Autophagy in Colon Cancer - Literature review Example

?Autophagy in Colon Cancer Background information Cancer cells are continuously proliferating and they need nutrients for their growth. Since the proliferation rate is more than the normal cells, their nutrient requirement is also more. One of the ways in which nutrient supply is increased is by way of increased blood supply through tumor angiogenesis. The increased blood supply increases oxygen and also nutrient supply for increased proliferation. However, some recent studies have thrown light upon the fact that the glucose and oxygen levels of tumors that are locally advanced are actually less than the levels necessary for normal tissue proliferation, despite establishment of tumor vessels. From this point, it is evident that the microvasculature of the tumor tissue is actually structurally and functionally deficient and hence is unable to provide blood supply that is prerequisite for appropriate tissue growth. To support this fact, there is evidence that some tumors, like the pancreas cancers are actually hypovascular (Sato et al, 2007). These conditions contribute to hyponutrient state of the tumor. However, for hypoproliferation, excess nutrient supply is mandatory and hence tumor cells are likely to use alternative source of energy and nutrients or some alternative metabolic process for the purpose. One such metabolic process is autophagy. There is evidence that some cancers, like the colon cancer, are resistant to nutrition depletion state and continue to thrive because of this metabolic process (Sato et al, 2007). Autophagy is a catabolic process that is conserved in which the organelles of the cells are self-digested. The first step in autophagy is development of isolation membrane, a lipid bilayer structure. This membrane sequesters various materials of the cytoplasm like the organelles to form autophagosomes. This step involves activation of LC3, a mammalian homologue of yeast ATG8 through an ubiquitination-like reaction that is regulated by ATG3 and 7. During activation, the proform of LC3 is cleaved into LC3-I which is soluble unlike the proform. This is then further modified into LC3-II which is membrane-bound form. This form is finally recruited by the autophagosomes which engulf the organelles (Rosenfeldt and Ryan, 2009). The engulfed organelles further fuse with the lysosomes and then mature into autolysosomes. This step also causes autodigestion and diminision of LC3 and also various other components of autophagosome. Thus autophagy has an important role to play in the provision of nutrition to cells during shortage of external supply of nutrition. Following autodigestion, aminoacids are released from the organelles and they are the alternative sources of energy to the proliferative and nutrient deficient cells. Though, theoretically, this explanation seems logical with reference to nutrition supply to cancer cells in unfavorable environment, several controversial arguments have arisen in this regard. Some researchers are of the opinion that autophagic machinery may not be activated in cancer tissue contexts (Sato et al, 2007). However, there is enough evidence to point the role of autophagy in the pathogenesis of colon cancers. In this literature review the role of autophagy in the proliferation and thriving of colon cancers will be discussed through suitable literature review. Pathogenesis of colon cancer Cancer of the colon (and rectum) is the third most common cancer in men and women. It has been estimated that 940,000 new cases of colorectal cancer and nearly 500,000 deaths are occur worldwide each year (El- Deiry, 2006). The frequency is same both in men and women. The risk of the disease increases after 40 years of age (El- Deiry, 2006). Colon cancer (colorectal cancer) is almost always adenocarcinoma. The most common predisposing condition leading to adenocarcinoma is adenomatous polyps. Alterations in the adenomatous polyposis or APC gene as a result of mutations is the beginning point of development of the cancer. This gene is mutated in individuals affected by familial adenomatous polyposis. APC gene encodes a protein that targets degradation of beta-catenin, a protein component of a transcriptional complex that activates growth-promoting oncogenes, such as cyclin D1 or c-myc. In mutation, the earliest event is DNA methylation and this can be detected even at the polyp stage. This leads to global hypomethylation and regional hypermethylation. While hypomethylation leads to oncogene activation, hypermethylation contributes to silencing of tumor supressor genes. One important mutation is ras gene mutation which is commonly noted in larger polyps and not in smaller polyps prompting the role of this mutated gene in polyp growth. Other mutations include chromosome arm 18q deletions, chromosome arm 17p losses, tumor suppressor p53 mutations, Bc12 over expression and 18q deletions. Chromosome arm 18q deletions are likely to involve the targets DPC4 (a gene deleted in pancreatic cancer and involved in the transforming growth factor [TGF]-beta growth-inhibitory signaling pathway) and DCC (a gene frequently deleted in colon cancer). While Bc12 over expression is an early event in colon cancer, chromosome arm 17p losses and tumor suppressor p53 mutations are late events. 