AMPK is involved in the mTOR signalling pathway, but his role in autophagy is not clarified yet. Other regulator molecules of autophagy are PTEN, p53, PI3K and akt.
Figure 2. Pathways of autophagy.cell signal.com
Stress and autophagy
Various stressors can induce autophagy, what helps the cells to survive under stress circumstances. The endoplasmatic reticulum (ER) is an important compartment of the cell to synthesize proteins and initiates the pathway of proteins to organelles to the cell surface. The ER is in mammals a Ca2+ reservoir. ER stressors are expression of aggregate prone proteins, glucose deprivation, Ca2+ efflux form the ER, oxidative stress and hypoxia. These types of stress leads to accumulation of unfolded proteins in the ER31. In yeast, the unfolded protein response (UPR) is mediated by Ire1. The UPR causes that an a member of the heat shock protein 70 family, activates Ire1. Activated Ire1 triggers the splicing of Hac1, which activates genes involved in protein modification, vesicle transport and ER-associated degradation32. In mammals, knockdown of the UPR regulator inhibits autophagosome formation, but has no influence non LC3-2, which suggest that knockdown of the UPR regulator is essential for autophagy33. The UPR response is more complex in mammals then in yeast. In mammals, there are three pathways of UPR that leads to autophagy: the IRE1, PERK and the ATF6 pathway. these factors recognize misfolded proteins and activate target genes. If ER stress autophagy had a prosurvival role in cells, or causes cell death is not known yet34.
Hypoxia is the condition in which low levels of oxygen are available for cells. Hypoxia exist in many pathological conditions, like tumorss, cadiovasculaire ischemia and brain injuries. Recent data shows that autophagy is a results of hypoxia in mammal cells. The study of the relation of autophagy and hypoxia is in the beginning stage, but pathways for autophagy mediated by hypoxia seems to be different for types of cells35. The Hypoxia inducible factor 1 (HEF1) is transcripted onder hypoxic conditions, and promotes transcription factors thath encodes for erythropoieses and angiogenesis. In mouse embryonic fibroblasts, mitochondria are mitophaged, dependent of HIF136 . Autophagy caused by hypoxia is partially TOR dependent, and ER caused by hypoxia might play a role in autophagy induction.
(ROS) are a common stress that leads to autophagy. The mitochondria are the major cell compartement in producing ROS, what will cause damge in cells. ROS producing molecules induce autophagic cell death in cancer cell lines. In healthy cells, ROS maintains at a stable level and cells can protect themselves for ROS, for example with superoxide dismutase (SOD). Over expression of SOD reduces autophagy37. Also had autophagy a major role in eliminating pathogens when the body is invaded. Toll like receptors recognize LPS, ssRNA and zymoson, which induces autophagy in the adaptive and the innate immune response, when bacteria and viruses are recognized38,39.
Autophagy and link with cancer (tumor suppression and oncogenes and regulation)
Autophagy had an important role in providing tumors cells from nutrients in periods during metabolic stress when the apoptose pathway is disrupted41. Due to autophagy, tumors can survive for weeks in vitro and in vivo in the periods of metabolic stress42. The role of autophagy is tumors was first discovered by a study of the beclin 1 gene, which was monoallelically deleted in a high percentage in prostate, ovarian and breast cancers. Expression of beclin1 is reduced in breast carcinomas43. In mice lacking one copy of beclin1, there was an high incidence of tumors, like B cell lymphoma and lung carcinomes44. This data suggest that beclin1 functions as a tumor suppression, and that autophagy has a role in tumor suppression. Next to beclin1, mutations in other genes that play a role autophagy, are associated with different types of cancer.
In autophagy defective cells more DNA double strand breaks are seen when metabolic stress occurs. This study was done with an inactivated p53, a tumor suppressor gene which normally maintains genome stability45. Incompetent autophagy cells might enhance DNA damage, that results in an increase of mutations in cells, which will lead to tumorgenesis. Autophagy Defective cells has as already said, defect in protein turnover, which leads to accumulation of scaffold protein p62, what is a major contributor to tumorgenesis46.
Another mechanism of how autophagy defected cells will lead to tumorgenesis is that autophagy defected cells causes necrose during metabolic stress, while cells which can induce normal autophagy, are more resistant to cell death when metabolic stress occurs47. In response to necrose, inflammatory cells invade tumor sites. When pro tumor inflammatory cells like macrophages invade, this has an poor prognoses for patients in clinic48. When autophagy in tumor cell necrosis is limited, this might serves as an mechanism for tumor suppression.
Autophagy and tumor cell survival
To proliferate, tumor cells have a high demand for oxygen and nutrients. This is why tumor cells, which often are poorly vascularized, often encounter hypoxia and metabolic stress. Cells on the inside of the tumor have a higher incidence of autophagy than cells on the edge of the tumor. This protects the cells from apoptosis and necrosis49. Autophagy has a pro survival effect on tumors, and also might has influence on metastasis, which is the leading cause of mortality in patients.
Autophagy and disturbed regulation in cancer
The most important regulator of autophagy, mTORC1 is deregulated in most human cancer50. Genes that regulates mTORC1 are mostly identified as oncogenes and tumor suppressor genes. An example of a gene that regulates is Rheb, which is over expressed in prostate cancer. Loss of TSC, caused high Rheb activity, which inhibits autophagy. PI3K and akt, are activators of mTORC1. In cancer, PI3K activity is increased, which should inhibit autophagy. However, in colon cancer the products of PI3K suppresses autophagy51. The regulator of the PI3K-akt pathway is the tumor suppressor gene PTEN, and when PTEN is over expressed this leads to inducing of autophagy52. Another tumor suppressor gene, ARHI, which function is lost in ovarian cancers, also regulates the PI3K pathway, which induces autophagy53.
Autophagy pathways as drug targets/ autophagy and cancer therapy