Autophagy: A Janus Faced Pathway

AUTOPHAGY: Autophagy (aka Macroautophagy) is a conserved intracellular pathway concerned with delivering organelles (i.e mitochondria), bulk cytoplasm, infectious agents and aggregate-prone proteins in double-membrane vesicles named autophagosomes to lysozomes for degradation (1). In mammalian cells, macroautophagy is one of the three autophagic processes, the other two being chaperone-mediated autophagy and microautophagy (2,3). The process initiates with phagophores bending at the edges to create autophagosomes. The formation occurs throughout the cytoplasm in most mammalian cells, and autophagosomes with captured content are then trafficked via the dynein machinery towards the microtubule-organizing center, where they are within reach of the degrading lysosomes1 (see Figure 1). An autophagosome fused with lysosome forms an autophagolysosome1. Autophagy functions are preserved through yeast to man, and they are to protect cells from starvation as well as the related stress. Through autophagy upregulation in such conditions, macromolecules are degraded into building blocks, which can then be used to generate energy (1). The degraded materials are recycled out of the lysosome into the cytoplasm, where they can be utilized for new metabolic and biosynthetic processes.

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Autophagy has been a subject of study in context of many diseases. It is an applicable target in neurodegenerative diseases, where certain aggregate-prone intracytoplasmic proteins like α-synuclein (Parkinson’s disease), tau proteins (dementia) or mutant huntingtin (Huntington’s disease) need to be degraded to ameliorate patient’s health (1). Autophagy also protects from some infectious diseases caused by pathogens such as M. tuberculosis and S.typhi (1). Additionally, autophagy can be beneficial under these conditions by alleviating inflammatory responses and protecting against cell death (4). The process plays an ambiguous role in cancer. Generally, the recycling function of autophagy allows established tumors to survive. However, in cancer, the role of the process is context-dependent. At early stages of the disease, autophagy suppresses tumor growth initiation. At later stages, autophagy improves the survival abilities of an established cancer by protecting it from metabolic stresses, like hypoxia, as well as therapeutic stresses, such as radiotherapy and chemotherapy (5). Tumors can be autophagy-dependent, and such characteristic makes it a potential drug target. Autophagy has been the scope of many preclinical and clinical trials, but thus far the only approved autophagy inhibitor has been the anti-malarial chloroquine (CQ) and its derivatives, for example hydroxychloroquine (HCQ) (6). The mechanism of CQ and derivatives is still not very well understood (7), although HCQ is known to inhibit lysosomal acidification, thus preventing autophagosome degradation and ultimately suppressing autophagy (7,8).

CLINICAL TRIALS: First clinical trials focusing on autophagy were motivated by the successful preclinical data recorded whilst experimenting with HCQ in renal cell carcinoma and breast cancer cell lines. The first clinical trial studied antineoplastic activity and safety of HCQ in 20 patients suffering from metastatic pancreatic cancer who were not responsive to conventional chemotherapy (9). The medicine showed promising results, however, two out of twenty subjects developed grade 3/4 side effects stemming from the treatment. Six other phase I clinical trials followed and were published in 2014, studying HCQ as an autophagy inhibitor (10-15.) The trials used HCQ in combination with different cancer chemotherapy and targeted therapies, such as bortezomib, temozolomide, doxorubicin, vorinostat or temsirolimus. There are also currently recruiting or scheduled clinical trials that aim to examine the role of HCQ in different conditions.

A currently recruiting non-randomized phase 2 study (NCT03037437) at the University of Texas Health Science Center in San Antonio aims to compare the efficacy of sorafenib (a drug inhibiting multiple kinases (16)) combined with HCQ against sorafenib on its own in hepatocellular cancer. It is predicted to enroll 68 patients, has started in February 2018, and final data collection is planned for December 2018.

Another phase 2 open label, randomized, interventional study at The University of Texas Health Science Center in San Antonio focuses on studying autophagy modulation in patients with histological documentation of metastatic colorectal cancer (NCT02316340). HCQ is combined with vorinostat (a histone deacetylase inhibitor (17)), and the active comparator is regorafenib (a protein kinase inhibitor (18)). The study started in February 2015 and is currently recruiting up to 76 eligible participants with the estimated completion date being June 2018.