18q deletions detected in Dukes stage B colon cancers are associated with an increased risk of recurrence following surgery (El- Deiry, 2006). Rarely, adenocarcinoma of the colon is predisposed by hereditary nonpolyposis colon cancer. Individuals affected by this condition inherit a mutation in one of several genes involved in DNA mismatch repair, including MSH2, MLH1, and PMS2 (El- Deiry, Emedicine). Familial nonpolyposis colon cancer accounts for 1-5% of colon cancers (Lynch & Chapelle, 1999, p. 801). There is some evidence to suggest that autophagy plays a major role in the tumorigenesis and pathogenesis of colon cancers and their predisposing tumors, the adenomas. Literature review Autophagy and colon cancer Autophagy is basically a multistep process and is orchestrated by certain subset of genes. These genes were originally identified in yeast and were called autophage-related genes or ATG. Many of these genes have mammalian orthologues (Rosenfeldt, and Ryan, 2009). During initiation in mammalian cells, the serine/threonine kinase complex is activated. This complex contains ULK1/2, FIP200 and ATG13. The complex transfers necessary signals from mammalian/mechanistic target of rapamycin (mTOR) kinase to cause initiation of autophagy. mTOR induced phosphorylation of ULK and ATG3 is inhibited and this causes liberation of kinase activity of ULK. This liberated form of ULK than phosphorylates itself and also FIP200 and ATG13. Then, the ULK complex accumulates and causes initiation of vesicle formation. The focus of vesicle formation is known as phagophore or isolation membrane (Rosenfeldt, and Ryan, 2009). After initiation, the next step is autophagosome formation. Further development of the phagophore is dependent on the class III phosphoinositide 3-kinase (PI3K-III) activity, activity of hVps34 (PIK3C3; the orthologue of yeast Vps34) and formation of complex between Beclin 1 and p150/hVps35. Beclin 1 is yeast ATG6 and p150/hVps35 is yeast Vps15. The vesicle is then elongated and completed from the initial phagophore and this is dependent on the ubiquitin-like conjugation systems, ATG8 and ATG12 systems. The phagophore turns into nascent autophagosome. ATG12 gets activated by ATG7. The activation causes binding of ATG12 to E2-like enzyme ATG10. This binding is temporary and ATG10 is transferred to ATG5. This then reacts with ATG16 and forms a complex ATG12–ATG5–ATG16 (Rosenfeldt, and Ryan, 2009). There are several orthologues of ATG8 and they are GABARAPL1 (ATG8L), MAP1LC3 (LC3), GABARAP and GABARAPL2 (GATE16). These orthologues too are subjected to modification as described above. Of these, the most thoroughly investigated orthologue is LC3. The precursor form of LC3 is proLC3. However, this, soon after its formation is is cleaved at its C-terminal aminoacid by ATG4 and converted to LC3-1. LC3-1 is then reversibly conjugated with phosphatidylethanolamine (PE) by ATG7 and ATG3 at the C-terminus to form LC3-II. This completes the process of maturation of LC3 (Rosenfeldt, and Ryan, 2009). Maturation involves fusion of autophagosomes with lysosomes to form autolysosome which is the end-stage autophagy vesicle. There is not much research on the molecular mechanism of maturation. Some emerging reports have suggested the role of lysosomal proteins LAMP1 and 2, UVRAG, the small GTPase Rab7 (RAB7A) and others in the the maturation of autophagosome. UVRAG is basically tumor suppressor and regulates the interaction between Beclin 1 and hVps34 during vesicle nucleation. It also directs the tethering proteins to the membrane of the autophagosome and then activates Rab7 to facilitate fusion of it with lysosomes (Rosenfeldt, and Ryan, 2009). This is the final autolysosome and this is basically an acidic vesicle. The intracellular material is degraded by the hydrolases of the lysosome, mostly by the set of hydrolases called cathepsins. This catabolic process generates aminoacids and other sources of energy which are released to be used as fuel for other cells (Rosenfeldt, and Ryan, 2009). Figure-1: Mechanisms of autophagy (Rosenfeldt, and Ryan, 2009) Role of autophagy in cancer Transformation of normal cells to malignant cell involves overriding of various cellular mechanisms like oncogene-induced senescence and apoptosis that safeguard the normal cells. Such a change occurs mainly due to accumulation of significant mutations. There is overwhelming evidence of link between cancer and autophagy, especially colon cancer and autophagy (Lorin et al, 2008). Such an evidence has been accumulated through research in mouse models and animal models. One of the important autophagy-regulating gene has