**1. Introduction**

#### **1.1. Brief history of autophagy**

The term autophagy, or sometimes known as autophagocytosis, comes from the Greek language for "self-eating." This term was coined by a Belgian biochemist, Christian de Duve in

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1963. Prior to this term, however, autophagy was first observed, or at least hinted in as early as 1955 [1]. Kleinfeld and his colleagues found that the process involves three continuous stages of maturation and is seen as being used for the reutilization of cellular materials and organelle disposal. In Duve's definition, autophagy is a part of lysosomal function and glucagon being the main inducer of hepatic cell degradation. Together with his student, Russell Deter, they were the first to demonstrate that lysosomes play a central role for intracellular autophagy [2, 3]. Using Duve's work as reference, independent scientist groups discovered autophagy-related genes in yeast. In that period, Ohsumi and Michael Thumm studied on nonselective autophagy induced by starvation [4, 5]. At the same time, Klionsky discovered a form of selective autophagy called the cytoplasm-to-vacuole targeting (CVT) pathway [6, 7]. Not longer after, they discovered that their independent work actually revolves around the same pathway. Through collaborating, they published a paper titled "Cytoplasm-to-vacuole targeting and autophagy employ the same machinery to deliver proteins to the yeast vacuole" [8]. In 2003, a unified nomenclature was advocated by scientists in the field to use ATG for autophagy-related genes [9]. More than a decade later, in 2016, Ohsumi was finally awarded a Nobel Prize in Physiology or Medicine for his contribution toward the field of autophagy. While it is undeniable that Ohsumi deserves the Nobel Prize, some individuals have pointed out that the prize should have been more inclusive of other researchers who made Ohsumi's work possible [10]. In the second millennium, there was an accelerated growth of research in autophagy thanks to the work and contribution by these scientists on ATG genes. With the fundamentals on autophagy set strong, scientists began to study its association with human health and diseases. The first breakthrough discovery associating autophagy with cancer was landmarked by Beth Levine's group in 1999 [11]. To date, the link between cancer and

Cell-Based Assays for Evaluation of Autophagy in Cancers

http://dx.doi.org/10.5772/intechopen.80088

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Autophagy is an evolutionary conserved mechanism involved in maintaining homeostasis and metabolism at the cellular level by degrading proteins with long half-life and clearing cytoplasmic organelles through lysosomes. Lipids, nucleotides, and glycogen are also subjected to lysosomal degradation via autophagy. Like other pathways involved in homeostasis, the cellular pathways are highly regulated. There are evidences that the recycling of proteins and other macromolecules may contribute to protective roles in normal development, senescence, cell death, and as a defense mechanism against intracellular infections. **Figure 1** shows the detailed processes involved in the autophagy pathway. The dysregulation of autophagy has been shown to cause cancer, inflammatory, metabolic, and neurodegenerative diseases [12, 13].

The suppression of autophagy has also been linked to an increase in oxidative stress, genome instability, and activation of the DNA damage response. The increase in oxidative stress leads to a cascade of reaction which may promote tumor growth [14]. A lack of autophagy in hepatocytes can also cause cell death and inflammation, which are known to progress to liver cancer [14]. Deficiency in autophagy has also been shown to promote the accumulation of p62, an autophagy substrate used as a reporter for autophagy activity [15, 16]. The aberrant accumulation of p62 is linked to increase toxicity and tumorigenesis [15]. In other conditions, the expression of p62 increased oxidative stress and tumor growth [17], while a suppression of p62 has been shown to suppress tumorigenesis in modified mouse with hereditary lung cancer [18]. Besides being associated to increased oxidative stress, p62 also acts as a signaling adaptor for the regulation of many oncogenic pathways, including NRF2, mTOR, and NF-κB [19]. That being said, how the dysregulation of p62 contributes toward tumorigenesis is still not known. Other than BECN1 and p62, other well-known oncogenes and tumor suppressor genes have also been reported to interrupt upon autophagic pathways, which include the PTEN/PI3K/ Akt pathway, Ras, Myc, and DAPk. The autophagy-impinging activities from these genes may have been caused by malignant transformation [20]. PTEN is a tumor suppressor gene that inhibits the pro-proliferation PI3K/Akt pathway and has been shown to promote autophagy in HT-29 colon cancer cells [21]. Thus, the loss of PTEN or upregulation of PI3K can contribute to malignancy through the inhibition of both autophagy and apoptosis. Myc on the other hand has been shown to promote apoptosis, autophagic cell death, and even oncogenesis. In rat fibroblasts, the overexpression of Myc improved autophagic activities [22]. It is interesting to note that the autophagic-inducing domain of the Myc gene is different to that of apoptosis and oncogenesis. The oncogenic Ras protein, including KRAS and NRAS, has been implicated in the promotion of p53-independent non-apoptotic autophagic cell death, which cannot be blocked by Bcl-2 overexpression. This is exemplified in a study where non-apoptotic

autophagy remains to be the top focus of autophagy researchers.

**1.2. Autophagy pathway**

**2. Role of autophagy in cancers**

**2.1. Autophagy as tumor suppressor**

**Figure 1.** Cellular autophagy processes. (1) Phagophore forms and elongates to package the cargoes comprising damaged organelles and proteins. (2) LC3B proteins are then recruited and together with phagophore, it forms autophagosome. (3) LC3B proteins are then dissociated, and the cytoplasmic cargo is sequestered to fuse with endosome-derived lysosome to form autolysosome. (4) Hydrolytic enzymes then degrade the cargoes and release metabolic products and building blocks such as amino acids and fatty acids for nutrient recycling.

was landmarked by Beth Levine's group in 1999 [11]. To date, the link between cancer and autophagy remains to be the top focus of autophagy researchers.