A study that is yet to start recruiting at Maastricht Radiation Oncology in the Netherlands plans to test the effects of incorporating CQ into the regular treatment regimen for glioblastoma (GBM) (NCT02432417) (See Glioblsatoma Review). It is a multi-centre controlled randomized, interventional phase II clinical trial aimed at new cases of GBM. Its estimated enrollment is 156 participants, planned to start in January 2020 and finish by January 2022.

FUTURE PROSPECTS: Autophagy research is an exciting area that is gaining increasingly more recognition and poses many possibilities for the future. The 2016 Nobel Prize in Physiology or Medicine has been awarded to Yoshinori Ohsumi for ‘’discoveries of the mechanisms for autophagy’’ – a crowning achievement highlighting the importance of autophagy research (19). Although it has been actively studied for decades, we are just now beginning to understand the importance and potential that autophagy has. Alterations to autophagy and mutations in autophagy genes, known as the ATG genes, have been linked to such human conditions as autoimmune disease, neurological diseases, cancer, infectious diseases and metabolic disorders (2). Therapeutic interventions aiming to increase or inhibit autophagy thus appear as a natural next step in research. In the years to come autophagy altering drugs and therapies may pave the way to a whole new approach to treating a variety of diseases, such as Parkinson’s disease or pancreatic cancer.

LABORATORY RESOURCES: Autophagy is a dynamic process, and therefore its study requires constant monitoring. ThermoFisher Scientific offers a set of tools designed specifically for tracking autophagy at different stages. The tools include chemical sensors sensitive to LC3B and p62 protein detection (two proteins associated with early stages of autophagy) from the PremoTM product line:

https://www.thermofisher.com/order/catalog/product/P36235

https://www.thermofisher.com/order/catalog/product/P36236

https://www.thermofisher.com/order/catalog/product/P36239

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Antibody products play an important role in autophagy research. Utilized in techniques such as western blotting, immunocytochemistry or immunohistochemistry, antibodies allow detection and analysis of the presence of specific proteins. Novus Biologicals offers autophagy antibody packs containing relevant antibodies required to perform such operations in the context of autophagy research:

https://www.novusbio.com/products/autophagy-antibody-pack_nb910-94159

https://www.novusbio.com/products/autophagy-antibody-pack_nb910-94877

Invivogen is a company that offers a vast array of autophagy research products that may be of interest to anyone pursuing a study in autophagy. Their offer includes a variety of products, ranging from autophagy inducers to autophagy reporter cells:

https://www.invivogen.com/autophagy

 References:

1.     Bento, C., Renna, M., Ghislat, G., Puri, C., Ashkenazi, A., Vicinanza, M., Menzies, F. and Rubinsztein, D. (2016). Mammalian Autophagy: How Does It Work?. Annual Review of Biochemistry, 85(1), pp.685-713.

2.     Rubinsztein, D., Codogno, P. and Levine, B. (2012). Autophagy modulation as a potential therapeutic target for diverse diseases. Nature Reviews Drug Discovery, 11(9), pp.709-730.

3.     Ozpolat, B. and Benbrook, D. (2015). Targeting autophagy in cancer management – strategies and developments. Cancer Management and Research, pp.291-299.

4.     Rubinsztein, D., Bento, C. and Deretic, V. (2015). Therapeutic targeting of autophagy in neurodegenerative and infectious diseases. The Journal of Experimental Medicine, 212(7), pp.979-990.

5.     Rubinsztein, D., Codogno, P. and Levine, B. (2012). Autophagy modulation as a potential therapeutic target for diverse diseases. Nature Reviews Drug Discovery, 11(9), pp.709-730.

6.     Renna, M., Bento, C., Fleming, A., Menzies, F., Siddiqi, F., Ravikumar, B., Puri, C., Garcia-Arencibia, M., Sadiq, O., Corrochano, S., Carter, S., Brown, S., Acevedo-Arozena, A. and Rubinsztein, D. (2013). IGF-1 receptor antagonism inhibits autophagy. Human Molecular Genetics, 22(22), pp.4528-4544.