been Beclin 1 or BECN 1 (Ahn et al, 2007). Infact, this gene provided the first link between autophagy and cancer. Studies have shown that BECN1 is deleted in more than 50 percent of prostate, ovarian and breast cancers monoallelically (Rosenfeldt, and Ryan, 2009).. In various brain tumors, it is only expressed in low levels. Bialleic loss of this gene has been found to be lethal in mice. Also, those who have one gene copy are viable, but have higher incidence of carcinomas of the liver, lung, breast and hemotopoietic system. There is evidence that reintroduction of this gene into breast cancer cells of MCF7 which have only traces of this gene causes restoration of autophagic capacity and inhibition of their tumor forming potentia (Rosenfeldt, and Ryan, 2009).. Thus, from these evidences, it can be said that Beclin 1 is a tumour suppressor gene of haplosufficient type (Ahn et al, 2007). Another set of important autophagy regulators are ATG5 and ATG7. In mouse models with deficiency of these regulators, there seemed to be no lethality and the mice were born normally. However complete deletion of these regulators in mice caused lethality within 24 hours because of lack of compensation for neonatal starvation. Also, those hemizygous with these regulators appeared to be viable and normal, but unlike BECN-1 deficiency, did not develop tumors. Another autophagy regulator, AT4 has four mammalian orthologues. Of these, the most widely expressed is ATG4C or autophagin-3. Animals with knockout of this gene are born viable, thrive in the neonatal starvation period and also develop normally. When nutrients are restricted in these animals, autophagy is seen in the diaphragms of these animals. These prototypes are at risk of development of fibrosarcomas. According to Marino et al (2007; cited in Rosenfeldt, and Ryan, 2009), "a functional redundancy of the different ATG4 proteins in mammals might explain the notion that ATG4C deficiency does not alter basal autophagic activity in vivo and the fact that ATG4C-deficient mice survive the neonatal starvation period." There is some evidence that the tumor suppressor gene UVRAG is deleted monoallelically in a significant number of colorectal cancers in humans. There is also proof that expression of UVRAG in human HCT116 colon carcinoma cells causes reduction of the tumorigenic potential of the cells. Research has shown that in several gastric and colorectal cancers with instability of the microsatellite, UVRAG is the main target for frameshift mutations. Thus, deficiency of autophagy is one of the causes for intestinal tumorigenesis. This fact is further supported by the point that in more than 28 percent of colorectal and gastric cancers, frameshift mutations have been seen in other autophagy related genes also like ATG 5 and 12, and ATG 2B and (Rosenfeldt, and Ryan, 2009). BIF1 is also one of the regulators of autophagy and this protein exerts its action mainly through interaction with other autophagy regulators, Beclin 1 and UVRAG. Mice with knock out of this regulator are born normal, thrive through neonatal starvation period and also develop normally, but have enlarged spleen and higher risk of development of spontaneous cancers like lymphomas. Thus, BIF1 is an important autophagy regulator and this a potential cancer suppressor (Rosenfeldt, and Ryan, 2009). Figure-2: Contrasting roles for autophagy during cancer development, progression, and treatment. Hippert et al, 2006) Oncogenes and tumor suppressors Several oncogenes and tumor suppressors have opposing effects on the autophagy and apoptosis due to either direct or indirect regulation of Beclin1 and mTOR and also several other unknown mechanisms. PI3K-I (class I phosphoinositide 3-kinase), a lipid kinase, causes cell growth and proliferation and at the same time leads to inhibition of autophagy by activating mTOR (Gozuacik and Kimchi, 2004). PTEN (phosphatase with tensin homologue) mutation is seen in several malignancies, specifically in the cancer predisposition syndrome Cowden disease. Inactivation of PTEN through mutation causes suppression of autophagy through negative regulation of PI3K-I (Meley et al, 2006). Infact, PTEN loss is the most common cause of PI3K-I activation and it is the second most frequently mutated cancer suppressor in humans (Meley et al, 2006). Ras–MEK–ERK pathway has a major role to play in mTOR activation and this pathway is deregulated in several human cancers. STK11 (serine/threonine kinase 11; also known as LKB1) is another tumor suppressor gene that has been reported to be mutated in patients with intestinal polyposis, which is a major predisposing factor for development of colon cancer. This gene regulates autophagy positively through mTOR and AMP-activated protein kinase. Similarly, even DAPK1 (death-associated protein kinase 1), ARHI (aplasia Ras homologue member I), p53, ARF [‘alternative reading frame’ of the CDKN2A (INK4A) locus], NF1 (neurofibromin), REDD1 (regulated in development and DNA-damage responses 1) and Deptor (DEPDC6) are both a tumor suppressors and positive regulators of autophagy. ARHI is down regulated in more than 60 percent of ovarian cancers (Gozuacik and Kimchi, 2004). Role of autophagy in tumorigenesis Autophagy is responsible both for de novo tumorigenesis and also tumor suppression. Mechanism of both the actions are yet unclear. However, based on some research-based reports, it is possible to proposed certain mechanisms. Autophagy is known to promote survival of cells in nutrient deficient states. In some cases, it also accompanies cell death. There are reports about ATG dependent cell deaths in apoptosis inhibited systems. In the sense, inhibition of ATGs like ATG5 and 7 and Beclin1 suppressed cell death (Rosenfeldt, and Ryan, 2009). Autophagy has been strongly linked to programmed cell death. Another association between autophagy and tumorogenesis is during metabolic stress during which there is decreased blood supply or nutrients or both to the tumors in the context of rapid requirement of these substances due to increased proliferation. There is evidence of increased autophagy in hypoxic regions of cancerous tissues. The other side of the argument is that hypoxia and deficient nutrition leads to necrosis of tissue, thus triggering release of inflammatory mediators which inturn enhance tumor growth (Eisenberg-Lerner and Kimchi, 2009). Thus, autophagy, due to its capacity to impede necrosis, becomes tumor suppressive. There is some argument that autophagy actually reduces oxidative stress, maintains the quality control of protein and organelle and limits cellular damage (Degenhardt et al, 2005). It has been substantiated that during metabolic stress, ROS is accumulated along with damaged organelles and proteins which are additional sources of ROS. Due to ROS, autophagy arises as an important countermeasure. Different studies have however observed that tumor cells with defective autophagy are more susceptible to cell death, but, at the same time were more susceptible to genetic damage and thus had increased tumorogenic potential when compared to cells that were autophagy competent. One of the critical molecular link that has been identified between genomic instability, autophagy and tumorogenecity is sequestome 1. Tumor cells with impairment of autophagy and apoptosis accumulate p62 under metabolic stress, thereby promoting tumorogenesis (Rosenfeldt, and Ryan, 2009). In a study by Sato et al (2007), the researchers found formation of autophagosome by using ectopically expressed green fluorescent protein-LC3 fusion proteins in DLD-1 and SW480 cells. From these data it was suggested that autophagosomes were produced actively and then consumed promptly is colorectal cancer cells in situations of nutrient starvation. Suppressors of autophagosome formation, 3-methyl adenine and autolysosome inhibitors enhanced apoptosis remarkably under conditions of aminoacid and glucose deprivation. Similar results were obtained in cells in which ATG7 levels was decreased with RNA interference. From this study, it is evident that autophagy is a pivotal process for the survival of colon cancer cells, especially those which have acquired austerity. Another important observation noted in this study was the fact that formation of autophagosome was seen only in cancer cells and not in noncancerous epithelial cells of specimens for colorectal cancer. In toto, it was observed that autophagy in colorectal cancers was observed both in vivo and in vitro, suggesting the role of autophagy in the survival of cancer cells in microenvironment. Through such an understanding of autophagy, researchers are now beginning to look at manipulation of autophagy as a preventive and treatment strategy for cancer (Kondo and Kondo, 2006). Drugs like rapamycin induce autophagy and there are several drugs which are being developed in mouse models to target inactivation of autophagy (Hippert et al, 2006). Many cancer cells have mutations in the apoptotic machinery resulting in resistance to even advanced anti-cancer treatments. Treatment of cancer in such a scenario can be instituted by facilitating enhanced autodigestion and destruction through induction of enhanced autophagy (Moretti et al, 2007). Figure-3: Autophagy manages cellular stress to counteract tumour growth (Rosenfeldt, and Ryan, 2009) References Ahn, C.H., Jeong, E.G., Lee, J.W. et al. (2007). Expression of beclin-1, an autophagy-related protein, in gastric and colorectal cancers. APMIS., 115(12), 1344-9. Degenhardt, K., Mathew, R., Beaudoin, B., et al. (2005). Autophagy promotes tumor cell survival and restricts necrosis, inflammation and tumorigenesis. Cancer Cell, 10, 51-64. Eisenberg-Lerner, A., and Kimchi, A. (2009). The paradox of autophagy and its implication in cancer etiology and therapy. Apoptosis, 14(4), 376-91. El- Deiry, W.F. (2006). Colon Cancer, Adenocarcinoma. Emedicine from WebMD. 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