7.     Zavodszky, E., Seaman, M., Moreau, K., Jimenez-Sanchez, M., Breusegem, S., Harbour, M. and Rubinsztein, D. (2014). Mutation in VPS35 associated with Parkinson’s disease impairs WASH complex association and inhibits autophagy. Nature Communications, 5(1).

8.     Moreau, K., Ghislat, G., Hochfeld, W., Renna, M., Zavodszky, E., Runwal, G., Puri, C., Lee, S., Siddiqi, F., Menzies, F., Ravikumar, B. and Rubinsztein, D. (2015). Transcriptional regulation of Annexin A2 promotes starvation-induced autophagy. Nature Communications, 6(1).

9.     Liu, J., Xia, H., Kim, M., Xu, L., Li, Y., Zhang, L., Cai, Y., Norberg, H., Zhang, T., Furuya, T., Jin, M., Zhu, Z., Wang, H., Yu, J., Li, Y., Hao, Y., Choi, A., Ke, H., Ma, D. and Yuan, J. (2011). Beclin1 Controls the Levels of p53 by Regulating the Deubiquitination Activity of USP10 and USP13. Cell, 147(1), pp.223-234.

10.  Maiuri, M., Le Toumelin, G., Criollo, A., Rain, J., Gautier, F., Juin, P., Tasdemir, E., Pierron, G., Troulinaki, K., Tavernarakis, N., Hickman, J., Geneste, O. and Kroemer, G. (2007). Functional and physical interaction between Bcl-XL and a BH3-like domain in Beclin-1. The EMBO Journal, 26(10), pp.2527-2539.

11.  Platta, H., Abrahamsen, H., Thoresen, S. and Stenmark, H. (2012). Nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor Beclin 1. Biochemical Journal, 441(1), pp.399-406.

12.  Sun, T., Li, X., Zhang, P., Chen, W., Zhang, H., Li, D., Deng, R., Qian, X., Jiao, L., Ji, J., Li, Y., Wu, R., Yu, Y., Feng, G. and Zhu, X. (2015). Acetylation of Beclin 1 inhibits autophagosome maturation and promotes tumour growth. Nature Communications, 6(1).

13.  Yang, Y., Fiskus, W., Yong, B., Atadja, P., Takahashi, Y., Pandita, T., Wang, H. and Bhalla, K. (2013). Acetylated hsp70 and KAP1-mediated Vps34 SUMOylation is required for autophagosome creation in autophagy. Proceedings of the National Academy of Sciences, 110(17), pp.6841-6846.

14.  Xu, D., Zhang, T., Xiao, J., Zhu, K., Wei, R., Wu, Z., Meng, H., Li, Y. and Yuan, J. (2015). Modification of BECN1 by ISG15 plays a crucial role in autophagy regulation by type I IFN/interferon. Autophagy, 11(4), pp.617-628.

15.  Codogno, P., Mehrpour, M. and Proikas-Cezanne, T. (2011). Canonical and non-canonical autophagy: variations on a common theme of self-eating?. Nature Reviews Molecular Cell Biology, 13(1), pp.7-12.

16.  Bnf.nice.org.uk. (2018). SORAFENIB | Drug | BNF Provided by NICE. [online] Available at: https://bnf.nice.org.uk/drug/sorafenib.html [Accessed 27 Jun. 2018].

17.  Chemocare.com. (n.d.). Vorinostat - Chemotherapy Drugs - Chemocare. [online] Available at: http://chemocare.com/chemotherapy/drug-info/Vorinostat.aspx [Accessed 27 Jun. 2018].

18.  Bnf.nice.org.uk. (n.d.). REGORAFENIB | Drug | BNF Provided by NICE. [online] Available at: https://bnf.nice.org.uk/drug/regorafenib.html [Accessed 27 Jun. 2018].

19.  Levine, B. and Klionsky, D. (2017). Autophagy wins the 2016 Nobel Prize in Physiology or Medicine: Breakthroughs in baker's yeast fuel advances in biomedical research. Proceedings of the National Academy of Sciences, 114(2), pp.201-205.

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